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WO2024112813A2 - Systems for amplification of aav cap protein - Google Patents

Systems for amplification of aav cap protein Download PDF

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Publication number
WO2024112813A2
WO2024112813A2 PCT/US2023/080783 US2023080783W WO2024112813A2 WO 2024112813 A2 WO2024112813 A2 WO 2024112813A2 US 2023080783 W US2023080783 W US 2023080783W WO 2024112813 A2 WO2024112813 A2 WO 2024112813A2
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Prior art keywords
polynucleotide
sequence
aav
proteins
promoter
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WO2024112813A3 (en
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Sandhya PANDE
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Shape Therapeutics Inc
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Shape Therapeutics Inc
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Priority to EP23895432.5A priority Critical patent/EP4623087A2/en
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Publication of WO2024112813A3 publication Critical patent/WO2024112813A3/en
Anticipated expiration legal-status Critical
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
    • C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
    • C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • Recombinant adeno-associated virus is the preferred vehicle for in vivo gene delivery.
  • AAV has no known disease associations, infects dividing and non-dividing cells, rarely if ever integrates into the mammalian cell genome, and can persist essentially for the lifetime of infected cells as a transcriptionally active nuclear episome.
  • the FDA has recently approved several rAAV gene therapy products and many other rAAV-based gene therapy and gene editing products are in development.
  • the most widely used method for producing rAAV virions is based on the helper-virus- free transient transfection of multiple plasmids, typically a triple transfection, into adherent cell lines.
  • the polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids, and cells, such as, cell lines stably integrated with the polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids provided herein enable increased production of recombinant AAV (rAAV) virions.
  • the increased production of rAAV is hypothesized to be achieved by providing an overall increase in the levels of AAV capsid proteins.
  • the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins.
  • the increase in levels of AAV capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins.
  • the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins.
  • polyA polyadenylation
  • the increase in levels of AAV capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of the native promoter and another under the control of an inducible promoter.
  • rAAV recombinant AAV
  • the polynucleotide constructs may be introduced into cells, e.g., stably integrated into the nuclear genome of the cells and used to express components for formation of rAAV in an inducible manner thereby avoiding the toxicity of AAV Rep proteins and AAV Cap proteins when constitutively expressed.
  • Methods for generating such cells and cell lines useful for producing rAAV and methods for producing rAAV from the cells and the cell lines are also disclosed.
  • the present disclosure provides that polynucleotides comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter; and (ii) a polyadenylation signal sequence.
  • the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3' of the sequence encoding AAV Cap proteins.
  • the present disclosure provides polynucleotides comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence.
  • the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.
  • the present disclosure provides that systems of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, and one or more of c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload.
  • the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.
  • constructs that are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell.
  • the constructs may or may not be integrated into the genome of the cell.
  • a stable mammalian cell line and constructs wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is inducible upon addition of a triggering agent.
  • rAAV recombinant AAV
  • the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is not conditioned on the presence of a plasmid within the cell.
  • a set of nucleic acids comprising: (i) a first recombinant nucleic acid sequence encoding AAV Rep proteins and AAV Cap proteins; (ii) a second recombinant nucleic acid sequence encoding the AAV Cap proteins; and (iii) a third recombinant nucleic acid sequence encoding one or more adenoviral helper proteins wherein when the one or more nucleic acids are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • a set of nucleic acids comprising: (i) a first recombinant nucleic acid sequence encoding AAV Rep proteins; (ii) a second recombinant nucleic acid sequence encoding the AAV Cap proteins; and (iii) a third recombinant nucleic acid sequence encoding one or more adenoviral helper proteins wherein when the one or more nucleic acids are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.
  • a cell e.g., a mammalian cell
  • a first polynucleotide comprises the first recombinant nucleic acid sequence and a second polynucleotide comprises the second recombinant nucleic acid sequence. In some embodiments, a first polynucleotide comprises the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence. In some embodiments, the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are separated by a transcriptional blocking element.
  • conditional expression of the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins is controlled by one or more excisable elements present in the first and third nucleic acids.
  • the one or more excisable elements comprise one or more introns and/or one or more exons.
  • the first recombinant nucleic acid sequence comprises: a) a first part of the AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep proteins coding sequence; and d) the AAV Cap proteins coding sequence.
  • the excisable element comprises a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell.
  • the first recombinant nucleic acid sequence comprises: a) a first part of the AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; and c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep proteins coding sequence.
  • the excisable element comprises a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell.
  • the excisable element comprises from 5’ to 3’: a) a 5’ splice site; b) a first spacer segment comprising a first intron; c) a second spacer segment comprising: i) a first lox sequence; ii) a 3’ splice site; iii) an exon; iv) a stop signaling sequence; and v) a second lox sequence; and d) a third spacer segment comprising a second intron and another 3’ splice site.
  • the detectable marker is a luminescent marker, a radiolabel or a fluorescent marker, optionally a fluorescent marker which is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • a) the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1; and/or b) the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2; and/or c) the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3.
  • the second spacer segment is capable of being excised by a Cre polypeptide.
  • the expression of the AAV Rep proteins and/or the AAV Cap proteins is driven by native promoters.
  • a) the native promoters P5 and/or P19 drive the expression of the AAV Rep proteins; and/or b) the native promoter P40 drives the expression of the AAV Cap proteins.
  • a) the native promoters P5 and/or P19 drive the expression of the AAV Rep proteins; and/or b) an inducible promoter drives the expression of the AAV Cap proteins.
  • the second recombinant nucleic acid sequence encodes the AAV Cap proteins under the control of an inducible promoter and a selectable marker under the control of a constitutive promoter.
  • the selectable marker is an antibiotic resistance gene, such as, hygromycin.
  • the inducible promoter is a tetracycline inducible promoter and the constitutive promoter is a CMV or EF1alpha promoter.
  • the third recombinant nucleic acid sequence encodes: a) one or more adenoviral helper proteins; b) a conditionally self-excising element; and c) an inducible promoter; wherein, once integrated into the nuclear genome of a mammalian cell, the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter.
  • the one or more adenoviral helper proteins comprise E2A and E4.
  • the self-excising element comprises a sequence which encodes a polypeptide, e.g., a recombinase polypeptide, such as, a Cre polypeptide.
  • the polypeptide encoded by the self-excising element is conditionally expressible and is expressed only in the presence of a triggering agent.
  • the triggering agent is a hormone, e.g., tamoxifen.
  • the inducible promoter is a tetracycline- inducible promoter (“Tet inducible promoter”).
  • the third recombinant nucleic acid sequence further comprises a sequence that encodes a Tet responsive activator protein, e.g., Tet-on 3G.
  • a Tet responsive activator protein e.g., Tet-on 3G.
  • the expression of Tet-on 3G activator protein is driven by an EF1alpha promoter.
  • the third recombinant nucleic acid sequence comprises a sequence with at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, or a sequence identical to SEQ ID NO: 11 or SEQ ID NO: 12.
  • the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further comprises a nucleic acid sequence encoding a viral associated RNA (“VA-RNA”) sequence.
  • VA-RNA viral associated RNA
  • the third recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a VA-RNA sequence.
  • the expression of VA-RNA is constitutive.
  • the expression of VA-RNA is inducible.
  • the VA-RNA sequence comprises one or more mutations in the VA-RNA internal promoter, preferably G16A and G60A.
  • the expression of VA-RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter. In some embodiments, the expression of VA-RNA is driven by a U6 promoter or a U7 promoter. In some embodiments, the U6 promoter or the U7 promoter comprises a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide.
  • a serotype of the AAV Cap proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, A
  • the serotype is an AAV5 Cap protein which comprises one or more mutations or insertions.
  • the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further encode a fourth recombinant nucleic acid sequence encoding a payload, optionally wherein the payload is: (a) a polynucleotide payload, such as a guide RNA for RNA editing, a guide RNA for Cas protein-directed DNA editing, a tRNA suppressor, or a gene for replacement gene therapy; or (b) a protein such as a therapeutic antibody or a vaccine immunogen.
  • the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein comprise one or more mammalian cell selection elements.
  • one or more of the mammalian cell selection elements encodes an antibiotic resistance gene, optionally a blasticidin resistance gene.
  • one or more of the mammalian cell selection elements is an auxotrophic selection element which encodes an active protein.
  • the auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • one or more of the mammalian cell selection elements is a first auxotrophic selection element which encodes an inactive protein that requires expression of a second inactive protein from a second auxotrophic selection coding sequence for activity.
  • the first auxotrophic selection coding sequence encodes for DHFR Z-Cter (SEQ ID NO: 5) activity, and/or wherein the second auxotrophic selection coding sequence encodes for DHFR Z-Nter (SEQ ID NO: 4).
  • the first recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., a blasticidin resistance gene; b) the second recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., hygromycin; and c) the third recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., puromycin.
  • the elements of the previous embodiments are capable of being in one or more separate constructs (e.g., polynucleotide constructs), in any combination, wherein the one or more constructs are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell.
  • rAAV recombinant AAV
  • the nuclear genome of the cell comprises a plurality of integrated recombinant nucleic acid constructs which together encode for a recombinant adeno-associated virus (rAAV) virions, wherein the rAAV virions can be conditionally expressed from the cell.
  • the plurality of integrated recombinant nucleic acid constructs comprises the one or more recombinant nucleic acids of any one of previous embodiments, wherein the AAV Rep proteins, the AAV Cap proteins and/or the adenoviral helper proteins can be conditionally expressed from the cell.
  • the cell is from a cell line that expresses adenoviral helper proteins E1A and E1B, e.g., from nucleic acids integrated into the nuclear genome of the cell.
  • the plurality of integrated recombinant nucleic acid constructs comprises a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part and the second part of the AAV Rep proteins coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap proteins coding sequence; wherein the AAV Rep proteins and the AAV Cap proteins expression is driven by the native promoters P5, P19, and P40.
  • the plurality of integrated recombinant nucleic acid constructs comprises a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part and the second part of the AAV Rep proteins coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap proteins coding sequence; wherein the AAV Rep proteins expression is driven by native promoters P5 and P19, and the AAV Cap proteins coding sequence is driven by an inducible promoter.
  • the plurality of integrated recombinant nucleic acid constructs further comprises a second integrated polynucleotide construct comprising a sequence encoding the same AAV Cap proteins as the first integrated polynucleotide construct. In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a second integrated polynucleotide construct comprising a sequence encoding the same AAV Cap proteins as the first integrated polynucleotide construct, wherein the AAV Cap proteins are operably linked to an inducible promoter.
  • the plurality of integrated recombinant nucleic acid constructs further comprises a third integrated polynucleotide construct comprising a) a conditionally expressible VA-RNA coding sequence which comprises a mutation in the VA-RNA internal promoter, wherein the expression of VA-RNA is driven by a U6 or a U7 promoter, optionally wherein the VA-RNA sequence comprises G16A and G60A mutations; b) one or more adenoviral helper protein coding sequences, wherein the adenoviral helper proteins are E2A and E4; c) a conditionally self-excising element which encodes a Cre polypeptide which translocates to the nucleus and self-excises only in the presence of a triggering agent which is tamoxifen, and d) an inducible promoter which is a Tet inducible promoter, and wherein the expression of the one or more adenoviral helper protein coding sequences is under the
  • the plurality of integrated recombinant nucleic acid constructs further comprises a fourth integrated polynucleotide construct comprising encodes for the payload, wherein the payload is a polynucleotide payload.
  • a method of producing a population of rAAV virions comprises: (a) culturing the cell of any one of the embodiments disclosed herein in conditions which allow for the expression of the rAAV virions; and (b) isolating the rAAV virions from the cell culture.
  • a method of preparing the cell of any one of the previous embodiments comprises: i) providing a mammalian cell and the one or more nucleic acids of any one of the previous embodiments; and ii) selecting for integration of the one or more nucleic acids of any one of the previous embodiments into the nuclear genome of the mammalian cell.
  • cells comprising: a) a first polynucleotide construct comprising a first polynucleotide coding for AAV Rep proteins and AAV Cap proteins; b) a second polynucleotide construct coding the AAV Cap proteins; c) a third polynucleotide construct coding for one or more adenoviral helper proteins; wherein when the one or more nucleic acids are integrated into the nuclear genome of a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide coding for AAV Rep proteins and AAV Cap proteins comprises a transcriptional blocking element separating the sequence coding for the AAV Rep proteins and the sequence coding for the AAV Cap proteins.
  • the third polynucleotide construct comprises a sequence coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element.
  • the self-excising element is operably linked to the inducible promoter.
  • expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the second recombinant nucleic acid sequence is driven by the same inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the second polynucleotide construct is driven by the same inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the first polynucleotide construct is driven by the same inducible promoter.
  • the inducible promoter is a tetracycline-responsive promoter element (TRE).
  • the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
  • the minimal promoter is a human cytomegalovirus promoter.
  • the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68.
  • the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62.
  • transcription is activated from the inducible promoter upon binding of an activator.
  • the activator binds to the inducible promoter in the presence of a first triggering agent.
  • the third polynucleotide construct further comprises a sequence coding for an activator.
  • the activator is operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the EF1alpha promoter comprises at least one mutation.
  • the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20.
  • the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.
  • the rTA comprises four mutations in the tetR DNA binding moiety.
  • the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-85, or variants thereof.
  • the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter.
  • the constitutive promoter is a human cytomegalovirus promoter.
  • the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent.
  • the inducible promoter is activated in the presence of a first triggering agent.
  • the first triggering agent binds to the cymR repressor.
  • the third polynucleotide construct further comprises a cymR repressor.
  • the cymR repressor is operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter.
  • the EF1alpha promoter comprises at least one mutation.
  • the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20.
  • the first triggering agent is a cumate.
  • the sequence coding for the self-excising element comprises a poly A sequence.
  • the self-excising element is a recombinase.
  • the recombinase is fused to a ligand binding domain.
  • the recombinase is Cre polypeptide or flippase polypeptide.
  • the Cre polypeptide is fused to a ligand binding domain.
  • the ligand binding domain is a hormone receptor.
  • the recombinase is a Cre-ERT2 polypeptide.
  • the self-excising element translocates to the nucleus in the presence of a second triggering agent.
  • the second triggering agent is an estrogen receptor ligand.
  • the second triggering agent is a selective estrogen receptor modulator (SERM).
  • SERM selective estrogen receptor modulator
  • the second triggering agent is tamoxifen.
  • the recombinase is flanked by recombination sites.
  • the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites.
  • the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the E2A is FLAG-tagged E2A. In some embodiments, the sequence coding for E2A and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A. [0045] In some embodiments, the third polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance protein.
  • the selectable marker is an auxotrophic protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic protein or leucine zipper linked to a C-terminus of the auxotrophic protein. In some embodiments, the auxotrophic protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • GS glutamine synthetase
  • TYMS thymidylate synthase
  • PAH phenylalanine hydroxylase
  • DHFR dihydrofolate reductase
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the third polynucleotide construct further comprises a sequence coding for VA-RNA.
  • the sequence coding for VA-RNA is a transcriptionally dead sequence.
  • the sequence coding for VA-RNA comprises at least two mutations in the internal promoter.
  • the third polynucleotide construct further comprises upstream of the sequence coding for VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; and e) a second part of a U6 or U7 promoter sequence.
  • the stuffer sequence is excisable by the recombinase.
  • the stuffer sequence comprises a sequence encoding a gene.
  • the stuffer sequence comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a CMV promoter.
  • the first polynucleotide construct comprises: a) a sequence of a first part of a Rep gene; b) a sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene.
  • the excisable element comprises a stop signaling sequence.
  • the excisable element comprises a rabbit beta globin intron.
  • the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron. [0049] In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5’ splice site and a 3’ splice site. In some embodiments, the 5’ splice site is a rabbit beta globin 5’ splice site. In some embodiments, the 3’ splice site is a rabbit beta globin 3’ splice site.
  • three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene.
  • the three splice sites are a 5’ splice site, a first 3’ splice site, and a second 3’ splice site.
  • a first 3’ splice site is a duplicate of the second 3’ splice site.
  • the first 3’ splice site is a rabbit beta globin 3’ splice site.
  • the second 3’ splice site is a rabbit beta globin 3’ splice site.
  • the excisable element comprises a recombination site.
  • the recombination site is a lox site or FRT site.
  • the lox site is a loxP site.
  • the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first recombination site; c) the first 3’ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3’ splice site.
  • the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3’ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3’ splice site.
  • the first spacer sequence comprises an intron.
  • the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1.
  • the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2.
  • the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3.
  • the third spacer segment comprises an intron.
  • the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery.
  • the second spacer segment comprises an exon.
  • the second spacer segment further comprises a polyA sequence. In some embodiments, the polyA sequence is 3’ of the exon.
  • the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence.
  • the second spacer segment comprises from 5’ to 3’: a) a first recombination site; b) the first 3’ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site.
  • the first recombination site is a first lox sequence and the second recombination site is a second lox sequence.
  • the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence.
  • the first recombination site is a first FRT site and the second recombination site is a second FRT site.
  • the stop signaling sequence is a termination codon of the exon or a polyA sequence.
  • the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence.
  • the exon encodes a detectable marker or a selectable marker.
  • the detectable marker comprises a luminescent marker or a fluorescent marker.
  • the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • the second spacer segment is excisable by a recombinase.
  • the recombinase is a Cre polypeptide or a Flippase polypeptide.
  • the Cre polypeptide is fused to a ligand binding domain.
  • the ligand binding domain is a hormone receptor.
  • the recombinase is a Cre-ERT2 polypeptide.
  • the Rep gene codes for Rep polypeptides.
  • the Cap gene codes for Cap polypeptides.
  • transcription of the Rep gene and the Cap gene are driven by native promoters.
  • the native promoters comprise P5, P19, and P40.
  • the Rep proteins are wildtype Rep polypeptides.
  • the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40.
  • a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase.
  • the Cap polypeptides are wildtype Cap polypeptides.
  • the Cap polypeptides are AAV capsid proteins.
  • the AAV capsid proteins comprise VP1, VP2, and VP3.
  • a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.
  • the first polynucleotide construct further comprises a sequence coding for a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is DHFR, GS, TYMS, or PAH.
  • the selectable marker is a split intein linked to an N-terminus of the auxotrophic selection element or split intein linked to a C-terminus of the auxotrophic selection element.
  • the selectable marker is a split intein linked to an N-terminus of the active protein or split intein linked to a C-terminus of the active protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic selection element or leucine zipper linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the active protein or leucine zipper linked to a C-terminus of the active protein. In some embodiments, the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138.
  • the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, but wherein these sequences lack SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.
  • the second polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the second polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 148.
  • the third polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the third polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are stably integrated in the cell’s genome.
  • the cell further comprises a payload construct, wherein the payload construct is a polynucleotide coding for a payload.
  • the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33.
  • the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the payload construct comprises a sequence of a payload flanked by ITR sequences.
  • the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 139.
  • sequence of the payload is driven by a constitutive promoter.
  • the constitutive promoter and sequence of the payload are flanked by ITR sequences.
  • the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146.
  • sequence of the payload comprises a polynucleotide sequence coding for a gene.
  • the gene codes for a selectable marker or detectable marker.
  • the gene codes for a therapeutic polypeptide or transgene.
  • the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide.
  • the therapeutic polynucleotide is a tRNA suppressor or a guide RNA.
  • the guide RNA is a polyribonucleotide capable of binding to a protein.
  • the protein is nuclease.
  • the protein is a Cas protein, an ADAR protein, or an ADAT protein.
  • the Cas protein is catalytically inactive Cas protein.
  • the payload construct is stably integrated into the genome of the cell.
  • a plurality of the payload construct is stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs is separately stably integrated into the genome of the cell. In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, the payload construct is integrated into the genome of the cell. In some embodiments, the selectable marker is a first part of a split blasticidin and wherein the first polynucleotide sequence encodes the second part of the split blasticidin.
  • a method for increasing production of rAAV virions from a cell comprises amplifying expression of AAV Rep and capsid proteins, helper proteins, and/or payload in the cell, wherein the amplifying comprises: increasing copy number of a polynucleotide construct comprising a sequence encoding one or more AAV Rep proteins and a sequence encoding one or more AAV cap proteins, a polynucleotide construct comprising a sequence encoding the one or more AAV cap proteins, a polynucleotide construct comprising a sequence encoding one or more AAV helper proteins, and/or a polynucleotide construct comprising a sequence encoding the payload by introducing an agent to amplify expression of the Rep/Cap genes, helper genes, and/or payload.
  • the increasing copy number of the polynucleotide construct(s) comprises culturing the cell under conditions that select for the presence of the selectable marker encoded by the polynucleotide construct(s) under the control of an attenuated promoter, thereby producing the cell comprising an increased copy number of the polynucleotide construct(s) compared to a cell comprising the polynucleotide construct comprising a selectable marker operably linked to a nonattenuated promoter.
  • the attenuated promoter is an attenuated EF1alpha promoter and the nonattenuated promoter is an EF1alpha promoter; optionally, wherein the attenuated EF1alpha promoter is SEQ ID NO: 132 and the EF1alpha promoter is SEQ ID NO: 133.
  • the polynucleotide construct further comprises a mutated selectable marker having decreased enzymatic activity compared to an unmutated selectable marker.
  • the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the mutated selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the unmutated selectable marker.
  • the mutated selectable marker is a mutated GS and the unmutated selectable marker is GS; optionally, wherein the mutated GS having a R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112 and the GS is SEQ ID NO: 112; optionally, wherein the mutated GS is SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144.
  • the polynucleotide construct further comprises a selectable marker.
  • the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the selectable marker and in the presence of an inhibitor of the selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the selectable marker cultured in the absence of the inhibitor of the selectable marker.
  • the polynucleotide construct is any polynucleotide construct as described herein. [0069] Also provided herein are methods of producing a stable cell line comprising expanding a cell described above.
  • Also provided herein are methods of producing a plurality of rAAV virion comprising culturing a cell described above in the presence of a first triggering agent and a second triggering agent.
  • the first triggering agent is doxycycline and the second triggering agent is tamoxifen.
  • the plurality of rAAV virion has an encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification.
  • the plurality of rAAV virion has a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion have a concentration of greater than 1 ⁇ 10 11 or no less than 5 ⁇ 10 11 , 1 ⁇ 10 12 , 5 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 viral genomes per milliliter prior to purification. In some embodiments, the plurality of rAAV virion have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1 ⁇ 10 5 vg/target cell or less.
  • the culturing is in a bioreactor.
  • pharmaceutical compositions comprising the rAAV virion produced by the cell or the method described above and a pharmaceutically acceptable carrier.
  • methods of treating a condition or disorder comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need thereof.
  • FIG.1A depicts an exemplary system of polynucleotides for inducibly producing rAAV. In absence of first and second triggering agents, the system is in an off state. This system includes Construct 1, Construct 3, Construct 4, and Construct 2, and is referred to as v1.2 system.
  • the v1.2 system is distinct from the v1.0 system in that the v1.2 system comprises a recombinant nucleic acid that expresses Cap protein driven by an inducible promoter (see, e.g., Construct 2).
  • Construct 1 and Construct 2 are two separate polynucleotides which are configured for inducibly expressing AAV capsid proteins.
  • Construct 1 in addition to inducibly expressing AAV Cap proteins also inducibly expresses full-length AAV Rep proteins, and both AAV Cap proteins and AAV Rep proteins are driven by their native promoters (p40, and p5 and p19, respectively).
  • FIG.1B depicts the post-triggered state (also referred to as the on state) of Constructs 1- 4 shown in FIG.1A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen.
  • the Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen.
  • ER2 estrogen response elements
  • FIG.2A depicts an exemplary system of polynucleotides for inducibly producing rAAV, which is also an exemplary v1.2 system.
  • the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet-On promoter).
  • an inducible promoter for example, Tet-On promoter.
  • the coding sequences for the Rep and Cap proteins are oriented in opposite directions such that the internal p40 promoter in Rep coding sequence which controls expression of the Cap proteins is spatially separated from the Cap coding sequence and does not control Cap proteins expression.
  • the inducible promoter (for example, Tet-On promoter) controlling the expression of the Cap proteins is stronger than the native p40 promoter, resulting in increased Cap proteins expression as compared to the v1.0 system.
  • the coding sequences and promoters for the Rep proteins are separated from the coding sequence and the inducible promoter for the Cap proteins by a transcription blocking element (TBE). This construct may also be referred to as a Rep-Cap construct.
  • FIG.2B depicts the on state of the system of polynucleotides for inducibly producing rAAV depicted in FIG.2A, after induction by a first triggering agent, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen.
  • the Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen.
  • ER2 estrogen response elements
  • Cre Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed and Cap proteins are expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG.2B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep coding sequences expression under control of native promoters.
  • a first triggering agent e.g., doxycycline
  • FIG.3A shows an exemplary system of polynucleotides for inducibly producing rAAV, which is also an exemplary v1.2 system. In absence of a first triggering agent and a second triggering agent, the system is in an off state.
  • the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet- On promoter).
  • the coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE).
  • TBE transcription blocking element
  • This system includes an additional polynucleotide, depicted as Construct 4, for expressing AAV Cap proteins driven by an inducible promoter.
  • Construct 4 may be referred to as a Cap construct and is the same as Construct 2 shown in FIGs.1A and 1B.
  • FIG.3B depicts the on state of the system of polynucleotides for inducibly producing rAAV depicted in FIG.3A, after induction by the agents, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen.
  • the Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen.
  • ER2 estrogen response elements
  • a first triggering agent e.g., doxycycline
  • Cre is expressed and Cap proteins are expressed
  • the second triggering agent e.g., tamoxifen
  • Cre translocates to the nucleus.
  • the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG. 2B. Therefore, adenoviral E2A and E4 helper proteins are expressed.
  • FIG.4 shows an exemplary method for generating a pool of P2 cell clone that includes Construct 3 configured to express Adenovirus helper proteins, Construct 1 configured to express AAV Rep and Cap proteins and Construct 4 to express a payload (GOI, e.g., GFP or progranulin).
  • Constructs 1, 3, and 4 are those depicted in FIG.1A.
  • FIG.5 shows an exemplary method for generating a P3 cell pool from the P2 cell pool by introducing a polynucleotide (e.g., Construct 2, as depicted in FIG.1A) that encodes for AAV Cap proteins “T231 (P3)” into the P2 polyclonal cell pool of FIG.4.
  • a polynucleotide e.g., Construct 2, as depicted in FIG.1A
  • a polynucleotide that does not encode for AAV Cap proteins is used as a control to generate “T232 (P3)”.
  • FIG.6 provides data for the titer of AAV Capsid proteins (Capsid Titer) and encapsidated payload, progranulin (PGRN), (PGRN titer) produced in T231 (P3) and T232 (P3) cells. Also shown is a table depicting percent packaging of the gene encoding PGRN for T231 (P3) cells and T232 (P3) cells.
  • FIG.7 depicts an exemplary v1.2 system of polynucleotide Constructs 1, 2, 3, and 4 that includes a first polynucleotide for encoding AAV Cap proteins under the control of the native promoter, p40, (see Construct 1) and a second polynucleotide for encoding AAV Cap proteins (Construct 2) under the control of an inducible promoter to achieve an overall increase in AAV Cap proteins expression as compared to a system of constructs that includes only the first polynucleotide for encoding AAV Cap proteins under the control of the native promoter, p40 (e.g., only Construct 1 and not including Construct 2).
  • FIG.8 provides a schematic summarizing induction of cells that include the v1.2 system of polynucleotides “v1.2 cells” and characterization of the Capsid levels, excision kinetics for production of AAV Rep and Cap, GOI levels, and levels of encapsidated GOI.
  • FIG.10 provides data for characterization of rAAV produced by v1.2 cells and v1.0 cells.
  • the ratio of “encapsidated GOI/total GOI” in v1.2 cells was measured and compared to the ratio of “encapsidated GOI/total GOI” in v1.0 cells.
  • Fig.10 shows that in v1.0 cells, the production of genome of interest (GOI) is higher than Capsid production, indicating that capsid production could be a rate limiting step for producing encapsidated GOI (e.g., a payload).
  • Increased capsid production in the V1.2 cells resulted in increased generation of rAAV.
  • FIG.11 shows a time course of production of viral particles over a 7-day period after induction of v1.2 (pool) and v1.0 (clone) cells with tamoxifen and doxycycline.
  • v1.2 cells produce viral particles that encapsidate a gene for expressing PRGN while v1.0 cells produce viral particles that encapsidate a gene for expressing green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • FIG.11 shows a time course of production of viral particles over a 7-day period after induction of v1.2 (pool) and v1.0 (clone) cells with tamoxifen and doxycycline.
  • v1.2 cells produce viral particles that encapsidate a gene for expressing PGRN while v1.0 cells produce viral particles that encapsidate a gene for expressing green fluorescent protein (GFP).
  • GFP green fluorescent protein
  • FIG. 12 encapsidated GOI (either the gene for expressing PGRN or the gene for expressing GFP) was measured (vg/ml).
  • FIG.13 shows high titer produced after induction of v1.2 cells from the T231 stable cell pool wherein the payload is progranulin (“v1.2 Pool (PGRN)”).
  • PGRN progranulin
  • the graph shows the capsid titer (vp/ml) from cell lysate produced after induction of v1.2 Pool (PGRN) cells compared to after induction of a v1.0 stable cell line pool wherein the payload is progranulin (“v1.0 Pool (PGRN)”), compared to after induction of a v1.0 stable cell line pool wherein the payload is eGFP (“v1.2 Pool (eGFP)”), and compared to after transient transfection of cells wherein the payload is eGFP (“Transient Transfection (eGFP)”).
  • v1.0 Pool PGRN
  • eGFP v1.2 Pool
  • Transient Transfection eGFP
  • FIG.14A shows high capsid titer produced after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)).
  • the v1.2 Pool (PGRN) capsid titer and v1.2 Clones (PGRN) capsid titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) capsid titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) capsid titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) capsid titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) capsid titer, indicating the v1.2 stable cells have successfully increased capsid titer >2 log.
  • FIG.14B shows high vector genome production after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)).
  • PGRN progranulin
  • PGRN progranulin
  • the v1.2 Pool (PGRN) vector genome titer and v1.2 Clones (PGRN) vector genome titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) vector genome titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) vector genome titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) vector genome titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) vector genome titer, indicating the v1.2 stable cells have successfully increased vector genome titer.
  • FIG.15 shows the parent cell line is T205 CL23 (Top clone from stable cells v1.0 pool, in which the payload is progranulin).
  • T318 was generated from T205 CL23 clone that has integrated the sequence for Tet inducible capsid (Construct 2 of FIG.1A) and selected based on hygromycin-resistance to make a v1.2 pool. Selecting a top clone with v1.0 system integrated in the genome and introducing Construct 2 into the top clone provides for increased vector genome titer and capsid titer as compared to introducing Construct 2 into a pool of clones with v1.0 system integrated in the genome.
  • FIG.16 depicts an exemplary v1.0 system in the pre-triggered state. Cap proteins are expressed under the control of a native AAV p40 promoter. The brackets in construct 3 indicate the position of the flanking ITRs.
  • V1.0 system is described in U.S. Patent Application Publication No.2022/0145328 which is incorporated herein.
  • V1.0 cells are a pool of cells or a cell line that includes the constructs of the V1.0 system.
  • polynucleotide constructs and cells such as, cell lines stably integrated with the polynucleotide constructs that enable increased production of recombinant AAV (rAAV) virions.
  • the increased production is hypothesized to be achieved by providing an overall increase in the levels of capsid proteins which in turn increases production of rAAV.
  • the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins.
  • the increase in levels of capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins.
  • the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins.
  • AAV adeno-associated virus
  • Rep and Cap flanked by two 145 base inverted terminal repeats
  • Rep gene encodes multiple distinct proteins including Rep78, Rep68, Rep52, and Rep40. These proteins are also referred to herein as Rep proteins or Rep and may encompass one or more of Rep78, Rep68, Rep52, and Rep40 and functional variants thereof and homologs thereof. Rep78 and Rep68 and functional variants thereof and homologs thereof are referred to herein as large Rep. Rep52, and Rep40 and functional variants thereof and homologs thereof are referred to herein as small Rep.
  • Rep proteins from an AAV of a particular serotype may also be referred to as Rep1, Rep2, etc. where the Rep protein is derived from an AAV1 or an AAV2 serotype, respectively.
  • Cap gene encodes capsid proteins VP1, VP2, and VP3 required for production of rAAV capsids. These proteins are also referred to herein as Cap proteins or Cap and may encompass one or more of VP1, VP2, and VP3 and functional variants thereof and homologs thereof. Cap proteins from an AAV of a particular serotype may also be referred to as Cap1, Cap2, Cap4, etc. where the Rep protein is derived from an AA1, an AAV2, or an AAV5 serotype, respectively.
  • AAV requires a helper plasmid containing genes from a helper virus such as adenovirus, including E1a, E1b, E4, E2a, and VA genes for AAV replication.
  • Recombinant as applied to an AAV virion, means that the rAAV virion (synonymously, rAAV virus particle) is the product of one or more procedures that result in an AAV particle Construct that is distinct from an AAV virion in nature. The procedure may be genetic alteration, e.g., by the addition or insertion of a heterologous nucleic acid Construct into the virus.
  • Recombinant virus is meant to describe a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the virus.
  • AAV serotype may be AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, AAV-7m8, AAV-6.2, AAV- Dj, AAV-DJ/8, AAV2-retro, AAV2-QuadYF and AAV2.7m8, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, AAV-PHP.eS, evolved capsids that are less immunogenic to mice and humans
  • Prime AAV refers to AAV isolated from a primate
  • non-primate AAV refers to AAV isolated from a non-primate mammal
  • bovine AAV refers to AAV isolated from a bovine mammal (e.g., a cow)
  • An "rAAV vector” comprises a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a polynucleotide sequence of interest for introducing into a target cell.
  • the heterologous polynucleotide is flanked by at least one, and usually by two AAV inverted terminal repeat sequences (ITRs).
  • the heterologous polynucleotide can also be referred to as a polynucleotide payload.
  • the term rAAV vector encompasses both rAAV virions and rAAV vector plasmids.
  • An "AAV virus” or “AAV viral particle” or “rAAV vector particle” refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector.
  • the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle” or simply an “rAAV vector”.
  • production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector contained within an rAAV particle.
  • "Packaging” refers to a series of intracellular events that result in the assembly, encapsidation, and production of an AAV particle.
  • AAV "rep” and “cap” genes refer to polynucleotide sequences encoding replication and capsid proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes.”
  • AAV Rep coding region or “sequence encoding one or more Rep proteins” or “Rep encoding sequence” and grammatical equivalents thereof is meant the art-recognized region of the AAV genome which encodes the replication proteins of the virus which are required to replicate the viral genome and/or a payload flanked by ITRs.
  • the rep coding region as used herein, may be derived from any viral serotype, such as those described above.
  • the region need not include all of the wild-type genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the rep genes provide for expression Rep proteins.
  • Rep coding sequences are further described below.
  • the terms “AAV Rep coding sequence” and “AAV Rep proteins coding sequence” are used interchangeably herein.
  • the terms “AAV Rep proteins”, “Rep proteins”, “AAV Rep polypeptide”, and “Rep polypeptide” are interchangeably used herein.
  • AAV cap coding region or “sequence encoding one or more cap proteins,” or “Cap encoding sequence” and grammatical equivalents thereof it is meant the art-recognized region of the AAV genome which encodes the coat proteins of the virus which are required for the capsid that viral genome or a payload is packaged into by the Rep proteins.
  • AAV cap coding region see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158, 97-129; Kotin, R. M. (1994) Human Gene Therapy 5, 793-801.
  • the AAV cap coding region may be derived from any AAV serotype, as described above.
  • the region need not include all of the wild-type cap genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the genes provide for sufficient packaging functions.
  • Cap coding sequences are further described below.
  • the term “AAV Cap coding sequence” and “AAV Cap proteins coding sequence” are interchangeably used herein.
  • the terms “AAV Cap proteins”, “Cap proteins”, “AAV Cap polypeptide”, and “Cap polypeptide” are interchangeably used herein.
  • Capsid” and “Cap” are interchangeably used herein.
  • AAV ITRs adeno-associated virus inverted terminal repeats
  • AAV ITRs the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the viral genome.
  • the nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5, 793-801; Berns, K. I. "Parvoviridae and their Replication” in Fundamental Virology, 2d ed., (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 ITRs sequence.
  • an "AAV ITR” need not have a wild-type nucleotide sequence, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides.
  • the AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc.
  • 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate.
  • the ITRs may be single stranded (ssITRs) or self-complementary (scITRs).
  • a "helper virus” for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell.
  • a variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia.
  • the adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used.
  • Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC.
  • helper virus function may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans.
  • An "infectious" virus or viral particle is one that comprises a polynucleotide component which the particle is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus.
  • an “infectious” virus or viral particle is one that may access a target cell, may infect a target cell, and may express a heterologous nucleic acid in a target cell.
  • infectivity refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity may refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles may be expressed as the number of viral genome (vg) copies.
  • the ability of a viral particle to express a heterologous nucleic acid in a cell may be referred to as “transduction.”
  • the ability of a viral particle to express a heterologous nucleic acid in a cell may be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA).
  • GFP green fluorescent protein
  • ELISA enzyme-linked immunosorbent assay
  • Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles.
  • polynucleotide refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof.
  • polynucleotide and nucleic acid are interchangeably used herein.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form.
  • polynucleotide construct refers to a DNA segment of any size that includes one or more sequences encoding an RNA or protein and at least one promoter for driving expression from the one or more sequences.
  • polynucleotide construct and “nucleic acid construct” are interchangeably used herein.
  • a polynucleotide construct may be a circular DNA or a linear DNA.
  • a polynucleotide construct may be single stranded or double stranded.
  • the term "vector” includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which may transfer gene sequences into and between cells.
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • the use of the term “vector” throughout this specification encompasses plasmid or viral vectors, which permit the desired components to be transferred to the host cell via transfection or infection.
  • an adeno-associated viral (AAV) vector is a plasmid comprising a recombinant AAV genome.
  • useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter.
  • a vector may be linear or circular, single stranded or double stranded, DNA or RNA.
  • the vector may be circular, double stranded DNA.
  • the term “vector system” refers to two or more vectors that are used together, e.g., by simultaneous or sequential introduction into a cell, to provide at least two different components into the cell. The two different components may then work together in the cell.
  • a polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences.
  • percent “sequence identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • sequence identity can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared.
  • the percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence.
  • test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary.
  • the percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence.
  • the BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against.
  • the BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence.
  • the scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment.
  • the alignment scores for nucleic acids can be scored by set match/mismatch scores.
  • the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution.
  • the substitution matrix used is the BLOSUM62 matrix.
  • the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms.
  • the BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value.
  • Percent Identity (“Percent Identity” output value) ⁇ (“Query Coverage” output value)
  • the following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences.
  • test sequence 1 has 50% sequence identity to reference sequence 2.
  • Test sequence 3 has 50% sequence identity to reference sequence 4.
  • test sequence 5 has 50% sequence identity to reference sequence 6.
  • Test sequence 5 has 100% sequence identity to reference sequence 6.
  • polynucleotide sequence e.g., a DNA sequence or an RNA sequence
  • a sequence of AAAAAGGGGG also encompasses a sequence of CCCCCTTTTT.
  • a "gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated.
  • host cell denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that may be, or have been, used as recipients of an AAV vector system as described herein, or other transfer DNA.
  • the term includes the progeny of the original cell which has been transfected.
  • a "host cell” as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.
  • the disclosure provides transfected host cells.
  • transfection is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197.
  • cell line refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants.
  • cell culture refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as the components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants.
  • Large scale approaches such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture.”
  • Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure.
  • the terms “recombinant cell” refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced.
  • the term “intermediate cell line” refers to a cell line that contains the AAV rep and cap components integrated into the host cell genome or a cell line that contains the adenoviral helper functions integrated into the host cell genome.
  • the term “packaging cell line” refers to a cell line that contains the AAV rep and cap components and the adenoviral helper functions integrated into the host cell genome.
  • a payload construct must be added to the packaging cell line to generate rAAV virions.
  • the term “production cell line” refers to a cell line that contains the AAV rep and cap components, the adenoviral helper functions, and a payload construct.
  • the rep and cap components and the adenoviral helper functions are integrated into the host cell genome.
  • the payload construct can be stably integrated into the host cell genome or transiently transfected.
  • rAAV virions can be generated from the production cell line upon the introduction of one or more triggering agents in the absence of any plasmid or transfection agent.
  • downstream purification refers to the process of separating rAAV virions from cellular and other impurities. Downstream purification processes include chromatography-based purification processes, such as ion exchange (IEX) chromatography and affinity chromatography.
  • prepurification yield refers to the rAAV yield prior to the downstream purification processes.
  • postpurification yield refers to the rAAV yield after the downstream purification processes. rAAV yield can be measured as viral genome (vg)/L.
  • the encapsidation ratio of a population of rAAV virions can be measured as the ratio of rAAV viral particle (VP) to viral genome (VG).
  • the rAAV viral particle includes empty capsids, partially full capsids (e.g., comprising a partial viral genome), and full capsids (e.g., comprising a full viral genome).
  • the F:E ratio of a population of rAAV virions can be measured as the ratio of rAAV full capsids to empty capsids.
  • the rAAV full capsid particle includes partially full capsids (e.g., comprising a partial viral genome) and full capsids (e.g., comprising a full viral genome).
  • the empty capsids lack a viral genome.
  • the potency or infectivity of a population of rAAV virions can be measured as the percentage of target cells infected by the rAAV virions at a multiplicity of infection (MOI; viral genomes/target cell).
  • MOI multiplicity of infection
  • Exemplary MOI values are 1 ⁇ 10 1 , 1 ⁇ 10 2 , 2 ⁇ 10 3 , 5 ⁇ 10 4 , or 1 ⁇ 10 5 vg/target cell.
  • An MOI can be a value chosen from the range of 1 ⁇ 10 1 to 1 ⁇ 10 5 vg/target cell.
  • expression vector or construct or “synthetic construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene.
  • auxotrophic or “auxotrophic selectable marker” as used herein refers to the usage of a medium lacking a supplement, such as a medium lacking an essential nutrient such as the purine precursors hypoxanthine and thymidine (HT), or the like, for selection of a functional enzyme which allows for growth in the medium lacking the essential nutrient, e.g., a functional dihydrofolate reductase or the like.
  • a medium lacking a supplement such as a medium lacking an essential nutrient such as the purine precursors hypoxanthine and thymidine (HT), or the like.
  • tetracycline is used generically herein to refer to all antibiotics that are structurally and functionally related to tetracycline, including tetracycline, doxycycline, demeclocycline, minocycline, sarecycline, oxytetracycline, omadacycline, or eravacycline.
  • the terms “constitutive” or “constitutive expression” are used interchangeably herein. They refer to genes that are transcribed in an ongoing manner. Such gene are driven by a constitutive promoter. In some embodiments, the terms refer to the expression of a therapeutic payload or a nucleic acid sequence that is not conditioned on addition of an triggering agent to the cell culture medium.
  • a constitutive promoter is capable of directing continuous gene expression in a cell. Constitutive promoters regulate expression of basal genes, like housekeeping genes. In contrast, an inducible promoter directs gene expression in the presence of an external stimulus. Thus, an inducible promoter can be controlled by providing or withdrawing the stimulus.
  • the term “polynucleotide payload” refers to a polynucleotide sequence that is packaged into a rAAV virion for delivery by the rAAV virion into a cell. A polynucleotide payload is flanked by AAV inverted terminal repeats (ITRs).
  • the polynucleotide payload may be available to the cell as a DNA (e.g., a homology region for homology-directed repair), transcribed into an RNA (e.g., a guide RNA (gRNA), a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO)), or transcribed and translated into a polypeptide (e.g., an antibody, a hormone, a site-specific endonuclease, a reporter gene, a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, a ribozyme, or a DNAzyme.
  • a DNA e.g., a homology region for homology-directed repair
  • Recombinant as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature.
  • a recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct.
  • control element or "control sequence” is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature.
  • Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers.
  • a promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter.
  • a promoter is usually upstream of a gene whose expression is controlled by the promoter.
  • “Operatively linked” or “operably linked” refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.
  • "Heterologous” means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide.
  • sequences or sequence motifs operably linked to a sequence where it is not naturally found are also heterologous; such sequences or sequence motifs include polyA signal sequences, introns, and/or any other relevant sequence.
  • an rAAV that includes a heterologous nucleic acid encoding a heterologous payload is an rAAV that includes a nucleic acid not normally included in a naturally occurring, wild-type AAV, and the encoded heterologous payload is a payload not normally encoded by a naturally- occurring, wild-type AAV.
  • a Capsid proteins coding sequence operably linked to a heterologous polyA signal sequence refers to a AAV Cap coding sequence operably linked to a sequence not native to AAV.
  • a cell is said to be "stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro.
  • such a cell is "heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.
  • a gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell.
  • a cell is said to be “stably” altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro.
  • such a cell is "heritably” altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell.
  • a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro.
  • a gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell.
  • polypeptide refers to polymers of amino acids of any length.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component.
  • Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a payload to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein.
  • references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a payload to a mammalian subject include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function.
  • An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from.
  • an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment may be measured on an absolute basis, such as weight per volume of solution, or it may be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated.
  • An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some cases purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure.
  • treatment used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease.
  • treatment encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s).
  • Those in need of treatment may include those already afflicted (e.g., those with a neurological disorder) as well as those in which prevention is desired (e.g., those with increased susceptibility to a neurological disorder; those suspected of having a neurological disorder; those having one or more risk factors for a neurological disorder, etc.).
  • a “therapeutically effective amount” or “efficacious amount” means the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient, in combination with another agent, or alone in one or more doses, to effect such treatment for the disease.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated.
  • the terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses, camels, etc.); mammalian farm animals (e.g., sheep, goats, cows, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human.
  • the terms “v1.1 system” and “v1.2” system are interchangeable and are used herein to refer to the same version of system of polynucleotides.
  • hybridize and “hybridization” refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson Crick base pairing.
  • homologous region refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a “homologous region” is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term “homologous, region,” as used herein, refers to the ability of nucleic acid molecules to hybridize to each other.
  • a single-stranded nucleic acid molecule may have two homologous regions which are capable of hybridizing to each other.
  • the term "homologous region” includes nucleic acid segments with complementary sequences. Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400, from about 400 to about 440, etc.).
  • nucleotides e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to
  • complementary refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands may base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine.
  • uracil when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated.
  • “Complementarity” may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be “complementary” and able to form a duplex despite having less than perfect or less than 100% complementarity.
  • Two sequences are "perfectly complementary” or “100% complementary” if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region.
  • Two or more sequences are considered “perfectly complementary” or “100% complementary” even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other.
  • "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other.
  • recombination site denotes a region of a nucleic acid molecule comprising a binding site or sequence-specific motif recognized by a site-specific recombinase that binds at the target site and catalyzes recombination of specific sequences of DNA at the target site.
  • Site-specific recombinases catalyze recombination between two such target sites.
  • the relative orientation of the target sites determines the outcome of recombination. For example, translocation occurs if the recombination sites are on separate DNA molecules.
  • v1.2. Overview To solve the problems associated with low rAAV titers, disclosed herein are polynucleotide constructs and cells, such as, cell lines stably integrated with the polynucleotide constructs that enable increased production of recombinant AAV (rAAV) virions.
  • FIG.10 shows that in a v1.0 system, the production of payload sequences is higher than capsid production, indicating that capsid production could be a rate limiting step for producing encapsidated payloads, i.e., rAAV.
  • FIG.10 demonstrates that increased encaspidated payload titer, i.e., increased rAAV titer was acheived by increasing total capsid production.
  • increased production of rAAV is achieved by providing an overall increase in the levels of capsid proteins which in turn increases production of rAAV.
  • Disclosed herein are various methods and constructs for increasing capsid production and therefore also increasing the titer of encapsidated genomes.
  • the increase in levels of capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3' to the sequence encoding the capsid proteins.
  • the increase in levels of capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins.
  • the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins.
  • the increase in levels of capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of a native promoter and another under the control of an inducible promoter.
  • One or more of these embodiments can also be combined to increase capsid proteins expression and enable increased production of recombinant AAV (rAAV). These embodiments are further described in detail below.
  • rAAV recombinant AAV
  • the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence is 3' of the sequence encoding AAV Cap proteins are provided. In certain embodiments, the polynucleotides comprise (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3' of the sequence encoding AAV Cap proteins. Suitable polyadenylation signals are further described below in the section “Polyadenylation Signals”.
  • Polyadenylation (polyA) signal sequences generally include a short sequence that triggers polyadenylation of an mRNA. In certain instances, RNA stability, expression, and/or function can be enhanced with additional sequences surrounding a shorter sequence. Various polyA signaling sequences can be used for the coding sequences of various embodiments.
  • the polyadenylation signal sequence is a SV40 polyadenylation signal sequence (SV40 polyA). In other embodiments, the polyadenylation signal sequence is a bovine growth hormone polyadenylation signal sequence (bGH polyA). In still other embodiments, the polyadenylation signal sequence is a Rabbit Beta Globin polyadenylation signal sequence.
  • the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 (SV40 poly A). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 152 (SV40 poly A). In certain embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151 (bGH polyA).
  • the polyadenylation signal sequence has sequence of SEQ ID NO: 151 (bGH polyA). In some embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 170 (Rabbit Beta Globin PolyA). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 170 (Rabbit Beta Globin PolyA).
  • the polyadenylation signal sequence is any polyadenylation signal sequence that encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence.
  • the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162.
  • the native AAV Cap polyadenylation signal sequence has sequence of SEQ ID NO: 162.
  • the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof.
  • the polynucleotide described herein is not flanked by inverted terminal repeat sequences. [0165] Additional details regarding polyadenylation signaling sequences are disclosed in one or more of: US Pat. Nos.11,793,180, 11,752,181, 10,912,826, 8,975,391, 7,557,197, and 5,122,458; US Pat. Pub.
  • AAV Cap proteins [0166] In some embodiments, the AAV Cap proteins comprise VP1, VP2, and VP3.
  • a serotype of the AAV Cap proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, A
  • the serotype is an AAV5 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV5 Cap proteins. In some embodiments, the AAV Cap proteins encode for AAV9 Cap proteins. In some embodiments, the serotype is an AAV9 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for PhP.EB Cap proteins. In some embodiments, the serotype is an PhP.EB Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV8 Cap proteins. In some embodiments, the serotype is an AAV8 Cap protein which comprises one or more mutations or insertions.
  • the AAV Cap proteins encode for AAV2 proteins. In some embodiments, the serotype is an AAV2 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV6 Cap proteins. In some embodiments, the serotype is an AAV6 Cap protein which comprises one or more mutations or insertions.
  • polynucleotides comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is 3' of the sequence encoding AAV Cap proteins are provided.
  • Any suitable inducible promoter that is stronger than the native p40 promoter may be used for increasing AAV Cap proteins expression. Suitable inducible promoters are further described below in the section "Inducible Promoters”.
  • the inducible promoters are selected on the basis of the regulatory sequence that allows for control of the promoter.
  • the regulatory sequence may be operably linked to the promoter and positioned upstream of the promoter.
  • Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • the regulatory sequence used to control expression may be endogenous or exogenous to the host cell.
  • bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression.
  • mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides.
  • inclusion of a bacterial tetracycline response element (TRE) in a construct allows mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline).
  • TRE bacterial tetracycline response element
  • the inducible promoter is a tetracycline-inducible promoter. In other embodiments, the inducible promoter is an ecdysone-inducible promoter. In still other embodiments, the inducible promoter is a cumate-inducible promoter.
  • the inducible promoter comprises a tetracycline-responsive promoter element (TRE).
  • the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
  • the minimal promoter is a human cytomegalovirus promoter.
  • the TRE comprises seven repeats of a 19 base pair operator sequence (tetO).
  • the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal human cytomegalovirus (CMV) promoter.
  • the inducible promoter is a Tet-On promoter.
  • the inducible promoter comprises a first inducible promoter.
  • the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148.
  • the polynucleotide has sequence of SEQ ID NO: 148.
  • the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163.
  • the polynucleotide has sequence of SEQ ID NO: 163.
  • the Cap coding sequence is operably linked to a promoter.
  • the AAV Cap proteins comprise VP1, VP2, and VP3.
  • the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter.
  • a single construct or separate constructs comprise these sequences, in any combination.
  • the promoter is an inducible promoter.
  • the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter.
  • the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter.
  • an excisable element e.g., a sequence flanked by recombination sites and comprising a stop signal
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the Cap protein encoding sequence provided in a separate polynucleotide construct can be expressed under the control of an inducible promoter.
  • the Cap coding sequence may include the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to the inducible promoter.
  • the Cap protein encoding sequence provided in a separate polynucleotide construct or in the same polynucleotide comprising the Rep coding sequence can be expressed under the control of an inducible promoter.
  • the Cap protein encoding sequence may be operably linked to a polyadenylation (polyA) signal sequence.
  • the polyA signal sequence may be a polyA signal sequence functional in the cells used for producing the rAAV.
  • the polyA signal sequence may be a bovine Growth Hormone polyA (bGH-PolyA) signal sequence, a SV40 polyA signal sequence, or a Rabbit Beta Globin PolyA signal sequence.
  • the polyadenylation signal sequence is any polyadenylation signal sequence that encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162.
  • the bGH-PolyA signal sequence may include a nucleotide sequence that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 151.
  • the SV40 polyA signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 152.
  • the SV40 polyA sequence is shorter than SEQ ID NO: 152. In some embodiments, the SV40 polyA sequence is longer than SEQ ID NO: 152.
  • the Rabbit Beta Globin signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 170.
  • the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof.
  • the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, A
  • the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC
  • capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, A
  • the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety. [0187] In particular embodiments, the capsid is an AAV1 capsid. In particular embodiments, the capsid is an AAV5 capsid. In particular embodiments, the capsid is an AAV9 capsid.
  • polynucleotides comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence are provided.
  • the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.
  • the second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above.
  • transcription of the first sequence is driven by native AAV promoters.
  • transcription of the first sequence is driven by the P5 and P19 native AAV promoters.
  • the one or more promoters comprise P5 and P19 native promoters.
  • a First Sequence Encoding AAV Rep Proteins nce encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161.
  • the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 160 or 161.
  • the first sequence encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136.
  • the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 136. In other embodiments, the first sequence encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.
  • the first sequence encoding AAV Rep proteins is separated from the second sequence by an intervening sequence.
  • the intervening sequence comprises a transcriptional blocking element (TBE).
  • TBE transcriptional blocking element
  • a sequence of the TBE has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 169.
  • a sequence of the TBE has sequence of SEQ ID NO: 169.
  • the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element, and (iii) a second part of the AAV Rep proteins coding sequence.
  • the excisable element comprises a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction.
  • the first sequence encoding AAV Rep proteins comprises from 5' to 3': one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a first recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3' splice site, and a sequence comprising a second part of the AAV Rep coding sequence.
  • the first recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element.
  • the first recombination site and the second recombination site are oriented in the same direction.
  • the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence.
  • the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element.
  • the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.
  • the coding sequence encoding the stop signaling sequence of the first sequence encodes for from 5' to 3': an exon and the stop signaling sequence.
  • the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.
  • the polynucleotide of the present disclosure further comprises a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker.
  • the polynucleotide of the present disclosure is a polynucleotide construct. In some embodiments, both the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins are on single construct. [0198] In some embodiments, the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins are on separate constructs.
  • the polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the promoter is a constitutive promoter.
  • polynucleotides comprising: a first sequence encoding AAV Rep proteins and a second sequence encoding AAV Cap proteins, wherein the first sequence is operably linked to one or more promoters and the second sequence is operably linked to an inducible promoter are provided.
  • the one or more promoters comprise P5 and P19 native promoters and the inducible promoter comprises a first inducible promoter.
  • the first inducible promoter comprises a tetracycline-responsive promoter element (TRE).
  • the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
  • the minimal promoter is a human cytomegalovirus promoter.
  • the first sequence and the one or more promoters operably linked to the first sequence are separated from the second sequence and the inducible promoter operably linked to the second sequence by an intervening sequence.
  • the intervening sequence comprises a transcriptional blocking element (TBE).
  • TBE transcriptional blocking element
  • the inducible promoter is a Tet-On promoter.
  • the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence.
  • the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.
  • the polypeptides further comprise a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker. 1.4.
  • systems of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload are provided.
  • the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence.
  • the polyadenylation signal sequence is 3' of the sequence encoding AAV Cap proteins are provided.
  • the b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above.
  • transcription of a first polynucleotide is driven by native AAV promoters. In other embodiments, optionally, transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters.
  • the one or more promoters comprise a P5 native AAV promoter and a P19 native AAV promoter.
  • the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence.
  • the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises from 5' to 3': one or more promoters operably linked to a first sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a first recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3' splice site, and a second sequence comprising a second part of the AAV Rep coding sequence, allowing expression of AAV Rep proteins.
  • the first recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element.
  • the first recombination site and the second recombination site are oriented in the same direction.
  • the one or more promoters are not operably linked to the second sequence comprising the second part of the AAV Rep coding sequence.
  • the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element.
  • the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.
  • the coding sequence encoding the stop signaling sequence of the first polynucleotide encodes for from 5' to 3': an exon and the stop signaling sequence.
  • the coding sequence encoding the stop signaling sequence of the first polynucleotide further comprises a sequence encoding a protein marker.
  • the sequence encoding the protein marker is in-frame with the stop signaling sequence.
  • the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161.
  • a set of polynucleotides comprises: (i) a first polynucleotide encoding AAV Rep proteins; (ii) a second polynucleotide encoding the AAV Cap proteins; and (iii) a third polynucleotide encoding one or more adenoviral helper proteins wherein when the one or more polynucleotides are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide further comprises a sequence encoding AAV Cap proteins.
  • transcription of the sequence encoding the AAV Cap proteins on the first polynucleotide is driven by a native AAV Cap proteins promoter.
  • the native AAV Cap proteins promoter is a P40 native AAV promoter.
  • the Rep coding sequence is 5’ to the Cap coding sequence.
  • the Cap coding sequence is operatively linked to an endogenous P40 promoter.
  • the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136. In certain embodiments, the first polynucleotide has sequence of SEQ ID NO: 136.
  • the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.
  • the first polynucleotide has sequence of SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.
  • a set of polynucleotides comprises: (i) a first polynucleotide encoding AAV Rep proteins and AAV Cap proteins; (ii) a second polynucleotide encoding the AAV Cap proteins; and (iii) a third polynucleotide encoding one or more adenoviral helper proteins wherein when the one or more polynucleotides are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the Rep sequence can encode Rep from any desired AAV serotype.
  • the encoded Rep protein is drawn from the same serotype as the Cap protein.
  • the encoded Rep protein is drawn from a different serotype from the Cap protein.
  • the encoded Rep protein includes, but is not limited to, a Rep protein from AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11, or chimeric combinations thereof.
  • the nucleotide sequences of the genomes of the AAV serotypes are known.
  • the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV- 2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol, 45: 555-564 (1983); the complete genome of AAV- 3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos.
  • the AAV-9 genome is provided in Gao et al. Virol, 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol Ther, 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004).
  • the Rep coding sequence is interrupted by an excisable element. Addition of both the first triggering agent and the second triggering agent are required for excision of the excisable element.
  • the excisable element is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (-) indicates the point of insertion of the excisable element, in the Rep coding sequence, and the excisable element is inserted downstream of the p19 promoter.
  • the excisable element is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (-) indicates the point of insertion of the excisable element, in the Rep coding sequence, and the excisable element is inserted downstream of the p19 promoter and upstream of the p40 promoter.
  • the excisable element comprises, from 5’ to 3’, a first spacer segment, a second spacer segment, and a third spacer segment.
  • the first spacer segment comprises a 5’ splice site (5’SS) 5’ to the first spacer element.
  • the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1.
  • the second spacer segment comprises a polynucleotide encoding a detectable protein marker flanked by lox sites.
  • the detectable protein marker is a fluorescent protein.
  • the fluorescent protein is a green or blue fluorescent protein (GFP of BFP).
  • GFP green or blue fluorescent protein
  • the GFP is EGFP.
  • the fluorescent protein is a blue fluorescent protein (BFP). Screening for the fluorescent marker can be used to confirm integration of the construct into the cell genome, and can subsequently be used to confirm excision of the intervening spacer segment.
  • the second spacer segment further comprises a polyA signal sequence.
  • the poly A signal sequence comprises a rabbit beta globin (RBG) polyA signal sequence.
  • the second spacer segment further comprises a first 3’ splice site (3’SS) between the first lox site and the polynucleotide encoding the protein marker.
  • the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2.
  • the third spacer segment further comprises a second 3’ splice site (3’SS). In particular embodiments, the second 3’ splice site is positioned 3’ to the second lox site.
  • the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3.
  • the Rep coding sequences are operatively linked to an endogenous P5 promoter. In various embodiments, the Rep coding sequences are operatively linked to an endogenous P19 promoter.
  • the Rep coding sequences are operably linked to an inducible promoter.
  • the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter.
  • the Rep coding sequences are operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • Second Polynucleotide Encoding Inducible Cap Proteins [0233] In some embodiments, the second polynucleotide comprises a sequence encoding AAV Cap proteins operably linked to an inducible promoter.
  • the second polynucleotide comprises (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is 3' of the sequence encoding AAV Cap proteins are provided.
  • the second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and optionally, (ii) a polyadenylation signal sequence, is described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above.
  • the first polynucleotide and second polynucleotide are in a first polynucleotide construct.
  • a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide.
  • a first polynucleotide construct comprises the Rep coding sequence is linked to native promoters and the Cap coding sequence is linked to an inducible promoter and the Rep coding sequence and the native promoters are separated from the Cap coding sequence and the inducible promoter by an intervening sequence, such as, a TBE.
  • an intervening sequence such as, a TBE.
  • the TBE comprises a nucleotide sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 169.
  • the first polynucleotide lacks a sequence encoding AAV Cap proteins and the second polynucleotide comprises a sequence encoding AAV Cap proteins.
  • the sequence encoding the AAV Cap proteins of the first polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide.
  • Third Polynucleotide Encoding Adenoviral Helper Proteins [0238]
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase; a first constitutive promoter operably linked to a sequence encoding an activator.
  • the third recombination site and the fourth recombination site are oriented in the same direction.
  • the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins.
  • the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a first triggering agent.
  • a second polynucleotide construct comprises the third polynucleotide as described herein.
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: (i) a first sequence comprising from 5' to 3': a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self- excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site; and a sequence encoding one or more adenoviral helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; (ii) a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator.
  • the third recombination site and the fourth recombination site are oriented in the same direction.
  • the cell constitutively expresses the activator, and the activator is unable to activate the second inducible promoter in absence of a first triggering agent.
  • the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed.
  • the inducible recombinase in the presence of a second triggering agent, translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins.
  • the one or more adenoviral helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein.
  • the one or more adenoviral helper proteins comprises E2A protein and E4 protein.
  • the third polynucleotide comprising the sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins.
  • the SEQ ID NO: 30 comprises the third polynucleotide.
  • the one or more adenoviral helper proteins are separated by a bicistronic open reading frame.
  • the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
  • the second inducible promoter operably linked to the self- excising element in the third polynucleotide is a tetracycline-inducible promoter, an ecdysone- inducible promoter, or a cumate-inducible promoter.
  • the first inducible promoter and the second inducible promoter are the same.
  • the first inducible promoter and the second inducible promoter are a tetracycline-inducible promoter.
  • the tetracycline- inducible promoter comprises a tetracycline-responsive promoter element (TRE).
  • the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
  • the minimal promoter is a human cytomegalovirus promoter.
  • the first constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.
  • a triggering agent for inducing the tetracycline-inducible promoter is tetracycline.
  • a triggering agent for inducing the tetracycline- inducible promoter is doxycycline.
  • the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen.
  • the recombination sites in the first polynucleotide and the third polynucleotide are lox sites and the inducible recombinase is a Cre recombinase.
  • the recombination sites in the first polynucleotide and the third polynucleotide are flippase recognition target (FRT) sites and the inducible recombinase is a flippase (Flp) recombinase.
  • FRT flippase recognition target
  • Flp flippase
  • presence of the triggering agent activates the activator for activation of the first inducible promoter to express AAV Cap proteins from the second polynucleotide encoding the AAV Cap proteins.
  • presence of the triggering agent activates the activator for activation of the second inducible promoter to express the Rep proteins of the first polynucleotide, if present the Cap proteins of the first polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins.
  • the inducible recombinase upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide and if present the Cap Proteins of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding
  • the third polynucleotide further comprises a third selectable marker operably linked to a third promoter.
  • Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA [0254]
  • the third polynucleotide further comprises a sequence encoding a viral associated RNA (VA-RNA).
  • VA-RNA is a mutated VA-RNA.
  • the VA-RNA is wild-type VA-RNA.
  • VA-RNA comprises one or more mutations in the VA-RNA internal promoter.
  • a second polynucleotide construct comprises the third polynucleotide as described herein.
  • the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a second constitutive promoter and a second part of the second constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, and excision of the second excisable element by the inducible recombinase generates a functional complete second constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.
  • the fifth and sixth recombination sites are oriented in the same direction.
  • the first part of the second constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter
  • the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter
  • the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter
  • the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter.
  • the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter
  • the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.
  • the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further comprises a nucleic acid sequence encoding a viral associated RNA (“VA-RNA”) sequence.
  • VA-RNA viral associated RNA
  • the third recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a VA-RNA sequence.
  • the expression of VA-RNA is constitutive.
  • the expression of VA-RNA is inducible.
  • the VA-RNA sequence comprises one or more mutations in the VA-RNA internal promoter, preferably G16A and G60A.
  • the expression of VA-RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter.
  • the expression of VA-RNA is driven by a U6 promoter or a U7 promoter.
  • the U6 promoter or the U7 promoter comprises a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide.
  • the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.
  • the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 153. In certain embodiments, the third polynucleotide has sequence of SEQ ID NO: 153.
  • the payload of the fourth polynucleotide comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest.
  • the payload of the fourth polynucleotide is progranulin.
  • a third polynucleotide construct comprises the fourth polynucleotide as described herein.
  • the sequence encoding the payload of the fourth polynucleotide comprises a sequence encoding a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest.
  • the sequence encoding the payload of the fourth polynucleotide is a sequence encoding progranulin.
  • the sequence encoding progranulin has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 166.
  • the sequence encoding progranulin has sequence of SEQ ID NO: 166.
  • the sequence encoding the payload comprises a sequence encoding a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair.
  • the fourth polynucleotide comprising the sequence encoding the payload comprises the sequence encoding the payload flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR).
  • the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146.
  • the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has sequence of SEQ ID NO: 146.
  • the sequence encoding the payload has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the sequence encoding the payload has sequence of SEQ ID NO: 156.
  • the sequence encoding the payload has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158. In certain embodiments, the sequence encoding the payload has sequence of SEQ ID NO: 158. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the fourth integrated synthetic construct (also referred to as Construct 4) comprises the coding sequence for an expressible payload and a third mammalian cell selection element.
  • the expressible payload is under the control of a constitutive promoter. This construct can be referred to as construct 4 or payload construct, interchangeably.
  • the expressible payload encodes a guide RNA.
  • the guide RNA directs RNA editing.
  • the guide RNA directs Cas-mediated DNA editing.
  • the guide RNA directs ADAR- mediated RNA editing.
  • the fourth integrated synthetic construct comprises a sequence encoding for any of the expressible payloads disclosed herein.
  • said sequence can encode for any therapeutic.
  • the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof.
  • the transgene encodes for progranulin.
  • the tRNA suppressor is capable of suppressing an opal stop codon.
  • the tRNA suppressor is capable of suppressing an ochre stop codon.
  • the tRNA suppressor is capable of suppressing an amber stop codon.
  • the fourth integrated synthetic construct comprises sequences encoding for more than one of the expressible payloads disclosed herein.
  • the fourth integrated synthetic construct comprises 2 gRNA, 3 gRNA, 4 gRNA, 5 gRNA, 6 gRNA, 7 gRNA, 8 gRNA, 9 gRNA, or 10 gRNA. These gRNAs can all be the same, all be different, or any combination of the same and different.
  • the fourth integrated synthetic construct comprises 2 suppressor tRNAs, 3 suppressor tRNAs, 4 suppressor tRNAs, 5 suppressor tRNAs, 6 suppressor tRNAs, 7 suppressor tRNAs, 8 suppressor tRNAs, 9 suppressor tRNAs, or 10 suppressor tRNAs.
  • These suppressor tRNAs can all be the same, all be different, or any combination of the same and different.
  • the expressible payload encodes a protein.
  • the expressible payload is an enzyme, useful for replacement gene therapy.
  • the protein is a therapeutic antibody.
  • the protein is a vaccine immunogen.
  • the vaccine immunogen is a viral protein.
  • the expressible payload is a homology construct for homologous recombination.
  • the third mammalian cell selection element is an auxotrophic selection element.
  • the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139.
  • the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139, wherein SEQ ID NO: 34 in SEQ ID NO: 33 or SEQ ID NO: 139 is replaced with a sequence of the payload of interest.
  • the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, or SEQ ID NO: 159.
  • the payload construct is a plasmid comprising at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159.
  • the payload construct comprises a sequence of a payload flanked by ITR sequences.
  • expression of the sequence of the payload is driven by a constitutive promoter or an inducible promoter.
  • the promoter and sequence of the payload are flanked by ITR sequences.
  • the payload construct flanked by ITRs comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146.
  • the sequence of the payload comprises a polynucleotide sequence coding for a gene.
  • the gene codes for a selectable marker or detectable marker.
  • the gene codes for a therapeutic polypeptide or transgene.
  • the therapeutic polypeptide or transgene is progranulin.
  • the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide.
  • the therapeutic polynucleotide is a tRNA suppressor or a guide RNA.
  • the tRNA suppressor is capable of suppressing an opal stop codon.
  • the tRNA suppressor is capable of suppressing an ochre stop codon.
  • the tRNA suppressor is capable of suppressing an amber stop codon.
  • the guide RNA is a polyribonucleotide capable of binding to a protein.
  • the protein is nuclease.
  • the protein is a Cas protein, an ADAR protein, or an ADAT protein.
  • the guide RNA when bound to a target RNA, recruits an ADAR protein for editing of the target RNA.
  • the Cas protein is catalytically inactive Cas protein.
  • the payload construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the payload construct are stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs are separately stably integrated into the genome of the cell. [0275] In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences.
  • expression of the selectable marker or detectable marker outside of the ITR sequences is driven by a promoter.
  • the promoter can be a constitutive promoter or an inducible promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.
  • the selectable marker is a mammalian cell selection element (e.g., a third mammalian cell selection element). In some embodiments, the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z- Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the payload construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the payload construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the selectable marker is outside of the ITR sequences on the payload construct.
  • the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C- terminus of the auxotrophic protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the auxotrophic or leucine zipper linked to a C-terminus of the auxotrophic. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the payload construct further comprises a spacer between the 5’ ITR and the promoter/selectable marker or promoter/detectable marker outside of the ITR sequences.
  • the payload construct further comprises a spacer between the 3’ ITR and the promoter/selectable marker or promoter/detectable marker outside of the ITR sequences.
  • the spacer ranges in length from 500 base pairs to 5000 base pairs, including any length within this range such as 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 base pairs.
  • the spacer length is a sufficient length for decreasing reverse packaging of the selectable marker or detectable marker that is outside the ITR sequences.
  • the fourth integrated synthetic construct comprising the coding sequence for a payload and a selectable marker or detectable marker is further engineered to remove locations having the potential for Rep-mediated nicking.
  • a location having the potential for Rep-mediated nicking is a location having the sequence CAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 87); a sequence comprising GAGC (SEQ ID NO: 88) repeats; or the sequence GATGGAGTTGGCCACTCCCTC (SEQ ID NO: 89). These sequences can be engineered to prevent binding of Rep proteins for Rep-mediated nicking.
  • the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 100 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 200 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 300 nucleotides of an ITR sequence.
  • the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 400 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 500 nucleotides of an ITR sequence.
  • a payload construct comprises a polynucleotide construct coding for a VA-RNA.
  • the VA-RNA is operably linked to a constitutive promoter or an inducible promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.
  • a payload construct comprises a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter.
  • a separate polynucleotide construct codes for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter.
  • the sequence coding for the VA-RNA comprises a sequence coding for a transcriptionally dead VA-RNA.
  • the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region.
  • the sequence coding for the VA-RNA comprises at least one mutation.
  • the at least one mutation is in the A Box promoter region.
  • the at least one mutation is in the B Box promoter region.
  • the at least one mutation is G16A and G60A.
  • the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter.
  • the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence.
  • the stuffer sequence is excisable by a recombinase.
  • the stuffer sequence comprises a sequence encoding a gene.
  • the stuffer sequence comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a CMV promoter.
  • the gene encodes a detectable marker or a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z- Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GT—CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the detectable marker comprises a luminescent marker or a fluorescent marker.
  • the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided.
  • the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide.
  • the first recombination site is a first lox sequence and the second recombination site is a second lox sequence.
  • the first lox sequence is a first loxP site and the second lox sequence is a second loxP site.
  • the first recombination site is a first FRT site and the second recombination site is a second FRT site.
  • the construct comprising the VA-RNA as described herein further comprises a sequence coding for a selectable marker.
  • the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the construct comprising VA-RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the construct comprising the VA-RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a vector.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is a synthetic nucleic acid construct.
  • an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26.
  • a VA-RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26.
  • a VA-RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26.
  • Payloads [0287] Disclosed herein are payloads that may be encoded for by polynucleotide construct 4, which encodes for a payload.
  • This fourth polynucleotide is referred to herein as a “payload construct” or “therapeutic payload.”
  • the payload may be an expressible payload.
  • the polynucleotide may encode for any therapeutic.
  • the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof.
  • the payload is guide RNA, wherein the guide RNA, when bound to a target RNA, recruits an ADAR enzyme for editing of the target RNA.
  • the payload is progranulin.
  • the stable mammalian cell lines disclosed herein can conditionally produce rAAV virions that encapsidate more than one payload. Any combination of payloads disclosed herein is contemplated. [0288]
  • the sequence encoding a payload as disclosed herein encompasses any nucleotide sequence that is to be delivered to a cell.
  • the nucleotide sequence may be utilized in the cell for, e.g., insertion of the nucleotide sequence or a part thereof.
  • the nucleotide sequence may be used to repair an endogenous DNA.
  • the nucleotide sequence itself is the payload being delivered by the rAAV to a cell.
  • the polynucleotide payload is transcribed in the cell into an RNA which is not translated into a protein.
  • the RNA is the payload that is delivered by the polynucleotide payload present in the rAAV.
  • the polynucleotide payload is transcribed in the cell into an mRNA which is translated into a protein.
  • the protein is the payload that is delivered by the polynucleotide payload present in the rAAV.
  • the polynucleotide payload may include a promoter operably linked to a DNA sequence.
  • the promoter may be any promoter that allows for transcription of the DNA in the cell.
  • a payload disclosed herein may be a therapeutic payload.
  • the DNA sequence may be transcribed to produce RNA in the cell.
  • the RNA may be mRNA.
  • the RNA may be a guide RNA (gRNA), a tRNA, a suppressor tRNA, an mRNA, or a circular RNA.
  • the RNA may be a regulatory RNA of interest such as, but not limited to, a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a small nuclear RNA (snRNA), a long non-coding RNA (lncRNA), an antisense nucleic acid, and the like.
  • miRNA microRNA
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • snRNA small nuclear RNA
  • lncRNA long non-coding RNA
  • antisense nucleic acid and the like.
  • the polynucleotide payload may be a gene encoding a polypeptide, such as, an antibody, a hormone, a site-specific endonuclease, a reporter gene, a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, a ribozyme, a DNAzyme, or any combination thereof.
  • ADAR adenosine deaminase acting on RNA
  • a payload may include any one or combination of the following: a transgene, a tRNA suppressor, a guide RNA, or any other target binding/modifying oligonucleotide or derivative thereof, or payloads may include immunogens for vaccines, and elements for any gene editing machinery (DNA or RNA editing). Payloads may also include those that deliver a transgene encoding antibody chains or fragments that are amenable to viral vector-mediated expression (also referred to as “vectored or vectorized antibody” for gene delivery). See, e.g., Curr Opinion HIV AIDS.2015 May; 10(3): 190–197, describing vectored antibody gene delivery for the prevention or treatment of HIV infection. See also, U.S. Pat.
  • a payload disclosed herein may not be a therapeutic payload (e.g., a coding for a detectable marker such as GFP).
  • the polynucleotide payload refers to a polynucleotide that may be a homology element for homology-directed repair, or polynucleotide transcribed into a guide RNA to be delivered for a variety of purposes.
  • the transgene refers to a nucleic acid sequence coding for expression of guide RNA for ADAR editing or ADAT editing.
  • the transgene refers to a transgene packaged for gene therapy. In some embodiments, the transgene refers to synthetic constructs packaged for vaccines.
  • a polynucleotide payload may be described as encoding an RNA, which is meant to refer to the RNA transcribed from the polynucleotide.
  • the sequence encoding the payload comprises two expressible sequences, wherein a first expressible sequence encodes for a first gRNA and a second expressible sequence encodes for a second gRNA. In some embodiments, the first gRNA and the second gRNA are different. In some embodiments, the first gRNA and the second gRNA are the same.
  • the sequence encoding the payload comprises two or more expressible sequences.
  • the two or more expressible sequences encode for two or more gRNA.
  • the two or more gRNA are all different gRNA, all the same gRNA, or a combination of the same and different gRNA.
  • the sequence encoding the payload comprises an expressible sequence encoding both a heterologous RNA and a heterologous polypeptide.
  • the expressible sequence encodes two or more heterologous payloads. Where the expressible sequence encodes two heterologous payloads, in some cases, the nucleotide sequences encoding the two heterologous payloads are operably linked to the same promoter.
  • sequence encoding the payload comprises an expressible sequence encoding three heterologous payloads.
  • sequence encoding the payload comprises an expressible sequence encoding three heterologous payloads.
  • the nucleotide sequences encoding the three heterologous payloads are operably linked to the same promoter.
  • the nucleotide sequences encoding the three heterologous payloads are operably linked to two or three different promoters.
  • the fourth polynucleotide construct of the present disclosure comprises two or more expressible sequences, each comprising a nucleotide sequence encoding a heterologous payload.
  • the expressible sequence encodes a polypeptide of interest.
  • the polypeptide of interest may be any type of protein/peptide including, without limitation, an enzyme, an extracellular matrix protein, a receptor, transporter, ion channel, or other membrane protein, a hormone, a neuropeptide, an antibody, or a cytoskeletal protein; or a fragment thereof, or a biologically active domain of interest.
  • the payload is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit.
  • RNAi interfering RNA
  • suitable RNAi include RNAi that decrease the level of an apoptotic or angiogenic factor in a cell.
  • an RNAi may be an shRNA or siRNA that reduces the level of a payload that induces or promotes apoptosis in a cell.
  • a payload may be a gene whose gene product induces or promotes apoptosis are referred to herein as “pro-apoptotic genes” and the products of those genes (mRNA; protein) are referred to as “pro-apoptotic gene products.”
  • Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad gene products.
  • the RNAi specifically reduces the level of an RNA and/or a polypeptide product of a defective allele.
  • the payload is an aptamer.
  • the aptamer is a therapeutic aptamer.
  • the aptamer may function as an antagonist by blocking interactions at a disease-associated target (e.g., receptor-ligand interactions).
  • an aptamer may serve as an agonist for activating the function of a target receptor.
  • Exemplary aptamers of interest include aptamers against growth factor receptors and growth factors such as aptamers that bind to epidermal growth factor receptor (see, e.g., Wang et al. (2014) Biochem. Biophys. Res. Commun.453(4):681-5), transforming growth factor-beta type III receptor (see, e.g., Ohuchi et al. (2006) Biochimie 88(7):897-904.), vascular endothelial growth factor (VEGF) (see, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee et al. (2005) Proc. Natl. Acad. Sci.
  • VEGF vascular endothelial growth factor
  • the expressible sequence encodes a sequence-specific endonuclease for use in genome editing.
  • the sequence specific endonuclease may be used to create a double-stranded break at a specific site in the genome.
  • the double stranded breaks may then be repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR) pathways.
  • NHEJ non-homologous end joining
  • MMEJ microhomology-mediated end joining
  • HDR homology-directed repair
  • Desired genome edits may be introduced into the genome using donor DNA to repair double-strand breaks by homologous recombination.
  • Various sequence-specific endonucleases may be used in genome editing for creation of double-strand breaks in DNA, including, without limitation, engineered zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) Cas9.
  • ZFNs engineered zinc-finger nucleases
  • TALENs transcription activator-like effector nucleases
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a payload of interest is a site-specific endonuclease that provides for site- specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a disease.
  • a site-specific endonuclease may be targeted to the defective allele and knock out the defective allele.
  • a site-specific endonuclease is an RNA-guided endonuclease.
  • a site-specific nuclease may also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele.
  • a subject rAAV virion may be used to deliver a site-specific endonuclease that knocks out a defective allele and also be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional gene product.
  • the payload is an RNA-guided endonuclease.
  • the payload is an RNA comprising a nucleotide sequence encoding an RNA-guided endonuclease.
  • the payload is a guide RNA, e.g., a single-guide RNA.
  • the payloads are: 1) a guide RNA; and 2) an RNA-guided endonuclease.
  • the guide RNA may comprise: a) a protein- binding region that binds to the RNA-guided endonuclease; and b) a region that binds to a target nucleic acid.
  • RNA-guided endonuclease is also referred to herein as a “genome editing nuclease.”
  • Examples of RNA-guided endonucleases are CRISPR/Cas endonucleases (e.g., class 2 CRISPR/Cas endonucleases such as a type II, type V, or type VI CRISPR/Cas endonucleases).
  • a suitable genome editing nuclease is a CRISPR/Cas endonuclease (e.g., a class 2 CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Cas endonuclease).
  • a suitable RNA-guided endonuclease is a class 2 CRISPR/Cas endonuclease. In some cases, a suitable RNA-guided endonuclease is a class 2 type II CRISPR/Cas endonuclease (e.g., a Cas9 protein). In some cases, a genome targeting composition includes a class 2 type V CRISPR/Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein).
  • a suitable RNA-guided endonuclease is a class 2 type VI CRISPR/Cas endonuclease (e.g., a C2c2 protein; also referred to as a “Cas13a” protein). Also suitable for use is a CasX protein. Also suitable for use is a CasY protein. [0304] In some cases, the genome-editing endonuclease is a Type II CRISPR/Cas endonuclease. In some cases, the genome-editing endonuclease is a Cas9 polypeptide.
  • the Cas9 protein is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by virtue of its association with the protein-binding segment of the Cas9 guide RNA.
  • a target nucleic acid sequence e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.
  • the Cas9 polypeptide used in a composition or method of the present disclosure is a Staphylococcus aureus Cas9 (saCas9) polypeptide.
  • a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide.
  • the genome-editing endonuclease is a type V CRISPR/Cas endonuclease. In some cases a type V CRISPR/Cas endonuclease is a Cpf1 protein. In some cases, the genome-editing endonuclease is a CasX or a CasY polypeptide. CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237.
  • a genome editing nuclease is a fusion protein that is fused to a heterologous polypeptide (also referred to as a “fusion partner”).
  • a genome editing nuclease is fused to an amino acid sequence (a fusion partner) that provides for subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.).
  • a fusion partner e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.
  • NLSs nuclear localization signals
  • Also suitable for use is an RNA-guided endonuclease with reduced enzymatic activity.
  • RNA-guided endonuclease is referred to as a “dead” RNA-guided endonuclease; for example, a Cas9 polypeptide that comprises certain amino acid substitutions such that it exhibits substantially no endonuclease activity, but such that it still binds to a target nucleic acid when complexed with a guide RNA, is referred to as a “dead” Cas9 or “dCas9.”
  • a “dead” Cas9 protein has a reduced ability to cleave both the complementary and the non- complementary strands of a double stranded target nucleic acid.
  • a “nuclease defective” Cas9 lacks a functioning RuvC domain (i.e., does not cleave the non-complementary strand of a double stranded target DNA) and lacks a functioning HNH domain (i.e., does not cleave the complementary strand of a double stranded target DNA).
  • a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid.
  • a Cas9 protein that may not cleave target nucleic acid is referred to as a “nuclease defective Cas9”, “dead Cas9” or simply “dCas9.”
  • Other residues may be mutated to achieve the above effects (i.e. inactivate one or the other nuclease portions).
  • the genome-editing endonuclease is an RNA-guided endonuclease (and its corresponding guide RNA) known as Cas9-synergistic activation mediator (Cas9-SAM).
  • RNA-guided endonuclease (e.g., Cas9) of the Cas9-SAM system is a “dead” Cas9 fused to a transcriptional activation domain (wherein suitable transcriptional activation domains include, e.g., VP64, p65, MyoD1, HSF1, RTA, and SET7/9) or a transcriptional repressor domain (where suitable transcriptional repressor domains include, e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, and a SID4X domain).
  • the guide RNA of the Cas9-SAM system comprises a loop that binds an adapter protein fused to a transcriptional activator domain (e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7/9) or a transcriptional repressor domain (e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, or a SID4X domain).
  • a transcriptional activator domain e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7/9
  • a transcriptional repressor domain e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, or a SID4X domain.
  • the guide RNA is a single-guide RNA comprising an MS2 RNA aptamer inserted into one or two loops of the sgRNA;
  • the dCas9 is a fusion polypeptide comprising dCas9 fused to VP64;
  • the adaptor/functional protein is a fusion polypeptide comprising: i) MS2; ii) p65; and iii) HSF1. See, e.g., U.S. Patent Publication No.2016/0355797.
  • a chimeric polypeptide comprising: a) a dead RNA-guided endonuclease; and b) a heterologous fusion polypeptide.
  • suitable heterologous fusion polypeptides include a polypeptide having, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity.
  • a nucleic acid that binds to a class 2 CRISPR/Cas endonuclease e.g., a Cas9 protein; a type V or type VI CRISPR/Cas protein; a Cpf1 protein; etc.
  • a guide RNA or “CRISPR/Cas guide nucleic acid” or “CRISPR/Cas guide RNA.”
  • a guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid.
  • a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.”
  • the guide RNA is one molecule (e.g., for some class 2 CRISPR/Cas proteins, the corresponding guide RNA is a single molecule; and in some cases, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.”
  • the guide RNA is at least partially complementary to a target RNA sequence and is capable of recruiting an ADAR enzyme for RNA editing of the target RNA sequence.
  • the payload may modify a target nucleic acid.
  • a target nucleic acid comprises a deleterious mutation in a defective allele (e.g., a deleterious mutation in a neural cell target nucleic acid)
  • the RNA-guided endonuclease/guide RNA complex together with a donor nucleic acid comprising a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid comprising a nucleotide sequence that encodes a functional copy of the protein encoded by the defective allele)
  • HDR homology-directed repair
  • the payloads are an RNA-guided endonuclease and 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non-homologous end joining (NHEJ).
  • the payloads are: i) an RNA-guided endonuclease; and ii) one guide RNA.
  • the guide RNA is a single-molecule (or “single guide”) guide RNA (an “sgRNA”).
  • the guide RNA is a dual-molecule (or “dual-guide”) guide RNA (“dgRNA”).
  • the payloads are: i) an RNA-guided endonuclease; and ii) 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non- homologous end joining (NHEJ).
  • the guide RNAs are sgRNAs.
  • the guide RNAs are dgRNAs.
  • the payloads are: i) a Cpf1 polypeptide; and ii) a guide RNA precursor; in these cases, the precursor may be cleaved by the Cpf1 polypeptide to generate 2 or more guide RNAs.
  • VA-RNA viral associated RNA
  • the VA-RNA is a mutated VA-RNA.
  • a second polynucleotide construct or a fourth comprises the fifth polynucleotide as described herein.
  • the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.
  • the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter.
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.
  • the VA-RNA comprises a G16A mutation.
  • the VA-RNA comprises a G60A mutation.
  • the VA-RNA comprises a G16A mutation and a G60A mutation.
  • Selectable Markers for Polynucleotides [0323]
  • a) the first polynucleotide further comprises a sequence encoding a first selectable marker operably linked to a first promoter.
  • the second polynucleotide further comprises a sequence encoding a second selectable marker operably linked to a second promoter.
  • c) the third polynucleotide further comprises a sequence encoding a third selectable marker operably linked to a third promoter.
  • d) the fourth polynucleotide further comprises a sequence encoding a fourth selectable marker operably linked to a fourth promoter.
  • e) the fifth polynucleotide further comprises a sequence encoding a fifth selectable marker operably linked to a fifth promoter.
  • the system of polynucleotides comprises any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker.
  • any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker are different selectable markers; the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, the fifth selectable marker, or any combinations thereof are the same selectable marker but as a different split portion of the selectable marker; or any combinations thereof.
  • any combinations of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are the same constitutive promoter or different constitutive promoters.
  • the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is an antibiotic resistance gene.
  • the antibiotic resistance gene is a blasticidin resistance gene, a hygromycin resistance gene, or a puromycin resistance gene.
  • the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a first split portion of an antibiotic resistance gene. In certain embodiments, the first split portion of the antibiotic resistance gene is a first split portion of the blasticidin resistance gene. [0333] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a second split portion of an antibiotic resistance gene. In certain embodiments, the second split portion of the antibiotic resistance gene is a second split portion of the blasticidin resistance gene.
  • the first promoter, the second promoter, the third promoter, the fourth promoter, the fifth promoter, or any combination thereof is an EF1alpha promoter or an attenuated version thereof.
  • the attenuated version comprises a mutation in the TATA box.
  • the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter.
  • the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5' ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof.
  • the first polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker;
  • the second polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter;
  • the third polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter; and/or
  • the fourth polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker.
  • the constitutive promoter is an EF1 alpha promoter and/or the split selectable marker is a split antibiotic resistance protein. In some embodiments, the constitutive promoter is an EF1 alpha promoter and/or the selectable marker is a first antibiotic resistance protein. In some embodiments, the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein. In some embodiments, the constitutive promoter is an EF1 alpha promoter.
  • the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5' ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof.
  • the spacer ranges in length from 500 base pairs to 5000 base pairs.
  • suitable markers include genes which confer resistance to antibiotics or toxins, or sensitivity, or impart color, or change the antigenic characteristics when cells, which have been transfected with the nucleic acid constructs, are grown in an appropriate selective medium.
  • Exemplary selectable marker genes include, without limitation, the neomycin resistance gene (neo encoding aminoglycoside phosphotransferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (Geneticin), the hygromycin-B resistance gene (hygB encoding hygromycin-B-phosphotransferase (HPH)) that confers resistance to hygromycin-B, the puromycin resistance gene (pac encoding puromycin-N-acetyltransferase) that confers resistance to puromycin, Zeocin resistance gene (Sh bla encodes a protein that binds to Zeocin) that prevents Zeocin from binding DNA and damaging it, and the blasticidin resistance gene (BSD) that confers resistance to blasticidin.
  • APH aminoglycoside phosphotransferase
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split selectable marker that allows for selection of cells retaining two different polynucleotides using a single selective pressure.
  • the antibiotic resistance protein is split into two portions that can associate to form a functional antibiotic resistance protein.
  • a first portion of the antibiotic resistance protein is encoded by a first polynucleotide and a second portion of the antibiotic resistance protein is encoded by a second polynucleotide.
  • a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure is used.
  • Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond.
  • Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form.
  • split inteins For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein.
  • Split intein systems are described in: Cheriyan et al, J. Biol.
  • a split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • a first portion of the antibiotic resistance protein is the N- terminal portion which is fused to a N-terminal intein at the C-terminus and a second portion of the antibiotic resistance protein is the C-terminal portion which is fused to a C-terminal intein at the N-terminus.
  • the N-terminal intein associates with the C- terminal intein resulting in excision of the inteins and splicing of the C-terminus of the N- terminal portion of the antibiotic resistance protein to the N-terminus of the C-terminal portion of the antibiotic resistance protein, thereby forming a functional antibiotic resistance proteins.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter and DHFR Z-Nter.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • a split auxotrophic selection system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure can be used.
  • One construct encodes the N-terminal fragment of mammalian dihydrofolate reductase (DHFR) fused to a leucine zipper peptide (“Nter-DHFR”). This N-terminal fragment is enzymatically nonfunctional.
  • DHFR mammalian dihydrofolate reductase
  • the other construct encodes the C-terminal fragment of DHFR fused to a leucine zipper peptide (“Cter-DHFR”).
  • Cter-DHFR leucine zipper peptide
  • This C-terminal fragment is enzymatically nonfunctional.
  • a functional DHFR enzyme complex is formed through association of the leucine zipper peptides.
  • Both constructs can be stably retained in the genome of a DHFR null cell by growth in a medium lacking hypoxanthine and thymidine.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3.
  • the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3.
  • Polynucleotides Encoding AAV Capsid Proteins above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.
  • systems of polynucleotides comprising: (a) the polynucleotide of any one of of the embodiments disclosed here, wherein the polynucleotide is a first polynucleotide; and one or more of: b) a second polynucleotide comprising a sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload are provided.
  • the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide.
  • the second polynucleotide comprises an inducible promoter operably linked to the sequence encoding the AAV Cap proteins.
  • the inducible promoter is a second inducible promoter.
  • the second polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the selectable marker is a second selectable marker and the promoter is a constitutive promoter, optionally wherein the constitutive promoter is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutation in the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter.
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: an inducible promoter operably linked to a self- excising element, wherein the inducible promoter is a third inducible promoter; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the third inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins; a constitutive promoter operably linked to a sequence encoding an activator, [0355] wherein a cell comprising the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter, if present the second inducible promoter
  • the one or more helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein.
  • the fourth polynucleotide comprising the sequence encoding the payload further comprises: a selectable marker or a second part or a first part of the split selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR); optionally, wherein the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at
  • the fourth polynucleotide comprising the sequence encoding the payload further comprises further comprises a spacer between the 5' ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the selectable marker and the 3' ITR, or a combination thereof; optionally wherein SEQ ID NO: 146 or SEQ ID NO: 147 comprises the fourth polynucleotide.
  • the third polynucleotide comprises a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA.
  • the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.
  • the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.
  • the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.
  • the first polynucleotide comprising the sequence encoding the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the split selectable marker is a split antibiotic resistance protein;
  • the second polynucleotide comprising the sequence encoding the AAV Cap proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally wherein the constitutive promoter is an EF1 alpha promoter and/or the selectable marker is a first antibiotic resistance protein;
  • polynucleotide Construct System [0365] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3.
  • Polynucleotides Encoding AAV Capsid Proteins above; and one or more of: b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided.
  • system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above are provided.
  • systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3.
  • polynucleotides Encoding AAV Capsid Proteins above; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above are provided.
  • the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.
  • systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided.
  • system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.
  • systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3.
  • the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above.
  • the sequence of the first polynucleotide is separated from the sequence of the second polynucleotide by an intervening sequence.
  • the intervening sequence comprises a transcriptional blocking element (TBE).
  • TBE transcriptional blocking element
  • the first polynucleotide construct comprises a sequence encoding a single selectable marker.
  • the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker.
  • the constitutive promoter is an EF1 alpha promoter and/or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein.
  • the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter.
  • the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein.
  • the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker.
  • the constitutive promoter is an EF1 alpha promoter and/or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein.
  • the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein;
  • the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or
  • the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the second part of the split selectable maker is a second
  • the first antibiotic resistance protein is a blasticidin resistance protein.
  • the second antibiotic resistance protein is a puromycin resistance protein.
  • AAV Rep Cap Construct(s) and Cap Construct [0377] Disclosed herein are polynucleotide constructs encoding for a AAV Rep protein and Cap protein, where Cap protein is expressed from a native promoter and polynucleotide constructs encoding for the Cap protein, where Cap protein is expressed from an inducible promoter. [0378] Disclosed herein is a polynucleotide construct encoding for a Rep and Cap protein, where Cap protein is expressed from an inducible promoter.
  • a polynucleotide construct encoding for a Rep and Cap protein, where Cap protein is expressed from an inducible promoter and lacks expression from a native promoter.
  • a first polynucleotide construct which encodes for Rep and Cap proteins and comprises an excisable element within the Rep coding sequence.
  • This first polynucleotide construct (Construct 1) is also referred to as a Rep Cap construct, and/or “AAV Rep Cap Construct.”
  • the elements of this first polynucleotide construct can be in one or more separate constructs.
  • the AAV Rep Cap Construct is Construct 1 of FIG.2A.
  • FIG.1A shows two separate polynucleotide constructs that each express AAV Capsid proteins, thereby resulting in a higher AAV capsid proteins level as compared to those expressed by cells having a single polynucleotide construct for encoding AAV Capsid proteins.
  • the level of AAV Capsids produced by the cells having two separate polynucleotide constructs expressing the AAV Capsids is at least 2-fold higher, e.g., at least 5-fold to at least 100-fold higher than level of AAV Capsids produced by cells not having two separate polynucleotide constructs expressing the AAV Capsids.
  • cells with two separate polynucleotide constructs encoding AAV Capsid proteins produce a level of AAV Capsids that is at least 10-fold higher, 20-fold higher, 30-fold higher, 35-fold higher, 50-fold higher, or 100-fold higher than the level of AAV Capsids produced by cells with only a single polynucleotide construct encoding the AAV Capsids.
  • the percent encapsidation of a payload in virus particles produced by the cells having two separate polynucleotide constructs expressing the AAV Capsids is at least 2-fold higher, e.g., at least 5-fold to at least 20-fold higher than the percent encapsidation of a payload in virus particles produced by cells not having two separate polynucleotide constructs expressing the AAV Capsids.
  • the Cap coding sequence is operably linked to a promoter.
  • the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter.
  • the promoter is an inducible promoter.
  • the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter.
  • the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the Cap coding sequence may include the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to the inducible promoter.
  • the Cap protein encoding sequence provided in a separate polynucleotide construct or in the same polynucleotide comprising the Rep coding sequence can be expressed under the control of an inducible promoter.
  • the Cap protein encoding sequence may be operably linked to a polyadenylation signal sequence.
  • the polyA signal sequence may be a polyA signal sequence functional in the cells used for producing the rAAV.
  • the polyA signal sequence may be a bovine Growth Hormone polyA (bGH-PolyA) signal sequence, a SV40 polyA signal sequence, or a Rabbit Beta Globin PolyA signal sequence.
  • the bGH-PolyA signal sequence may include a nucleotide sequence that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 151.
  • the SV40 polyA signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 152.
  • the SV40 polyA sequence is shorter than SEQ ID NO: 152. In some embodiments, the SV40 polyA sequence is longer than SEQ ID NO: 152.
  • the Rabbit Beta Globin signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 170.
  • the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof.
  • the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, A
  • the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC
  • capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, A
  • the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety.
  • the capsid is an AAV1 capsid.
  • the capsid is an AAV5 capsid.
  • the capsid is an AAV9 capsid.
  • the first integrated construct further comprises a first mammalian cell selection element.
  • an inducible Rep and Cap construct is as shown in FIG.1A, FIG. 2A, or FIG.3A.
  • the inducible polynucleotide construct comprises one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence, a
  • the Rep polypeptide is a wildtype Rep polypeptide. In other embodiments, the Rep polypeptide is a mutant Rep polypeptide. In some embodiments, the Cap polypeptide is a wildtype Cap polypeptide. In other embodiments, the Cap polypeptide is a mutant Cap polypeptide.
  • the intron may be spliced out by endogenous cellular machinery.
  • the excisable element is excised by a recombinase.
  • a recombinase can be Cre. Cre may be provided as any form of exogenous Cre, such as Cre vesicles.
  • Cre may also be encoded for by a third polynucleotide construct or by any separate polynucleotide construct.
  • a construct encoding for adenoviral helper proteins also encodes for Cre.
  • the third polynucleotide construct is also inducible, for example, as described below.
  • a construct encoding for Rep/Cap proteins also encodes for Cre. [0399]
  • expression of the Rep and Cap are driven by native promoters, including P5, P19, P40, or any combination thereof.
  • expression of the Rep and Cap are driven by inducible promoters.
  • expression of the Rep and Cap are driven by constitutive promoters.
  • the exon of the excisable element may be any detectable marker.
  • detectable markers contemplated herein include luminescent markers, fluorescent markers, or radiolabels. Fluorescent markers include, but are not limited to, EGFP, GFP, BFP, RFP, or any combination thereof.
  • the Rep Cap construct is a polynucleotide construct comprising: a) a sequence of a first part of a Rep gene; b) sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene.
  • the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron. In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5’ splice site and a 3’ splice site. In some embodiments, the 5’ splice site is a rabbit beta globin 5’ splice site.
  • the 3’ splice site is a rabbit beta globin 3’ splice site.
  • three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene.
  • the three splice sites are a 5’ splice site, a first 3’ splice site, and a second 3’ splice site.
  • a first 3’ splice site is a duplicate of the second 3’ splice site.
  • the first 3’ splice site is a rabbit beta globin 3’ splice site.
  • the second 3’ splice site is a rabbit beta globin 3’ splice site.
  • the excisable element comprises a recombination site.
  • the recombination site is a lox site or FRT site.
  • the lox site is a loxP site.
  • the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first recombination site; c) the first 3’ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3’ splice site.
  • the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3’ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3’ splice site.
  • a recombinase recombines the first and second recombination sites.
  • a transcript produced from the recombined sequence includes an intron flanked by the 5’ splice site and the second 3 splice site and is processed by the endogenous cellular machinery to produce a mRNA in which the intron has been spliced out.
  • the first spacer sequence comprises an intron.
  • the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1.
  • the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2.
  • the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3.
  • the third spacer segment comprises an intron.
  • the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery.
  • the second spacer segment comprises an exon.
  • the second spacer segment further comprises a polyA sequence.
  • the polyA sequence is 3’ of the exon.
  • the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence.
  • the second spacer segment comprises from 5’ to 3’: a) a first recombination site; b) the first 3’ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site.
  • the first recombination site is a first lox sequence and the second recombination site is a second lox sequence.
  • the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence.
  • the first recombination site is a first FRT site and the second recombination site is a second FRT site.
  • the stop signaling sequence is a termination codon of the exon or a polyA sequence.
  • the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence.
  • the exon encodes a detectable marker or a selectable marker.
  • the detectable marker comprises a luminescent marker or a fluorescent marker.
  • the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • the second spacer segment is excisable by a recombinase.
  • the recombinase is a site-specific recombinase.
  • the recombinase is a Cre polypeptide or a Flippase polypeptide.
  • the Cre polypeptide is fused to a ligand binding domain.
  • the ligand binding domain is a hormone receptor.
  • the hormone receptor is an estrogen receptor.
  • the estrogen receptor comprises a point mutation.
  • the estrogen receptor is ERT2.
  • the recombinase is a ER2 Cre polypeptide.
  • the recombinase is encoded by a third polynucleotide construct or exogenously provided.
  • the Rep gene codes for Rep polypeptides.
  • the Cap gene codes for Cap polypeptides.
  • transcription of the Rep gene and the Cap gene are driven by native promoters.
  • the native promoters comprise P5, P19, and P40.
  • transcription of the Rep gene and/or the Cap gene are driven by inducible promoters.
  • the Rep polypeptides are wildtype Rep polypeptides.
  • the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40.
  • a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase.
  • the Cap polypeptides are wildtype Cap polypeptides.
  • the Cap polypeptides are AAV capsid proteins.
  • the AAV capsid proteins comprise VP1, VP2, and VP3.
  • a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.
  • the Rep Cap construct further comprises a sequence coding for a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z- Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the Rep Cap construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the Rep Cap construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the Rep Cap construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • a selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the Rep Cap construct is in a vector. In some embodiments, the Rep Cap construct is in a plasmid.
  • the Rep Cap construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, the Rep Cap construct is a synthetic nucleic acid construct. In some embodiments, the Rep Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, or any combination thereof.
  • the Rep Cap construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, or any combination thereof.
  • the Rep Cap construct comprises SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.
  • the Rep Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.
  • the Rep Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, but wherein the Rep Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins.
  • the Rep Cap construct further comprises a sequence coding for VA-RNA.
  • a sequence coding for VA-RNA is in separate construct or in any separate construct coding for an element of the Rep Cap construct.
  • a payload construct comprises a polynucleotide construct coding for a VA-RNA.
  • the VA-RNA is operably linked to a constitutive promoter or an inducible promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the inducible promoter is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.
  • the sequence coding for VA-RNA is a transcriptionally dead sequence.
  • the sequence coding for VA-RNA comprises at least two mutations in the internal promoter.
  • the expression of the VA-RNA is under the control of an RNA polymerase III promoter.
  • the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter.
  • the expression of the VA-RNA is under the control of a U6 or U7 promoter.
  • the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter.
  • the polynucleotide construct comprises upstream of the sequence coding for VA- RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence.
  • the stuffer sequence is excisable by the recombinase.
  • the stuffer sequence comprises a sequence encoding a gene.
  • the stuffer sequence comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a CMV promoter.
  • the gene codes for a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • a major advantage of the inducible polynucleotide constructs disclosed herein encoding for Rep and Cap include that upon stable integration into a mammalian cell line, expression of Rep and Cap is inducible even in the absence of a transfection agent or a plasmid.
  • the stable cell line populations disclosed herein are homogeneous.
  • the second polynucleotide construct may include a Cap protein encoding sequence identical or substantially identical to the Cap protein encoding sequence present in the Rep/Cap encoding polynucleotide construct.
  • the second polynucleotide construct may include a Cap protein encoding sequence identical or substantially identical to the Cap protein encoding sequence present in the Rep/Cap encoding polynucleotide construct, but the Cap protein encoding sequence is driven by an inducible promoter.
  • the second polynucleotide construct may be referred to as an inducible Cap construct.
  • the inducible promoter may be any inducible promoter that is described herein in the below Adenoviral Helper Construct(s) section and is activated by any triggering agent and activator as described herein in the below Adenoviral Helper Construct(s) section.
  • the inducible promoter is a Tet inducible promoter.
  • the Tet inducible promoter is activated when bound to Tet responsive activator protein, e.g., Tet-on 3G, in the presence of a trigger agent, such as doxycycline.
  • a polynucleotide encoding a sequence comprising an inducible promoter Cap protein sequence may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 148.
  • the second polynucleotide construct may be provided as a plasmid.
  • the plasmid may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 149.
  • the second polynucleotide construct may be provided as a vector.
  • Adenoviral Helper Construct(s) [0411]
  • a third polynucleotide construct also referred to as Construct 3
  • Construct 3 encodes for one or more adenoviral helper proteins.
  • Construct 3 is also referred to as Ad helper/Cre construct.
  • This third polynucleotide construct is also referred to as an inducible helper construct (e.g., Adenoviral Helper Construct provides one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) to be used in production of rAAV virions.
  • the sequence encoding E4 is a sequence encoding E4orf6.
  • the elements of an inducible helper construct are in one or more separate constructs to be used in production of rAAV virions.
  • the host cell provides, one, two, or three of the four helper proteins.
  • the adenoviral helper construct provides E2A and E4.
  • the adenoviral helper construct provides E1A and E1B.
  • the adenoviral helper construct provides E1A, E2A and E4.
  • the adenoviral helper construct For a host cell expressing E2A, the adenoviral helper construct provides E1B, E1A and E4. For a host cell expressing E4, the adenoviral helper construct provides E1B, E2A and E1A. For a host cell expressing E1A, E2A and E4, the adenoviral helper construct provides E1B. For a host cell expressing E1B, E1A and E4, the adenoviral helper construct provides E2A. For a host cell expressing E1B, E2A and E1A, the adenoviral helper construct provides E4. In some embodiments, E4 is E4orf6.
  • the sequences coding for E1A, E1B, E2A, and E4 are operably linked to separate promoters. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to one promoter. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked, in any combination, to one promoter or separate promoters.
  • the separate promoters can be the same promoters or different promoters. A combination of the separate promoters and the one promoter can be the same promoters or different promoters.
  • the one promoter can be a native promoter, a constitutive promoter, or an inducible promoter.
  • the separate promoters can be native promoters, constitutive promoters, inducible promoters, or any combination thereof.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the inducible promoter is a tetracycline-inducible promoter, a cumate-inducible promoter, or an ecdysone-inducible promoter.
  • the sequence coding for E1A and E1B are separated by an IRES sequence of P2A sequence and are operably linked to one promoter.
  • the sequence coding for E1A and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter.
  • the sequence coding for E1A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E2A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequences coding for the helper proteins are in different orientations.
  • the sequences coding for the helper proteins are bidirectional.
  • the E1A is operably linked to a natural or constitutive promoter
  • E1B is operably linked to a natural or constitutive promoter
  • E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the constitutive promoter.
  • an excisable element e.g., a sequence flanked by recombination sites and comprising a stop signal
  • the E1A is operably linked to a natural or constitutive promoter
  • E1B is operably linked to a natural or constitutive promoter
  • E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the inducible promoter.
  • the E2A is operably linked to a natural or constitutive promoter
  • E4 is operably linked to a natural or constitutive promoter
  • E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the constitutive promoter.
  • an excisable element e.g., a sequence flanked by recombination sites and comprising a stop signal
  • the E2A is operably linked to a natural or constitutive promoter
  • E4 is operably linked to a natural or constitutive promoter
  • E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the inducible promoter.
  • the adenoviral helper construct provides inducible production of one or more of the helper proteins.
  • an adenoviral helper protein further comprises a protein tag.
  • a protein tag can be a FLAG tag.
  • E2A is a FLAG tagged E2A.
  • E4 is a FLAG tagged E4.
  • a protein tag, such as a FLAG tag can be used to screen for or to confirm integration of the third polynucleotide construct and expression of the adenoviral helper protein from the third polynucleotide construct in a cell after induction.
  • the third integrated synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins.
  • the third synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins.
  • the one or more separate integrated constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the third integrated synthetic construct comprises conditionally expressible Cre recombinase and conditionally expressible adenovirus helper proteins.
  • the third integrated construct comprises, from 5’ to 3’: an inducible promoter, a Cre coding sequence, a first polyA sequence, adenoviral helper protein coding sequences, a second polyA sequence, a constitutive promoter, a coding sequence for a protein that is responsive to the first triggering agent, and a second mammalian cell selection element.
  • the Cre coding sequence is operatively linked to the inducible promoter.
  • the inducible promoter comprises an element responsive to the third triggering agent.
  • the inducible promoter contains a regulatory sequence that allows for control of the promoter.
  • the regulatory sequence can be operably linked to the promoter and positioned upstream of the promoter.
  • Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • the regulatory sequence used to control expression may be endogenous or exogenous to the host cell.
  • bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression.
  • mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides.
  • inducible expression systems see, e.g., Weber et al. (2004) Methods Mol. Biol.267:451-66, Das et al. (2016) Curr. Gene Ther.16(3):156-67, Chruscicka et al. (2015) J. Biomol. Screen.20(3):350-8, Yarranton (1992) Curr. Opin.
  • a bacterial tetracycline response element is included in a construct to allow mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline).
  • the inducible promoter comprises a plurality of tetracycline (Tet) operator elements capable of binding to a Tet responsive activator protein in the presence of a tetracycline.
  • the plurality of tetracycline (Tet) operator elements form a Tetracycline Responsive element (TRE).
  • the TRE comprises seven repeats of a 19 base pair operator sequence. In further embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal CMV promoter sequence.
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0418] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFD LDMLPADALDDFDLDMLPG (SEQ ID NO:40).
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0420] ATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCA ATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTT GAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCT GCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCTGGAAGGCGAGT CATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCAC ATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAAACAGTACGA AACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGAACGTTCTGGAGAACG CACTGTACGCTCTGT
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0422] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATA
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0424] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATA
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0426] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACGGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATA
  • a Tet responsive activator protein encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0428] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACGGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCA
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0430] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAGCAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGGTGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATA
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0432] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATAGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATA
  • a Tet responsive activator protein is encoded a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0434] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGAACATTTAGGTACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATATGC
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0436] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGTGCTTTACTAAGTCATC GCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACT CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTA TATGCACTCA
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0438] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGAACATTTAGGTACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATAT
  • a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0440] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGAACATTTAGGTACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATAT
  • a Tet responsive activator protein is a variant of a Tet responsive activator protein and comprises the sequence set forth in SEQ ID NO:40 but with the following amino acid substitutions:86Y A209T; V9I F86Y A209T; F67S F86Y A209T; G138D F86Y A209T; E157K F86Y A209T; R171K F86Y A209T; V9I G138D F86Y A209T; V9I E157K F86Y A209T; V9I R171K F86Y A209T; F67S R171K F86Y A209T; V9I F67S F86Y A209T; F67S G138D F86Y A209T; F67S E157K F86Y A209T; V9I F67S E157K F86Y A209T; V9I F67S E157K F86Y A209T; V9I F67S E157K F86Y A2
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0443] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:41).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0445] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:42).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0447] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:43).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0449] MSRLDKSKVINSALELLNEVGIEGLATRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRGGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:44).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0451] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDRAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:45).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0453] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKEHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 80).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0455] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFSALLSHRDGAKVHLGTRPTEKQYET LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLR QAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 81).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0457] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKEHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCKSGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 82).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0459] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLKGESWQDFLRNNAKSFRCALLSHRNGAKVHSDTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 83).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0461] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 84).
  • a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0463] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 85).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0465] GAATTCCTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG AGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCT ATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGA AAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGA GTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTA TCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTA TCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTA TCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTA
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0467] GAATTCCTCGACCCGGGTACCGAGCTCGACTTTCACTTTTCTCTATCACTGAT AGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACT CGACTTTCACTTTTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCT CTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGG AGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGAC TTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGAGTAGGCGTGTACGGTG GGAGGCCTATATAAGCAGAGCTCGTTTAGTCAGATCTCGTTTAGTCAGATCTCGTTTA
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0469] GAGCTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTT TCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCA CTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTATCACTGATAGGGAGTGGT AAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCAC TTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGA TAGGTAAACTCGAGATCCGGCGAATTCGAACACGCAGATGCAGTCGGGGC GGCGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGTGT
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0471] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTC CATAGAAGA (SEQ ID NO: 50).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0473] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA (SEQ ID NO: 51).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0475] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGGTAATCAACTACCAATTC CAGCTCTCTTTTGACAACTGGTCTTATACCAACTTTCCGTACCACTTCTCTCAG;
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0477] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTG TCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAA (SEQ ID
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0479] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCTAATCAACTACCAATTC CAGCTCTCTTTTGACAACTGGTCTTATACCAACTTTCCGTACCACTTCTCAG:
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0481] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTGCCTATGTTCTTTTGGAATCTATCCAAG TCTTATGTAAATGCTTATGTAAACCATAATATAAAAGAGTGCTGATTTTTTGAGTAA ACTTGCAACAGTCCTAACATTCTTC
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0483] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTCCATAATATAAAAGAGTGCTGATTTTTT GAGTAAACTTGCAACAGTCCTAACATTCTTCTCTCGTGTGTTTGTCTGTTCGCCAT CCCGTCTCCGCTCGTCACTTATCCTTCACT
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0485] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTCCAGGGCGCCTATAAAAGAGTGCTGAT TTTTTGAGTAAACTTGCAACAGTCCTAACATTCTTCTCTCGTGTGTTTGTCTGTTC GCCATCCCGTCTCCGCTCGTCACTTATCCTTCACTTTTCAGAGGG
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0487] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTGCTTATGTAAACCAGGGCGCCTATAA AAGAGTGCTGATTTTTTGAGTAAACTTCAATTCCACAACACTTTTGTCTTATACCAA CTTTCCGTACCACTTCCTACCCTCGTAAA (SEQ ID NO: 58).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0489] GAATTCTTTACTCCCTATCAGTGATAGAGAATGTATGAAGAGTTTACTCCCTA TCAGTGATAGAGAATGTATGCAGACTTTACTCCCTATCAGTGATAGAGAATGTATA AGGAGTTTACTCCCTATCAGTGATAGAGAATGTATGACCAGTTTACTCCCTATCAGT GATAGAGAATGTATCTACAGTTTACTCCCTATCAGTGATAGAGAATGTATATCCAGT TTACTCCCTATCAGTGATAGAGAATGTATAAGCTTTAGG (SEQ ID NO: 59).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0491] CATGTACAGTGGGCACCTATAAAAGCAGAGCTCATTTAGTGAACTGTCAGAT TGCCTGGAGCAATTCCACAACACTTTTGTCTTATACCAACTTTCCATACCACTTCCTA CCCTCATAAAGTGCACACCATGG (SEQ ID NO: 60).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0493] CCATGGTGTGCACTTTATGAGGGTAGGAAGTGGTATGGAAAGTTGGTATAAG ACAAAAGTGTTGTGGAATTGCTCCAGGCAATCTGACAGTTCACTAAATGAGCTCTG CTTATAGGTGCCCACTGTACATGCCTAAGAATTCTTTACT (SEQ ID NO: 61).
  • a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0495] CCCTATCAGTGATAGAGAATGTATGAAGAGTTTACTCCCTATCAGTGATAGA GAATGTATGCAGACTTTACTCCCTATCAGTGATAGAGAATGTATAAGGAGTTTACTC CCTATCAGTGATAGAGAATGT ATCTACAGTTTACTCCCTATCAGTGATAGAGAATGTATATCCAGTTTACTCCCTATC AGTGATAGAGAATGTATAAGCTTTAGG (SEQ ID NO: 62).
  • a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0497] GGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACC GTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGG GACCGATCCAGCCTCCGCGG (SEQ ID NO: 63).
  • a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0499] GGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCG TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGG ACCGATCCAGCCTCCGCG (SEQ ID NO: 64).
  • a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0501] TAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTC AGATCGCCTGGAGA (SEQ ID NO: 65).
  • a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0503] GTAATCAACTACCAATTCCAGCTCTCTTTTGACAACTGGTCTTATACCAACTT TCCGTACCACTTGCAACCCTCGTAAGACAATTGCAA (SEQ ID NO: 66).
  • a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0505] AATTCCACAACACTTTTGTCTTATACCAACTTTCCGTACCACTTCCTACCCTC GTAAA (SEQ ID NO: 67).
  • a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0507] GCCTATGTTCTTTTGGAATCTATCCAAGTCTTATGTAAATGCTTATGTAAACC ATAATATAAAAGAGTGCTGATTTTTTGAGTAAACTTGCAACAGTCCTAACATTCTTC TCTCGTGTGTTTGTCTGTTCGCCATCCCGTCTCCGCTCGTCACTTATCCTTCACTTT TCAGAGGGTCCCCCCGCAGATCCCGGTCACCCTCAGGTCGG (SEQ ID NO: 68).
  • a Tet Repressor binding protein may comprise a sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0509] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLKGESWQDFLRNKAKSFRCALLSHRNGAKVHSDTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS (SEQ ID NO: 49).
  • a Tet Repressor binding protein may comprise a sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0511] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACACTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAAAAGGGGAAA GCTGGCAAGATTTTTTACGCAATAAGGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCAATGGAGCAAAAGTACATTCAGATACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATATGCACTCAGCTGTGGG
  • the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68.
  • the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62.
  • the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-86, or variants thereof.
  • the Tet responsive activator protein or variant thereof, the Tet Repressor binding protein or variant thereof, TRE sequence, or any tetracycline-inducible promoter sequence or variant thereof can be any of those disclosed in US 7,541,446; US 8,383,364; US 6,136,954; US 5,814,618; US 6,271,348; US 5,789,156; US 7,666,668; US 6,914,124; US 5,650,298; US 5,922,927; US 5,464,758; US 5,866,755; US 5,589,362; US 5,654,168; US 6,242,667; US 5,912,411; US 6,783,756; US 5,888,981; US 6,004,941; US 6,252,136; US 5,859,310; US 6,271,341; US 6,087,166; US2003022315; US20050037335; US 9,181,556; and WO03056021, which are each herein incorporated by reference in their entirety
  • any of the proteins described herein may be expressed from a nucleotide sequence that has been codon-optimized to increase expression in a host cell, e.g., a mammalian cell or a human cell line.
  • an insect gene control element is used to provide mammalian inducible expression.
  • an ecdysone-responsive element and a gene encoding the ecdysone receptor can be included in a construct to allow mammalian expression to be induced by the insect hormone ecdysone or analogs or derivatives thereof, such as ponasterone.
  • the ecdysone receptor heterodimerizes with the retinoid X receptor (RXR).
  • the ecdysone-responsive element comprises a binding site for the RXR-ecdysone receptor heterodimer, which is typically a synthetic recognition site for the heterodimer that preferably does not bind any endogenous transcription factors or natural nuclear hormone receptors.
  • the RXR-ecdysone receptor heterodimer binds to the ecdysone-responsive element to activate transcription from the promoter.
  • the third construct or an additional separate construct comprises an element responsive to a fourth triggering agent.
  • the fourth triggering agent-responsive element comprises a plurality of hormone-response elements.
  • the hormone-response elements are estrogen responsive elements (EREs).
  • the third triggering agent is the same as the first triggering agent, and the fourth triggering agent is the same as the second triggering agent.
  • the inducible promoter comprises a plurality of Tet operator elements capable of binding to a Tet responsive activator protein in the presence of a third triggering agent.
  • the third triggering agent is the same as the first triggering agent.
  • the recombinase coding sequence is flanked by a first recombinase site and a second recombinase site.
  • the recombinase is Cre.
  • the Cre coding sequence is flanked by a first lox site and a second lox site.
  • the first polyA sequence is positioned between the Cre coding sequence and adenoviral helper protein coding sequences that encode one or both of adenovirus E2A and E4.
  • the strong 3’ polyadenylation signal positioned upstream (5’ to) the coding sequences for the adenovirus helper proteins prevents basal expression of the downstream adenoviral helper genes, E2A and E4.
  • helper construct does not comprise a recombinase that is self- excising.
  • the recombinase is a site-specific recombinase.
  • the recombinase is fused to a ligand binding domain.
  • the recombinase is Cre polypeptide or flippase polypeptide.
  • the Cre polypeptide is fused to a ligand binding domain.
  • the ligand binding domain is a hormone receptor.
  • the hormone receptor is an estrogen receptor.
  • the estrogen receptor comprises a point mutation.
  • the estrogen receptor is ERT2.
  • the recombinase is a Cre- ERT2 polypeptide.
  • the recombinase translocates to the nucleus in the presence of a triggering agent.
  • the triggering agent is an estrogen receptor ligand. In some embodiments, the triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the triggering agent is tamoxifen.
  • SERM selective estrogen receptor modulator
  • the sequence or sequences encoding one or more helper proteins can be operably linked to an inducible promoter.
  • the sequence or sequences encoding one or more helper proteins can be downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequence or sequences encoding one or more helper proteins are operably linked to the constitutive promoter.
  • an excisable element e.g., a sequence flanked by recombination sites and comprising a stop signal
  • constitutive promoter e.g., a sequence flanked by recombination sites and comprising a stop signal
  • the sequence or sequences encoding one or more helper proteins are operably linked to the constitutive promoter.
  • the further segment shown in FIG.1A provides for inducible production of VA-RNA from construct 3.
  • the further segment includes a Cre-inducible U6 or U7 promoter.
  • the U6 or U7 promoter is split into 2 parts separated by a Lox flanked stuffer sequence.
  • the U6 or U7 promoter is inactive because of the presence of the stuffer sequence. Cre mediated excision of the stuffer activates the U6 or U7 promoter.
  • the U6 or U7 promoter drives the expression of transcriptionally dead mutants of VA-RNA1 (a preferred embodiment is a double point mutant G16A- G60A). Other embodiments provide for alternative sources of VA-RNA.
  • the coding sequence for the first triggering agent-responsive protein is operatively linked to a CMV promoter.
  • the coding sequence for the first triggering agent-responsive protein comprises a coding sequence for the Tet responsive activator protein.
  • the Tet responsive activator protein is Tet-on 3G activator protein.
  • the second mammalian cell selection element confers antibiotic resistance.
  • the antibiotic resistance conferring element is a blasticidin resistance gene.
  • Multiple inducible helper polynucleotide constructs are contemplated herein.
  • the elements of the inducible helper polynucleotide constructs can be in one or more separate constructs.
  • said inducible helper polynucleotide constructs encode for one or more adenoviral helper proteins, such as VA-RNA, E2A, E4, or any combination thereof.
  • the present disclosure provides for an inducible polynucleotide construct encoding for a mutated VA-RNA gene sequence.
  • the mutations to VA-RNA render its internal promoters inactive.
  • the inducible helper polynucleotide construct may comprise from 5’ to 3’ a first part of a U6 promoter sequence, a first lox sequence, a stuffer sequence, a second lox sequence, and a second part of a U6 promoter sequence.
  • the stuffer sequence may be any polynucleotide sequence and is excised by Cre. Cre may be exogenously provided, such as in the form of Cre gesicles.
  • Cre may also be encoded for in the same inducible helper polynucleotide construct and expression of Cre may be conditioned on the presence of at least two triggering agents, such as doxycycline and tamoxifen. Cre may be a hormone activated Cre.
  • the inducible helper constructs instead of a mutated VA-RNA gene sequence, may comprise a constitutively expressed VA-RNA that is not mutated.
  • the inducible helper polynucleotide construct also encodes for one or more helper proteins, a self-excising element upstream of the one or more helper proteins, and an inducible promoter upstream of the self-excising element.
  • Expression of the self-excising element may be driven by a Tet-on 3G system.
  • the construct may comprise a Tet- On 3G gene sequence, wherein expression is driven by an EF1alpha promoter.
  • the EF1alpha promoter may be a mutated EF1alpha promoter.
  • the mutated EF1alpha promoter can have a sequence of: ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattg aacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaac cgtatgtaagtgcagtagtcgccgtgaacgttctttttcgcaacgggttttgcgccagaacacagctgaagctttcgaggggctcgcgccagaacacagctgaagcttcgaggggctcggggg
  • Tet-On 3G is able to bind the Tet inducible promoter.
  • the Tet inducible promoter drives expression of the self-excising element.
  • the self-excising element is a hormone activated Cre.
  • Cre self-excises itself leading to expression of downstream adenoviral helper proteins.
  • an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; and b) a self-excising element upstream of the one or more helper proteins.
  • the self-excising element is operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • a sequence coding for the self- excising element comprises a poly A sequence.
  • the self-excising element is a recombinase.
  • the recombinase is a site-specific recombinase.
  • the recombinase is fused to a ligand binding domain.
  • the recombinase is Cre polypeptide or flippase polypeptide.
  • the Cre polypeptide is fused to a ligand binding domain.
  • the ligand binding domain is a hormone receptor.
  • the hormone receptor is an estrogen receptor.
  • the estrogen receptor comprises a point mutation.
  • the estrogen receptor is ERT2.
  • the recombinase is a Cre- ERT2 polypeptide.
  • the self-excising element translocates to the nucleus in the presence of a triggering agent.
  • the triggering agent is an estrogen receptor ligand.
  • the triggering agent is a selective estrogen receptor modulator (SERM).
  • SERM selective estrogen receptor modulator
  • the triggering agent is tamoxifen.
  • the recombinase is flanked by recombination sites.
  • the recombination sites are lox sites or flippase recognition target (FRT) sites.
  • the lox sites are loxP sites.
  • the self-excising element is excised upon administration of the triggering agent, thereby operably linking the constitutive promoter to the one or more helper proteins.
  • the inducible helper construct lacks sequences coding for a tetracycline inducible system (e.g., a tetracycline- responsive promoter element (TRE) and/or a reverse tetracycline-controlled transactivator (rTA)).
  • TRE tetracycline- responsive promoter element
  • rTA reverse tetracycline-controlled transactivator
  • the inducible helper construct lacks sequences coding for a tetracyline-inducible system, an ecdysone-inducible system, or a cumate-inducible system.
  • an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element.
  • the self-excising element is operably linked to the inducible promoter.
  • expression of the self-excising element is driven by the inducible promoter.
  • an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; b) a recombinase; and c) an inducible promoter upstream of the one or more helper proteins; and d) an inducible promoter upstream the recombinase.
  • the recombinase is operably linked to the inducible promoter.
  • the one or more helper proteins are operably linked to the inducible promoter.
  • expression of the recombinase is driven by the inducible promoter.
  • the inducible promoter is a tetracycline-responsive promoter element (TRE).
  • the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
  • the minimal promoter is a human cytomegalovirus promoter.
  • the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68.
  • the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62.
  • transcription is activated from the inducible promoter upon binding of an activator.
  • the activator binds to the inducible promoter in the presence of a first triggering agent.
  • the activator is operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the EF1alpha promoter comprises at least one mutation.
  • the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20.
  • the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.
  • the rTA comprises four mutations in the tetR DNA binding moiety.
  • the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-86, or variants thereof.
  • the inducible promoter is bound by a repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the repressor. In some embodiments, the repressor is a tetracycline-controlled transactivator. In some embodiments, further comprising the repressor. In some embodiments, the repressor is operably linked to a constitutive promoter. In some embodiments, further comprising a tetracycline-controlled transactivator. In some embodiments, the tetracycline-controlled transactivator is operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the tetracycline-controlled transactivator is unbound in the presence of a first triggering agent. In some embodiments, the tetracycline-controlled transactivator does not bind to the inducible promoter in the presence of a first triggering agent. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation.
  • the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20.
  • transcription is activated from the inducible promoter upon binding of the first triggering agent to the repressor.
  • the repressor binds to the first triggering agent.
  • the first triggering agent is a tetracycline.
  • the tetracycline is doxycycline.
  • the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter.
  • the constitutive promoter is a human cytomegalovirus promoter. In some embodiments, wherein the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the cymR repressor. In some embodiments, the cumate inducible system further comprises a cymR repressor. In some embodiments, the cymR repressor is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter.
  • the EF1alpha promoter comprises at least one mutation.
  • the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20.
  • the first triggering agent is a cumate.
  • a sequence coding for the self-excising element comprises a poly A sequence.
  • the self-excising element is a recombinase.
  • the recombinase is a site-specific recombinase.
  • the recombinase is fused to a ligand binding domain.
  • the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ER2. In some embodiments, the recombinase is a ER2 Cre polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand.
  • the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites. [0535] In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the one or more adenoviral helper proteins further comprises a protein tag. In some embodiments, the protein tag is a FLAG-tag.
  • the E2A is FLAG-tagged E2A.
  • the sequence coding for E2 and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A.
  • the inducible helper construct further comprises a sequence coding for a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the helper construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the helper construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • an inducible helper construct further comprises a sequence coding for VA-RNA.
  • the VA-RNA is on a separate construct from the sequences encoding one or more helper proteins.
  • the VA-RNA is operably linked to a constitutive promoter or an inducible promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.
  • the sequence coding for VA-RNA is a transcriptionally dead sequence.
  • the sequence coding for VA-RNA comprises at least two mutations in the internal promoter.
  • the expression of the VA-RNA is under the control of an RNA polymerase III promoter.
  • the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter.
  • the expression of the VA-RNA is under the control of a U6 or U7 promoter.
  • the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter.
  • the polynucleotide construct comprises upstream of the sequence coding for VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence.
  • the stuffer sequence is excisable by the recombinase.
  • the stuffer sequence comprises a sequence encoding a gene.
  • the stuffer sequence comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a CMV promoter.
  • the gene encodes a detectable marker or a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z- Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C- terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the detectable marker comprises a luminescent marker or a fluorescent marker.
  • the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • the first recombination site is a first lox sequence and the second recombination site is a second lox sequence.
  • the first lox sequence is a first loxP site and the second lox sequence is a second loxP site.
  • an inducible helper construct is in a vector. In some embodiments, an inducible helper construct is in a plasmid. In some embodiments, an inducible helper construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct is a synthetic nucleic acid construct.
  • an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138, or any combination thereof.
  • an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138, or any combination thereof.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA.
  • the VA-RNA is on a separate construct from the sequences encoding one or more helper proteins.
  • the VA-RNA is operably linked to a constitutive promoter or an inducible promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the inducible promoter is a tetracycline-inducible promoter, an ecdysone- inducible promoter, or a cumate-inducible promoter.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter.
  • a separate polynucleotide construct codes for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter.
  • the sequence coding for the VA-RNA comprises a sequence coding for a transcriptionally dead VA-RNA.
  • the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region.
  • the sequence coding for the VA-RNA comprises at least one mutation.
  • the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A.
  • the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA- RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter.
  • the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence.
  • the stuffer sequence is excisable by a recombinase.
  • the stuffer sequence comprises a sequence encoding a gene.
  • the stuffer sequence comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a CMV promoter.
  • the gene encodes a detectable marker or a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z- Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C- terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the detectable marker comprises a luminescent marker or a fluorescent marker.
  • the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided.
  • the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide.
  • the first recombination site is a first lox sequence and the second recombination site is a second lox sequence.
  • the first lox sequence is a first loxP site and the second lox sequence is a second loxP site.
  • the first recombination site is a first FRT site and the second recombination site is a second FRT site.
  • the construct comprising the VA-RNA as described herein further comprises a sequence coding for a selectable marker.
  • the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the construct comprising the VA-RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the construct comprising the VA-RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a vector.
  • an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is a synthetic nucleic acid construct.
  • an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26.
  • a VA-RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26.
  • a VA-RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26.
  • Synthetic nucleic acid constructs [0553]
  • the nuclear genome of the cell of the stable cell line comprises a plurality of integrated synthetic nucleic acid constructs.
  • each of the plurality of synthetic nucleic acid constructs is separately integrated into the nuclear genome of the cell. In some embodiments, only a single non-auxotrophic selection is required to maintain all of the plurality of synthetic nucleic acid constructs stably within the nuclear genome of the cells. In some embodiments, antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, both a non-auxotrophic selection and antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, auxotrophic selection and antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells.
  • auxotrophic selection is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells.
  • the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences;
  • the second integrated synthetic construct comprises a conditionally expressible AAV Cap coding sequence, where the AAV Cap coding sequence is operably linked to a native or an inducible promoter;
  • the third integrated synthetic construct comprises a conditionally expressible Cre coding sequence and conditionally expressible adenoviral helper protein coding sequences;
  • the fourth integrated synthetic construct comprises expressible coding sequences for the payload.
  • FIG.1A An example of this embodiment is depicted in FIG.1A.
  • the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences, where the AAV Cap coding sequence is operably linked to an inducible promoter; the second integrated synthetic construct comprises a conditionally expressible Cre coding sequence and conditionally expressible adenoviral helper protein coding sequences; and the third integrated synthetic construct comprises expressible coding sequences for the payload.
  • An example of this embodiment is depicted in FIG.2A. Production of Single-Stranded or Self-Complementary rAAV Virion DNA
  • the region of the fourth polynucleotide construct between the two inverted terminal repeats (3’ ITR and 5’ ITR) is packaged into rAAV virions.
  • the rAAV virions comprise wild-type inverted terminal repeats, wherein the rAAV virion DNA that is generated is single-stranded (i.e., ssAAV virion).
  • a terminal resolution site in the 3' ITR is deleted, resulting in formation of an rAAV virion comprising DNA that is self-complementary (i.e., scAAV virion).
  • the scAAV forms a single-stranded DNA molecule during replication in which two single-stranded genomes comprising a plus DNA strand and a minus DNA strand are concatenated to form a self- complementary intramolecular dsDNA genome.
  • the scAAV virions do not need to perform second-strand DNA synthesis, which increases the efficiency of scAAV transgene expression relative to ssAAV.
  • the maximum cargo capacity of scAAV i.e., maximum length of region between the 5' ITR and 3' ITR of the fourth polynucleotide construct
  • the scAAV DNA packaged into a viral particle is a concatemer of two single-stranded genomes of opposite strands.
  • a ssAAV plasmid encoding a sequence of a payload can be SEQ ID NO: 159.
  • a scAAV plasmid encoding a sequence of a payload can be SEQ ID NO: 157.
  • each integrated nucleic acid construct comprises a mammalian cell selection element.
  • the stable cell line comprises four integrated nucleic acid constructs, wherein the first nucleic acid construct comprises a first mammalian cell selection element, the second nucleic acid construct comprises a second mammalian cell selection element, the third nucleic acid construct comprises a third mammalian cell selection element and the fourth nucleic acid construct comprises a fourth mammalian cell selection element.
  • the stable cell line comprises three integrated nucleic acid constructs, wherein the first nucleic acid construct comprises a first mammalian cell selection element, the second nucleic acid construct comprises a second mammalian cell selection element, and the third nucleic acid construct comprises a third mammalian cell selection element.
  • the mammalian selection elements are components of a split auxotrophic selection system or a split antibiotic resistance gene.
  • a split auxotrophic system can be a leucine zipper based system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure.
  • components of the split auxotrophic selection system described herein comprise a C-terminal fragment of the auxotrophic protein Z-Cter and an N-terminal fragment of an auxotrophic protein Z-Nter.
  • a split auxotrophic system is a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form.
  • split inteins the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein.
  • Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288: 6202-6211 (2013); Stevens et al, PNAS 114: 8538-8543 (2017); Jillette et al., Nat Comm 10: 4968 (2019); US 2020/0087388 A1; and US 2020/0263197 A1.
  • components of the split auxotrophic selection system described herein comprises a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein.
  • This N-terminal fragment is enzymatically nonfunctional and this C-terminal fragment is enzymatically nonfunctional.
  • both constructs can catalyze the joining of the N-terminal fragment of the auxotrophic protein and a C-terminal fragment of the auxotrophic protein to form a functional enzyme, such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR).
  • a functional enzyme such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR).
  • both constructs can be stably retained in the genome of a cell by growth in a medium lacking the product produced by the enzyme.
  • a construct encoding for a component of a split auxotrophic system further encodes a helper enzyme, wherein expression of the helper enzyme facilitates growth of the host cell in conjunction with the functional enzyme upon application of the single selective pressure.
  • the first nucleic acid construct comprises a first mammalian cell selection element, and the first mammalian cell selection element is a first auxotrophic selection element.
  • the first auxotrophic selection element encodes an active protein.
  • the first auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the first auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the first auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z- Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the first auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the first auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the first auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N- terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • first nucleic acid construct further comprises a sequence coding for a first auxotrophic selection element and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the first nucleic acid construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a first auxotrophic selection element comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the first auxotrophic selection element and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the second nucleic acid construct comprises a second mammalian cell selection element, and the second mammalian cell selection element encodes antibiotic resistance.
  • the antibiotic resistance gene is a blasticidin resistance gene.
  • the second mammalian cell selection element encodes an active protein.
  • the second mammalian cell selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the third nucleic acid construct comprises a third mammalian cell selection element.
  • the third mammalian cell selection element is a second auxotrophic selection element.
  • the second auxotrophic selection element encodes an active protein.
  • the second auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the second auxotrophic selection element codes for an inactive protein that requires expression of a first auxotrophic selection element for activity. In some embodiments, the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the first auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the second auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the first auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein.
  • the second auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the second auxotrophic selection element codes for an N- terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C- terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the third nucleic acid construct further comprises a sequence coding for second auxotrophic element and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the third nucleic acid construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a second auxotrophic selection element comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the second auxotrophic selection element and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the selectable marker of the nucleic acid constructs is interchangeable between each other.
  • the stable mammalian cell line can be propagated in growth media lacking hypoxanthine and thymidine.
  • the average concentration of Rep protein within the cells is less than between 1-99%, 10-90%, 20-80%, 30-70%, 40-60% prior to addition of the at least first triggering agent to the cell culture medium. In some embodiments, the average concentration of Rep protein within the cells is less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% prior to addition of the at least first triggering agent to the cell culture medium.
  • the mammalian cell line is selected from the group consisting of a human embryonic kidney (HEK) 293 cell line, a human HeLa cell line, and a Chinese hamster ovary (CHO) cell line.
  • the mammalian cell line is a HEK293 cell line.
  • the mammalian cell line expresses adenovirus helper functions E1A and E1B.
  • Alternative constructs as described herein can be used in a complete system. In some embodiments, the complete system can be integrated into the host cell genome to produce a stable cell line.
  • the complete system can be transfected into the host cell and then conditional production of AAV virion from the plasmids can be induced.
  • the complete system comprises episomes in the host cell and conditional production of AAV virion from the episomes is induced.
  • the cell is conditionally capable of producing rAAV virions having a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99.
  • the rAAV virions have a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the rAAV virions have a concentration of greater than 1 ⁇ 10 11 or no less than 5 ⁇ 10 11 , 1 ⁇ 10 12 , 5 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 viral genomes per milliliter prior to purification.
  • the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a titer of greater than 1 ⁇ 10 11 or no less than 5 ⁇ 10 11 , 1 ⁇ 10 12 , 5 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 viral genomes per milliliter. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1 ⁇ 10 11 or no less than 5 ⁇ 10 11 , 1 ⁇ 10 12 , 5 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 viral genomes per milliliter prior to purification. In some embodiments, this cell is expanded to produce a population of cells.
  • the population of cells produces a stable cell line as described herein. In some embodiments, this cell is passaged at least three times. In some embodiments, this cell can be passaged up to 60 times. In some embodiments, this cell can be passage more than 60 times. In some embodiments, the cell maintains the ability to be conditionally induced after each passage.
  • Vector/Vector System Vector [0574] In some aspects, vectors comprising the polynucleotide described in the present disclosure are provided. The first polynucleotide is described in section "First Polynucleotide encoding AAV Rep Protein" above. The second polynucleotide is described in section "AAV Cap Encoding Sequence linked to PolyA Signal Sequence" above.
  • the third polynucleotide is described in sections "Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above.
  • the fourth polynucleotide is described in section “Fourth Polynucleotide Encoding Payload” above.
  • the fifth polynucleotide is described in section "Fifth Polynucleotide Encoding VA-RNA" above.
  • vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; b) a second vector comprising the sequence of the second polynucleotide described in section “1.3.
  • vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; b) a second vector comprising the sequence of the second polynucleotide described in section “1.3.
  • the first vector is a first plasmid
  • the second vector is a second plasmid
  • the third vector is a third plasmid
  • the fourth vector is a fourth plasmid
  • the fifth vector is a fifth plasmid.
  • the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 160 or 161
  • the first plasmid has sequence of SEQ ID NO: 32 or sequence of SEQ ID NO: 160 or 161.
  • the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149. In certain embodiments, the second plasmid has sequence of SEQ ID NO: 149. [0580] In some embodiments, the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154.
  • the third plasmid has sequence of SEQ ID NO: 30 or sequence of SEQ ID NO: 154.
  • the fourth plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159.
  • the fourth plasmid has sequence of SEQ ID NO: 157 or sequence of SEQ ID NO: 159.
  • vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3.
  • the vector systems further comprise a fourth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above or the fourth polynucleotide construct described in section “Polynucleotide Construction System” above.
  • vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3.
  • the vector systems further comprise a fourth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above or the fourth polynucleotide construct described in section “Polynucleotide Construction System” above.
  • vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3.
  • the vector systems further comprise a fourth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above or the fourth polynucleotide construct described in section “Polynucleotide Construction System” above.
  • the first vector is a first plasmid
  • the second vector is a second plasmid
  • the third vector is a third plasmid
  • the fourth vector is a fourth plasmid.
  • the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. In certain embodiments, the first plasmid has sequence of SEQ ID NO: 164. [0587] In some embodiments, the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154.
  • the second plasmid has sequence of SEQ ID NO: 30 or sequence of SEQ ID NO: 154.
  • the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159.
  • the third plasmid has sequence of SEQ ID NO: 157 or sequence of SEQ ID NO: 159. 1.7.
  • Host production cell [0589] The present disclosure further provides host cells comprising the vector system described herein.
  • a subject host cell can be an isolated cell, e.g., a cell in in vitro culture.
  • a subject host cell is useful for producing rAAV virions, as described below.
  • a subject host cell useful for producing rAAV virions can be any cell that is capable of expressing proteins from a p5 promoter. Where a subject host cell is used to produce rAAV virions, it is referred to as a “packaging cell.”
  • a subject host cell is stably genetically modified with the vector system.
  • a subject host cell is transiently genetically modified with the vector system.
  • vector systems that comprise the first polynucleotide described herein; the second polynucleotide described herein; the third polynucleotide described herein; and the fourth polynucleotide described herein are provided. [0591] In some aspects, vector systems that comprise the first polynucleotide described herein; the second polynucleotide described herein; the third polynucleotide described herein; the fourth polynucleotide described herein; and the fifth polynucleotide described herein are provided.
  • vector systems that comprise the first polynucleotide described herein; the second polynucleotide described herein; the third polynucleotide described herein; the fourth polynucleotide described herein; the fifth polynucleotide described herein; and the sixth polynucleotide described herein are provided.
  • the vector system described herein can be used in a variety of host cells for rAAV virion production. For example, suitable host cells that have been transfected with the vector system are rendered capable of producing rAAV virions.
  • the first, second, third and fourth polynucleotide constructs of the vector system can be introduced into a host cell, either simultaneously or serially, using established transfection techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like.
  • the first, second, and third polynucleotide constructs of the vector system are introduced into a host cell, and the fourth polynucleotide construct comprising the expressible payload is introduced later when production of the payload is desired.
  • a subject host cell is generated by introducing the vector system into any of a variety of cells, e.g., mammalian cells, including, without limitation, murine cells, and primate cells (e.g., human cells).
  • Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and the like.
  • suitable host cells include, e.g., HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos.
  • CRL9618, CCL61, CRL9096 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like.
  • a subject host cell can also be made using a baculovirus to infect insect cells such as Sf9 cells, which produce AAV (see, e.g., U.S. Patent Nos.7,271,002 and 8,945,918).
  • a host cell is any cell capable of activating a p5 promoter of sequence encoding a Rep protein.
  • the production host cell is a mammalian cell line that expresses adenovirus E1A and E1B.
  • the cell is a human embryonic kidney (HEK) 293 cell line or derivatives thereof (HEK293T cells, HEK293F cells), a human HeLa cell line that expresses E1A and E1B, a Chinese hamster ovary (CHO) cell line that expresses E1A and E1B, or a Vero cell that expresses adenovirus E1A and E1B.
  • the host cell is a HEK293 cell line.
  • the host cell is or is genetically altered to be deficient in an enzyme required for production of a molecule required for cell growth, for example, an enzyme required for catalyzing production of a cofactor or nutrient.
  • the host cell is DHFR null. In specific embodiments, the host cell is a DHFR null HEK293 cell. In some embodiments, the host cell is GS null. In some embodiments, the host cell is a GS null HEK293. [0597] In some embodiments, the host cell expresses or is genetically modified to express GTP- CH1. [0598] In some embodiments, the HEK293 cell expresses AAV E1A and E1B. In the presence of doxycycline and tamoxifen, the ER2 Cre is excised from the first integrated synthetic construct, thereby permitting expression of AAV E2A and E4.
  • the self-excised ER2 Cre recombines by virtue of the lox sites flanking the EGFP cassette in the second integrated synthetic construct, thereby removing the EGFP segment from the second spacer element in the integrated second synthetic construct.
  • any cells comprising only the second integrated synthetic construct will be EGFP signal positive whereas cells comprising both the first and second integrated synthetic constructs will be EGFP signal negative, following the addition of the triggering agents. Absence of EGFP signal indicates successful transfection of both the first and second integrated synthetic constructs in a cell. This is further ensured by antibiotic resistance selection, e.g., blasticidin resistance.
  • the first selectable marker and second selectable marker can be any selectable marker as described herein, wherein expression of the first selectable marker and expression of the second selectable marker form an active molecule (e.g., a functional enzyme) that allows the cell to survive in a selection medium (e.g., a selection media deficient in the product produced by the functional enzyme).
  • the fourth integrated synthetic construct comprises a payload.
  • the payload can be a guide RNA, an HDR homology region, or a gene of interest.
  • one or more of the synthetic nucleic acid constructs are integrated into the genome of a production host cell. In some embodiments, the integration of a construct into a chromosome is site-specific.
  • a polynucleotide construct can be cloned into a lentivirus vector that integrates into the nuclear genome of the cell.
  • a transposon system a clustered regularly interspersed short palindromic repeats (CRISPR) system, or a site-specific recombinase can be used to integrate a polynucleotide construct into the host cell genome, as described further below.
  • CRISPR clustered regularly interspersed short palindromic repeats
  • a site-specific recombinase can be used to integrate a polynucleotide construct into the host cell genome, as described further below.
  • a polynucleotide construct is integrated into the genome using a transposon system comprising a transposase and transposon donor DNA.
  • the transposase can be provided to a host cell with an expression vector or mRNA comprising a coding sequence encoding the transposase.
  • the transposon donor DNA can be provided with a vector comprising transposon terminal inverted repeats (TIRs).
  • TIRs transposon terminal inverted repeats
  • the polynucleotide construct is cloned into the transposon donor vector between the TIRs.
  • the host cell is cotransfected with an expression vector or mRNA encoding the transposase and the transposon donor vector containing the polynucleotide construct insert, wherein the polynucleotide construct is excised from the transposon donor vector and integrated into the genome of the host cell at a target transposon insertion site.
  • Transposition efficiency may be improved in a host cell by codon optimization of the transposase, using engineered hyperactive transposases, and/or introduction of mutations in the transposon terminal repeats.
  • Any suitable transposon system can be used including, without limitation, the piggyBac, Tol2, or Sleeping Beauty transposon systems.
  • the piggyBac Tol2, or Sleeping Beauty transposon systems.
  • For a description of various transposon systems see, e.g., Kawakami et al. (2007) Genome Biol.8 Suppl 1(Suppl 1):S7, Tipanee et al. (2017) Biosci Rep.37(6):BSR20160614, Yoshida et al. (2017) Sci Rep. 7:43613, Yusa et al. (2011) Proc. Natl.
  • a construct is integrated at a target chromosomal locus by homologous recombination using site-specific nucleases or site-specific recombinases.
  • a construct can be integrated into a double-strand DNA break at the target chromosomal site by homology-directed repair.
  • a DNA break may be created by a site-specific nuclease, such as, but not limited to, a Cas nuclease (e.g., Cas9, Cpf1, or C2c1), an engineered RNA-guided FokI nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector- based nuclease (TALEN), a restriction endonuclease, a meganuclease, a homing endonuclease, and the like.
  • a site-specific nuclease such as, but not limited to, a Cas nuclease (e.g., Cas9, Cpf1, or C2c1), an engineered RNA-guided FokI nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector- based nuclease (TALEN), a restriction endonuclease
  • a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence can be introduced into a polynucleotide construct.
  • the homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms in the polynucleotide construct.
  • the 5' and 3' homology arms hybridize to regions within the target locus where the construct is integrated, which are referred to herein as the "5' target sequence" and "3' target sequence,” respectively.
  • the homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the construct and genomic DNA at the target locus.
  • a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein construct is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.
  • the corresponding homologous nucleotide sequences in the genomic target sequence flank a specific site for cleavage and/or a specific site for integrating the construct.
  • the distance between the specific cleavage site and the homologous nucleotide sequences can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate.
  • a homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc.
  • the 5' and 3' homology arms are substantially equal in length to one another, e.g., one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm.
  • the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.
  • RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence for insertion of a polynucleotide construct) by altering its guide RNA sequence.
  • a target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site.
  • the gRNA can be designed to selectively bind to the chromosomal target site where integration of the construct is desired.
  • the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease.
  • CRISPR clustered regularly interspersed short palindromic repeats
  • RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of polynucleotide constructs by the HDR mechanism can be used for selective integration at a target chromosomal site, including CRISPR system type I, type II, or type III Cas nucleases.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Cs
  • a type II CRISPR system Cas9 endonuclease is used.
  • Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks
  • the Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced.
  • Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma ta
  • sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res.42(4):2577-90; Kapitonov et al. (2015) J.
  • the CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA.
  • the bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break.
  • Cas9 endonuclease
  • targeting of Cas9 typically further relies on the presence of a 5′ protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.
  • the genomic target site may comprise a nucleotide sequence that is complementary to the gRNA and may further comprise a protospacer adjacent motif (PAM).
  • the target site comprises 20-30 base pairs in addition to a 3 base pair PAM.
  • the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen.
  • Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide.
  • the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele.
  • the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length.
  • the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.
  • Cpf1 the CRISPR nuclease from Prevotella and Francisella 1
  • Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9. Cpf1 is capable of cleaving either DNA or RNA.
  • the PAM sites recognized by Cpf1 have the sequences 5'-YTN-3' (where "Y” is a pyrimidine and “N” is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9.
  • Cpf1 cleavage of DNA produces double- stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang.
  • C2c1 is another class II CRISPR/Cas system RNA-guided nuclease that may be used.
  • C2c1 similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites.
  • RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI.
  • dCas9 inactive Cas9
  • FokI-dCas9 FokI endonuclease
  • the RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector) that is introduced into the host cell.
  • Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism.
  • a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence.
  • the protein can be transiently, conditionally, or constitutively expressed in the cell.
  • a polynucleotide construct is site-specifically integrated into the genome of a host cell using a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, wherein the construct is integrated into a Cas9-induced double-strand break at the target chromosomal site.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a vector encoding Cas9 and a gRNA targeting the desired chromosomal site for integration is introduced into the host cell.
  • sequences with homology to the target locus are introduced into the polynucleotide construct to allow for integration by homology-directed repair.
  • a target chromosomal site for integration of one or more polynucleotide constructs disclosed herein may include transcriptionally active chromosomal sites.
  • transcriptionally active chromosomal sites include DNaseI hypersensitive sites (DHSs).
  • a polynucleotide construct can be site-specifically integrated into the genome of a host cell by introducing a first recombination site into the construct and expressing a site-specific recombinase in the host cell.
  • the target chromosomal site of the host cell comprises a second recombination site, wherein recombination between the first and second recombination sites mediated by the site-specific recombinase results in integration of the vector at the target chromosomal locus.
  • the target chromosomal site may comprise either a recombination site native to the genome of the host cell or an engineered recombination site recognized by the site-specific recombinase.
  • various recombinases may be used for site-specific integration of vector constructs, including, but not limited to phi C31 phage recombinase, TP901-1 phage recombinase, and R4 phage recombinase.
  • a recombinase engineered to improve the efficiency of genomic integration at the target chromosomal site may be used.
  • one or more of the polynucleotide constructs are not integrated into the genome of the production host cell, and instead are maintained in the cell extrachromosomally.
  • extrachromosomal polynucleotide constructs include those that persist as stable/persistent plasmids or episomal plasmids.
  • a construct comprises Epstein-Barr virus (EBV) sequences, including the EBV origin of replication. oriP, and the EBV gene, EBNA1, to provide stable extrachromosomal maintenance and replication of the construct.
  • EBV sequences including the EBV origin of replication. oriP, and the EBV gene, EBNA1
  • EBV sequences including the EBV origin of replication. oriP, and the EBV gene, EBNA1
  • the polynucleotide constructs of the present disclosure may be introduced into a cell in manner similar to the currently used triple-transfection method for production of rAAV virions.
  • this system requires only one antibiotic resistance marker, and two split auxotrophic constructs for selection of all three plasmids, each being transformed just once into the DHFR knockout strain- producing a master cell line for virion production which can be stored and then utilized for scaled-up production without further transformations.
  • This approach provides inducible control over expression of the Rep/Cap products avoiding the toxicity typically associated with Rep/Cap production and also avoids selection with multiple antibiotics, which is not preferred for therapeutic products.
  • both overexpression of Rep/Cap and selection with multiple antibiotics can be toxic and result in diminished virion yield.
  • the transformed cells can be frozen for storage and thawed for subsequent applications.
  • Plasmids [0620] In some aspects, plasmids comprising a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 are provided.
  • plasmids comprising a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149 are provided. [0622] In some aspects, plasmids comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 are provided.
  • plasmids comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 are provided.
  • plasmids comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 and a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 or 165 are provided.
  • Cells Cells Comprising Polynucleotides [0625] In some aspects, cells comprising the polynucleotide described in the present disclosure are provided.
  • the first polynucleotide is described in section "First Polynucleotide encoding AAV Rep Protein” above.
  • the second polynucleotide is described in section "AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above.
  • the third polynucleotide is described in sections "Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above.
  • the fourth polynucleotide is described in section “Fourth Polynucleotide Encoding Payload” above.
  • the fifth polynucleotide is described in section "Fifth Polynucleotide Encoding VA-RNA” above.
  • the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof.
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof.
  • the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof.
  • the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein.
  • the cell comprises an adenovirus E2A protein and E4 protein
  • the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein.
  • the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome.
  • the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.
  • the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins.
  • the third polynucleotide comprises SEQ ID NO: 30.
  • the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
  • the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B.
  • the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient.
  • the cell expresses adenoviral helper proteins E1A and E1B.
  • recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter
  • the inducible recombinase upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one
  • the cell after induction, comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0639] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158.
  • cells that comprise a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; and d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins are provided.
  • cells that comprise a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins; and e) a fifth polynucleotide comprising a fifth sequence encoding a payload are provided.
  • cells that comprise a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins; e) a fifth polynucleotide comprising a fifth sequence encoding a payload; and f) a sixth polynucleotide comprising a sixth sequence encoding a viral associated RNA (VA-RNA) are provided.
  • VA-RNA viral associated RNA
  • the VA- RNA is a mutated VA-RNA.
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions that encapsidates the payload of the fifth polynucleotide.
  • rAAV recombinant AAV
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide, and the sixth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions that encapsidates the payload of the fifth polynucleotide.
  • rAAV recombinant AAV
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are, in any combination, on one or more polynucleotide constructs.
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and fifth polynucleotide are, in any combination, on one or more polynucleotide constructs.
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide, and the sixth polynucleotide are, in any combination, on one or more polynucleotide constructs.
  • cells for inducibly producing rAAV comprise a single copy of each of the first polynucleotide, the second polynucleotide, and the third polynucleotide. In other embodiments, cells for inducibly producing rAAV comprise a single copy of each of the first polynucleotide construct and the third polynucleotide construct, and comprise two or more copies of the second polynucleotide construct.
  • the first polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the second polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the third polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the fourth polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the fifth polynucleotide further comprises a selectable marker operably linked to a promoter.
  • the sixth polynucleotide further comprises a selectable marker operably linked to a promoter.
  • any combination of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise a selectable marker.
  • the selectable marker of any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide are different selectable markers, the same selectable marker but as a different split portion of the selectable marker, or a combination thereof.
  • the promoter is a constitutive promoter.
  • a) the first polynucleotide comprising the first sequence comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence comprising a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence comprising the stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, (iii) a second part of the AAV Rep proteins coding sequence; and (iv) one or more promoters operably linked to the first sequence.
  • the coding sequence comprising the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker operably linked to the stop signaling sequence.
  • the first sequence comprises from 5’ to 3’: [0656] one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form the excisable element, wherein the first recombination
  • the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to a promoter present in the second part of the AAV Rep proteins coding sequence or the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to an inducible promoter.
  • the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to a native AAV promoter and the third polynucleotide comprising the third sequence encoding the AAV Cap proteins is operably linked to a first inducible promoter.
  • the third polynucleotide further comprises a second constitutive promoter operably linked to a second selectable marker.
  • the fourth polynucleotide comprising the fourth sequence encoding the one or more adenoviral helper proteins further comprises a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the fourth sequence encoding the one or more AAV helper proteins; a third constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter
  • the sequence encoding one or more adenoviral helper proteins comprises from 5’ to 3’: a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site, wherein the third recombination site and the fourth recombination site are oriented in the same direction; and a sequence encoding one or more AAV helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the second inducible promoter in absence of a first triggering agent, wherein in the presence
  • the fifth polynucleotide further comprises a fourth selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR); optionally, wherein the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146, SEQ ID NO: 156, or SEQ ID NO: 158; optionally, wherein SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159 comprises the sequence of the fifth polynucleotide.
  • the fifth polynucleotide further comprises a spacer between the 5' ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof; optionally wherein SEQ ID NO: 147 comprises the fifth polynucleotide.
  • the spacer ranges in length from 500 base pairs to 5000 base pairs.
  • the fourth polynucleotide comprising the fourth sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins; optionally, wherein SEQ ID NO: 30 or SEQ ID NO: 154 comprises the fourth polynucleotide or the fourth polynucleotide comprises SEQ ID NO: 153.
  • the two AAV helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein; optionally, wherein the any two proteins are E2A and E4 or E1A and E1B.
  • the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
  • the first inducible promoter operably linked to the Cap in the third polynucleotide construct is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.
  • the tetracycline-inducible promoter comprises a tetracycline-responsive promoter element (TRE).
  • the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
  • the minimal promoter is a human cytomegalovirus promoter.
  • the second inducible promoter operably linked to the self- excising element in the third polynucleotide construct is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same; further optionally, wherein the first inducible promoter and the second inducible promoter are a tetracyline-inducible promoter.
  • the inducible recombinase upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to
  • the fifth polynucleotide comprising the fifth sequence encoding the payload comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the sequence encoding the payload a sequence encoding progranulin.
  • the fifth polynucleotide comprising the fifth sequence encoding the payload comprises a suppressor tRNA, a guide RNA, or a homology region for homology- directed repair.
  • the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.
  • the sixth polynucleotide comprising the sixth sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, wherein the fifth and sixth recombination sites are oriented in the same direction, and wherein excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter.
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter.
  • the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter
  • the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.
  • the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.
  • cells of interest for inducibly producing recombinant AAV (rAAV) virions comprise a payload.
  • transcription of the first polynucleotide and the second polynucleotide are driven by native AAV promoters.
  • transcription of the first polynucleotide is driven by native AAV promoters and transcription of the second polynucleotide is driven by a first inducible promoter.
  • the first polynucleotide and the second polynucleotide are separated by a transcriptional blocking element.
  • transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters and transcription of the second polynucleotide is driven by the P40 native AAV promoter.
  • transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters and transcription of the second polynucleotide is driven by a first inducible promoter.
  • the first polynucleotide and the second polynucleotide are separated by a transcriptional blocking element.
  • transcription of the third polynucleotide is driven by a first inducible promoter.
  • the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof; optionally, wherein the fourth polynucleotide comprising the fourth sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof; further optionally, wherein the third polynucleotide
  • the cell comprises an adenovirus E1A protein and E1B protein
  • the one or more AAV helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein or wherein the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the third polynucleotide construct are an adenovirus E1A protein and E1B protein.
  • the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome; optionally, wherein the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.
  • the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell.
  • the HEK293 cell is DHFR-deficient or GS-deficient.
  • Cells Comprising Polynucleotide Systems [0684] In some aspects, cells comprising the polynucleotide system described in the present disclosure are provided. The polynucleotide system is described in section “1.4. Polynucleotide System” above.
  • the first polynucleotide is described in section "First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above; the third polynucleotide is described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; the fourth polynucleotide is described in section “Fourth Polynucleotide Encoding Payload” above; and the fifth polynucleotide is described in section “Fifth Polynucleotide Encoding VA- RNA” above.
  • one or more of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell.
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are, in any combination, on one or more polynucleotide constructs.
  • the one or more polynucleotide constructs are described in section “1.5. Polynucleotide Construct System”.
  • the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof.
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof.
  • the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof.
  • the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein.
  • the cell comprises an adenovirus E2A protein and E4 protein
  • the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein.
  • the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome.
  • the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.
  • the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins.
  • the third polynucleotide comprises SEQ ID NO: 30.
  • the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
  • the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B.
  • the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient.
  • the cell expresses adenoviral helper proteins E1A and E1B.
  • recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter
  • the inducible recombinase upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one
  • the cell after induction, comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0702] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158.
  • cells comprising the polynucleotide construct system described in section “1.5. Polynucleotide Construct System” above are provided.
  • one or more of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, and the fourth polynucleotide are integrated into the nuclear genome of the cell.
  • the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof.
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof.
  • the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof.
  • the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein.
  • the cell comprises an adenovirus E2A protein and E4 protein
  • the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein.
  • the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome.
  • the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.
  • the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins.
  • the third polynucleotide comprises SEQ ID NO: 30.
  • the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
  • the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B.
  • the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient.
  • the cell expresses adenoviral helper proteins E1A and E1B.
  • recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter
  • the inducible recombinase upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one
  • the cell after induction, comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0722] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158.
  • stable mammalian cell lines that include a) a first polynucleotide construct comprising a first sequence encoding AAV Rep proteins and a second sequence encoding AAV Cap proteins; b) a second polynucleotide construct comprising a third sequence encoding the AAV Cap proteins; and c) a third polynucleotide construct comprising a fourth sequence encoding one or more adenoviral helper proteins are provided.
  • the stable mammalian cell lines further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs).
  • stable mammalian cell lines that include a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins; b) a polynucleotide construct comprising a sequence encoding the AAV Cap proteins; and c) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided.
  • the stable mammalian cell lines further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • stable mammalian cell lines that include a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins encoding sequence and a sequence encoding AAV Cap proteins; and b) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided.
  • the stable mammalian cell lines further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • cells that comprise a) a first polynucleotide construct comprising a first sequence encoding AAV Rep proteins encoding sequence and a second sequence encoding AAV Cap proteins; b) a second polynucleotide construct comprising a third sequence encoding the AAV Cap proteins; and c) a third polynucleotide construct comprising a fourth sequence encoding one or more adenoviral helper proteins are provided.
  • the cells further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • cells that comprise a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins encoding; b) a polynucleotide construct comprising a sequence encoding the AAV Cap proteins; and c) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided.
  • the cells further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • cells that comprise a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins encoding and a sequence encoding the AAV Cap proteins; and b) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided.
  • the cells further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs).
  • ITRs inverted terminal repeats
  • a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; further optionally, wherein the first polynucleotide sequence lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.
  • a second polynucleotide construct comprises the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148.
  • a third polynucleotide construct comprises the fourth polynucleotide; optionally, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11.
  • a) a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32.
  • a second polynucleotide construct comprises the third polynucleotide; optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149.
  • a third polynucleotide construct comprises the fourth; optionally, wherein the third polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30.
  • a fourth polynucleotide construct comprises the fifth polynucleotide; optionally, wherein the fourth polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • cells for inducibly producing AAV comprise a) the first polynucleotide construct comprising the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; further optionally, wherein the first polynucleotide sequence lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136; b) the second polynucleotide construct comprising the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%
  • cells for inducibly producing AAV comprise a) a first polynucleotide construct comprising the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; b) a second polynucleotide construct comprising the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149; c) a third polynucleotide construct comprising the fourth, optionally, wherein the third polynucleot
  • the first polynucleotide construct, second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide construct, second polynucleotide construct, the third polynucleotide construct and fourth polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first polynucleotide construct, second polynucleotide construct, the third polynucleotide construct, fourth polynucleotide construct and fifth polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • cells of interest comprise a plurality of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, the fourth polynucleotide construct, or the fifth polynucleotide construct, or any combination thereof.
  • the first polynucleotide construct further comprises a first constitutive promoter operably linked to a sequence encoding a first selectable marker.
  • the third polynucleotide construct comprises a sequence encoding the activator. In certain embodiments, the sequence encoding the activator is operably linked to a constitutive promoter.
  • the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
  • the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.
  • rTA reverse tetracycline-controlled transactivator
  • TetR Tet Repressor binding protein
  • the triggering agent for inducing the tetracycline-inducible promoter is tetracycline or doxycycline.
  • the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen.
  • ER estrogen response element
  • the recombination sites in the first polynucleotide construct and the third polynucleotide construct are lox sites and the recombinase is a cre recombinase or wherein the recombination sites in the first polynucleotide construct and the third polynucleotide are flippase recognition target (FRT) sites and the recombinase is a flippase (Flp) recombinase.
  • presence of the triggering agent activates the activator for activation of the first inducible promoter to express Cap proteins from the third polynucleotide comprising the third sequence encoding the AAV Cap proteins.
  • the cells or stable cells may further include a fourth polynucleotide construct encoding a payload.
  • these cells provide for an increased levels of AAV capsid proteins thereby leading to increased rAAV production as compared to cells that include the first and third polynucleotide construct but do not include a second polynucleotide construct comprising a third sequence encoding the AAV Cap proteins.
  • the third polynucleotide construct permits inducible expression of a hormone-activated excising element.
  • the excising element can be a recombinase.
  • the recombinase can be a site-specific recombinase.
  • the site-specific recombinase can be a Cre polypeptide or a flippase.
  • Triggering of Cre expression via an inducible promoter leads to recombination at lax sites, which in turn lead to expression of adenovirus helper proteins, expression of AAV Rep and Cap proteins, and production of rAAV, optionally, encapsidating a therapeutic payload (e.g., transgene, a tRNA suppressor, a guide RNA, or other oligonucleotide).
  • a therapeutic payload e.g., transgene, a tRNA suppressor, a guide RNA, or other oligonucleotide.
  • FIG.1A depicts an exemplary system of polynucleotides for inducibly producing rAAV.
  • four synthetic polynucleotide constructs are separately integrated into the nuclear genome of a cell line that expresses adenovirus E1A and E1B, such as HEK 293 cells.
  • adenovirus E1A and E1B such as HEK 293 cells.
  • transcriptional read-through of rep on Construct 1 is blocked by an excisable element comprising a first recombination site, a coding sequence comprising a stop signaling sequence, a second recombination site.
  • a first and second recombination sites flank the coding sequence comprising a stop signaling sequence and the first recombination site and the second recombination site are oriented in the same direction.
  • Construct 1 comprises from 5’ to 3’: one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence.
  • Construct 2 encodes a mammalian selectable marker under the control of a constitutive promoter and AAV capsid proteins under the control of an inducible promoter.
  • Each of Constructs 1 and 2 include sequences that encode for the same Cap proteins.
  • each of the Constructs 1 and 2 encodes for AAV5 cap proteins, VP1, VP2, and VP3.
  • Construct 3 includes a conditionally self-excising element which permits conditional expression of ER2 Cre under the control of an inducible promoter.
  • the inducible promoter is a Tet-inducible promoter (e.g., Tet-On promoter).
  • the inducible promoter in the absence of a triggering agent, is not active.
  • a triggering agent for Tet-inducible promoter is a tetracycline.
  • Tet activator protein TetOn3G expressed, under the control of a constitutive promoter, cannot bind and activate the basal Tet inducible promoter.
  • the localization of Cre is under control of estrogen response elements (“ER2”) that require binding of an estrogen agonist or selective modulator, such as tamoxifen, for the translocation from the cytoplasm to the nucleus.
  • the ER2 Cre element also comprises a strong 3’ polyadenylation signal, which prevents basal expression of the downstream adenoviral helper genes, E2A and E4.
  • the Cre element is split into two fragments, that can be fused in the presence of a chemical agent, such as rapamycin.
  • the Cre is a light inducible Cre.
  • the Construct 3 comprises a P2A sequence positioned between an E2A sequence and an E4 sequence.
  • the P2A sequence encodes a self-cleaving peptide such that E2A and E4 proteins are generated after cleavage of a fusion protein comprising E4, P2A, E2A amino acid sequences.
  • the Construct 3 comprises an internal ribosomal entry site (IRES) sequence positioned between an E2A sequence and an E4 sequence.
  • IRES can comprise at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135.
  • the inducible promoter system of Construct 3 is a tetracycline- inducible promoter (e.g., a Tet On inducible promoter that is part of a Tet On inducible promoter system). In some embodiments, the inducible promoter system of Construct 3 is a Tet Off inducible promoter system. In some embodiments, the inducible promoter system of Construct 3 is a cumate inducible promoter system. [0758] In this embodiment, in Construct 3, the activator TetOn3G is expressed under the control of the constitutive EF1alpha promoter and a gene encoding for puromycin resistance is under the control of the constitutive CMV promoter.
  • Construct 3 includes an optional insert which includes a Cre inducible U6 promoter that drives the expression of transcriptionally dead mutants of VA RNA1 (VA RNA).
  • the U6 promoter is split into two parts separated by a Lox flanked stuffer sequence. The U6 promoter is inactive because of the presence of the stuffer sequence. Cre mediated excision of the stuffer sequence activates the U6 promoter, which then drives the expression of VA RNA.
  • the payload polynucleotide on Construct 4 is flanked by AAV ITRs, represented by the brackets.
  • Construct 4 also includes a constitutive promoter operably linked to a sequence encoding a selectable marker or a first portion or a second portion of a split selectable marker.
  • Construct 1 includes a sequence encoding a first portion or a second portion of a split selectable marker and Construct 4 includes a sequence encoding the other portion of the split selectable marker.
  • a first triggering agent e.g., doxycycline, which is also referred to as Dox
  • a second triggering agent e.g., tamoxifen
  • Tet-on 3G and Dox bind the Tet responsive basal promoter and ER2 Cre is expressed and translocates to the nucleus.
  • ER2 Cre excises its own coding sequence from Construct 3, leaving the integrated construct shown in FIG.1B. Excision of the self-excising ER2 Cre coding sequence allows expression of E2A and E4 helper proteins under the control of the tetracycline-inducible promoter.
  • Construct 1 is designed to prevent expression of AAV Rep prior expression and translocation of ER2 Cre and permit expression of AAV Rep and Cap proteins from their endogenous promoters after translocation of ER2 Cre to the nucleus.
  • the excisable element in Construct 1 which interrupts the rep coding sequence, blocking transcriptional read-through of the full-length rep coding sequence results in production of a pre-triggered transcript that includes the 5’ portion of AAV Rep coding sequence fused to a blue fluorescent marker protein (BFP) coding sequence.
  • BFP blue fluorescent marker protein
  • the transcript contains a single intron flanked by 5’ and 3’ splice sites. Routine splicing produces a transcript that encodes a fusion protein that includes the N-terminal portion of rep fused to BFP.
  • the fusion protein lacks the toxicity of full-length Rep protein, and presence of pre-triggered Construct 1 in the cell genome can be detected by BFP fluorescence for quality control.
  • the excisable element comprises a first spacer segment, a second spacer segment, and a third spacer segment.
  • the excisable element is inserted at an insertion site between the p19 internal promoter and the p40 internal promoter of the AAV Rep coding sequence.
  • the insertion site is CAG-G, CAG-A, AAG-G, or AAG-A, wherein the dash (-) indicates the point of insertion of the excisable spacer.
  • Construct 1 also includes a sequence encoding a selectable marker.
  • the selectable marker may be a split selectable marker, e.g., split antibiotic resistance protein expressed under the control of a constitutive promoter.
  • FIG.1B shows the conversion of the pre-triggered Construct 1 to a post-triggered state “on state” upon exposure to ER2 Cre within the cell nucleus. ER2 Cre excises the excisable element, which includes the BFP marker coding sequence and the upstream 3’ splice site. As rearranged, the construct now allows expression of functional Rep and Cap transcripts from their respective endogenous promoters. Loss of BFP expression indicates successful Cre-mediated genomic recombination.
  • Construct 4 shown in FIG.1A and FIG.1B encodes a payload.
  • Construct 4 includes a polynucleotide encoding a payload, which can be a gene of interest flanked by ITRs, and a second portion of the selectable marker expressed from a constitutive promoter.
  • the polynucleotide payload can be any payload for which rAAV is an appropriate vehicle, including a transgene encoding a protein of interest, a homology element for homology-directed repair, or a guide RNA.
  • the polynucleotide payload is flanked by AAV ITRs, represented by the brackets.
  • the sequence encoding the payload may be under control of a constitutive promoter. In certain embodiments, the sequence encoding the payload is under control of a tissue-specific promoter active in the tissue where the payload is to be delivered.
  • FIG.1B depicts the post-triggered state “on state” of the constructs shown in FIG.1A following the addition of tamoxifen and doxycycline to the cell medium.
  • Adenoviral E2A and E4 helper proteins are expressed from integrated Construct 3 under control of the inducible promoter (e.g., a Tet-On promoter activated in the presence of Dox).
  • AAV rep and cap coding sequences are expressed from Construct 1 under control of endogenous promoters.
  • FIGS.1A and 1B depict polynucleotides for selection of cells based in antibiotics resistance.
  • the split blasticidin is used in Constructs 1 and 4.
  • a separate exemplary antibiotic selection approach, puromycin resistance is used in Construct 3.
  • a separate exemplary antibiotic selection approach, hygromycin resistance is used in Construct 2.
  • the cell line stably maintains all 4 constructs in the nuclear genome with two antibiotic selections, plus a single auxotrophic selection can also be utilized.
  • the cell line stably maintains all 4 constructs in the nuclear genome with no antibiotic selection and instead utilizes auxotrophic protein selection.
  • a split auxotrophic selection system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure can be used.
  • One construct encodes the N-terminal fragment of mammalian dihydrofolate reductase (DHFR) fused to a leucine zipper peptide (“Nter-DHFR”). This N-terminal fragment is enzymatically nonfunctional.
  • the other construct encodes the C-terminal fragment of DHFR fused to a leucine zipper peptide (“Cter-DHFR”). This C-terminal fragment is enzymatically nonfunctional.
  • a functional DHFR enzyme complex is formed through association of the leucine zipper peptides.
  • Both constructs can be stably retained in the genome of a DHFR null cell by growth in a medium lacking hypoxanthine and thymidine.
  • FIG.1A An exemplary deployment of this split auxotrophic selection design in the multi- construct system of FIG.1A is disclosed.
  • the split auxotrophic selection elements are deployed on Constructs 1 and 4.
  • a separate exemplary antibiotic selection approach, puromycin resistance is deployed on Construct 3.
  • a separate exemplary antibiotic selection approach, hygromycin resistance is deployed on Construct 2. This results in the ability to stably maintain all four constructs in the mammalian cell line using two antibiotics, culturing in medium with puromycin and hygromycin, lacking thymidine and hypoxanthine.
  • the split selection system is a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure.
  • Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond.
  • Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form.
  • the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein.
  • Split intein systems are described in: Cheriyan et al, J. Biol.
  • the split auxotrophic selection system described herein comprises a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein.
  • this N-terminal fragment is enzymatically nonfunctional and this C-terminal fragment is enzymatically nonfunctional.
  • the split inteins can catalyze the joining of the N-terminal fragment of the auxotrophic protein and a C-terminal fragment of the auxotrophic protein to form a functional enzyme, such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR).
  • both constructs can be stably retained in the genome of a cell by growth in a medium lacking the product produced by the enzyme.
  • the split auxotrophic selection elements e.g., a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein
  • Constructs 1 and 4 are part of a split intein system.
  • a separate exemplary auxotrophic selection approach e.g., a full length auxotrophic protein, can be deployed on Construct 3.
  • a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein or a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein further encodes a helper enzyme, wherein expression of the helper enzyme facilitates growth of the host cell in conjunction with the functional enzyme upon application of the single selective pressure.
  • the selection elements following triggering and Cre-mediated genomic rearrangement, the selection elements remain unchanged, allowing continued maintenance of the four post- triggering integrated constructs using two antibiotics in medium lacking hypoxanthine and thymidine.
  • viral proteins needed for AAV virion formation are inhibited by host cell mechanisms. Inhibition of these host cell mechanisms to maximize AAV viral titers in the stable cell lines described herein include, but are not limited to: knocking out PKR (PKR KO) (pathway is responsible for inhibition of viral proteins) in the starting cell line (P0), introducing a mutant EIF2alpha (in the PKR pathway) in the starting cell line (P0), and/or manipulating or modulating virus-associated (VA) RNAs (VA-RNAs, an inhibitor of PKR).
  • PKR KO knocking out PKR
  • VA-RNAs a mutant EIF2alpha
  • VA-RNAs an inhibitor of PKR
  • VA-RNAs can trigger the innate immune response.
  • VA-RNAs are processed to functional viral miRNAs and disturb the expression of numerous cellular genes. Therefore, VA-deleted adenoviral vector production constructs (AdVs) lacking VA-RNA genes, or having modified VA-RNA, would be advantageous.
  • AdVs VA-deleted adenoviral vector production constructs
  • VA- deleted AdVs do not produce commercially sufficient quantities of AAV titers (e.g., resulting in fewer and poor-quality virions).
  • overexpressing VA-RNA also results in a low titer of AAV production that would not be commercially feasible for scale-up.
  • VA-RNA is also an inhibitor of PKR, which is involved in a pathway responsible for inhibiting AAV viral protein synthesis.
  • PKR phosphorylates EIF2alpha, which results in inhibition of viral protein synthesis.
  • PKR is a major kinase that may self-phosphorylate and EIF2alpha may be phosphorylated by other kinases.
  • three strategies (PKR KO, EIF2alpha mutation, manipulation of VA- RNA) are being developed for use in any combination in the AAV production systems described herein.
  • the constructs of this system can also be used in a vector system, wherein the constructs do not integrate into the genome of the cell.
  • FIG.2A an exemplary system of polynucleotides for inducibly producing rAAV is depicted.
  • Construct 1 depicted in this embodiment, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter.
  • the coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE).
  • TBE transcription blocking element
  • the coding sequence for the Rep proteins is similar to the coding sequence for the Rep proteins described in FIG.1A. This construct may also be referred to as a Rep-Cap construct.
  • This construct comprises from 5’ to 3’: one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence.
  • Construct 2 is similar to Construct 4 depicted in FIGS.1A and 1B.
  • Construct 3 is similar to Construct 3 depicted in FIGS.1A and 1B. This system does not include Construct 2 depicted in FIGS.1A and 1B.
  • the Cap proteins coding sequence may include a strong polyA signal sequence positioned 3’ to the Cap proteins coding sequence.
  • the strong polyA signal sequence may be SV40, bGH, or RBG polyA signal sequence.
  • an exemplary system of polynucleotides for inducibly producing rAAV is depicted.
  • the system in absence of a first triggering agent and a second triggering agent, the system is in an off state.
  • Construct 1 depicted in this embodiment, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet-On promoter).
  • the coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE).
  • this system includes an additional polynucleotide, depicted as Construct 4, for expressing AAV Cap proteins.
  • Construct 4 may be referred to as a Cap construct and is the same as Construct 2 shown in FIGS.1A and 1B.
  • FIG.3B the system of polynucleotides for inducibly producing rAAV depicted in FIG.3A is shown in the on-state, after induction by the first triggering agent and the second triggering agent.
  • a cell comprises one construct (Rep Cap construct, inducible helper construct, and the payload construct).
  • the one construct is stably integrated into the genome of the cell.
  • the one construct is not stably integrated into the genome of the cell.
  • a plurality of the one construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the one construct is not stably integrated into the genome of the cell.
  • a cell comprises two constructs (any combination of Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct). In some embodiments, the two constructs are stably integrated into the genome of the cell. In some embodiments, the two constructs are not stably integrated into the genome of the cell. In some embodiments, the two constructs are separately stably integrated into the genome of the cell. In some embodiments, the two constructs are not separately stably integrated into the genome of the cell.
  • a plurality of the two constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not separately stably integrated into the genome of the cell. In some embodiments, a cell comprises the Rep Cap construct and the inducible Cap construct. In some embodiments, a cell comprises the Rep Cap construct and the payload construct. In some embodiments, a cell comprises inducible Cap construct and the payload construct. In some embodiments, a cell comprises the inducible helper construct and the payload construct.
  • a cell comprises the Rep Cap construct and the inducible helper construct.
  • a cell comprises the Rep Cap construct (s) as disclosed herein, the inducible Cap construct(s) as disclosed herein, the inducible helper construct(s) as disclosed herein, payload construct(s) as disclosed herein, or any combination thereof.
  • the inducible helper construct comprises a VA-RNA construct as described herein.
  • the cell further comprises the VA-RNA construct as described herein.
  • the VA-RNA construct is stably integrated into the genome of the cell. In some embodiments, the VA-RNA construct is not stably integrated into the genome of the cell.
  • a cell comprises three constructs (any combination of Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct).
  • the constructs of FIG.2A are stably integrated into the genome of the cell.
  • the three constructs are not stably integrated into the genome of the cell.
  • the three constructs are separately stably integrated into the genome of the cell.
  • the three constructs are not separately stably integrated into the genome of the cell.
  • a plurality of the three constructs are stably integrated into the genome of the cell.
  • a plurality of the three constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are not separately stably integrated into the genome of the cell. In some embodiments, a cell comprises the Rep Cap construct, the inducible Cap construct, and the inducible helper construct. In some embodiments, a cell comprises the Rep Cap construct, the inducible Cap construct, and the payload construct. In some embodiments, a cell comprises the Rep Cap construct, the inducible helper construct, and the payload.
  • a cell comprises the inducible Cap construct, the inducible helper construct, and the payload.
  • a cell comprises the Rep Cap construct (s) as disclosed herein, the inducible Cap construct(s) as disclosed herein, the inducible helper construct(s) as disclosed herein, payload construct(s) as disclosed herein, or any combination thereof.
  • the inducible helper construct comprises a VA- RNA construct as described herein.
  • a cell further comprises the VA-RNA construct as described herein.
  • the VA-RNA construct is stably integrated into the genome of the cell. In some embodiments, the VA-RNA construct is not stably integrated into the genome of the cell.
  • a cell comprises three constructs (any combination of Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct).
  • the constructs of FIG.2A the constructs of FIG.2A.
  • the one of the three constructs is stably integrated into the genome of the cell.
  • Construct 3 is stably integrated into the cell and Construct 1 and Construct 2 are transiently transfected into that cell.
  • the two of the three constructs are stably integrated into the genome of the cell.
  • Construct 3 and Construct 1 are is stably integrated into the cell and Construct 2 (e.g., but also with a different selectable marker than shown in FIG.2A) is transiently transfected into that cell.
  • Construct 2 e.g., but also with a different selectable marker than shown in FIG.2A
  • the three constructs are not stably integrated into the genome of the cell, but are instead all transiently transfected into the cell.
  • a cell comprises all four constructs (Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct). For example, the constructs of FIG.1A, FIG.3A, or FIG.7.
  • the four constructs are stably integrated into the genome of the cell. In some embodiments, the four constructs are not stably integrated into the genome of the cell. In some embodiments, the four constructs are separately stably integrated into the genome of the cell. In some embodiments, the four constructs are not separately stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are separately stably integrated into the genome of the cell.
  • a plurality of the four constructs are not separately stably integrated into the genome of the cell.
  • the cell, the inducible helper construct comprises a VA-RNA construct as described herein.
  • cell further comprises the VA-RNA construct.
  • a VA-RNA construct is a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter.
  • the sequence coding for the VA-RNA comprises a sequence coding for a transcriptionally dead VA-RNA.
  • the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region.
  • the sequence coding for the VA-RNA comprises at least one mutation.
  • the at least one mutation is in the A Box promoter region.
  • the at least one mutation is in the B Box promoter region.
  • the at least one mutation is G16A and G60A.
  • the expression of the VA-RNA is under the control of an RNA polymerase III promoter.
  • the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter.
  • the expression of the VA-RNA is under the control of a U6 or U7 promoter.
  • the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter.
  • the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence.
  • the stuffer sequence is excisable by a recombinase.
  • the stuffer sequence comprises a sequence encoding a gene.
  • the stuffer sequence comprises a promoter.
  • the promoter is a constitutive promoter.
  • the promoter is a CMV promoter.
  • the gene encodes a detectable marker or a selectable marker.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa.
  • the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter.
  • the selectable marker is DHFR Z-Nter or DHFR Z-Cter.
  • the DHFR Z- Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.
  • the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.
  • the detectable marker comprises a luminescent marker or a fluorescent marker.
  • the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry.
  • the VA-RNA construct further comprises a sequence coding for a recombinase.
  • the recombinase is exogenously provided.
  • the recombinase is a site-specific recombinase.
  • the recombinase is a Cre polypeptide or a Flippase polypeptide.
  • the Cre polypeptide is fused to a ligand binding domain.
  • the ligand binding domain is a hormone receptor.
  • the hormone receptor is an estrogen receptor.
  • the estrogen receptor comprises a point mutation.
  • the estrogen receptor is ERT2.
  • the recombinase is a Cre-ERT2 polypeptide.
  • the first recombination site is a first lox sequence and the second recombination site is a second lox sequence.
  • the first lox sequence is a first loxP site and the second lox sequence is a second loxP site.
  • the first recombination site is a first FRT site and the second recombination site is a second FRT site.
  • the selectable marker is a mammalian cell selection element.
  • the selectable marker is an auxotrophic selection element.
  • the auxotrophic selection element codes for an active protein.
  • the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR).
  • PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90.
  • GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112.
  • TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123.
  • the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter.
  • the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4.
  • the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5.
  • the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein.
  • the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein.
  • the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein.
  • the selectable marker is an antibiotic resistance protein.
  • the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein.
  • the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein.
  • the antibiotic resistance protein is for puromycin resistance or blasticidin resistance.
  • the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa).
  • Npu Nostoc punctiforme
  • Ssp Synechocystis species
  • strain PCC6803 Ssp
  • Cfa consensus DnaE intein
  • an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140.
  • a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141.
  • the polynucleotide construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker.
  • the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth.
  • the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth.
  • the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity.
  • the polynucleotide construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine.
  • the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine.
  • the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine.
  • the helper enzyme is GTP cyclohydrolase I (GTP-CH1).
  • the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99.
  • the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine.
  • expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure.
  • a stable cell line is produced from the cell or the population of cells as described herein.
  • the stable cell line is derived from a single cell and is monoclonal.
  • the stable cell line can be a mammalian stable cell line. The stable cell line can be produced by expanding or passaging a cell as described herein.
  • a stable cell line comprises the population of cells as disclosed herein.
  • the population of cells are derived from a single cell.
  • at least 70%, 80%, 90%, 95%, 99%, or 100% of the cells of the stable cell line are the population of cells as disclosed herein.
  • Cells Comprising Vector/Vector System [0793] In some aspects, cells comprising vector system described in section “1.6. Vector/Vector System” above are provided. [0794] In some embodiments, one or more of the first vector, the second vector, the third vector, and the fourth vector are integrated into the nuclear genome of the cell.
  • the first vector, the second vector, and the third vector are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • rAAV recombinant AAV
  • the first vector, the second vector, and the third vector are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
  • the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof.
  • the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof.
  • the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof.
  • the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein.
  • the cell comprises an adenovirus E2A protein and E4 protein
  • the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein.
  • the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome.
  • the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.
  • the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins.
  • the third polynucleotide comprises SEQ ID NO: 30.
  • the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
  • the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B.
  • the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient.
  • the cell expresses adenoviral helper proteins E1A and E1B.
  • recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter
  • the inducible recombinase upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one
  • the cell after induction, comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0810] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147.
  • the cell in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158. 1.10.
  • stable cell lines e.g., mammalian stable cell lines, capable of conditionally producing recombinant AAV (rAAV) virions.
  • the rAAV virions package an expressible payload, such as, a sequence encoding a therapeutic nucleic acid or protein or a complex therefore.
  • production of virions is not conditioned on the presence of an episome or independent plasmid within the cell.
  • the plasmids are provided comprising the constructs as disclosed herein.
  • the plasmids further comprise Epstein-Barr virus (EBV) sequences to stably maintain the constructs extrachromosomally.
  • EBV Epstein-Barr virus
  • expression of AAV Rep and Cap proteins is conditional.
  • expression of AAV Cap proteins is conditioned on addition of at least a first triggering agent to the cell culture medium.
  • expression of AAV Rep proteins is conditioned on addition of a first triggering agent and a second triggering agent to the cell culture medium.
  • the first triggering agent is .
  • doxycycline is used to the control a Tet inducible promoter.
  • the expression of the AAV cap proteins is under the control of a Tet inducible promoter.
  • expression of AAV Rep proteins is conditional.
  • expression of AAV Rep proteins is conditioned on addition of at least a first triggering agent to the cell culture medium. In certain embodiments, expression of AAV Rep proteins is conditioned on addition of a first triggering agent and a second triggering agent to the cell culture medium. In certain embodiments, a first triggering agent is used to the control a Tet inducible promoter. In certain embodiments, the expression of the AAV cap proteins is under the control of a Tet inducible promoter. In some embodiments, a first triggering agent is doxycycline.
  • inducible promoters can be utilized instead of a Tet inducible promoter, such as, but not limited to, a cumate inducible promoter system, which is under the control of cumate as the triggering agent or an ecdysone-inducible promoter, which is under the control of ecdysone or ponasterone as the triggering agent.
  • Site-Specific Recombinase System any suitable inducible excising agent (e.g., recombinase) can be utilized.
  • an excising agent can be a recombinase.
  • an excising agent can be a site-specific recombinase.
  • site-specific recombinase systems include, without limitation, Cre-loxP, Flp-FRT, PhiC31-att, Dre-rox, and Tre-loxLTR site- specific recombinase systems.
  • the Cre-loxP system uses a Cre recombinase to catalyze site-specific recombination between two loxP sites.
  • the Flp-FRT system uses a flippase (FLP) recombinase to catalyze site-specific recombination between two flippase recognition target (FRT) sites.
  • FLP flippase
  • the PhiC31-att system uses a phiC31 recombinase to catalyze site-specific recombination between two attachment (att) sites referred to as attB and attP.
  • the Dre-rox system uses a DreO recombinase to catalyze site-specific recombination between two rox sites.
  • the Tre-loxLTR system uses a Tre recombinase to catalyze site-specific recombination between two loxP sites that are modified with HIV long terminal repeats (loxLTR).
  • an excising agent can target a recombination site.
  • Suitable inducible excising agents include Cre and a flippase.
  • the Cre element can be hormone activated Cre, or light inducible Cre.
  • a recombination site can be a lox site.
  • a lox site can be a loxP site.
  • a recombination site can be an FRT site.
  • the Flippase recombinase system is based on Flp-FRT recombination, a site-directed recombination technology used to manipulate DNA under controlled conditions in vivo.
  • the cells do not express cytotoxic levels of Rep protein prior to addition of both the first expression and second triggering agents to the cell culture medium. In certain embodiments, the cells do not express cytostatic levels of Rep protein prior to addition of both the first and second triggering agents to the cell culture medium.
  • the average concentration of Rep protein within the cells is less than the amount prior to addition of both of the first and second triggering agents to the cell culture medium.
  • expression of Rep and Cap proteins becomes constitutive after addition of all of the at least first triggering agents to the cell culture medium.
  • expression of at least one adenoviral helper protein is conditional.
  • expression of the at least one adenoviral helper protein is conditioned on addition of at least a third triggering agent to the cell culture medium.
  • the third triggering agent is the same as the first triggering agent.
  • expression of adenoviral helper proteins is conditioned on addition of a third triggering agent and a fourth triggering agent to the cell culture medium.
  • the fourth triggering agent is the same as the second triggering agent.
  • the third triggering agent is the same as the first triggering agent and the fourth triggering agent is the same as the second triggering agent.
  • the first triggering agent and the third triggering agent are both doxycycline.
  • the second triggering agent and the fourth triggering agent are both tamoxifen.
  • adenoviral helper proteins following triggering of expression by contact of the cell with the at least third triggering agent requires the presence of only the third triggering agent in the cell culture medium.
  • the third triggering agent is the same as the first triggering agent.
  • the first triggering agent and the third triggering agent are both doxycycline.
  • expression of at least one adenoviral helper RNA is conditional.
  • the adenoviral helper proteins comprise Ad E2A.
  • the adenoviral helper proteins comprise Ad E4.
  • the adenoviral helper protein is tagged.
  • a tag can be a protein tag.
  • a protein tag can be a FLAG tag.
  • E2A is FLAG-tagged.
  • E4 is FLAG-tagged.
  • the adenoviral helper RNA is a VA-RNA.
  • the adenoviral helper RNA is expressed from an inducible VA-RNA construct.
  • the VA-RNA is a mutant VA-RNA.
  • the VA-RNA is a transcriptionally dead VA-RNA.
  • the VA-RNA is under the control of an RNA polymerase III promoter.
  • the VA-RNA is under the control of an interrupted RNA polymerase III promoter.
  • the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the VA-RNA is under the control of an interrupted U6 or U7 promoter.
  • the third triggering agent is a tetracycline. In certain embodiments, the tetracycline is doxycycline (“Dox”).
  • the fourth triggering agent is an estrogen receptor ligand. In certain embodiments, the estrogen receptor ligand is a selective estrogen receptor modulator (SERM). In particular embodiments, the estrogen receptor ligand is tamoxifen.
  • SERM selective estrogen receptor modulator
  • expression of the payload is conditioned on addition of at least a fifth triggering agent to the cell culture medium. In some embodiments, expression of the payload is not conditioned on addition of an triggering agent to the cell culture medium.
  • expression of Rep and Cap proteins, Cap proteins, adenoviral helper proteins, and the payload becomes constitutive after addition of only one triggering agent to the cell culture medium. In certain embodiments, expression of Rep and Cap proteins, Cap proteins, and the adenoviral helper proteins becomes constitutive after addition of only one triggering agent to the cell culture medium.
  • the one triggering agent is the first triggering agent.
  • the first triggering agent is a tetracycline. In particular embodiments, the first triggering agent is doxycycline. 1.11. Cell Cultures and Bioreactors [0831] In some embodiments, a cell, population of cells, or stable cell line as disclosed herein is in a cell culture. In some embodiments, a cell culture composition comprises: a) suspension- adapted cells, b) serum-free cell culture media, and c) recombinant AAV (rAAV) virions, wherein the cell culture composition is free of herpes simplex virus, baculovirus, and adenovirus, and wherein the cell culture composition is free of plasmid and transfection agent.
  • rAAV recombinant AAV
  • the cell culture composition is free of polyethylenimine (PEI).
  • the suspension-adapted cells are suspension-adapted mammalian cells.
  • the suspension-adapted cells are suspension-adapted HEK293 cells or derivatives thereof.
  • the suspension-adapted mammalian cells are cells from the stable cell line of as disclosed herein, the population of cells as disclosed herein, or comprise a cell as disclosed herein.
  • the cell culture composition has a prepurification rAAV concentration of no less than 1 ⁇ 10 14 , 2 ⁇ 10 14 , 3 ⁇ 10 14 , 4 ⁇ 10 14 , 5 ⁇ 10 14 , 6 ⁇ 10 14 , 7 ⁇ 10 14 , 8 ⁇ 10 14 , 9 ⁇ 10 14 , 1 ⁇ 10 15 , or 5 ⁇ 10 15 viral genome (vg)/L.
  • the cell culture composition has a prepurification rAAV encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99.
  • rAAV virion from the stable cells as disclosed herein is produced in a bioreactor.
  • a bioreactor comprises the stable cell line as disclosed herein. In some embodiments, a bioreactor comprising the population of cells of as disclosed herein. In some embodiments, a bioreactor comprising the cell as disclosed herein. In some embodiments, a bioreactor contains the cell culture as disclosed herein. In some embodiments, the bioreactor is a 1L bioreactor. In some embodiments, the 1L bioreactor has a total rAAV yield of greater than 1 ⁇ 10 14 viral genome (vg). In some embodiments, the bioreactor is a 5L bioreactor. In some embodiments, the 5L bioreactor has a total rAAV yield of greater than 5 ⁇ 10 14 viral genome (vg).
  • the bioreactor is a 50L bioreactor. In some embodiments, the 50L bioreactor has a total rAAV yield of greater than 5 ⁇ 10 15 viral genome (vg). In some embodiments, the bioreactor is a 100L bioreactor. In some embodiments, the 100L bioreactor has a total rAAV yield of greater than 1 ⁇ 10 16 viral genome (vg). In some embodiments, the bioreactor is a 500L bioreactor. In some embodiments, the 500L bioreactor has a total rAAV yield of greater than 5 ⁇ 10 16 viral genome (vg). In some embodiments, the bioreactor is a 2000L bioreactor.
  • the 2000L bioreactor has a total rAAV yield of greater than 2 ⁇ 10 17 viral genome (vg).
  • a bioreactor comprises a plurality of rAAV virions having a concentration of greater than 1 ⁇ 10 14 , 2 ⁇ 10 14 , 3 ⁇ 10 14 , 4 ⁇ 10 14 , 5 ⁇ 10 14 , 6 ⁇ 10 14 , 7 ⁇ 10 14 , 8 ⁇ 10 14 , 9 ⁇ 10 14 , 1 ⁇ 10 15 , or 5 ⁇ 10 15 viral genome (vg)/L.
  • a bioreactor comprises a plurality of rAAV virions having a prepurification concentration of greater than 1 ⁇ 10 14 , 2 ⁇ 10 14 , 3 ⁇ 10 14 , 4 ⁇ 10 14 , 5 ⁇ 10 14 , 6 ⁇ 10 14 , 7 ⁇ 10 14 , 8 ⁇ 10 14 , 9 ⁇ 10 14 , 1 ⁇ 10 15 , or 5 ⁇ 10 15 viral genome (vg)/L.
  • the bioreactor is a 1L, 5L, 50L, 100L, 500L, or 2000L bioreactor.
  • the bioreactor is a single use bioreactor. 1.12.
  • compositions of rAAV [0833]
  • the cell, population of cells, or stable cell line as disclosed herein is induced (as disclosed herein, e.g., after administration of a first and a second triggering agent in a bioreactor) to produce a plurality of rAAV virions.
  • a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification concentration of greater than 1 ⁇ 10 11 or no less than 5 ⁇ 10 11 , 1 ⁇ 10 12 , 5 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 viral genomes per milliliter.
  • a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99.
  • a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99.
  • a composition comprises an rAAV virion encapsidating a viral genome, wherein the composition has an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1 ⁇ 10 5 vg/target cell or less.
  • the rAAV virion has an increased infectivity compared an rAAV virion produced by an otherwise comparable cell capable of producing rAAV virions upon transient transfection at the same MOI.
  • the rAAV virion has at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared an rAAV virion produced by an otherwise comparable cell capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virion has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to an AAV virion produced by a cell having wildtype AAV at the same MOI.
  • the rAAV virion has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared an AAV virion produced by a cell having wildtype AAV at the same MOI.
  • the compositions further comprise a plurality of the rAAV virion.
  • the plurality of rAAV virions have a prepurification concentration of greater than 1 ⁇ 10 11 or no less than 5 ⁇ 10 11 , 1 ⁇ 10 12 , 5 ⁇ 10 12 , 1 ⁇ 10 13 or 1 ⁇ 10 14 viral genomes per milliliter.
  • the plurality of rAAV virions have a prepurification encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the plurality of rAAV virions have a prepurification F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the plurality of rAAV virions have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99%.
  • the plurality of rAAV virions have an increased infectivity compared to a plurality of rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the plurality of rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared a plurality of rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI.
  • the plurality of rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared a plurality of AAV virions produced by a cell having wildtype AAV at the same MOI.
  • the MOI is 1 ⁇ 10 1 , 1 ⁇ 10 2 , 2 ⁇ 10 3 , 5 ⁇ 10 4 , or 1 ⁇ 10 5 vg/target cell.
  • the MOI is selected from a range of 1 ⁇ 10 1 to 1 ⁇ 10 5 vg/target cell.
  • the viral genome comprises a sequence coding for a payload.
  • sequence of the payload comprises a polynucleotide sequence coding for a gene.
  • the gene codes for a selectable marker or detectable marker.
  • the gene codes for a therapeutic polypeptide or transgene.
  • the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide.
  • the therapeutic polynucleotide is a tRNA suppressor or a guide RNA.
  • guide RNA is a polyribonucleotide capable of binding to a protein.
  • the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the rAAV virion comprises a Cap polypeptide. In some embodiments, the Cap polypeptide is an AAV capsid protein. In some embodiments, the AAV capsid protein is VP1, VP2, or VP3.
  • a serotype of the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.
  • rAAV virions as disclosed herein are in a first composition and a second composition.
  • the first composition and the second composition have an encapsidation ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition and the second composition have an F:E ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition and the second composition have a concentration of viral genomes per milliliter that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition and the second composition have an infectivity that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition is a first dose and the second composition is a second dose.
  • the first composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days before the second composition is produced.
  • a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days before a plurality of rAAV virions of the second composition is produced.
  • the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the second composition is produced.
  • a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the second composition is produced. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the second composition is produced. In some embodiments, the first composition is produced from a plurality of virions from a first bioreactor and the second composition is produced from a plurality of virions from a second bioreactor.
  • a third composition or more compositions are produced from the rAAV as disclosed herein.
  • the first composition, the second composition, and the third composition have an encapsidation ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition, the second composition, and the third composition have an F:E ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition, the second composition, and the third composition have a concentration of viral genomes per milliliter that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the first composition, the second composition, and the third composition have an infectivity that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the third composition is a third dose.
  • the third composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days after the second composition is produced.
  • a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days after a plurality of rAAV virions of the second composition is produced.
  • the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second composition is produced.
  • a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second composition is produced. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after the second composition is produced. In some embodiments, the third composition is produced from a plurality of virions from a third bioreactor. 1.13.
  • compositions comprising the polynucleotides, or vector system described herein or an rAAV virion encapsidating a polynucleotide payload (e.g., for encoding a therapeutic protein, such as an antibody or any fragment or derivative thereof), produced from such a vector system, and a pharmaceutically acceptable carrier, diluent, excipient, or buffer.
  • a pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human.
  • excipients, carriers, diluents, and buffers include any pharmaceutical agent that may be administered without undue toxicity.
  • Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, and ethanol.
  • Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • a wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein.
  • compositions have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 2 0t h edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7t h ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3r d ed. Amer. Pharmaceutical Assoc. Certain facilitators of nucleic acid uptake and/or expression may also be included in the compositions or coadministered.
  • a pharmaceutical composition comprises the plurality of rAAV virions of any one of the embodiments as disclosed herein as disclosed herein and a pharmaceutically acceptable carrier.
  • a plurality of pharmaceutical doses each independently comprise the plurality of rAAV virions of any one of the embodiments as disclosed herein as disclosed herein and a pharmaceutically acceptable carrier.
  • the encapsidation ratio has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses.
  • the F:E ratio has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses.
  • the concentration of viral genomes has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses.
  • the concentration of vector genomes has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses.
  • the rAAV virion infectivity has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. 1.14.
  • methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload comprising: (i) introducing into a cell the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; (ii) selecting for a cell expressing a selectable marker of the third polynucleotide; (iii) introducing into the cell expressing the selectable marker of the third polynucleotide, the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above, the second polynucleotide described in section “1.3.
  • the methods further comprise contacting a cell of the cell line to a first triggering agent and a second triggering agent.
  • the activator activates the second inducible promoter resulting in expression of the recombinase.
  • recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase.
  • the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In certain embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein. [0841] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.
  • the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide.
  • the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.
  • a method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload comprising: (i) introducing into a cell the second polynucleotide construct described in section “1.5. Polynucleotide Construct System” above; (ii) selecting for a cell expressing a selectable marker of the second polynucleotide construct; (iii) introducing into the cell expressing the selectable marker of the second polynucleotide construct, the first polynucleotide construct described in section “1.5.
  • Polynucleotide Construct System above, and the third polynucleotide construct described in section “1.5. Polynucleotide Construct System” above; (iv) selecting for a cell expressing the selectable marker of the second polynucleotide construct, a selectable marker of the first polynucleotide construct, and a selectable marker of the third polynucleotide construct; (v) expanding the cell expressing the selectable marker of the second polynucleotide construct, the selectable marker of the first polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions are provided.
  • the method further comprises contacting a cell of the cell line to a first triggering agent and a second triggering agent.
  • the activator activates the second inducible promoter resulting in expression of the recombinase.
  • recombination between the first recombination site and the second recombination site in the second polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase.
  • the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In certain embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein. [0848] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.
  • the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide construct.
  • the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.
  • methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: (i) introducing into a cell the third vector described in section “1.6. Vector/Vector System”; (ii) selecting for a cell expressing a selectable marker of the third vector; (iii) introducing into the cell expressing the selectable marker of the third vector, the first vector described in section “1.6. Vector/Vector System”, the second vector described in section “1.6. Vector/Vector System”, and the fourth vector described in section “1.6.
  • the methods further comprise contacting a cell of the cell line to a first triggering agent and a second triggering agent.
  • the activator activates the second inducible promoter resulting in expression of the recombinase.
  • recombination between the first recombination site and the second recombination site in the first vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the third vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase.
  • the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In some embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein. [0854] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.
  • the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector.
  • the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.
  • methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: (i) introducing into a cell the second vector described in section “1.6.
  • Vector/Vector System selecting for a cell expressing a selectable marker of the second vector; (iii) introducing into the cell expressing the selectable marker of the second vector, the first vector described in section “1.6.
  • Vector/Vector System and the third vector described in section “1.6.
  • the methods further comprise contacting a cell of the cell line to a first triggering agent and a second triggering agent.
  • the activator activates the second inducible promoter resulting in expression of the recombinase.
  • recombination between the first recombination site and the second recombination site in the second vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the second vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase.
  • the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In some embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein. [0861] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.
  • the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector.
  • the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen.

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Abstract

Described herein are polynucleotides for increasing production of rAAV by increasing levels of AAV Capsid proteins, which is achieved by including a strong polyadenylation (polyA) signal sequence 3' to the sequence encoding the AAV capsid proteins and/or using an inducible promoter to drive expression of capsid proteins. Also provided are polynucleotide constructs, vectors, and system thereof, plasmids, and cells, such as, cell lines stably integrated with the polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids that enable increased production of recombinant AAV (rAAV) virions.

Description

SYSTEMS FOR AMPLIFICATION OF AAV CAP PROTEIN CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application No.63/542,480, filed on October 4, 2023; U.S. Provisional Application No.63/536,899, filed on September 6, 2023; U.S. Provisional Application No.63/522,304, filed on June 21, 2023; U.S. Provisional Application No.63/437,562, filed on January 6, 2023; and U.S. Provisional Application No.63/427,040, filed on November 21, 2022, the disclosures of which applications are herein incorporated by reference in their entirety. INTRODUCTION [0002] Recombinant adeno-associated virus (rAAV) is the preferred vehicle for in vivo gene delivery. AAV has no known disease associations, infects dividing and non-dividing cells, rarely if ever integrates into the mammalian cell genome, and can persist essentially for the lifetime of infected cells as a transcriptionally active nuclear episome. The FDA has recently approved several rAAV gene therapy products and many other rAAV-based gene therapy and gene editing products are in development. [0003] The most widely used method for producing rAAV virions is based on the helper-virus- free transient transfection of multiple plasmids, typically a triple transfection, into adherent cell lines. Although there is ongoing investment to increase production capacity, current AAV manufacturing processes are inefficient and expensive. In addition, they result in variable product quality, with low levels of encapsidation of a payload, such as a therapeutic payload. There is, therefore, a need for improved methods for producing rAAV. SUMMARY [0004] The polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids, and cells, such as, cell lines stably integrated with the polynucleotides, polynucleotide constructs, vectors, and system thereof, plasmids provided herein enable increased production of recombinant AAV (rAAV) virions. [0005] The increased production of rAAV is hypothesized to be achieved by providing an overall increase in the levels of AAV capsid proteins. In one embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins. In another embodiment, the increase in levels of AAV capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In a further embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In another embodiment, the increase in levels of AAV capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of the native promoter and another under the control of an inducible promoter. One or more of these embodiments can also be combined to further increase capsid proteins expression and enable increased production of recombinant AAV (rAAV). [0006] In certain embodiments, the polynucleotide constructs may be introduced into cells, e.g., stably integrated into the nuclear genome of the cells and used to express components for formation of rAAV in an inducible manner thereby avoiding the toxicity of AAV Rep proteins and AAV Cap proteins when constitutively expressed. Methods for generating such cells and cell lines useful for producing rAAV and methods for producing rAAV from the cells and the cell lines are also disclosed. [0007] In some aspects, the present disclosure provides that polynucleotides comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter; and (ii) a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3' of the sequence encoding AAV Cap proteins. [0008] In some aspects, the present disclosure provides polynucleotides comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins. [0009] In some aspects, the present disclosure provides that systems of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, and one or more of c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins. [0010] In other aspects, also disclosed herein are constructs that are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell. In some embodiments, the constructs may or may not be integrated into the genome of the cell. [0011] In still other aspects, further provided herein is a stable mammalian cell line and constructs, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is inducible upon addition of a triggering agent. [0012] In some aspects, further provided herein is a stable mammalian cell line and constructs, wherein the cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload; and production of virions is not conditioned on the presence of a plasmid within the cell. In some aspects, a set of nucleic acids is provided, comprising: (i) a first recombinant nucleic acid sequence encoding AAV Rep proteins and AAV Cap proteins; (ii) a second recombinant nucleic acid sequence encoding the AAV Cap proteins; and (iii) a third recombinant nucleic acid sequence encoding one or more adenoviral helper proteins wherein when the one or more nucleic acids are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. [0013] In some aspects, a set of nucleic acids is provided, comprising: (i) a first recombinant nucleic acid sequence encoding AAV Rep proteins; (ii) a second recombinant nucleic acid sequence encoding the AAV Cap proteins; and (iii) a third recombinant nucleic acid sequence encoding one or more adenoviral helper proteins wherein when the one or more nucleic acids are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. In some embodiments, a first polynucleotide comprises the first recombinant nucleic acid sequence and a second polynucleotide comprises the second recombinant nucleic acid sequence. In some embodiments, a first polynucleotide comprises the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence. In some embodiments, the first recombinant nucleic acid sequence and the second recombinant nucleic acid sequence are separated by a transcriptional blocking element. [0014] In some embodiments, the conditional expression of the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins is controlled by one or more excisable elements present in the first and third nucleic acids. [0015] In some embodiments, the one or more excisable elements comprise one or more introns and/or one or more exons. In some embodiments, the first recombinant nucleic acid sequence comprises: a) a first part of the AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep proteins coding sequence; and d) the AAV Cap proteins coding sequence. In some embodiments, the excisable element comprises a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell. [0016] In some embodiments, the first recombinant nucleic acid sequence comprises: a) a first part of the AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; and c) an excisable element between the first part of the AAV Rep protein coding sequence and the second part of the AAV Rep proteins coding sequence. In some embodiments, the excisable element comprises a) a first spacer segment comprising a first intron, b) a second spacer segment comprising a coding sequence of a detectable marker; and c) a third spacer segment comprising a second intron, and wherein the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery of a mammalian cell. [0017] In some embodiments, the excisable element comprises from 5’ to 3’: a) a 5’ splice site; b) a first spacer segment comprising a first intron; c) a second spacer segment comprising: i) a first lox sequence; ii) a 3’ splice site; iii) an exon; iv) a stop signaling sequence; and v) a second lox sequence; and d) a third spacer segment comprising a second intron and another 3’ splice site. [0018] In some embodiments, the detectable marker is a luminescent marker, a radiolabel or a fluorescent marker, optionally a fluorescent marker which is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. [0019] In some embodiments, a) the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1; and/or b) the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2; and/or c) the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3. In some embodiments, the second spacer segment is capable of being excised by a Cre polypeptide. [0020] In some embodiments, the expression of the AAV Rep proteins and/or the AAV Cap proteins is driven by native promoters. In some embodiments, a) the native promoters P5 and/or P19 drive the expression of the AAV Rep proteins; and/or b) the native promoter P40 drives the expression of the AAV Cap proteins. In some embodiments, a) the native promoters P5 and/or P19 drive the expression of the AAV Rep proteins; and/or b) an inducible promoter drives the expression of the AAV Cap proteins. [0021] In some embodiments, the second recombinant nucleic acid sequence encodes the AAV Cap proteins under the control of an inducible promoter and a selectable marker under the control of a constitutive promoter. In some embodiments, the selectable marker is an antibiotic resistance gene, such as, hygromycin. In some embodiments, the inducible promoter is a tetracycline inducible promoter and the constitutive promoter is a CMV or EF1alpha promoter. [0022] In some embodiments, the third recombinant nucleic acid sequence encodes: a) one or more adenoviral helper proteins; b) a conditionally self-excising element; and c) an inducible promoter; wherein, once integrated into the nuclear genome of a mammalian cell, the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter. [0023] In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the self-excising element comprises a sequence which encodes a polypeptide, e.g., a recombinase polypeptide, such as, a Cre polypeptide. In some embodiments, the polypeptide encoded by the self-excising element is conditionally expressible and is expressed only in the presence of a triggering agent. In some embodiments, the triggering agent is a hormone, e.g., tamoxifen. In some embodiments, the inducible promoter is a tetracycline- inducible promoter (“Tet inducible promoter”). In some embodiments, the third recombinant nucleic acid sequence further comprises a sequence that encodes a Tet responsive activator protein, e.g., Tet-on 3G. In some embodiments, the expression of Tet-on 3G activator protein is driven by an EF1alpha promoter. In some embodiments, the third recombinant nucleic acid sequence comprises a sequence with at least 80% homology, at least 90% homology, at least 95% homology, at least 99% homology, or a sequence identical to SEQ ID NO: 11 or SEQ ID NO: 12. [0024] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further comprises a nucleic acid sequence encoding a viral associated RNA (“VA-RNA”) sequence. In some embodiments, the third recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a VA-RNA sequence. In some embodiments, the expression of VA-RNA is constitutive. In some embodiments, the expression of VA-RNA is inducible. In some embodiments, the VA-RNA sequence comprises one or more mutations in the VA-RNA internal promoter, preferably G16A and G60A. In some embodiments, the expression of VA-RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter. In some embodiments, the expression of VA-RNA is driven by a U6 promoter or a U7 promoter. In some embodiments, the U6 promoter or the U7 promoter comprises a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide. [0025] In some embodiments, a serotype of the AAV Cap proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 and AAVhu68. In some embodiments, the serotype is an AAV5 Cap protein which comprises one or more mutations or insertions. [0026] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further encode a fourth recombinant nucleic acid sequence encoding a payload, optionally wherein the payload is: (a) a polynucleotide payload, such as a guide RNA for RNA editing, a guide RNA for Cas protein-directed DNA editing, a tRNA suppressor, or a gene for replacement gene therapy; or (b) a protein such as a therapeutic antibody or a vaccine immunogen. [0027] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein comprise one or more mammalian cell selection elements. In some embodiments, one or more of the mammalian cell selection elements encodes an antibiotic resistance gene, optionally a blasticidin resistance gene. In some embodiments, one or more of the mammalian cell selection elements is an auxotrophic selection element which encodes an active protein. In some embodiments the auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, one or more of the mammalian cell selection elements is a first auxotrophic selection element which encodes an inactive protein that requires expression of a second inactive protein from a second auxotrophic selection coding sequence for activity. In some embodiments, the first auxotrophic selection coding sequence encodes for DHFR Z-Cter (SEQ ID NO: 5) activity, and/or wherein the second auxotrophic selection coding sequence encodes for DHFR Z-Nter (SEQ ID NO: 4). [0028] In some embodiments, a) the first recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., a blasticidin resistance gene; b) the second recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., hygromycin; and c) the third recombinant nucleic acid comprises a mammalian cell selection element which encodes an antibiotic resistance gene, e.g., puromycin. [0029] The elements of the previous embodiments are capable of being in one or more separate constructs (e.g., polynucleotide constructs), in any combination, wherein the one or more constructs are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload, when introduced into a cell. [0030] In some aspects, disclosed herein is a mammalian cell wherein the nuclear genome of the cell comprises a plurality of integrated recombinant nucleic acid constructs which together encode for a recombinant adeno-associated virus (rAAV) virions, wherein the rAAV virions can be conditionally expressed from the cell. [0031] In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprises the one or more recombinant nucleic acids of any one of previous embodiments, wherein the AAV Rep proteins, the AAV Cap proteins and/or the adenoviral helper proteins can be conditionally expressed from the cell. In some embodiments, the cell is from a cell line that expresses adenoviral helper proteins E1A and E1B, e.g., from nucleic acids integrated into the nuclear genome of the cell. [0032] In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprises a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part and the second part of the AAV Rep proteins coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap proteins coding sequence; wherein the AAV Rep proteins and the AAV Cap proteins expression is driven by the native promoters P5, P19, and P40. [0033] In some embodiments, the plurality of integrated recombinant nucleic acid constructs comprises a first integrated polynucleotide construct comprising: a) a first part of an AAV Rep proteins coding sequence; b) a second part of the AAV Rep proteins coding sequence; c) an excisable element between the first part and the second part of the AAV Rep proteins coding sequence, wherein the excisable element comprises: i) a first spacer segment comprising a first intron; ii) a second spacer segment comprising a coding sequence of a detectable marker, wherein the second spacer segment is capable of being excised by a Cre polypeptide; and iii) a third spacer segment comprising a second intron; and d) an AAV Cap proteins coding sequence; wherein the AAV Rep proteins expression is driven by native promoters P5 and P19, and the AAV Cap proteins coding sequence is driven by an inducible promoter. [0034] In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a second integrated polynucleotide construct comprising a sequence encoding the same AAV Cap proteins as the first integrated polynucleotide construct. In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a second integrated polynucleotide construct comprising a sequence encoding the same AAV Cap proteins as the first integrated polynucleotide construct, wherein the AAV Cap proteins are operably linked to an inducible promoter. [0035] In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a third integrated polynucleotide construct comprising a) a conditionally expressible VA-RNA coding sequence which comprises a mutation in the VA-RNA internal promoter, wherein the expression of VA-RNA is driven by a U6 or a U7 promoter, optionally wherein the VA-RNA sequence comprises G16A and G60A mutations; b) one or more adenoviral helper protein coding sequences, wherein the adenoviral helper proteins are E2A and E4; c) a conditionally self-excising element which encodes a Cre polypeptide which translocates to the nucleus and self-excises only in the presence of a triggering agent which is tamoxifen, and d) an inducible promoter which is a Tet inducible promoter, and wherein the expression of the one or more adenoviral helper protein coding sequences is under the control of the conditionally self-excising element and the inducible promoter. [0036] In some embodiments, the plurality of integrated recombinant nucleic acid constructs further comprises a fourth integrated polynucleotide construct comprising encodes for the payload, wherein the payload is a polynucleotide payload. [0037] In some aspects, a method of producing a population of rAAV virions comprises: (a) culturing the cell of any one of the embodiments disclosed herein in conditions which allow for the expression of the rAAV virions; and (b) isolating the rAAV virions from the cell culture. [0038] In some aspects, a method of preparing the cell of any one of the previous embodiments comprises: i) providing a mammalian cell and the one or more nucleic acids of any one of the previous embodiments; and ii) selecting for integration of the one or more nucleic acids of any one of the previous embodiments into the nuclear genome of the mammalian cell. [0039] In other aspects, also provided herein are cells comprising: a) a first polynucleotide construct comprising a first polynucleotide coding for AAV Rep proteins and AAV Cap proteins; b) a second polynucleotide construct coding the AAV Cap proteins; c) a third polynucleotide construct coding for one or more adenoviral helper proteins; wherein when the one or more nucleic acids are integrated into the nuclear genome of a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. In some embodiments, the first polynucleotide coding for AAV Rep proteins and AAV Cap proteins comprises a transcriptional blocking element separating the sequence coding for the AAV Rep proteins and the sequence coding for the AAV Cap proteins. [0040] In some embodiments, the third polynucleotide construct comprises a sequence coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element. In some embodiments, the self-excising element is operably linked to the inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the second recombinant nucleic acid sequence is driven by the same inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the second polynucleotide construct is driven by the same inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter and expression of the AAV Cap proteins encoded by the first polynucleotide construct is driven by the same inducible promoter. [0041] In some embodiments, the inducible promoter is a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, transcription is activated from the inducible promoter upon binding of an activator. In some embodiments, the activator binds to the inducible promoter in the presence of a first triggering agent. In some embodiments, the third polynucleotide construct further comprises a sequence coding for an activator. In some embodiments, the activator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. In some embodiments, the rTA comprises four mutations in the tetR DNA binding moiety. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-85, or variants thereof. [0042] In some embodiments, the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter. In some embodiments, the constitutive promoter is a human cytomegalovirus promoter. In some embodiments, the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the cymR repressor. In some embodiments, the third polynucleotide construct further comprises a cymR repressor. In some embodiments, the cymR repressor is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the first triggering agent is a cumate. [0043] In some embodiments, the sequence coding for the self-excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites. [0044] In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the E2A is FLAG-tagged E2A. In some embodiments, the sequence coding for E2A and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A. [0045] In some embodiments, the third polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is an auxotrophic protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C-terminus of the auxotrophic protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic protein or leucine zipper linked to a C-terminus of the auxotrophic protein. In some embodiments, the auxotrophic protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal fragment of the split intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0046] In some embodiments, the third polynucleotide construct further comprises a sequence coding for VA-RNA. In some embodiments, the sequence coding for VA-RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA-RNA comprises at least two mutations in the internal promoter. In some embodiments, expression of VA-RNA is driven by a U6 or U7 promoter. In some embodiments, the third polynucleotide construct further comprises upstream of the sequence coding for VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; and e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. [0047] In some embodiments, the first polynucleotide construct comprises: a) a sequence of a first part of a Rep gene; b) a sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene. [0048] In some embodiments, the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron. [0049] In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5’ splice site and a 3’ splice site. In some embodiments, the 5’ splice site is a rabbit beta globin 5’ splice site. In some embodiments, the 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the three splice sites are a 5’ splice site, a first 3’ splice site, and a second 3’ splice site. In some embodiments, a first 3’ splice site is a duplicate of the second 3’ splice site. In some embodiments, the first 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, the second 3’ splice site is a rabbit beta globin 3’ splice site. [0050] In some embodiments, the excisable element comprises a recombination site. In some embodiments, the recombination site is a lox site or FRT site. In some embodiments, the lox site is a loxP site. [0051] In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first recombination site; c) the first 3’ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3’ splice site. [0052] In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3’ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3’ splice site. In some embodiments, the first spacer sequence comprises an intron. In some embodiments, the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the third spacer segment comprises an intron. In some embodiments, the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery. In some embodiments, the second spacer segment comprises an exon. In some embodiments, the second spacer segment further comprises a polyA sequence. In some embodiments, the polyA sequence is 3’ of the exon. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. [0053] In some embodiments, the second spacer segment comprises from 5’ to 3’: a) a first recombination site; b) the first 3’ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the stop signaling sequence is a termination codon of the exon or a polyA sequence. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. In some embodiments, the exon encodes a detectable marker or a selectable marker. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. [0054] In some embodiments, the second spacer segment is excisable by a recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. [0055] In some embodiments, the Rep gene codes for Rep polypeptides. In some embodiments, the Cap gene codes for Cap polypeptides. In some embodiments, transcription of the Rep gene and the Cap gene are driven by native promoters. In some embodiments, the native promoters comprise P5, P19, and P40. [0056] In some embodiments, the Rep proteins are wildtype Rep polypeptides. In some embodiments, the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40. In some embodiments, a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase. [0057] In some embodiments, the Cap polypeptides are wildtype Cap polypeptides. In some embodiments, the Cap polypeptides are AAV capsid proteins. In some embodiments, the AAV capsid proteins comprise VP1, VP2, and VP3. In some embodiments, a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68. [0058] In some embodiments, the first polynucleotide construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is DHFR, GS, TYMS, or PAH. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the auxotrophic selection element or split intein linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the active protein or split intein linked to a C-terminus of the active protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the auxotrophic selection element or leucine zipper linked to a C-terminus of the auxotrophic selection element. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the active protein or leucine zipper linked to a C-terminus of the active protein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. [0059] In some embodiments, the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138. In some embodiments, the first polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, but wherein these sequences lack SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. [0060] In some embodiments, the second polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the second polynucleotide construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 148. [0061] In some embodiments, the third polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the third polynucleotide construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138.In some embodiments, the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are stably integrated in the cell’s genome. [0062] In some embodiments, the cell further comprises a payload construct, wherein the payload construct is a polynucleotide coding for a payload. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter. In some embodiments, the constitutive promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146. [0063] In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. [0064] In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell. [0065] In some embodiments, a plurality of the payload construct is stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs is separately stably integrated into the genome of the cell. In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, the payload construct is integrated into the genome of the cell. In some embodiments, the selectable marker is a first part of a split blasticidin and wherein the first polynucleotide sequence encodes the second part of the split blasticidin. [0066] In some embodiments, a method for increasing production of rAAV virions from a cell, comprises amplifying expression of AAV Rep and capsid proteins, helper proteins, and/or payload in the cell, wherein the amplifying comprises: increasing copy number of a polynucleotide construct comprising a sequence encoding one or more AAV Rep proteins and a sequence encoding one or more AAV cap proteins, a polynucleotide construct comprising a sequence encoding the one or more AAV cap proteins, a polynucleotide construct comprising a sequence encoding one or more AAV helper proteins, and/or a polynucleotide construct comprising a sequence encoding the payload by introducing an agent to amplify expression of the Rep/Cap genes, helper genes, and/or payload. [0067] In some embodiments, the increasing copy number of the polynucleotide construct(s) comprises culturing the cell under conditions that select for the presence of the selectable marker encoded by the polynucleotide construct(s) under the control of an attenuated promoter, thereby producing the cell comprising an increased copy number of the polynucleotide construct(s) compared to a cell comprising the polynucleotide construct comprising a selectable marker operably linked to a nonattenuated promoter. In some embodiments, the attenuated promoter is an attenuated EF1alpha promoter and the nonattenuated promoter is an EF1alpha promoter; optionally, wherein the attenuated EF1alpha promoter is SEQ ID NO: 132 and the EF1alpha promoter is SEQ ID NO: 133. [0068] In some embodiments, the polynucleotide construct further comprises a mutated selectable marker having decreased enzymatic activity compared to an unmutated selectable marker. In some embodiments, the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the mutated selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the unmutated selectable marker. In some embodiments, the mutated selectable marker is a mutated GS and the unmutated selectable marker is GS; optionally, wherein the mutated GS having a R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112 and the GS is SEQ ID NO: 112; optionally, wherein the mutated GS is SEQ ID NO: 142, SEQ ID NO: 143, or SEQ ID NO: 144. In some embodiments, the polynucleotide construct further comprises a selectable marker. In some embodiments, the increasing copy number of the polynucleotide construct comprises culturing the cell under conditions that select for the presence of the selectable marker and in the presence of an inhibitor of the selectable marker, thereby producing the cell comprising an increased copy number of the polynucleotide construct compared to the polynucleotide construct further comprising the selectable marker cultured in the absence of the inhibitor of the selectable marker. In some embodiments, the polynucleotide construct is any polynucleotide construct as described herein. [0069] Also provided herein are methods of producing a stable cell line comprising expanding a cell described above. [0070] Also provided herein are methods of producing a plurality of rAAV virion comprising culturing a cell described above in the presence of a first triggering agent and a second triggering agent. In some embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. [0071] In some embodiments, the plurality of rAAV virion has an encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion has a F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the plurality of rAAV virion have a concentration of greater than 1 × 1011 or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013 or 1 × 1014 viral genomes per milliliter prior to purification. In some embodiments, the plurality of rAAV virion have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1 × 105 vg/target cell or less. In some embodiments, the culturing is in a bioreactor. [0072] Also provided herein are pharmaceutical compositions comprising the rAAV virion produced by the cell or the method described above and a pharmaceutically acceptable carrier. Also provided herein are methods of treating a condition or disorder, the method comprising administering a therapeutically effective amount of the pharmaceutical composition to a patient in need thereof. BRIEF DESCRIPTION OF THE DRAWINGS [0073] FIG.1A depicts an exemplary system of polynucleotides for inducibly producing rAAV. In absence of first and second triggering agents, the system is in an off state. This system includes Construct 1, Construct 3, Construct 4, and Construct 2, and is referred to as v1.2 system. The v1.2 system is distinct from the v1.0 system in that the v1.2 system comprises a recombinant nucleic acid that expresses Cap protein driven by an inducible promoter (see, e.g., Construct 2). In this schematic, which depicts an exemplary v1.2 system, Construct 1 and Construct 2 are two separate polynucleotides which are configured for inducibly expressing AAV capsid proteins. Construct 1, in addition to inducibly expressing AAV Cap proteins also inducibly expresses full-length AAV Rep proteins, and both AAV Cap proteins and AAV Rep proteins are driven by their native promoters (p40, and p5 and p19, respectively). [0074] FIG.1B depicts the post-triggered state (also referred to as the on state) of Constructs 1- 4 shown in FIG.1A following the addition of the first triggering agent, doxycycline, and the second triggering agent, tamoxifen. The Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen. Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG.1B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep and cap coding sequences expression under control of native promoters. rAAV virions encapsidating the payload, such as a GOI, are therefore produced. [0075] FIG.2A depicts an exemplary system of polynucleotides for inducibly producing rAAV, which is also an exemplary v1.2 system. In absence of a first triggering agent, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen, the system is in an off state. In Construct 1 of this schematic, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet-On promoter). This is different from the v1.0 system in which the AAV Cap proteins are expressed under the control of a native AAV promoter. In this v1.2 system, the coding sequences for the Rep and Cap proteins are oriented in opposite directions such that the internal p40 promoter in Rep coding sequence which controls expression of the Cap proteins is spatially separated from the Cap coding sequence and does not control Cap proteins expression. The inducible promoter (for example, Tet-On promoter) controlling the expression of the Cap proteins is stronger than the native p40 promoter, resulting in increased Cap proteins expression as compared to the v1.0 system. The coding sequences and promoters for the Rep proteins are separated from the coding sequence and the inducible promoter for the Cap proteins by a transcription blocking element (TBE). This construct may also be referred to as a Rep-Cap construct. [0076] FIG.2B depicts the on state of the system of polynucleotides for inducibly producing rAAV depicted in FIG.2A, after induction by a first triggering agent, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen. The Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen. Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed and Cap proteins are expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG.2B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep coding sequences expression under control of native promoters. rAAV virions encapsidating the payload, such as a GOI, are therefore produced. [0077] FIG.3A shows an exemplary system of polynucleotides for inducibly producing rAAV, which is also an exemplary v1.2 system. In absence of a first triggering agent and a second triggering agent, the system is in an off state. In Construct 1 of this schematic, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet- On promoter). The coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE). This system includes an additional polynucleotide, depicted as Construct 4, for expressing AAV Cap proteins driven by an inducible promoter. Construct 4 may be referred to as a Cap construct and is the same as Construct 2 shown in FIGs.1A and 1B. [0078] FIG.3B depicts the on state of the system of polynucleotides for inducibly producing rAAV depicted in FIG.3A, after induction by the agents, e.g., doxycycline, and a second triggering agent, e.g., tamoxifen. The Cre coding element is positioned between LoxP sites and is additionally fused to estrogen response elements (“ER2”), which allows for control over the localization of Cre in response to estrogen agonists, such as tamoxifen. Upon addition of a first triggering agent, e.g., doxycycline, Cre is expressed and Cap proteins are expressed, and upon addition of the second triggering agent, e.g., tamoxifen, Cre translocates to the nucleus. Following translation and translocation of the Cre protein into the cell nucleus, the Cre protein effects excision of its own coding sequence, leaving the integrated constructs as shown in FIG. 2B. Therefore, adenoviral E2A and E4 helper proteins are expressed. Cre also excises out the excisable element flanked by LoxP sites in Construct 1, allowing AAV rep and cap coding sequences expression under control of native promoters. rAAV virions encapsidating the payload, such as a GOI, are therefore produced. [0079] FIG.4 shows an exemplary method for generating a pool of P2 cell clone that includes Construct 3 configured to express Adenovirus helper proteins, Construct 1 configured to express AAV Rep and Cap proteins and Construct 4 to express a payload (GOI, e.g., GFP or progranulin). Constructs 1, 3, and 4 are those depicted in FIG.1A. [0080] FIG.5 shows an exemplary method for generating a P3 cell pool from the P2 cell pool by introducing a polynucleotide (e.g., Construct 2, as depicted in FIG.1A) that encodes for AAV Cap proteins “T231 (P3)” into the P2 polyclonal cell pool of FIG.4. A polynucleotide that does not encode for AAV Cap proteins is used as a control to generate “T232 (P3)”. [0081] FIG.6 provides data for the titer of AAV Capsid proteins (Capsid Titer) and encapsidated payload, progranulin (PGRN), (PGRN titer) produced in T231 (P3) and T232 (P3) cells. Also shown is a table depicting percent packaging of the gene encoding PGRN for T231 (P3) cells and T232 (P3) cells. [0082] FIG.7 depicts an exemplary v1.2 system of polynucleotide Constructs 1, 2, 3, and 4 that includes a first polynucleotide for encoding AAV Cap proteins under the control of the native promoter, p40, (see Construct 1) and a second polynucleotide for encoding AAV Cap proteins (Construct 2) under the control of an inducible promoter to achieve an overall increase in AAV Cap proteins expression as compared to a system of constructs that includes only the first polynucleotide for encoding AAV Cap proteins under the control of the native promoter, p40 (e.g., only Construct 1 and not including Construct 2). [0083] FIG.8 provides a schematic summarizing induction of cells that include the v1.2 system of polynucleotides “v1.2 cells” and characterization of the Capsid levels, excision kinetics for production of AAV Rep and Cap, GOI levels, and levels of encapsidated GOI. [0084] FIG.9 provides a timeline from the point of induction of cells with addition of tamoxifen and doxycycline (T=0) through 96 hours post-induction (T=96 hours). Expression of a protein marker, blue fluorescent protein, encoded by Construct 1 prior to the induction in v1.2 cells (T231) is measured and compared to a single cell clone generated after introduction and selection for integration of Construct 3 into viral producing cells and subsequent selection for that clone (T177CL9). Presence of the additional polynucleotide Construct 2 does not impact excision of the gene encoding BFP such that AAV Rep and Cap proteins can be expressed. [0085] FIG.10 provides data for characterization of rAAV produced by v1.2 cells and v1.0 cells. The ratio of “encapsidated GOI/total GOI” in v1.2 cells was measured and compared to the ratio of “encapsidated GOI/total GOI” in v1.0 cells. Fig.10 shows that in v1.0 cells, the production of genome of interest (GOI) is higher than Capsid production, indicating that capsid production could be a rate limiting step for producing encapsidated GOI (e.g., a payload). Increased capsid production in the V1.2 cells resulted in increased generation of rAAV. [0086] FIG.11 shows a time course of production of viral particles over a 7-day period after induction of v1.2 (pool) and v1.0 (clone) cells with tamoxifen and doxycycline. v1.2 cells produce viral particles that encapsidate a gene for expressing PRGN while v1.0 cells produce viral particles that encapsidate a gene for expressing green fluorescent protein (GFP). In FIG.11, the expression of capsids was measured (vp/ml). [0087] FIG.12 shows a time course of production of viral particles over a 7-day period after induction of v1.2 (pool) and v1.0 (clone) cells with tamoxifen and doxycycline. v1.2 cells produce viral particles that encapsidate a gene for expressing PGRN while v1.0 cells produce viral particles that encapsidate a gene for expressing green fluorescent protein (GFP). In FIG. 12, encapsidated GOI (either the gene for expressing PGRN or the gene for expressing GFP) was measured (vg/ml). [0088] FIG.13 shows high titer produced after induction of v1.2 cells from the T231 stable cell pool wherein the payload is progranulin (“v1.2 Pool (PGRN)”). The graph shows the capsid titer (vp/ml) from cell lysate produced after induction of v1.2 Pool (PGRN) cells compared to after induction of a v1.0 stable cell line pool wherein the payload is progranulin (“v1.0 Pool (PGRN)”), compared to after induction of a v1.0 stable cell line pool wherein the payload is eGFP (“v1.2 Pool (eGFP)”), and compared to after transient transfection of cells wherein the payload is eGFP (“Transient Transfection (eGFP)”). The v1.2 Pool (PGRN) cells show a greater than 10-fold improvement in viral titer over Transient Transfection (eGFP) titer and improvement in viral titer over both v1.0 Pool (PGRN) titer and v1.0 Pool (eGFP) titer. [0089] FIG.14A shows high capsid titer produced after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)). The v1.2 Pool (PGRN) capsid titer and v1.2 Clones (PGRN) capsid titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) capsid titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) capsid titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) capsid titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) capsid titer, indicating the v1.2 stable cells have successfully increased capsid titer >2 log. [0090] FIG.14B shows high vector genome production after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)). The v1.2 Pool (PGRN) vector genome titer and v1.2 Clones (PGRN) vector genome titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) vector genome titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) vector genome titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) vector genome titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) vector genome titer, indicating the v1.2 stable cells have successfully increased vector genome titer. [0091] FIG.15 shows the parent cell line is T205 CL23 (Top clone from stable cells v1.0 pool, in which the payload is progranulin). T318 was generated from T205 CL23 clone that has integrated the sequence for Tet inducible capsid (Construct 2 of FIG.1A) and selected based on hygromycin-resistance to make a v1.2 pool. Selecting a top clone with v1.0 system integrated in the genome and introducing Construct 2 into the top clone provides for increased vector genome titer and capsid titer as compared to introducing Construct 2 into a pool of clones with v1.0 system integrated in the genome. [0092] FIG.16 depicts an exemplary v1.0 system in the pre-triggered state. Cap proteins are expressed under the control of a native AAV p40 promoter. The brackets in construct 3 indicate the position of the flanking ITRs. V1.0 system is described in U.S. Patent Application Publication No.2022/0145328 which is incorporated herein. V1.0 cells are a pool of cells or a cell line that includes the constructs of the V1.0 system. DETAILED DESCRIPTION [0093] To solve the problems associated with low rAAV titers, disclosed herein are polynucleotide constructs and cells, such as, cell lines stably integrated with the polynucleotide constructs that enable increased production of recombinant AAV (rAAV) virions. The increased production is hypothesized to be achieved by providing an overall increase in the levels of capsid proteins which in turn increases production of rAAV. In one embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins In one embodiment, the increase in levels of capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins. In a further embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In another embodiment, the increase in levels of capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of a native promoter and another under the control of an inducible promoter. One or more of these embodiments can also be combined to increase capsid proteins expression and enable increased production of recombinant AAV (rAAV). [0094] In certain embodiments, the polynucleotide constructs may be stably integrated into the nuclear genome of the cells and may express components for formation of rAAV in an inducible manner thereby avoiding the toxicity of AAV Rep protein when constitutively expressed. 1.1. Definitions [0095] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which the invention pertains. [0096] The term "about", particularly in reference to a given quantity, is meant to encompass deviations of up to plus or minus five percent. [0097] "AAV" is an abbreviation for adeno-associated virus, and may be used to refer to the virus itself or derivatives thereof. The term covers all subtypes and both naturally occurring and recombinant forms, except where required otherwise. The components of the AAV DNA genome consist of two open reading frames, Rep and Cap, flanked by two 145 base inverted terminal repeats (ITRs). Rep gene encodes multiple distinct proteins including Rep78, Rep68, Rep52, and Rep40. These proteins are also referred to herein as Rep proteins or Rep and may encompass one or more of Rep78, Rep68, Rep52, and Rep40 and functional variants thereof and homologs thereof. Rep78 and Rep68 and functional variants thereof and homologs thereof are referred to herein as large Rep. Rep52, and Rep40 and functional variants thereof and homologs thereof are referred to herein as small Rep. Rep proteins from an AAV of a particular serotype may also be referred to as Rep1, Rep2, etc. where the Rep protein is derived from an AAV1 or an AAV2 serotype, respectively. Cap gene encodes capsid proteins VP1, VP2, and VP3 required for production of rAAV capsids. These proteins are also referred to herein as Cap proteins or Cap and may encompass one or more of VP1, VP2, and VP3 and functional variants thereof and homologs thereof. Cap proteins from an AAV of a particular serotype may also be referred to as Cap1, Cap2, Cap4, etc. where the Rep protein is derived from an AA1, an AAV2, or an AAV5 serotype, respectively. In addition to Rep and Cap, AAV requires a helper plasmid containing genes from a helper virus such as adenovirus, including E1a, E1b, E4, E2a, and VA genes for AAV replication. [0098] "Recombinant", as applied to an AAV virion, means that the rAAV virion (synonymously, rAAV virus particle) is the product of one or more procedures that result in an AAV particle Construct that is distinct from an AAV virion in nature. The procedure may be genetic alteration, e.g., by the addition or insertion of a heterologous nucleic acid Construct into the virus. [0099] "Recombinant virus" is meant to describe a virus that has been genetically altered, e.g., by the addition or insertion of a heterologous nucleic acid construct into the virus. [0100] The abbreviation "rAAV" refers to recombinant adeno-associated virus, also referred to as a recombinant AAV vector (or "rAAV vector"). The term “AAV” includes any AAV serotype as well as AAV vectors based on the combination of different serotypes (also referred to as "hybrid AAV vectors" or "pseudotype AAV vectors"). AAV serotype may be AAV type 1 (AAV-1), AAV type 2 (AAV-2), AAV type 3 (AAV-3), AAV type 4 (AAV-4), AAV type 5 (AAV-5), AAV type 6 (AAV-6), AAV type 7 (AAV-7), AAV type 8 (AAV-8), AAV type 9 (AAV-9), AAV type 10 (AAV-10), AAV type 11 (AAV-11), avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, ovine AAV, AAV-7m8, AAV-6.2, AAV- Dj, AAV-DJ/8, AAV2-retro, AAV2-QuadYF and AAV2.7m8, AAV-PHP.B, AAV-PHP.B2, AAV-PHP.B3, AAV-PHP.A, AAV-PHP.eB, AAV-PHP.eS, evolved capsids that are less immunogenic to mice and humans, and variants thereof and combinations thereof. “Primate AAV” refers to AAV isolated from a primate, “non-primate AAV” refers to AAV isolated from a non-primate mammal, “bovine AAV” refers to AAV isolated from a bovine mammal (e.g., a cow), etc. An "rAAV vector" comprises a polynucleotide sequence not of AAV origin (i.e., a polynucleotide heterologous to AAV), typically a polynucleotide sequence of interest for introducing into a target cell. In general, the heterologous polynucleotide is flanked by at least one, and usually by two AAV inverted terminal repeat sequences (ITRs). The heterologous polynucleotide can also be referred to as a polynucleotide payload. The term rAAV vector encompasses both rAAV virions and rAAV vector plasmids. [0101] An "AAV virus" or "AAV viral particle" or "rAAV vector particle" refers to a viral particle composed of at least one AAV capsid protein (typically by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide rAAV vector. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome, such as a transgene to be delivered to a mammalian cell), it is typically referred to as an "rAAV vector particle" or simply an "rAAV vector". Thus, production of a rAAV particle necessarily includes production of a rAAV vector, as such a vector contained within an rAAV particle. [0102] "Packaging" refers to a series of intracellular events that result in the assembly, encapsidation, and production of an AAV particle. [0103] AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and capsid proteins of adeno-associated virus. AAV rep and cap are referred to herein as AAV "packaging genes." [0104] By "AAV Rep coding region" or “sequence encoding one or more Rep proteins” or “Rep encoding sequence” and grammatical equivalents thereof is meant the art-recognized region of the AAV genome which encodes the replication proteins of the virus which are required to replicate the viral genome and/or a payload flanked by ITRs. The rep coding region, as used herein, may be derived from any viral serotype, such as those described above. The region need not include all of the wild-type genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the rep genes provide for expression Rep proteins. Rep coding sequences are further described below. The terms “AAV Rep coding sequence” and “AAV Rep proteins coding sequence” are used interchangeably herein. The terms “AAV Rep proteins”, “Rep proteins”, “AAV Rep polypeptide”, and “Rep polypeptide” are interchangeably used herein. [0105] By "AAV cap coding region" or “sequence encoding one or more cap proteins,” or “Cap encoding sequence” and grammatical equivalents thereof it is meant the art-recognized region of the AAV genome which encodes the coat proteins of the virus which are required for the capsid that viral genome or a payload is packaged into by the Rep proteins. For a further description of the cap coding region, see, e.g., Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol.158, 97-129; Kotin, R. M. (1994) Human Gene Therapy 5, 793-801. The AAV cap coding region, as used herein, may be derived from any AAV serotype, as described above. The region need not include all of the wild-type cap genes but may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the genes provide for sufficient packaging functions. Cap coding sequences are further described below. The term “AAV Cap coding sequence” and “AAV Cap proteins coding sequence” are interchangeably used herein. The terms “AAV Cap proteins”, “Cap proteins”, “AAV Cap polypeptide”, and “Cap polypeptide” are interchangeably used herein. The term “Capsid” and “Cap” are interchangeably used herein. [0106] By "adeno-associated virus inverted terminal repeats" or "AAV ITRs" is meant the art-recognized regions found at each end of the AAV genome which function together in cis as origins of DNA replication and as packaging signals for the viral genome. The nucleotide sequences of AAV ITR regions are known. See, e.g., Kotin, R. M. (1994) Human Gene Therapy 5, 793-801; Berns, K. I. "Parvoviridae and their Replication" in Fundamental Virology, 2d ed., (B. N. Fields and D. M. Knipe, eds.) for the AAV-2 ITRs sequence. As used herein, an "AAV ITR" need not have a wild-type nucleotide sequence, but may be altered, e.g., by the insertion, deletion or substitution of nucleotides. The AAV ITR may be derived from any of several AAV serotypes, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-7, etc. Furthermore, 5' and 3' ITRs which flank a selected nucleotide sequence in an AAV vector need not necessarily be identical or derived from the same AAV serotype or isolate. The ITRs may be single stranded (ssITRs) or self-complementary (scITRs). [0107] A "helper virus" for AAV refers to a virus that allows AAV (e.g., wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC. [0108] The term “adenoviral helper proteins” and “AAV helper proteins” are interchangeably used herein. [0109] "Helper virus function(s)" refers to function(s) encoded in a helper virus genome which allows AAV replication and packaging (in conjunction with other requirements for replication and packaging described herein). As described herein, "helper virus function" may be provided in a number of ways, including by providing helper virus or providing, for example, polynucleotide sequences encoding the requisite function(s) to a producer cell in trans. [0110] An "infectious" virus or viral particle is one that comprises a polynucleotide component which the particle is capable of delivering into a cell for which the viral species is tropic. The term does not necessarily imply any replication capacity of the virus. As used herein, an “infectious” virus or viral particle is one that may access a target cell, may infect a target cell, and may express a heterologous nucleic acid in a target cell. Thus, “infectivity” refers to the ability of a viral particle to access a target cell, infect a target cell, and express a heterologous nucleic acid in a target cell. Infectivity may refer to in vitro infectivity or in vivo infectivity. Assays for counting infectious viral particles are described elsewhere in this disclosure and in the art. Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Total viral particles may be expressed as the number of viral genome (vg) copies. The ability of a viral particle to express a heterologous nucleic acid in a cell may be referred to as “transduction.” The ability of a viral particle to express a heterologous nucleic acid in a cell may be assayed using a number of techniques, including assessment of a marker gene, such as a green fluorescent protein (GFP) assay (e.g., where the virus comprises a nucleotide sequence encoding GFP), where GFP is produced in a cell infected with the viral particle and is detected and/or measured; or the measurement of a produced protein, for example by an enzyme-linked immunosorbent assay (ELISA). Viral infectivity may be expressed as the ratio of infectious viral particles to total viral particles. Methods of determining the ratio of infectious viral particle to total viral particle are known in the art. See, e.g., Grainger et al. (2005) Mol. Ther.11:S337 (describing a TCID50 infectious titer assay); and Zolotukhin et al. (1999) Gene Ther.6:973. [0111] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, including deoxyribonucleotides or ribonucleotides, or analogs thereof. The terms “polynucleotide” and “nucleic acid” are interchangeably used herein. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The term polynucleotide, as used herein, refers interchangeably to double- and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention described herein that is a polynucleotide encompasses both the double-stranded form and each of two complementary single-stranded forms known or predicted to make up the double-stranded form. [0112] As used herein, the term “polynucleotide construct” refers to a DNA segment of any size that includes one or more sequences encoding an RNA or protein and at least one promoter for driving expression from the one or more sequences. The term “polynucleotide construct” and “nucleic acid construct” are interchangeably used herein. A polynucleotide construct may be a circular DNA or a linear DNA. A polynucleotide construct may be single stranded or double stranded. As used herein, the term "vector" includes any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, artificial chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which may transfer gene sequences into and between cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. The use of the term "vector" throughout this specification encompasses plasmid or viral vectors, which permit the desired components to be transferred to the host cell via transfection or infection. For example, an adeno-associated viral (AAV) vector is a plasmid comprising a recombinant AAV genome. In some embodiments, useful vectors are contemplated to be those vectors in which the nucleic acid segment to be transcribed is positioned under the transcriptional control of a promoter. A vector may be linear or circular, single stranded or double stranded, DNA or RNA. In certain aspects, the vector may be circular, double stranded DNA. [0113] As used herein, the term “vector system” refers to two or more vectors that are used together, e.g., by simultaneous or sequential introduction into a cell, to provide at least two different components into the cell. The two different components may then work together in the cell. [0114] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same when comparing the two sequences. The term percent “sequence identity,” in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the percent “sequence identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. [0115] For sequence comparison, typically one sequence acts as a reference sequence (also called the subject sequence) to which test sequences (also called query sequences) are compared. The percent sequence identity is defined as a test sequence’s percent identity to a reference sequence. For example, when stated “Sequence A having a sequence identity of 50% to Sequence B,” Sequence A is the test sequence and Sequence B is the reference sequence. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then aligns the sequences to achieve the maximum alignment, based on the designated program parameters, introducing gaps in the alignment if necessary. The percent sequence identity for the test sequence(s) relative to the reference sequence can then be determined from the alignment of the test sequence to the reference sequence. The equation for percent sequence identity from the aligned sequence is as follows: [0116] [(Number of Identical Positions)/(Total Number of Positions in the Test Sequence)] × 100% [0117] For purposes herein, percent identity and sequence similarity calculations are performed using the BLAST algorithm for sequence alignment, which is described in Altschul et al., J. Mol. Biol.215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov/). The BLAST algorithm uses a test sequence (also called a query sequence) and a reference sequence (also called a subject sequence) to search against, or in some cases, a database of multiple reference sequences to search against. The BLAST algorithm performs sequence alignment by finding high-scoring alignment regions between the test and the reference sequences by scoring alignment of short regions of the test sequence (termed “words”) to the reference sequence. The scoring of each alignment is determined by the BLAST algorithm and takes factors into account, such as the number of aligned positions, as well as whether introduction of gaps between the test and the reference sequences would improve the alignment. The alignment scores for nucleic acids can be scored by set match/mismatch scores. For protein sequences, the alignment scores can be scored using a substitution matrix to evaluate the significance of the sequence alignment, for example, the similarity between aligned amino acids based on their evolutionary probability of substitution. For purposes herein, the substitution matrix used is the BLOSUM62 matrix. For purposes herein, the public default values of April 6, 2023 are used when using the BLASTN and BLASTP algorithms. The BLASTN and BLASTP algorithms then output a “Percent Identity” output value and a “Query Coverage” output value. The overall percent sequence identity as used herein can then be calculated from the BLASTN or BLASTP output values as follows: [0118] Percent Sequence Identity = (“Percent Identity” output value) × (“Query Coverage” output value) [0119] The following non-limiting examples illustrate the calculation of percent identity between two nucleic acids sequences. The percent identity is calculated as follows: [(number of identical nucleotide positions)/(total number of nucleotides in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 1: AAAAAGGGGG (length = 10 nucleotides) to reference sequence 2: AAAAAAAAAA (length = 10 nucleotides). The percent identity between test sequence 1 and reference sequence 2 would be [(5)/(10)] ×100% = 50%. Test sequence 1 has 50% sequence identity to reference sequence 2. In another example, percent identity is calculated to compare test sequence 3: CCCCCGGGGGGGGGGCCCCC (length = 20 nucleotides) to reference sequence 4: GGGGGGGGGG (length = 10 nucleotides). The percent identity between test sequence 3 and reference sequence 4 would be [(10)/(20)] ×100% = 50%. Test sequence 3 has 50% sequence identity to reference sequence 4. In another example, percent identity is calculated to compare test sequence 5: GGGGGGGGGG (length = 10 nucleotides) to reference sequence 6: CCCCCGGGGGGGGGGCCCCC (length = 20 nucleotides). The percent identity between test sequence 5 and reference sequence 6 would be [(10)/(10)] ×100% = 100%. Test sequence 5 has 100% sequence identity to reference sequence 6. [0120] The following non-limiting examples illustrate the calculation of percent identity between two protein sequences. The percent identity is calculated as follows: [(number of identical amino acid positions)/(total number of amino acids in the test sequence)] × 100%. Percent identity is calculated to compare test sequence 7: FFFFFYYYYY (length = 10 amino acids) to reference sequence 8: YYYYYYYYYY (length = 10 amino acids). The percent identity between test sequence 7 and reference sequence 8 would be [(5)/(10)] ×100% = 50%. Test sequence 7 has 50% sequence identity to reference sequence 8. In another example, percent identity is calculated to compare test sequence 9: LLLLLFFFFFYYYYYLLLLL (length = 20 amino acids) to reference sequence 10: FFFFFYYYYY (length = 10 amino acids). The percent identity between test sequence 9 and reference sequence 10 would be [(10)/(20)] ×100% = 50%. Test sequence 9 has 50% sequence identity to reference sequence 10. In another example, percent identity is calculated to compare test sequence 11: FFFFFYYYYY (length = 10 amino acids) to reference sequence 12: LLLLLFFFFFYYYYYLLLLL (length = 20 amino acids). The percent identity between test sequence 11 and reference sequence 12 would be [(10)/(10)] ×100% = 100%. Test sequence 11 has 100% sequence identity to reference sequence 12. [0121] For purposes herein, reference to a polynucleotide sequence (e.g., a DNA sequence or an RNA sequence) also encompasses the reverse complement of the polynucleotide sequence. For example, a sequence of AAAAAGGGGG also encompasses a sequence of CCCCCTTTTT. [0122] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. [0123] The term "host cell" denotes, for example, microorganisms, yeast cells, insect cells, and mammalian cells, that may be, or have been, used as recipients of an AAV vector system as described herein, or other transfer DNA. The term includes the progeny of the original cell which has been transfected. Thus, a "host cell" as used herein generally refers to a cell which has been transfected with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation. In some aspects, the disclosure provides transfected host cells. [0124] The term "transfection" is used to refer to the uptake of foreign DNA by a cell, and a cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (1989) Molecular Cloning, a laboratory manual, Cold Spring Harbor Laboratories, New York, Davis et al. (1986) Basic Methods in Molecular Biology, Elsevier, and Chu et al. (1981) Gene 13:197. Such techniques can be used to introduce one or more exogenous nucleic acids, such as a nucleotide integration vector and other nucleic acid molecules, into suitable host cells. [0125] As used herein, the term "cell line" refers to a population of cells capable of continuous or prolonged growth and division in vitro. Often, cell lines are clonal populations derived from a single progenitor cell. It is further known in the art that spontaneous or induced changes may occur in karyotype during storage or transfer of such clonal populations. Therefore, cells derived from the cell line referred to may not be precisely identical to the ancestral cells or cultures, and the cell line referred to includes such variants. [0126] The term "cell culture," refers to cells grown adherent or in suspension, bioreactors, roller bottles, hyperstacks, microspheres, macrospheres, flasks and the like, as well as the components of the supernatant or suspension itself, including but not limited to rAAV particles, cells, cell debris, cellular contaminants, colloidal particles, biomolecules, host cell proteins, nucleic acids, and lipids, and flocculants. Large scale approaches, such as bioreactors, including suspension cultures and adherent cells growing attached to microcarriers or macrocarriers in stirred bioreactors, are also encompassed by the term "cell culture." Cell culture procedures for both large and small-scale production of proteins are encompassed by the present disclosure. [0127] As used herein, the terms "recombinant cell" refers to a cell into which an exogenous DNA segment, such as DNA segment that leads to the transcription of a biologically-active polypeptide or production of a biologically active nucleic acid such as an RNA, has been introduced. [0128] As used herein, the term “intermediate cell line” refers to a cell line that contains the AAV rep and cap components integrated into the host cell genome or a cell line that contains the adenoviral helper functions integrated into the host cell genome. [0129] As used herein, the term “packaging cell line” refers to a cell line that contains the AAV rep and cap components and the adenoviral helper functions integrated into the host cell genome. A payload construct must be added to the packaging cell line to generate rAAV virions. [0130] As used herein, the term “production cell line” refers to a cell line that contains the AAV rep and cap components, the adenoviral helper functions, and a payload construct. The rep and cap components and the adenoviral helper functions are integrated into the host cell genome. The payload construct can be stably integrated into the host cell genome or transiently transfected. rAAV virions can be generated from the production cell line upon the introduction of one or more triggering agents in the absence of any plasmid or transfection agent. [0131] As used herein, the term “downstream purification” refers to the process of separating rAAV virions from cellular and other impurities. Downstream purification processes include chromatography-based purification processes, such as ion exchange (IEX) chromatography and affinity chromatography. [0132] The term “prepurification yield” refers to the rAAV yield prior to the downstream purification processes. The term “postpurification yield” refers to the rAAV yield after the downstream purification processes. rAAV yield can be measured as viral genome (vg)/L. [0133] The encapsidation ratio of a population of rAAV virions can be measured as the ratio of rAAV viral particle (VP) to viral genome (VG). The rAAV viral particle includes empty capsids, partially full capsids (e.g., comprising a partial viral genome), and full capsids (e.g., comprising a full viral genome). [0134] The F:E ratio of a population of rAAV virions can be measured as the ratio of rAAV full capsids to empty capsids. The rAAV full capsid particle includes partially full capsids (e.g., comprising a partial viral genome) and full capsids (e.g., comprising a full viral genome). The empty capsids lack a viral genome. [0135] The potency or infectivity of a population of rAAV virions can be measured as the percentage of target cells infected by the rAAV virions at a multiplicity of infection (MOI; viral genomes/target cell). Exemplary MOI values are 1 × 101, 1 × 102, 2 × 103, 5 × 104, or 1 × 105 vg/target cell. An MOI can be a value chosen from the range of 1 × 101 to 1 × 105 vg/target cell. [0136] The term "expression vector or construct" or “synthetic construct” means any type of genetic construct containing a nucleic acid in which part or all of the nucleic acid encoding sequence is capable of being transcribed. In some embodiments, expression includes transcription of the nucleic acid, for example, to generate a biologically-active polypeptide product or functional RNA (e.g., guide RNA) from a transcribed gene. [0137] The term “auxotrophic” or “auxotrophic selectable marker” as used herein refers to the usage of a medium lacking a supplement, such as a medium lacking an essential nutrient such as the purine precursors hypoxanthine and thymidine (HT), or the like, for selection of a functional enzyme which allows for growth in the medium lacking the essential nutrient, e.g., a functional dihydrofolate reductase or the like. [0138] The terms “tetracycline” is used generically herein to refer to all antibiotics that are structurally and functionally related to tetracycline, including tetracycline, doxycycline, demeclocycline, minocycline, sarecycline, oxytetracycline, omadacycline, or eravacycline. [0139] The terms “constitutive” or “constitutive expression” are used interchangeably herein. They refer to genes that are transcribed in an ongoing manner. Such gene are driven by a constitutive promoter. In some embodiments, the terms refer to the expression of a therapeutic payload or a nucleic acid sequence that is not conditioned on addition of an triggering agent to the cell culture medium. A constitutive promoter is capable of directing continuous gene expression in a cell. Constitutive promoters regulate expression of basal genes, like housekeeping genes. In contrast, an inducible promoter directs gene expression in the presence of an external stimulus. Thus, an inducible promoter can be controlled by providing or withdrawing the stimulus. [0140] As used herein, the term “polynucleotide payload” refers to a polynucleotide sequence that is packaged into a rAAV virion for delivery by the rAAV virion into a cell. A polynucleotide payload is flanked by AAV inverted terminal repeats (ITRs). Upon delivery to a cell, the polynucleotide payload may be available to the cell as a DNA (e.g., a homology region for homology-directed repair), transcribed into an RNA (e.g., a guide RNA (gRNA), a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO)), or transcribed and translated into a polypeptide (e.g., an antibody, a hormone, a site-specific endonuclease, a reporter gene, a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, a ribozyme, or a DNAzyme. [0141] "Recombinant," as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction or ligation steps, and other procedures that result in a construct that is distinct from a polynucleotide found in nature. A recombinant virus is a viral particle comprising a recombinant polynucleotide. The terms respectively include replicates of the original polynucleotide construct and progeny of the original virus construct. [0142] A "control element" or "control sequence" is a nucleotide sequence involved in an interaction of molecules that contributes to the functional regulation of a polynucleotide, including replication, duplication, transcription, splicing, translation, or degradation of the polynucleotide. The regulation may affect the frequency, speed, or specificity of the process, and may be enhancing or inhibitory in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable under certain conditions of binding RNA polymerase and initiating transcription of a coding region usually located downstream (in the 3' direction) from the promoter. A promoter is usually upstream of a gene whose expression is controlled by the promoter. [0143] "Operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained. [0144] "Heterologous" means derived from a genotypically distinct entity from that of the rest of the entity to which it is being compared. For example, a polynucleotide introduced by genetic engineering techniques into a plasmid or vector derived from a different species is a heterologous polynucleotide. Additional sequences or sequence motifs operably linked to a sequence where it is not naturally found are also heterologous; such sequences or sequence motifs include polyA signal sequences, introns, and/or any other relevant sequence. Thus, for example, an rAAV that includes a heterologous nucleic acid encoding a heterologous payload is an rAAV that includes a nucleic acid not normally included in a naturally occurring, wild-type AAV, and the encoded heterologous payload is a payload not normally encoded by a naturally- occurring, wild-type AAV. As another example, a Capsid proteins coding sequence operably linked to a heterologous polyA signal sequence refers to a AAV Cap coding sequence operably linked to a sequence not native to AAV. [0145] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. For example, a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro. A gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell. [0146] A cell is said to be "stably" altered, transduced, genetically modified, or transformed with a genetic sequence if the sequence is available to perform its function during extended culture of the cell in vitro. Generally, such a cell is "heritably" altered (genetically modified) in that a genetic alteration is introduced which is also inheritable by progeny of the altered cell. For example, a gene integrated into the nuclear genome of the cell and is available to perform its function during extended culture of the cell in vitro. A gene integrated into the nuclear genome of the cell is inheritable by progeny of the cell. [0147] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, phosphorylation, or conjugation with a labeling component. Polypeptides such as anti-angiogenic polypeptides, neuroprotective polypeptides, and the like, when discussed in the context of delivering a payload to a mammalian subject, and compositions therefor, refer to the respective intact polypeptide, or any fragment or genetically engineered derivative thereof, which retains the desired biochemical function of the intact protein. Similarly, references to nucleic acids encoding anti-angiogenic polypeptides, nucleic acids encoding neuroprotective polypeptides, and other such nucleic acids for use in delivery of a payload to a mammalian subject (which may be referred to as "transgenes" to be delivered to a recipient cell), include polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative possessing the desired biochemical function. [0148] An "isolated" plasmid, nucleic acid, vector, virus, virion, host cell, or other substance refers to a preparation of the substance devoid of at least some of the other components that may also be present where the substance or a similar substance naturally occurs or is initially prepared from. Thus, for example, an isolated substance may be prepared by using a purification technique to enrich it from a source mixture. Enrichment may be measured on an absolute basis, such as weight per volume of solution, or it may be measured in relation to a second, potentially interfering substance present in the source mixture. Increasing enrichments of the embodiments of this invention are increasingly more isolated. An isolated plasmid, nucleic acid, vector, virus, host cell, or other substance is in some cases purified, e.g., from about 80% to about 90% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, or at least about 99%, or more, pure. [0149] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom(s) but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting development of a disease and/or the associated symptoms; or (c) relieving the disease and the associated symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment may include those already afflicted (e.g., those with a neurological disorder) as well as those in which prevention is desired (e.g., those with increased susceptibility to a neurological disorder; those suspected of having a neurological disorder; those having one or more risk factors for a neurological disorder, etc.). [0150] A "therapeutically effective amount" or "efficacious amount" means the amount of a compound that, when administered to a mammal or other subject for treating a disease, is sufficient, in combination with another agent, or alone in one or more doses, to effect such treatment for the disease. The "therapeutically effective amount" will vary depending on the compound, the disease and its severity and the age, weight, etc., of the subject to be treated. [0151] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses, camels, etc.); mammalian farm animals (e.g., sheep, goats, cows, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.). In some cases, the individual is a human. [0152] The terms “v1.1 system” and “v1.2” system are interchangeable and are used herein to refer to the same version of system of polynucleotides. [0153] The terms "hybridize" and "hybridization" refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson Crick base pairing. [0154] The term "homologous region" refers to a region of a nucleic acid with homology to another nucleic acid region. Thus, whether a "homologous region" is present in a nucleic acid molecule is determined with reference to another nucleic acid region in the same or a different molecule. Further, since a nucleic acid is often double-stranded, the term "homologous, region," as used herein, refers to the ability of nucleic acid molecules to hybridize to each other. For example, a single-stranded nucleic acid molecule may have two homologous regions which are capable of hybridizing to each other. Thus, the term "homologous region" includes nucleic acid segments with complementary sequences. Homologous regions may vary in length but will typically be between 4 and 500 nucleotides (e.g., from about 4 to about 40, from about 40 to about 80, from about 80 to about 120, from about 120 to about 160, from about 160 to about 200, from about 200 to about 240, from about 240 to about 280, from about 280 to about 320, from about 320 to about 360, from about 360 to about 400, from about 400 to about 440, etc.). [0155] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands may base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between an RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectly complementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non-complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. As used herein, the term "recombination site" denotes a region of a nucleic acid molecule comprising a binding site or sequence-specific motif recognized by a site-specific recombinase that binds at the target site and catalyzes recombination of specific sequences of DNA at the target site. Site-specific recombinases catalyze recombination between two such target sites. The relative orientation of the target sites determines the outcome of recombination. For example, translocation occurs if the recombination sites are on separate DNA molecules. DNA between two recombination sites oriented in the same direction on the same DNA molecule will be excised as a circular loop of DNA. DNA between two recombination sites that are orientated in the opposite direction on the same DNA molecule will be inverted. [0156] The terms "v1.1 system" and "v1.2 system” are interchangeable and are used herein to refer to the same version of system of polynucleotides. 1.2. Overview [0157] To solve the problems associated with low rAAV titers, disclosed herein are polynucleotide constructs and cells, such as, cell lines stably integrated with the polynucleotide constructs that enable increased production of recombinant AAV (rAAV) virions. FIG.10 shows that in a v1.0 system, the production of payload sequences is higher than capsid production, indicating that capsid production could be a rate limiting step for producing encapsidated payloads, i.e., rAAV. FIG.10 demonstrates that increased encaspidated payload titer, i.e., increased rAAV titer was acheived by increasing total capsid production. Thus, increased production of rAAV is achieved by providing an overall increase in the levels of capsid proteins which in turn increases production of rAAV. Disclosed herein are various methods and constructs for increasing capsid production and therefore also increasing the titer of encapsidated genomes. In one embodiment, the increase in levels of capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3' to the sequence encoding the capsid proteins. In another embodiment, the increase in levels of capsid proteins is achieved by using an inducible promoter to drive expression of capsid proteins. In a further embodiment, the increase in levels of AAV capsid proteins is achieved by including a strong polyadenylation (polyA) signal sequence 3’ to the sequence encoding the AAV capsid proteins and by using an inducible promoter to drive expression of capsid proteins which inducible promoter is stronger than the native promoter controlling expression of AAV capsid proteins. In another embodiment, the increase in levels of capsid proteins is achieved by using two separate polynucleotide constructs each encoding AAV capsid proteins, one under the control of a native promoter and another under the control of an inducible promoter. One or more of these embodiments can also be combined to increase capsid proteins expression and enable increased production of recombinant AAV (rAAV). These embodiments are further described in detail below. 1.3. Polynucleotides Encoding AAV Capsid Proteins AAV Cap Encoding Sequence linked to PolyA Signal Sequence [0158] In some aspects, polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence is 3' of the sequence encoding AAV Cap proteins are provided. In certain embodiments, the polynucleotides comprise (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3' of the sequence encoding AAV Cap proteins. Suitable polyadenylation signals are further described below in the section “Polyadenylation Signals”. Polyadenylation Signals [0159] Polyadenylation (polyA) signal sequences generally include a short sequence that triggers polyadenylation of an mRNA. In certain instances, RNA stability, expression, and/or function can be enhanced with additional sequences surrounding a shorter sequence. Various polyA signaling sequences can be used for the coding sequences of various embodiments. [0160] In some embodiments, the polyadenylation signal sequence is a SV40 polyadenylation signal sequence (SV40 polyA). In other embodiments, the polyadenylation signal sequence is a bovine growth hormone polyadenylation signal sequence (bGH polyA). In still other embodiments, the polyadenylation signal sequence is a Rabbit Beta Globin polyadenylation signal sequence. [0161] In some embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 (SV40 poly A). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 152 (SV40 poly A). In certain embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151 (bGH polyA). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 151 (bGH polyA). In some embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 170 (Rabbit Beta Globin PolyA). In certain embodiments, the polyadenylation signal sequence has sequence of SEQ ID NO: 170 (Rabbit Beta Globin PolyA). [0162] In some embodiments, the polyadenylation signal sequence is any polyadenylation signal sequence that encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. In some embodiments, the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162. In certain embodiments, the native AAV Cap polyadenylation signal sequence has sequence of SEQ ID NO: 162. [0163] In some embodiments, the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof. [0164] In some embodiments, the polynucleotide described herein is not flanked by inverted terminal repeat sequences. [0165] Additional details regarding polyadenylation signaling sequences are disclosed in one or more of: US Pat. Nos.11,793,180, 11,752,181, 10,912,826, 8,975,391, 7,557,197, and 5,122,458; US Pat. Pub. Nos.2023/0332169, 2023/0330265, and 2023/0323390; or Gene Volume 231, Issues 1–2, 29 April 1999, Pages 77-86, Mol Cell Biol.1989 Oct; 9(10): 4248– 4258, and Nucleic Acids Research, Volume 15, Issue 23, 10 December 1987, Pages 9627–9640; the disclosures of which are hereby incorporated by reference in their entireties. AAV Cap proteins [0166] In some embodiments, the AAV Cap proteins comprise VP1, VP2, and VP3. [0167] In some embodiments, a serotype of the AAV Cap proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 and AAVhu68. In some embodiments, the serotype is an AAV5 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV5 Cap proteins. In some embodiments, the AAV Cap proteins encode for AAV9 Cap proteins. In some embodiments, the serotype is an AAV9 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for PhP.EB Cap proteins. In some embodiments, the serotype is an PhP.EB Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV8 Cap proteins. In some embodiments, the serotype is an AAV8 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV2 proteins. In some embodiments, the serotype is an AAV2 Cap protein which comprises one or more mutations or insertions. In some embodiments, the AAV Cap proteins encode for AAV6 Cap proteins. In some embodiments, the serotype is an AAV6 Cap protein which comprises one or more mutations or insertions. AAV Cap Encoding Sequence linked to Inducible Promoter [0168] In some aspects, polynucleotides comprising (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is 3' of the sequence encoding AAV Cap proteins are provided. Any suitable inducible promoter that is stronger than the native p40 promoter may be used for increasing AAV Cap proteins expression. Suitable inducible promoters are further described below in the section "Inducible Promoters". Inducible Promoters [0169] In certain aspects, the inducible promoters are selected on the basis of the regulatory sequence that allows for control of the promoter. The regulatory sequence may be operably linked to the promoter and positioned upstream of the promoter. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The regulatory sequence used to control expression may be endogenous or exogenous to the host cell. In some embodiments, bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression. Examples of mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides. For example, inclusion of a bacterial tetracycline response element (TRE) in a construct allows mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline). See, e.g., Weber et al. (2004) Methods Mol. Biol.267:451-66, Das et al. (2016) Curr. Gene Ther.16(3):156-67, Chruscicka et al. (2015) J. Biomol. Screen.20(3):350-8, Yarranton (1992) Curr. Opin. Biotechnol.3(5):506- 11, Gossen & Bujard (1992) Proc. Natl.Acad. Sci. U.S.A.89(12):5547-51, Gossen et al. (1995) Science 268(5218):1766-9; herein incorporated by reference. [0170] In some embodiments, the inducible promoter is a tetracycline-inducible promoter. In other embodiments, the inducible promoter is an ecdysone-inducible promoter. In still other embodiments, the inducible promoter is a cumate-inducible promoter. [0171] In some embodiments, the inducible promoter comprises a tetracycline-responsive promoter element (TRE). In certain embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence (tetO). In further embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal human cytomegalovirus (CMV) promoter. [0172] In some embodiments, the inducible promoter is a Tet-On promoter. [0173] In some embodiments, the inducible promoter comprises a first inducible promoter. [0174] In some embodiments, the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148. In certain embodiments, the polynucleotide has sequence of SEQ ID NO: 148. In some embodiments, the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163. In certain embodiments, the polynucleotide has sequence of SEQ ID NO: 163. [0175] In some embodiments, the Cap coding sequence is operably linked to a promoter. [0176] In some embodiments, the AAV Cap proteins comprise VP1, VP2, and VP3. In some embodiments, the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter. In some embodiments, a single construct or separate constructs comprise these sequences, in any combination. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. [0177] The Cap protein encoding sequence provided in a separate polynucleotide construct can be expressed under the control of an inducible promoter. The Cap coding sequence may include the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to the inducible promoter. [0178] In various embodiments, the Cap protein encoding sequence provided in a separate polynucleotide construct or in the same polynucleotide comprising the Rep coding sequence can be expressed under the control of an inducible promoter. In various embodiments, the Cap protein encoding sequence may be operably linked to a polyadenylation (polyA) signal sequence. The polyA signal sequence may be a polyA signal sequence functional in the cells used for producing the rAAV. In some instances, the polyA signal sequence may be a bovine Growth Hormone polyA (bGH-PolyA) signal sequence, a SV40 polyA signal sequence, or a Rabbit Beta Globin PolyA signal sequence. In some embodiments, the polyadenylation signal sequence is any polyadenylation signal sequence that encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162. [0179] The bGH-PolyA signal sequence may include a nucleotide sequence that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 151. [0180] In certain cases, the SV40 polyA signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 152. [0181] In some embodiments, the SV40 polyA sequence is shorter than SEQ ID NO: 152. In some embodiments, the SV40 polyA sequence is longer than SEQ ID NO: 152. [0182] In certain cases, the Rabbit Beta Globin signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 170. [0183] In various embodiments, the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof. [0184] In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68 (described in WO2020/033842, incorporated herein by reference in its entirety). The hu68 capsid is described in WO 2018/160582, incorporated herein by reference in its entirety. [0185] In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68. [0186] In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 (described in WO2020/033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety. [0187] In particular embodiments, the capsid is an AAV1 capsid. In particular embodiments, the capsid is an AAV5 capsid. In particular embodiments, the capsid is an AAV9 capsid. AAV Cap and AAV Rep Encoding Polynucleotide [0188] In some aspects, polynucleotides comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence are provided. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins. The second sequence encoding the polynucleotide comprising a sequence encoding AAV Cap proteins operably linked to an inducible promoter and a polyadenylation signal sequence is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above. [0189] In some embodiments, transcription of the first sequence is driven by native AAV promoters. In other embodiments, optionally, transcription of the first sequence is driven by the P5 and P19 native AAV promoters. [0190] In some embodiments, the one or more promoters comprise P5 and P19 native promoters. A First Sequence Encoding AAV Rep Proteins
Figure imgf000046_0001
nce encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 160 or 161. In other embodiments, the first sequence encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 136. In other embodiments, the first sequence encoding AAV Rep proteins has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. In certain embodiments, the first sequence encoding AAV Rep proteins has sequence of SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. [0192] In some embodiments, the first sequence encoding AAV Rep proteins is separated from the second sequence by an intervening sequence. In some embodiments, the intervening sequence comprises a transcriptional blocking element (TBE). In other embodiments, optionally, a sequence of the TBE has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 169. In certain embodiments, a sequence of the TBE has sequence of SEQ ID NO: 169. [0193] In some embodiments, the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element, and (iii) a second part of the AAV Rep proteins coding sequence. In certain embodiments, the excisable element comprises a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction. [0194] In some embodiments, the first sequence encoding AAV Rep proteins comprises from 5' to 3': one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a first recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3' splice site, and a sequence comprising a second part of the AAV Rep coding sequence. In certain embodiments, the first recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element. In certain embodiments, the first recombination site and the second recombination site are oriented in the same direction. In certain embodiments, the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence. In certain embodiments, the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element. In certain embodiments, the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins. [0195] In some embodiments, the coding sequence encoding the stop signaling sequence of the first sequence encodes for from 5' to 3': an exon and the stop signaling sequence. In some embodiments, the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence. [0196] In some embodiments, the polynucleotide of the present disclosure further comprises a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker. [0197] In some embodiments, the polynucleotide of the present disclosure is a polynucleotide construct. In some embodiments, both the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins are on single construct. [0198] In some embodiments, the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins are on separate constructs. [0199] In some embodiments, the polynucleotide further comprises a selectable marker operably linked to a promoter. In certain embodiments, optionally the promoter is a constitutive promoter. [0200] In some aspects, polynucleotides comprising: a first sequence encoding AAV Rep proteins and a second sequence encoding AAV Cap proteins, wherein the first sequence is operably linked to one or more promoters and the second sequence is operably linked to an inducible promoter are provided. [0201] In some embodiments, the one or more promoters comprise P5 and P19 native promoters and the inducible promoter comprises a first inducible promoter. [0202] In some embodiments, the first inducible promoter comprises a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. [0203] In some embodiments, the first sequence and the one or more promoters operably linked to the first sequence are separated from the second sequence and the inducible promoter operably linked to the second sequence by an intervening sequence. In some embodiments, the intervening sequence comprises a transcriptional blocking element (TBE). [0204] In some embodiments, the inducible promoter is a Tet-On promoter. [0205] In some embodiments, the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence. [0206] In some embodiments, the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence. [0207] In some embodiments, the polypeptides further comprise a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker. 1.4. Polynucleotide System [0208] In some aspects, systems of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload are provided. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. [0209] In some embodiments, the polyadenylation signal sequence is 3' of the sequence encoding AAV Cap proteins are provided. The b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence is described in section “AAV Cap Encoding Sequence linked to PolyA Signal Sequence” above. First Polynucleotide encoding AAV Rep Protein [0210] In some embodiments, transcription of a first polynucleotide is driven by native AAV promoters. In other embodiments, optionally, transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters. [0211] In some embodiments, the one or more promoters comprise a P5 native AAV promoter and a P19 native AAV promoter. [0212] In some embodiments, the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence. [0213] In some embodiments, the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises from 5' to 3': one or more promoters operably linked to a first sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a first recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3' splice site, and a second sequence comprising a second part of the AAV Rep coding sequence, allowing expression of AAV Rep proteins. In certain embodiments, the first recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element. In certain embodiments, the first recombination site and the second recombination site are oriented in the same direction. In certain embodiments, the one or more promoters are not operably linked to the second sequence comprising the second part of the AAV Rep coding sequence. In certain embodiments, the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element. In certain embodiments, the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins. [0214] In some embodiments, the coding sequence encoding the stop signaling sequence of the first polynucleotide encodes for from 5' to 3': an exon and the stop signaling sequence. In some embodiments, the coding sequence encoding the stop signaling sequence of the first polynucleotide further comprises a sequence encoding a protein marker. In certain embodiments, the sequence encoding the protein marker is in-frame with the stop signaling sequence. [0215] In some embodiments, the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161. [0216] In certain embodiments, a set of polynucleotides comprises: (i) a first polynucleotide encoding AAV Rep proteins; (ii) a second polynucleotide encoding the AAV Cap proteins; and (iii) a third polynucleotide encoding one or more adenoviral helper proteins wherein when the one or more polynucleotides are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. [0217] In some embodiments, the first polynucleotide further comprises a sequence encoding AAV Cap proteins. In some embodiments, transcription of the sequence encoding the AAV Cap proteins on the first polynucleotide is driven by a native AAV Cap proteins promoter. In certain embodiments, the native AAV Cap proteins promoter is a P40 native AAV promoter. In some embodiments, the Rep coding sequence is 5’ to the Cap coding sequence. In certain embodiments, the Cap coding sequence is operatively linked to an endogenous P40 promoter. [0218] In some embodiments, the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136. In certain embodiments, the first polynucleotide has sequence of SEQ ID NO: 136. [0219] In some embodiments, the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. In certain embodiments, the first polynucleotide has sequence of SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. [0220] In certain embodiments, a set of polynucleotides comprises: (i) a first polynucleotide encoding AAV Rep proteins and AAV Cap proteins; (ii) a second polynucleotide encoding the AAV Cap proteins; and (iii) a third polynucleotide encoding one or more adenoviral helper proteins wherein when the one or more polynucleotides are integrated into the nuclear genome of a cell, e.g., a mammalian cell, the AAV Rep proteins, the AAV Cap proteins, and/or the one or more adenoviral helper proteins are conditionally expressible and thereby conditionally produce recombinant AAV (rAAV) virions. [0221] The Rep sequence can encode Rep from any desired AAV serotype. In some embody [0222] ments, the encoded Rep protein is drawn from the same serotype as the Cap protein. In some embodiments, the encoded Rep protein is drawn from a different serotype from the Cap protein. In particular embodiments, the encoded Rep protein includes, but is not limited to, a Rep protein from AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10 and AAV-11, or chimeric combinations thereof. [0223] The nucleotide sequences of the genomes of the AAV serotypes are known. For example, the complete genome of AAV-1 is provided in GenBank Accession No. NC_002077; the complete genome of AAV- 2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol, 45: 555-564 (1983); the complete genome of AAV- 3 is provided in GenBank Accession No. NC_1829; the complete genome of AAV-4 is provided in GenBank Accession No. NC_001829; the AAV-5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV-6 is provided in GenBank Accession No. NC_00 1862; at least portions of AAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos.7,282,199 and 7,790,449 relating to AAV-8); the AAV-9 genome is provided in Gao et al. Virol, 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol Ther, 13(1): 67-76 (2006); and the AAV-11 genome is provided in Virology, 330(2): 375-383 (2004). [0224] In the exemplary embodiments, prior to the cell being contacted with the first triggering agent and the second triggering agent, the Rep coding sequence is interrupted by an excisable element. Addition of both the first triggering agent and the second triggering agent are required for excision of the excisable element. In some embodiments, the excisable element is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (-) indicates the point of insertion of the excisable element, in the Rep coding sequence, and the excisable element is inserted downstream of the p19 promoter. In some embodiments, the excisable element is inserted at CAG-G, CAG-A, AAG-G, AAG-A, wherein the dash (-) indicates the point of insertion of the excisable element, in the Rep coding sequence, and the excisable element is inserted downstream of the p19 promoter and upstream of the p40 promoter. [0225] In certain embodiments, the excisable element comprises, from 5’ to 3’, a first spacer segment, a second spacer segment, and a third spacer segment. [0226] In particular embodiments, the first spacer segment comprises a 5’ splice site (5’SS) 5’ to the first spacer element. In some embodiments, the first spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 1. [0227] In some embodiments, the second spacer segment comprises a polynucleotide encoding a detectable protein marker flanked by lox sites. In certain embodiments, the detectable protein marker is a fluorescent protein. In particular embodiments, the fluorescent protein is a green or blue fluorescent protein (GFP of BFP). In specific embodiments, the GFP is EGFP. In particular embodiments, the fluorescent protein is a blue fluorescent protein (BFP). Screening for the fluorescent marker can be used to confirm integration of the construct into the cell genome, and can subsequently be used to confirm excision of the intervening spacer segment. In some embodiments, the second spacer segment further comprises a polyA signal sequence. In certain embodiments, the poly A signal sequence comprises a rabbit beta globin (RBG) polyA signal sequence. In some embodiments, the second spacer segment further comprises a first 3’ splice site (3’SS) between the first lox site and the polynucleotide encoding the protein marker. [0228] In some embodiments, the second spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 2. [0229] In some embodiments, the third spacer segment further comprises a second 3’ splice site (3’SS). In particular embodiments, the second 3’ splice site is positioned 3’ to the second lox site. [0230] In some embodiments, the third spacer segment comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO: 3. [0231] In various embodiments, the Rep coding sequences are operatively linked to an endogenous P5 promoter. In various embodiments, the Rep coding sequences are operatively linked to an endogenous P19 promoter. [0232] In some embodiments, the Rep coding sequences are operably linked to an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the Rep coding sequences are operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. Second Polynucleotide Encoding Inducible Cap Proteins [0233] In some embodiments, the second polynucleotide comprises a sequence encoding AAV Cap proteins operably linked to an inducible promoter. In some embodiments, the second polynucleotide comprises (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is 3' of the sequence encoding AAV Cap proteins are provided. The second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and optionally, (ii) a polyadenylation signal sequence, is described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above. Any suitable inducible promoter that is stronger than the native p40 promoter may be used for increasing AAV Cap proteins expression. Suitable inducible promoters are further described above in the section "Inducible Promoters". [0234] In some embodiments, the first polynucleotide and second polynucleotide are in a first polynucleotide construct. For example, a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide. In some embodiments, a first polynucleotide construct comprises the Rep coding sequence is linked to native promoters and the Cap coding sequence is linked to an inducible promoter and the Rep coding sequence and the native promoters are separated from the Cap coding sequence and the inducible promoter by an intervening sequence, such as, a TBE. For example, see the configuration of Construct 1 in FIG. 2A. [0235] In certain embodiments, the TBE comprises a nucleotide sequence at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical or 100% identical to the nucleotide sequence set forth in SEQ ID NO: 169. [0236] In some embodiments, the first polynucleotide lacks a sequence encoding AAV Cap proteins and the second polynucleotide comprises a sequence encoding AAV Cap proteins. [0237] In some embodiments, the sequence encoding the AAV Cap proteins of the first polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide. Third Polynucleotide Encoding Adenoviral Helper Proteins [0238] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase; a first constitutive promoter operably linked to a sequence encoding an activator. In some embodiments, the third recombination site and the fourth recombination site are oriented in the same direction. In some embodiments, the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins. In some embodiments, the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a first triggering agent. In some embodiments, in absence of activation of the first inducible promoter and the second inducible promoter, detectable levels of the Rep proteins from the first polynucleotide or if present the Cap proteins from the first polynucleotide, the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins are not expressed, and wherein the inducible recombinase is activated in the presence of a second triggering agent. In some embodiments, a second polynucleotide construct comprises the third polynucleotide as described herein. [0239] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: (i) a first sequence comprising from 5' to 3': a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self- excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site; and a sequence encoding one or more adenoviral helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; (ii) a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator. In some embodiments, the third recombination site and the fourth recombination site are oriented in the same direction. In some embodiments, the cell constitutively expresses the activator, and the activator is unable to activate the second inducible promoter in absence of a first triggering agent. In some embodiments, in the presence of the first triggering agent, the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed. In some embodiments, in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins. [0240] In some embodiments, the one or more adenoviral helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein. In certain embodiments, the one or more adenoviral helper proteins comprises E2A protein and E4 protein. [0241] In some embodiments, the third polynucleotide comprising the sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins. In certain embodiments, the SEQ ID NO: 30 comprises the third polynucleotide. [0242] In some embodiments, the one or more adenoviral helper proteins are separated by a bicistronic open reading frame. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. [0243] In some embodiments, the second inducible promoter operably linked to the self- excising element in the third polynucleotide is a tetracycline-inducible promoter, an ecdysone- inducible promoter, or a cumate-inducible promoter. [0244] In some embodiments, the first inducible promoter and the second inducible promoter are the same. In some embodiments, the first inducible promoter and the second inducible promoter are a tetracycline-inducible promoter. In certain embodiments, the tetracycline- inducible promoter comprises a tetracycline-responsive promoter element (TRE). In certain embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In certain embodiments, the minimal promoter is a human cytomegalovirus promoter. [0245] In some embodiments, the first constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. [0246] In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. [0247] In some embodiments, a triggering agent for inducing the tetracycline-inducible promoter is tetracycline. In other embodiments, a triggering agent for inducing the tetracycline- inducible promoter is doxycycline. [0248] In some embodiments, the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen. [0249] In some embodiments, the recombination sites in the first polynucleotide and the third polynucleotide are lox sites and the inducible recombinase is a Cre recombinase. In other embodiments, the recombination sites in the first polynucleotide and the third polynucleotide are flippase recognition target (FRT) sites and the inducible recombinase is a flippase (Flp) recombinase. [0250] In some embodiments, presence of the triggering agent activates the activator for activation of the first inducible promoter to express AAV Cap proteins from the second polynucleotide encoding the AAV Cap proteins. [0251] In some embodiments, presence of the triggering agent activates the activator for activation of the second inducible promoter to express the Rep proteins of the first polynucleotide, if present the Cap proteins of the first polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins. [0252] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide and if present the Cap Proteins of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins. [0253] In some embodiments, the third polynucleotide further comprises a third selectable marker operably linked to a third promoter. Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA [0254] In some embodiments, the third polynucleotide further comprises a sequence encoding a viral associated RNA (VA-RNA). In certain embodiments, the VA-RNA is a mutated VA-RNA. In some embodiments, the VA-RNA is wild-type VA-RNA. In other embodiments, VA-RNA comprises one or more mutations in the VA-RNA internal promoter. In some embodiments, a second polynucleotide construct comprises the third polynucleotide as described herein. [0255] In some embodiments, the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a second constitutive promoter and a second part of the second constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, and excision of the second excisable element by the inducible recombinase generates a functional complete second constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. In some embodiments, the fifth and sixth recombination sites are oriented in the same direction. [0256] In some embodiments, the first part of the second constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter. In other embodiments, the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter. In still other embodiments, the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. [0257] In some embodiments, the set of nucleic acids or any of the recombinant nucleic acids as disclosed herein further comprises a nucleic acid sequence encoding a viral associated RNA (“VA-RNA”) sequence. In some embodiments, the third recombinant nucleic acid sequence comprises a nucleic acid sequence encoding a VA-RNA sequence. In some embodiments, the expression of VA-RNA is constitutive. In some embodiments, the expression of VA-RNA is inducible. In some embodiments, the VA-RNA sequence comprises one or more mutations in the VA-RNA internal promoter, preferably G16A and G60A. In some embodiments, the expression of VA-RNA is driven by a EF1alpha promoter, a U6 promoter, or a U7 promoter. In some embodiments, the expression of VA-RNA is driven by a U6 promoter or a U7 promoter. In some embodiments, the U6 promoter or the U7 promoter comprises a) a first part of a U6 or U7 promoter sequence, b) a stuffer sequence, and c) a second part of a U6 or U7 promoter sequence, and wherein the stuffer sequence is capable of being excised by a Cre polypeptide. [0258] In some embodiments, the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. [0259] In some embodiments, the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 153. In certain embodiments, the third polynucleotide has sequence of SEQ ID NO: 153. Fourth Polynucleotide Encoding Payload [0260] In some embodiments, the payload of the fourth polynucleotide comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest. In certain embodiments, the payload of the fourth polynucleotide is progranulin. In some embodiments, a third polynucleotide construct comprises the fourth polynucleotide as described herein. [0261] In some embodiments, the sequence encoding the payload of the fourth polynucleotide comprises a sequence encoding a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest. In some embodiments, the sequence encoding the payload of the fourth polynucleotide is a sequence encoding progranulin. In some embodiments, the sequence encoding progranulin has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 166. In certain embodiments, the sequence encoding progranulin has sequence of SEQ ID NO: 166. [0262] In some embodiments, the sequence encoding the payload comprises a sequence encoding a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair. [0263] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload comprises the sequence encoding the payload flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR). [0264] In some embodiments, the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146. In certain embodiments, the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has sequence of SEQ ID NO: 146. [0265] In some embodiments, the sequence encoding the payload has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In certain embodiments, the sequence encoding the payload has sequence of SEQ ID NO: 156. [0266] In some embodiments, the sequence encoding the payload has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158. In certain embodiments, the sequence encoding the payload has sequence of SEQ ID NO: 158. In some embodiments, the payload construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147. Polynucleotide Encoding a Payload [0267] In some embodiments, the fourth integrated synthetic construct (also referred to as Construct 4) comprises the coding sequence for an expressible payload and a third mammalian cell selection element. In the exemplary embodiments, the expressible payload is under the control of a constitutive promoter. This construct can be referred to as construct 4 or payload construct, interchangeably. [0268] In some embodiments, the expressible payload encodes a guide RNA. In certain embodiments, the guide RNA directs RNA editing. In some embodiments, the guide RNA directs Cas-mediated DNA editing. In some embodiments, the guide RNA directs ADAR- mediated RNA editing. In some embodiments, the fourth integrated synthetic construct comprises a sequence encoding for any of the expressible payloads disclosed herein. For example, said sequence can encode for any therapeutic. For example, the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof. In some embodiments, the transgene encodes for progranulin. In some embodiments, the tRNA suppressor is capable of suppressing an opal stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an ochre stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an amber stop codon. In some embodiments, the fourth integrated synthetic construct comprises sequences encoding for more than one of the expressible payloads disclosed herein. For example, the fourth integrated synthetic construct comprises 2 gRNA, 3 gRNA, 4 gRNA, 5 gRNA, 6 gRNA, 7 gRNA, 8 gRNA, 9 gRNA, or 10 gRNA. These gRNAs can all be the same, all be different, or any combination of the same and different. For example, the fourth integrated synthetic construct comprises 2 suppressor tRNAs, 3 suppressor tRNAs, 4 suppressor tRNAs, 5 suppressor tRNAs, 6 suppressor tRNAs, 7 suppressor tRNAs, 8 suppressor tRNAs, 9 suppressor tRNAs, or 10 suppressor tRNAs. These suppressor tRNAs can all be the same, all be different, or any combination of the same and different. [0269] In some embodiments, the expressible payload encodes a protein. In certain embodiments, the expressible payload is an enzyme, useful for replacement gene therapy. In some embodiments, the protein is a therapeutic antibody. In some embodiments, the protein is a vaccine immunogen. In particular embodiments, the vaccine immunogen is a viral protein. [0270] In some embodiments, the expressible payload is a homology construct for homologous recombination. [0271] In various embodiments, the third mammalian cell selection element is an auxotrophic selection element. [0272] In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 33 or SEQ ID NO: 139, wherein SEQ ID NO: 34 in SEQ ID NO: 33 or SEQ ID NO: 139 is replaced with a sequence of the payload of interest. In some embodiments, the payload construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146, SEQ ID NO: 147, SEQ ID NO: 156, SEQ ID NO: 157, SEQ ID NO: 158, or SEQ ID NO: 159. In some embodiments, the payload construct is a plasmid comprising at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159. [0273] In some embodiments, the payload construct comprises a sequence of a payload flanked by ITR sequences. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter or an inducible promoter. In some embodiments, the promoter and sequence of the payload are flanked by ITR sequences. In some embodiments, the payload construct flanked by ITRs comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 146. [0274] In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the therapeutic polypeptide or transgene is progranulin. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the tRNA suppressor is capable of suppressing an opal stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an ochre stop codon. In some embodiments, the tRNA suppressor is capable of suppressing an amber stop codon. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the guide RNA, when bound to a target RNA, recruits an ADAR protein for editing of the target RNA. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the payload construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the payload construct are stably integrated into the genome of the cell. In some embodiments, the plurality of the payload constructs are separately stably integrated into the genome of the cell. [0275] In some embodiments, the payload construct further comprises a sequence coding for a selectable marker or detectable marker outside of the ITR sequences. In some embodiments, expression of the selectable marker or detectable marker outside of the ITR sequences is driven by a promoter. The promoter can be a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the selectable marker is a mammalian cell selection element (e.g., a third mammalian cell selection element). In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0276] In some embodiments, the payload construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the payload construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0277] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0278] . In some embodiments, the selectable marker is outside of the ITR sequences on the payload construct. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the auxotrophic protein or split intein linked to a C- terminus of the auxotrophic protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the auxotrophic or leucine zipper linked to a C-terminus of the auxotrophic. In some embodiments, the selectable marker outside of the ITR sequences is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker outside of the ITR sequences is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the payload construct further comprises a spacer between the 5’ ITR and the promoter/selectable marker or promoter/detectable marker outside of the ITR sequences. In some embodiments, the payload construct further comprises a spacer between the 3’ ITR and the promoter/selectable marker or promoter/detectable marker outside of the ITR sequences. In some embodiments, the spacer ranges in length from 500 base pairs to 5000 base pairs, including any length within this range such as 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 base pairs. In some embodiments, the spacer length is a sufficient length for decreasing reverse packaging of the selectable marker or detectable marker that is outside the ITR sequences. [0279] In some embodiments, the fourth integrated synthetic construct comprising the coding sequence for a payload and a selectable marker or detectable marker is further engineered to remove locations having the potential for Rep-mediated nicking. For example, a location having the potential for Rep-mediated nicking is a location having the sequence CAGTGAGCGAGCGAGCGCGCAG (SEQ ID NO: 87); a sequence comprising GAGC (SEQ ID NO: 88) repeats; or the sequence GATGGAGTTGGCCACTCCCTC (SEQ ID NO: 89). These sequences can be engineered to prevent binding of Rep proteins for Rep-mediated nicking. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 100 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 200 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 300 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 400 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 500 nucleotides of an ITR sequence. In some embodiments, the location having the potential for Rep-mediated nicking that is engineered to prevent binding of Rep proteins for Rep-mediated nicking is in a region within 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1250, 1500, 1750, 2000, 2225, 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4750, or 5000 nucleotides of an ITR sequence. [0280] In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA-RNA. In some embodiments, the VA-RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, a separate polynucleotide construct codes for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA-RNA comprises a sequence coding for a transcriptionally dead VA-RNA. In some embodiments, the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA-RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z- Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0281] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GT—CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0282] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0283] In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the construct comprising the VA-RNA as described herein further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0284] In some embodiments, the construct comprising VA-RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the construct comprising the VA-RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0285] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0286] In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a vector. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, a VA-RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, a VA-RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. Payloads [0287] Disclosed herein are payloads that may be encoded for by polynucleotide construct 4, which encodes for a payload. This fourth polynucleotide is referred to herein as a “payload construct” or “therapeutic payload.” Thus, disclosed herein are stable mammalian cell lines that encapsidate a payload. In some embodiments, the payload may be an expressible payload. In some embodiments, the polynucleotide may encode for any therapeutic. For example, the therapeutic may be a transgene, a guide RNA, an antisense RNA, an oligonucleotide, an mRNA, a miRNA, a shRNA, a tRNA suppressor, a CRISPR-Cas protein, any gene editing enzyme, or any combination thereof. In some embodiments, the payload is guide RNA, wherein the guide RNA, when bound to a target RNA, recruits an ADAR enzyme for editing of the target RNA. In some embodiments, the payload is progranulin. In some embodiments, the stable mammalian cell lines disclosed herein can conditionally produce rAAV virions that encapsidate more than one payload. Any combination of payloads disclosed herein is contemplated. [0288] The sequence encoding a payload as disclosed herein encompasses any nucleotide sequence that is to be delivered to a cell. The nucleotide sequence may be utilized in the cell for, e.g., insertion of the nucleotide sequence or a part thereof. For example, the nucleotide sequence may be used to repair an endogenous DNA. In such a case, the nucleotide sequence itself is the payload being delivered by the rAAV to a cell. [0289] In other cases, the polynucleotide payload is transcribed in the cell into an RNA which is not translated into a protein. In such a case, the RNA is the payload that is delivered by the polynucleotide payload present in the rAAV. In other cases, the polynucleotide payload is transcribed in the cell into an mRNA which is translated into a protein. In such a case, the protein is the payload that is delivered by the polynucleotide payload present in the rAAV. [0290] The polynucleotide payload may include a promoter operably linked to a DNA sequence. The promoter may be any promoter that allows for transcription of the DNA in the cell. A payload disclosed herein may be a therapeutic payload. [0291] The DNA sequence may be transcribed to produce RNA in the cell. The RNA may be mRNA. The RNA may be a guide RNA (gRNA), a tRNA, a suppressor tRNA, an mRNA, or a circular RNA. The RNA may be a regulatory RNA of interest such as, but not limited to, a microRNA (miRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a small nuclear RNA (snRNA), a long non-coding RNA (lncRNA), an antisense nucleic acid, and the like. [0292] The polynucleotide payload may be a gene encoding a polypeptide, such as, an antibody, a hormone, a site-specific endonuclease, a reporter gene, a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, a ribozyme, a DNAzyme, or any combination thereof. [0293] A payload may include any one or combination of the following: a transgene, a tRNA suppressor, a guide RNA, or any other target binding/modifying oligonucleotide or derivative thereof, or payloads may include immunogens for vaccines, and elements for any gene editing machinery (DNA or RNA editing). Payloads may also include those that deliver a transgene encoding antibody chains or fragments that are amenable to viral vector-mediated expression (also referred to as “vectored or vectorized antibody” for gene delivery). See, e.g., Curr Opinion HIV AIDS.2015 May; 10(3): 190–197, describing vectored antibody gene delivery for the prevention or treatment of HIV infection. See also, U.S. Pat. No.10,780,182, which describes AAV delivery of trastuzumab (Herceptin) for treatment of HER2+ brain metastases. A payload disclosed herein may not be a therapeutic payload (e.g., a coding for a detectable marker such as GFP). In particular, in some instances the polynucleotide payload refers to a polynucleotide that may be a homology element for homology-directed repair, or polynucleotide transcribed into a guide RNA to be delivered for a variety of purposes. In some embodiments, the transgene refers to a nucleic acid sequence coding for expression of guide RNA for ADAR editing or ADAT editing. In some embodiments, the transgene refers to a transgene packaged for gene therapy. In some embodiments, the transgene refers to synthetic constructs packaged for vaccines. In certain aspects, a polynucleotide payload may be described as encoding an RNA, which is meant to refer to the RNA transcribed from the polynucleotide. [0294] In certain examples, the sequence encoding the payload comprises two expressible sequences, wherein a first expressible sequence encodes for a first gRNA and a second expressible sequence encodes for a second gRNA. In some embodiments, the first gRNA and the second gRNA are different. In some embodiments, the first gRNA and the second gRNA are the same. In certain examples, the sequence encoding the payload comprises two or more expressible sequences. In some embodiments, the two or more expressible sequences encode for two or more gRNA. In some embodiments, the two or more gRNA are all different gRNA, all the same gRNA, or a combination of the same and different gRNA. [0295] In some cases, the sequence encoding the payload comprises an expressible sequence encoding both a heterologous RNA and a heterologous polypeptide. In other cases, the expressible sequence encodes two or more heterologous payloads. Where the expressible sequence encodes two heterologous payloads, in some cases, the nucleotide sequences encoding the two heterologous payloads are operably linked to the same promoter. Where the expressible sequence encodes two heterologous payloads, in some cases, the nucleotide sequences encoding the two heterologous payloads are operably linked to two different promoters. In some cases, sequence encoding the payload comprises an expressible sequence encoding three heterologous payloads. Where the expressible sequence encodes three heterologous payloads, in some cases, the nucleotide sequences encoding the three heterologous payloads are operably linked to the same promoter. Where the expressible sequence encodes three heterologous payloads, in some cases, the nucleotide sequences encoding the three heterologous payloads are operably linked to two or three different promoters. In some cases, the fourth polynucleotide construct of the present disclosure comprises two or more expressible sequences, each comprising a nucleotide sequence encoding a heterologous payload. [0296] In some embodiments, the expressible sequence encodes a polypeptide of interest. The polypeptide of interest may be any type of protein/peptide including, without limitation, an enzyme, an extracellular matrix protein, a receptor, transporter, ion channel, or other membrane protein, a hormone, a neuropeptide, an antibody, or a cytoskeletal protein; or a fragment thereof, or a biologically active domain of interest. In some cases, the payload is a therapeutic polypeptide, e.g., a polypeptide that provides clinical benefit. [0297] Where the payload is an interfering RNA (RNAi), suitable RNAi include RNAi that decrease the level of an apoptotic or angiogenic factor in a cell. For example, an RNAi may be an shRNA or siRNA that reduces the level of a payload that induces or promotes apoptosis in a cell. A payload may be a gene whose gene product induces or promotes apoptosis are referred to herein as “pro-apoptotic genes” and the products of those genes (mRNA; protein) are referred to as “pro-apoptotic gene products.” Pro-apoptotic gene products include, e.g., Bax, Bid, Bak, and Bad gene products. See, e.g., U.S. Patent No.7,846,730. In another example, the RNAi specifically reduces the level of an RNA and/or a polypeptide product of a defective allele. [0298] In some embodiments, the payload is an aptamer. In some cases, the aptamer is a therapeutic aptamer. For example, the aptamer may function as an antagonist by blocking interactions at a disease-associated target (e.g., receptor-ligand interactions). Alternatively, an aptamer may serve as an agonist for activating the function of a target receptor. Exemplary aptamers of interest include aptamers against growth factor receptors and growth factors such as aptamers that bind to epidermal growth factor receptor (see, e.g., Wang et al. (2014) Biochem. Biophys. Res. Commun.453(4):681-5), transforming growth factor-beta type III receptor (see, e.g., Ohuchi et al. (2006) Biochimie 88(7):897-904.), vascular endothelial growth factor (VEGF) (see, e.g., Ng et al. (2006) Nat. Rev. Drug Discovery 5:123; and Lee et al. (2005) Proc. Natl. Acad. Sci. USA 102:18902) or platelet-derived growth factor (PDGF), e.g., E10030 (see, e.g., Ni and Hui (2009) Ophthalmologica 223:401; and Akiyama et al. (2006) J. Cell Physiol. 207:407). [0299] In some embodiments, the expressible sequence encodes a sequence-specific endonuclease for use in genome editing. The sequence specific endonuclease may be used to create a double-stranded break at a specific site in the genome. The double stranded breaks may then be repaired by non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), or homology-directed repair (HDR) pathways. Desired genome edits may be introduced into the genome using donor DNA to repair double-strand breaks by homologous recombination. Various sequence-specific endonucleases may be used in genome editing for creation of double-strand breaks in DNA, including, without limitation, engineered zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered regularly interspaced short palindromic repeats (CRISPR) Cas9. See, e.g., Targeted Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System (T. Yamamoto ed., Springer, 2015); Genome Editing: The Next Step in Gene Therapy (Advances in Experimental Medicine and Biology, T. Cathomen, M. Hirsch, and M. Porteus eds., Springer, 2016); Aachen Press Genome Editing (CreateSpace Independent Publishing Platform, 2015); herein incorporated by reference. Precise control over the timing of production of the genome editing enzyme may be achieved by inducibly producing recombinant adenovirus associated virus (rAAV) virions with the vector system to allow turning on and off of expression as desired. [0300] In some cases, a payload of interest is a site-specific endonuclease that provides for site- specific knock-down of gene function, e.g., where the endonuclease knocks out an allele associated with a disease. For example, in a case where a dominant allele encodes a defective copy of a gene, and the wild-type gene provides for normal function, a site-specific endonuclease may be targeted to the defective allele and knock out the defective allele. In some cases, a site-specific endonuclease is an RNA-guided endonuclease. [0301] A site-specific nuclease may also be used to stimulate homologous recombination with a donor DNA that encodes a functional copy of the protein encoded by the defective allele. Thus, e.g., a subject rAAV virion may be used to deliver a site-specific endonuclease that knocks out a defective allele and also be used to deliver a functional copy of the defective allele, resulting in repair of the defective allele, thereby providing for production of a functional gene product. [0302] In some cases, the payload is an RNA-guided endonuclease. In some cases, the payload is an RNA comprising a nucleotide sequence encoding an RNA-guided endonuclease. In some cases, the payload is a guide RNA, e.g., a single-guide RNA. In some cases, the payloads are: 1) a guide RNA; and 2) an RNA-guided endonuclease. The guide RNA may comprise: a) a protein- binding region that binds to the RNA-guided endonuclease; and b) a region that binds to a target nucleic acid. An RNA-guided endonuclease is also referred to herein as a “genome editing nuclease.” [0303] Examples of RNA-guided endonucleases are CRISPR/Cas endonucleases (e.g., class 2 CRISPR/Cas endonucleases such as a type II, type V, or type VI CRISPR/Cas endonucleases). A suitable genome editing nuclease is a CRISPR/Cas endonuclease (e.g., a class 2 CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Cas endonuclease). In some cases, a suitable RNA-guided endonuclease is a class 2 CRISPR/Cas endonuclease. In some cases, a suitable RNA-guided endonuclease is a class 2 type II CRISPR/Cas endonuclease (e.g., a Cas9 protein). In some cases, a genome targeting composition includes a class 2 type V CRISPR/Cas endonuclease (e.g., a Cpf1 protein, a C2c1 protein, or a C2c3 protein). In some cases, a suitable RNA-guided endonuclease is a class 2 type VI CRISPR/Cas endonuclease (e.g., a C2c2 protein; also referred to as a “Cas13a” protein). Also suitable for use is a CasX protein. Also suitable for use is a CasY protein. [0304] In some cases, the genome-editing endonuclease is a Type II CRISPR/Cas endonuclease. In some cases, the genome-editing endonuclease is a Cas9 polypeptide. The Cas9 protein is guided to a target site (e.g., stabilized at a target site) within a target nucleic acid sequence (e.g., a chromosomal sequence or an extrachromosomal sequence, e.g., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.) by virtue of its association with the protein-binding segment of the Cas9 guide RNA. In some cases, the Cas9 polypeptide used in a composition or method of the present disclosure is a Staphylococcus aureus Cas9 (saCas9) polypeptide. In some cases, a suitable Cas9 polypeptide is a high-fidelity (HF) Cas9 polypeptide. Kleinstiver et al. (2016) Nature 529:490. In some cases, a suitable Cas9 polypeptide exhibits altered PAM specificity. See, e.g., Kleinstiver et al. (2015) Nature 523:481. In some cases, the genome-editing endonuclease is a type V CRISPR/Cas endonuclease. In some cases a type V CRISPR/Cas endonuclease is a Cpf1 protein. In some cases, the genome-editing endonuclease is a CasX or a CasY polypeptide. CasX and CasY polypeptides are described in Burstein et al. (2017) Nature 542:237. [0305] In some cases, a genome editing nuclease is a fusion protein that is fused to a heterologous polypeptide (also referred to as a “fusion partner”). In some cases, a genome editing nuclease is fused to an amino acid sequence (a fusion partner) that provides for subcellular localization, i.e., the fusion partner is a subcellular localization sequence (e.g., one or more nuclear localization signals (NLSs) for targeting to the nucleus, two or more NLSs, three or more NLSs, etc.). [0306] Also suitable for use is an RNA-guided endonuclease with reduced enzymatic activity. Such an RNA-guided endonuclease is referred to as a “dead” RNA-guided endonuclease; for example, a Cas9 polypeptide that comprises certain amino acid substitutions such that it exhibits substantially no endonuclease activity, but such that it still binds to a target nucleic acid when complexed with a guide RNA, is referred to as a “dead” Cas9 or “dCas9.” In some cases, a “dead” Cas9 protein has a reduced ability to cleave both the complementary and the non- complementary strands of a double stranded target nucleic acid. For example, a “nuclease defective” Cas9 lacks a functioning RuvC domain (i.e., does not cleave the non-complementary strand of a double stranded target DNA) and lacks a functioning HNH domain (i.e., does not cleave the complementary strand of a double stranded target DNA). Such a Cas9 protein has a reduced ability to cleave a target nucleic acid (e.g., a single stranded or double stranded target nucleic acid) but retains the ability to bind a target nucleic acid. A Cas9 protein that may not cleave target nucleic acid (e.g., due to one or more mutations, e.g., in the catalytic domains of the RuvC and HNH domains) is referred to as a “nuclease defective Cas9”, “dead Cas9” or simply “dCas9.” Other residues may be mutated to achieve the above effects (i.e. inactivate one or the other nuclease portions). [0307] In some cases, the genome-editing endonuclease is an RNA-guided endonuclease (and its corresponding guide RNA) known as Cas9-synergistic activation mediator (Cas9-SAM). The RNA-guided endonuclease (e.g., Cas9) of the Cas9-SAM system is a “dead” Cas9 fused to a transcriptional activation domain (wherein suitable transcriptional activation domains include, e.g., VP64, p65, MyoD1, HSF1, RTA, and SET7/9) or a transcriptional repressor domain (where suitable transcriptional repressor domains include, e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, and a SID4X domain). The guide RNA of the Cas9-SAM system comprises a loop that binds an adapter protein fused to a transcriptional activator domain (e.g., VP64, p65, MyoD1, HSF1, RTA, or SET7/9) or a transcriptional repressor domain (e.g., a KRAB domain, a NuE domain, an NcoR domain, a SID domain, or a SID4X domain). For example, in some cases, the guide RNA is a single-guide RNA comprising an MS2 RNA aptamer inserted into one or two loops of the sgRNA; the dCas9 is a fusion polypeptide comprising dCas9 fused to VP64; and the adaptor/functional protein is a fusion polypeptide comprising: i) MS2; ii) p65; and iii) HSF1. See, e.g., U.S. Patent Publication No.2016/0355797. [0308] Also suitable for use is a chimeric polypeptide comprising: a) a dead RNA-guided endonuclease; and b) a heterologous fusion polypeptide. Examples of suitable heterologous fusion polypeptides include a polypeptide having, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, DNA integration activity, or nucleic acid binding activity. [0309] A nucleic acid that binds to a class 2 CRISPR/Cas endonuclease (e.g., a Cas9 protein; a type V or type VI CRISPR/Cas protein; a Cpf1 protein; etc.) and targets the complex to a specific location within a target nucleic acid is referred to herein as a “guide RNA” or “CRISPR/Cas guide nucleic acid” or “CRISPR/Cas guide RNA.” A guide RNA provides target specificity to the complex (the RNP complex) by including a targeting segment, which includes a guide sequence (also referred to herein as a targeting sequence), which is a nucleotide sequence that is complementary to a sequence of a target nucleic acid. [0310] In some cases, a guide RNA includes two separate nucleic acid molecules: an “activator” and a “targeter” and is referred to herein as a “dual guide RNA”, a “double-molecule guide RNA”, a “two-molecule guide RNA”, or a “dgRNA.” In some cases, the guide RNA is one molecule (e.g., for some class 2 CRISPR/Cas proteins, the corresponding guide RNA is a single molecule; and in some cases, an activator and targeter are covalently linked to one another, e.g., via intervening nucleotides), and the guide RNA is referred to as a “single guide RNA”, a “single-molecule guide RNA,” a “one-molecule guide RNA”, or simply “sgRNA.” [0311] In some cases, the guide RNA is at least partially complementary to a target RNA sequence and is capable of recruiting an ADAR enzyme for RNA editing of the target RNA sequence. [0312] Where the payload is an RNA-guided endonuclease or is both an RNA-guided endonuclease and a guide RNA, the payload may modify a target nucleic acid. In some cases, e.g., where a target nucleic acid comprises a deleterious mutation in a defective allele (e.g., a deleterious mutation in a neural cell target nucleic acid), the RNA-guided endonuclease/guide RNA complex, together with a donor nucleic acid comprising a nucleotide sequence that corrects the deleterious mutation (e.g., a donor nucleic acid comprising a nucleotide sequence that encodes a functional copy of the protein encoded by the defective allele), may be used to correct the deleterious mutation, e.g., via homology-directed repair (HDR). [0313] In some cases, the payloads are an RNA-guided endonuclease and 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non-homologous end joining (NHEJ). [0314] In some cases, the payloads are: i) an RNA-guided endonuclease; and ii) one guide RNA. In some cases, the guide RNA is a single-molecule (or “single guide”) guide RNA (an “sgRNA”). In some cases, the guide RNA is a dual-molecule (or “dual-guide”) guide RNA (“dgRNA”). [0315] In some cases, the payloads are: i) an RNA-guided endonuclease; and ii) 2 separate sgRNAs, where the 2 separate sgRNAs provide for deletion of a target nucleic acid via non- homologous end joining (NHEJ). In some cases, the guide RNAs are sgRNAs. In some cases, the guide RNAs are dgRNAs. [0316] In some cases, the payloads are: i) a Cpf1 polypeptide; and ii) a guide RNA precursor; in these cases, the precursor may be cleaved by the Cpf1 polypeptide to generate 2 or more guide RNAs. [0317] The payloads as described herein may be flanked by ITRs. Fifth Polynucleotide Encoding VA-RNA [0318] In some embodiments, systems of polynucleotides comprising a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising a sequence of polynucleotides comprising (i) a sequence encoding AAV Cap proteins and (ii) a polyadenylation signal sequence; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload further comprise e) a fifth polynucleotide comprising a sequence encoding a viral associated RNA (VA-RNA). In certain embodiments, the VA-RNA is a mutated VA-RNA. In some embodiments, a second polynucleotide construct or a fourth comprises the fifth polynucleotide as described herein. [0319] In some embodiments, the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. [0320] In some embodiments, the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. [0321] In some embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter. In other embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter. In still other embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. [0322] In some embodiments, the VA-RNA comprises a G16A mutation. In other embodiments, the VA-RNA comprises a G60A mutation. In still other embodiments, the VA-RNA comprises a G16A mutation and a G60A mutation. Selectable Markers for Polynucleotides [0323] In some embodiments, a) the first polynucleotide further comprises a sequence encoding a first selectable marker operably linked to a first promoter. [0324] In some embodiments, b) the second polynucleotide further comprises a sequence encoding a second selectable marker operably linked to a second promoter. [0325] In some embodiments, c) the third polynucleotide further comprises a sequence encoding a third selectable marker operably linked to a third promoter. [0326] In some embodiments, d) the fourth polynucleotide further comprises a sequence encoding a fourth selectable marker operably linked to a fourth promoter. [0327] In some embodiments, e) the fifth polynucleotide further comprises a sequence encoding a fifth selectable marker operably linked to a fifth promoter. [0328] In some embodiments, the system of polynucleotides comprises any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker. [0329] In some embodiments, any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker are different selectable markers; the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, the fifth selectable marker, or any combinations thereof are the same selectable marker but as a different split portion of the selectable marker; or any combinations thereof. [0330] In some embodiments, any combinations of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are the same constitutive promoter or different constitutive promoters. [0331] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is an antibiotic resistance gene. In certain embodiments, the antibiotic resistance gene is a blasticidin resistance gene, a hygromycin resistance gene, or a puromycin resistance gene. [0332] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a first split portion of an antibiotic resistance gene. In certain embodiments, the first split portion of the antibiotic resistance gene is a first split portion of the blasticidin resistance gene. [0333] In some embodiments, the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a second split portion of an antibiotic resistance gene. In certain embodiments, the second split portion of the antibiotic resistance gene is a second split portion of the blasticidin resistance gene. [0334] In some embodiments, the first promoter, the second promoter, the third promoter, the fourth promoter, the fifth promoter, or any combination thereof, is an EF1alpha promoter or an attenuated version thereof. In some embodiments, the attenuated version comprises a mutation in the TATA box. In certain embodiments, the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter. [0335] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5' ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof. In some embodiments, (i) the first polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker; (ii) the second polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter; (iii) the third polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter; and/or (iv) the fourth polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker. In some embodiments, the constitutive promoter is an EF1 alpha promoter and/or the split selectable marker is a split antibiotic resistance protein. In some embodiments, the constitutive promoter is an EF1 alpha promoter and/or the selectable marker is a first antibiotic resistance protein. In some embodiments, the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein. In some embodiments, the constitutive promoter is an EF1 alpha promoter. [0336] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5' ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof. In some embodiments, the spacer ranges in length from 500 base pairs to 5000 base pairs. Selectable Marker [0337] In some embodiments, suitable markers include genes which confer resistance to antibiotics or toxins, or sensitivity, or impart color, or change the antigenic characteristics when cells, which have been transfected with the nucleic acid constructs, are grown in an appropriate selective medium. Exemplary selectable marker genes include, without limitation, the neomycin resistance gene (neo encoding aminoglycoside phosphotransferase (APH)) that allows selection in mammalian cells by conferring resistance to G418 (Geneticin), the hygromycin-B resistance gene (hygB encoding hygromycin-B-phosphotransferase (HPH)) that confers resistance to hygromycin-B, the puromycin resistance gene (pac encoding puromycin-N-acetyltransferase) that confers resistance to puromycin, Zeocin resistance gene (Sh bla encodes a protein that binds to Zeocin) that prevents Zeocin from binding DNA and damaging it, and the blasticidin resistance gene (BSD) that confers resistance to blasticidin. In addition, dihydrofolate reductase (DHFR)-based methotrexate (MTX) selection or glutamine synthetase (GS)-based methionine sulfoximine (MSX) selection may be used in mammalian cells. Other suitable markers and selection methods are known to those of skill in the art. [0338] In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split selectable marker that allows for selection of cells retaining two different polynucleotides using a single selective pressure. In some embodiments, the antibiotic resistance protein is split into two portions that can associate to form a functional antibiotic resistance protein. A first portion of the antibiotic resistance protein is encoded by a first polynucleotide and a second portion of the antibiotic resistance protein is encoded by a second polynucleotide. In some embodiments, a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure is used. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form. For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein. Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288: 6202-6211 (2013); Stevens et al, PNAS 114: 8538-8543 (2017); Jillette et al., Nat Comm 10: 4968 (2019); US 2020/0087388 A1; and US 2020/0263197 A1.In some embodiments, a split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, a first portion of the antibiotic resistance protein is the N- terminal portion which is fused to a N-terminal intein at the C-terminus and a second portion of the antibiotic resistance protein is the C-terminal portion which is fused to a C-terminal intein at the N-terminus. When both portions are present, the N-terminal intein associates with the C- terminal intein resulting in excision of the inteins and splicing of the C-terminus of the N- terminal portion of the antibiotic resistance protein to the N-terminus of the C-terminal portion of the antibiotic resistance protein, thereby forming a functional antibiotic resistance proteins. [0339] In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter and DHFR Z-Nter. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. [0340] In certain embodiments, a split auxotrophic selection system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure can be used. One construct encodes the N-terminal fragment of mammalian dihydrofolate reductase (DHFR) fused to a leucine zipper peptide (“Nter-DHFR”). This N-terminal fragment is enzymatically nonfunctional. The other construct encodes the C-terminal fragment of DHFR fused to a leucine zipper peptide (“Cter-DHFR”). This C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, a functional DHFR enzyme complex is formed through association of the leucine zipper peptides. Both constructs can be stably retained in the genome of a DHFR null cell by growth in a medium lacking hypoxanthine and thymidine. Embodiments of Polynucleotide System [0341] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above. [0342] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above. [0343] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above. [0344] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above. [0345] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above. [0346] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA- RNA” above. [0347] In some embodiments, the system of polynucleotide comprises the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; and the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above. [0348] In some aspects, systems of polynucleotides comprising: (a) the polynucleotide of any one of of the embodiments disclosed here, wherein the polynucleotide is a first polynucleotide; and one or more of: b) a second polynucleotide comprising a sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload are provided. [0349] In some embodiments, the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide. [0350] In some embodiments, the second polynucleotide comprises an inducible promoter operably linked to the sequence encoding the AAV Cap proteins. [0351] In some embodiments, the inducible promoter is a second inducible promoter. [0352] In some embodiments, the second polynucleotide further comprises a selectable marker operably linked to a promoter. [0353] In some embodiments, the selectable marker is a second selectable marker and the promoter is a constitutive promoter, optionally wherein the constitutive promoter is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutation in the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter. [0354] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: an inducible promoter operably linked to a self- excising element, wherein the inducible promoter is a third inducible promoter; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the third inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins; a constitutive promoter operably linked to a sequence encoding an activator, [0355] wherein a cell comprising the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter, if present the second inducible promoter, or the third inducible promoter in absence of a first triggering agent; wherein in absence of activation of the first inducible promoter, if present the second inducible promoter, and the third inducible promoter, the cell does not express detectable levels of the Rep proteins or the Cap proteins from the first polynucleotide, if present the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins, and wherein the inducible recombinase is activated in the presence of a second triggering agent. [0356] In some embodiments, the one or more helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein. [0357] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises: a selectable marker or a second part or a first part of the split selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR); optionally, wherein the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146 or SEQ ID NO: 147. [0358] In some embodiments, the fourth polynucleotide comprising the sequence encoding the payload further comprises further comprises a spacer between the 5' ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the selectable marker and the 3' ITR, or a combination thereof; optionally wherein SEQ ID NO: 146 or SEQ ID NO: 147 comprises the fourth polynucleotide. [0359] In some embodiments, the third polynucleotide comprises a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA. [0360] In some embodiments, the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. [0361] In some embodiments, the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. [0362] In some embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. [0363] In some embodiments, the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. [0364] In some embodiments, (i) the first polynucleotide comprising the sequence encoding the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the split selectable marker is a split antibiotic resistance protein; (ii) if present, the second polynucleotide comprising the sequence encoding the AAV Cap proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally wherein the constitutive promoter is an EF1 alpha promoter and/or the selectable marker is a first antibiotic resistance protein; (iii) the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the fourth polynucleotide comprising the sequence encoding the payload comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter. 1.5. Polynucleotide Construct System [0365] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and one or more of: b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above are provided. [0366] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above. [0367] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above. [0368] In some aspects, systems of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. In some embodiments, the system of polynucleotide further comprises d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above. [0369] In some embodiments, the sequence of the first polynucleotide is separated from the sequence of the second polynucleotide by an intervening sequence. In certain embodiments, the intervening sequence comprises a transcriptional blocking element (TBE). [0370] In some embodiments, the first polynucleotide construct comprises a sequence encoding a single selectable marker. [0371] In some embodiments, (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker. In certain embodiments, the constitutive promoter is an EF1 alpha promoter and/or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein. [0372] In some embodiments, (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter. In certain embodiments, the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein. [0373] In some embodiments, (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker. In certain embodiments, the constitutive promoter is an EF1 alpha promoter and/or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein. [0374] In some embodiments, (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein; (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein. [0375] In some embodiments, the first antibiotic resistance protein is a blasticidin resistance protein. [0376] In some embodiments, the second antibiotic resistance protein is a puromycin resistance protein. AAV Rep Cap Construct(s) and Cap Construct [0377] Disclosed herein are polynucleotide constructs encoding for a AAV Rep protein and Cap protein, where Cap protein is expressed from a native promoter and polynucleotide constructs encoding for the Cap protein, where Cap protein is expressed from an inducible promoter. [0378] Disclosed herein is a polynucleotide construct encoding for a Rep and Cap protein, where Cap protein is expressed from an inducible promoter. [0379] Disclosed herein is a polynucleotide construct encoding for a Rep and Cap protein, where Cap protein is expressed from an inducible promoter and lacks expression from a native promoter. [0380] Provided herein is a first polynucleotide construct, which encodes for Rep and Cap proteins and comprises an excisable element within the Rep coding sequence. This first polynucleotide construct (Construct 1) is also referred to as a Rep Cap construct, and/or “AAV Rep Cap Construct.” In some embodiments, the elements of this first polynucleotide construct can be in one or more separate constructs. For example, the AAV Rep Cap Construct is Construct 1 of FIG.2A. [0381] Provided herein is a second polynucleotide construct, which encodes for the Cap proteins under the control of an inducible promoter. The second polynucleotide construct (Construct 2) is also referred to as a “Cap construct,” and/or “AAV Cap Construct.” For example, the AAV Cap Construct is Construct 2 of FIG.1A. FIG.1A shows two separate polynucleotide constructs that each express AAV Capsid proteins, thereby resulting in a higher AAV capsid proteins level as compared to those expressed by cells having a single polynucleotide construct for encoding AAV Capsid proteins. These cells are capable of conditionally producing recombinant AAV (rAAV) virions within which are packaged an expressible payload. [0382] In some embodiments, the level of AAV Capsids produced by the cells having two separate polynucleotide constructs expressing the AAV Capsids is at least 2-fold higher, e.g., at least 5-fold to at least 100-fold higher than level of AAV Capsids produced by cells not having two separate polynucleotide constructs expressing the AAV Capsids. For example, cells with two separate polynucleotide constructs encoding AAV Capsid proteins produce a level of AAV Capsids that is at least 10-fold higher, 20-fold higher, 30-fold higher, 35-fold higher, 50-fold higher, or 100-fold higher than the level of AAV Capsids produced by cells with only a single polynucleotide construct encoding the AAV Capsids. [0383] In some embodiments, the percent encapsidation of a payload in virus particles produced by the cells having two separate polynucleotide constructs expressing the AAV Capsids is at least 2-fold higher, e.g., at least 5-fold to at least 20-fold higher than the percent encapsidation of a payload in virus particles produced by cells not having two separate polynucleotide constructs expressing the AAV Capsids. [0384] In some embodiments, the Cap coding sequence is operably linked to a promoter. In some embodiments, the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to a promoter. In some embodiments, a single construct or separate constructs comprise these sequences, in any combination. In some embodiments, the promoter is an inducible promoter. In some embodiments, the inducible promoter comprises a tetracycline-inducible promoter, a cumate-inducible promoter, or a cumate-inducible promoter. In some embodiments, the promoter is a constitutive promoter, wherein the sequences coding for the one or more cap proteins are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequences coding for the one or more cap proteins are operably linked to the constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. [0385] The Cap protein encoding sequence provided in a separate polynucleotide construct can be expressed under the control of an inducible promoter. The Cap coding sequence may include the sequence coding for VP1, the sequence coding for VP2, and the sequence coding for VP3 are operably linked to the inducible promoter. [0386] In various embodiments, the Cap protein encoding sequence provided in a separate polynucleotide construct or in the same polynucleotide comprising the Rep coding sequence can be expressed under the control of an inducible promoter. In various embodiments, the Cap protein encoding sequence may be operably linked to a polyadenylation signal sequence. The polyA signal sequence may be a polyA signal sequence functional in the cells used for producing the rAAV. In some instances, the polyA signal sequence may be a bovine Growth Hormone polyA (bGH-PolyA) signal sequence, a SV40 polyA signal sequence, or a Rabbit Beta Globin PolyA signal sequence. [0387] The bGH-PolyA signal sequence may include a nucleotide sequence that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 151. [0388] In certain cases, the SV40 polyA signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 152. [0389] In some embodiments, the SV40 polyA sequence is shorter than SEQ ID NO: 152. In some embodiments, the SV40 polyA sequence is longer than SEQ ID NO: 152. [0390] In certain cases, the Rabbit Beta Globin signal sequence may include a nucleotide sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or 100% sequence identity to nucleotide sequence set forth in SEQ ID NO: 170. [0391] In various embodiments, the Cap protein is selected from the capsid of an avian AAV, bovine AAV, canine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV, and modifications, derivatives, or pseudotypes thereof. [0392] In some embodiments, the capsid is a capsid selected from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4- 1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68 (described in WO2020/033842, incorporated herein by reference in its entirety). The hu68 capsid is described in WO 2018/160582, incorporated herein by reference in its entirety. [0393] In some embodiments, the capsid is a derivative, modification, or pseudotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV 13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10 , AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16 or AAVhu68. [0394] In some embodiments, capsid protein is a chimera of capsid proteins from two or more serotype selected from AAV1, AAV2, rAAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, and AAV.HSC16 (described in WO2020/033842, incorporated herein by reference in its entirety). In certain embodiments, the capsid is an rh32.33 capsid, described in US Pat. No.8,999,678, incorporated herein by reference in its entirety. [0395] In particular embodiments, the capsid is an AAV1 capsid. In particular embodiments, the capsid is an AAV5 capsid. In particular embodiments, the capsid is an AAV9 capsid. [0396] In various embodiments, the first integrated construct further comprises a first mammalian cell selection element. [0397] In some embodiments, an inducible Rep and Cap construct is as shown in FIG.1A, FIG. 2A, or FIG.3A. In some embodiments, the inducible polynucleotide construct comprises one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence. Without excision of the excisable element, Rep is minimally expressed or not expressed at all. In some embodiments, the Rep polypeptide is a wildtype Rep polypeptide. In other embodiments, the Rep polypeptide is a mutant Rep polypeptide. In some embodiments, the Cap polypeptide is a wildtype Cap polypeptide. In other embodiments, the Cap polypeptide is a mutant Cap polypeptide. The intron may be spliced out by endogenous cellular machinery. [0398] In some embodiments, the excisable element is excised by a recombinase. A recombinase can be Cre. Cre may be provided as any form of exogenous Cre, such as Cre vesicles. Cre may also be encoded for by a third polynucleotide construct or by any separate polynucleotide construct. In some embodiments, a construct encoding for adenoviral helper proteins also encodes for Cre. In some embodiments, the third polynucleotide construct is also inducible, for example, as described below. In some embodiments, a construct encoding for Rep/Cap proteins also encodes for Cre. [0399] In some embodiments, expression of the Rep and Cap are driven by native promoters, including P5, P19, P40, or any combination thereof. In some embodiments, expression of the Rep and Cap are driven by inducible promoters. In some embodiments, expression of the Rep and Cap are driven by constitutive promoters. In some embodiments the exon of the excisable element may be any detectable marker. For example, detectable markers contemplated herein include luminescent markers, fluorescent markers, or radiolabels. Fluorescent markers include, but are not limited to, EGFP, GFP, BFP, RFP, or any combination thereof. [0400] In some embodiments, the Rep Cap construct is a polynucleotide construct comprising: a) a sequence of a first part of a Rep gene; b) sequence of a second part of the Rep gene; c) a sequence of a Cap gene; and d) an excisable element positioned between the first part of the sequence of Rep gene and the second part of the sequence of the Rep gene. In some embodiments, the excisable element comprises a stop signaling sequence. In some embodiments, the excisable element comprises a rabbit beta globin intron. In some embodiments, the excisable element comprises an exon. In some embodiments, the excisable element comprises an intron and an exon. In some embodiments, the excisable element comprises an intron. In some embodiments, two splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the two splice sites are a 5’ splice site and a 3’ splice site. In some embodiments, the 5’ splice site is a rabbit beta globin 5’ splice site. In some embodiments, the 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, three splice sites are positioned between the sequence of the first part of the Rep gene and the sequence of the second part of the Rep gene. In some embodiments, the three splice sites are a 5’ splice site, a first 3’ splice site, and a second 3’ splice site. In some embodiments, a first 3’ splice site is a duplicate of the second 3’ splice site. In some embodiments, the first 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, the second 3’ splice site is a rabbit beta globin 3’ splice site. In some embodiments, the excisable element comprises a recombination site. In some embodiments, the recombination site is a lox site or FRT site. In some embodiments, the lox site is a loxP site. In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first recombination site; c) the first 3’ splice site; d) a stop signaling sequence; e) a second recombination site; and f) the second 3’ splice site. In some embodiments, the excisable element comprises from 5’ to 3’: a) the 5’ splice site; b) a first spacer segment; c) a second spacer segment comprising: i) a first recombination site; ii) the first 3’ splice site; iv) a stop signaling sequence; and v) a second recombination site; and d) a third spacer segment comprising the second 3’ splice site. A recombinase recombines the first and second recombination sites. A transcript produced from the recombined sequence includes an intron flanked by the 5’ splice site and the second 3 splice site and is processed by the endogenous cellular machinery to produce a mRNA in which the intron has been spliced out. In some embodiments, the first spacer sequence comprises an intron. In some embodiments, the first spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the second spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the third spacer segment comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the third spacer segment comprises an intron. In some embodiments, the first spacer segment and the third spacer segment are capable of being excised by endogenous cellular machinery. In some embodiments, the second spacer segment comprises an exon. In some embodiments, the second spacer segment further comprises a polyA sequence. In some embodiments, the polyA sequence is 3’ of the exon. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. The polynucleotide construct of any one of the embodiments disclosed herein, wherein the second spacer segment comprises from 5’ to 3’: a) a first recombination site; b) the first 3’ splice site; c) an exon; d) a stop signaling sequence; and e) a second recombination site. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP sequence and a second lox sequence is a second loxP sequence. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the stop signaling sequence is a termination codon of the exon or a polyA sequence. In some embodiments, the polyA sequence comprises a rabbit beta globin (RBG) polyA sequence. In some embodiments, the exon encodes a detectable marker or a selectable marker. In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, the second spacer segment is excisable by a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a ER2 Cre polypeptide. In some embodiments, the recombinase is encoded by a third polynucleotide construct or exogenously provided. In some embodiments, the Rep gene codes for Rep polypeptides. In some embodiments, the Cap gene codes for Cap polypeptides. In some embodiments, transcription of the Rep gene and the Cap gene are driven by native promoters. In some embodiments, the native promoters comprise P5, P19, and P40. In some embodiments, transcription of the Rep gene and/or the Cap gene are driven by inducible promoters. In some embodiments, the Rep polypeptides are wildtype Rep polypeptides. In some embodiments, the Rep polypeptides comprise Rep78, Rep68, Rep52, and Rep40. In some embodiments, a truncated replication associated protein comprising a polypeptide expressed from the sequence of first part of a Rep gene and the exon is capable of being expressed in the absence of the recombinase. In some embodiments, the Cap polypeptides are wildtype Cap polypeptides. In some embodiments, the Cap polypeptides are AAV capsid proteins. In some embodiments, the AAV capsid proteins comprise VP1, VP2, and VP3. In some embodiments, a serotype of the AAV capsid proteins is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68. [0401] In some embodiments, the Rep Cap construct further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0402] In some embodiments, the Rep Cap construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the Rep Cap construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the Rep Cap construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0403] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, a selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0404] In some embodiments, the Rep Cap construct is in a vector. In some embodiments, the Rep Cap construct is in a plasmid. In some embodiments, the Rep Cap construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, the Rep Cap construct is a synthetic nucleic acid construct. In some embodiments, the Rep Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, or any combination thereof. In some embodiments, the Rep Cap construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, or any combination thereof. In some embodiments, the Rep Cap construct comprises SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. In some embodiments, the Rep Cap construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 1 – SEQ ID NO: 3, SEQ ID 6 – SEQ ID NO: 8, SEQ ID NO: 32, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, or SEQ ID NO: 136 – SEQ ID NO: 138, but wherein the Rep Cap construct lacks SEQ ID NO: 145 downstream of the sequence encoding the AAV Cap proteins. [0405] In some embodiments, the Rep Cap construct further comprises a sequence coding for VA-RNA. In some embodiments, a sequence coding for VA-RNA is in separate construct or in any separate construct coding for an element of the Rep Cap construct. In some embodiments, a payload construct comprises a polynucleotide construct coding for a VA-RNA. In some embodiments, the VA-RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the sequence coding for VA-RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA-RNA comprises at least two mutations in the internal promoter. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the sequence coding for VA- RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. [0406] In some embodiments the gene codes for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0407] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0408] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0409] A major advantage of the inducible polynucleotide constructs disclosed herein encoding for Rep and Cap include that upon stable integration into a mammalian cell line, expression of Rep and Cap is inducible even in the absence of a transfection agent or a plasmid. In some embodiments, the stable cell line populations disclosed herein are homogeneous. For example, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the stable cell population comprises the stably integrated polynucleotide construct encoding for Rep and Cap proteins. [0410] The second polynucleotide construct may include a Cap protein encoding sequence identical or substantially identical to the Cap protein encoding sequence present in the Rep/Cap encoding polynucleotide construct. The second polynucleotide construct may include a Cap protein encoding sequence identical or substantially identical to the Cap protein encoding sequence present in the Rep/Cap encoding polynucleotide construct, but the Cap protein encoding sequence is driven by an inducible promoter. The second polynucleotide construct may be referred to as an inducible Cap construct. The inducible promoter may be any inducible promoter that is described herein in the below Adenoviral Helper Construct(s) section and is activated by any triggering agent and activator as described herein in the below Adenoviral Helper Construct(s) section. In some embodiments, the inducible promoter is a Tet inducible promoter. In some embodiments, the Tet inducible promoter is activated when bound to Tet responsive activator protein, e.g., Tet-on 3G, in the presence of a trigger agent, such as doxycycline. In some embodiments, a polynucleotide encoding a sequence comprising an inducible promoter Cap protein sequence may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 148. The second polynucleotide construct may be provided as a plasmid. The plasmid may have at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 149. The second polynucleotide construct may be provided as a vector. Adenoviral Helper Construct(s) [0411] In some aspects, provided herein is a third polynucleotide construct (also referred to as Construct 3), which encodes for one or more adenoviral helper proteins. Construct 3 is also referred to as Ad helper/Cre construct. This third polynucleotide construct is also referred to as an inducible helper construct (e.g., Adenoviral Helper Construct provides one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) to be used in production of rAAV virions. In some embodiments, the sequence encoding E4 is a sequence encoding E4orf6. In some embodiments, the elements of an inducible helper construct (e.g., one or more helper proteins selected from E1A, E1B, E2A and E4 absent from a host cell) are in one or more separate constructs to be used in production of rAAV virions. In some embodiments, the host cell provides, one, two, or three of the four helper proteins. For example, for a host cell expressing E1A and E1B, the adenoviral helper construct provides E2A and E4. For a host cell expressing E2A and E4, the adenoviral helper construct provides E1A and E1B. For a host cell expressing E1B, the adenoviral helper construct provides E1A, E2A and E4. For a host cell expressing E2A, the adenoviral helper construct provides E1B, E1A and E4. For a host cell expressing E4, the adenoviral helper construct provides E1B, E2A and E1A. For a host cell expressing E1A, E2A and E4, the adenoviral helper construct provides E1B. For a host cell expressing E1B, E1A and E4, the adenoviral helper construct provides E2A. For a host cell expressing E1B, E2A and E1A, the adenoviral helper construct provides E4. In some embodiments, E4 is E4orf6. [0412] In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to separate promoters. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked to one promoter. In some embodiments, the sequences coding for E1A, E1B, E2A, and E4 are operably linked, in any combination, to one promoter or separate promoters. The separate promoters can be the same promoters or different promoters. A combination of the separate promoters and the one promoter can be the same promoters or different promoters. The one promoter can be a native promoter, a constitutive promoter, or an inducible promoter. The separate promoters can be native promoters, constitutive promoters, inducible promoters, or any combination thereof. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, a cumate-inducible promoter, or an ecdysone-inducible promoter. For example, some the sequence coding for E1A and E1B are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1A and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E2A are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E1B and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequence coding for E2A and E4 are separated by an IRES sequence of P2A sequence and are operably linked to one promoter. In some embodiments, the sequences coding for the helper proteins are in different orientations. In some embodiments, the sequences coding for the helper proteins are bidirectional. In some embodiments, the E1A is operably linked to a natural or constitutive promoter, E1B is operably linked to a natural or constitutive promoter, and E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the constitutive promoter. In some embodiments, the E1A is operably linked to a natural or constitutive promoter, E1B is operably linked to a natural or constitutive promoter, and E2A and E4 are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E2A and E4 are operably linked to the inducible promoter. In some embodiments, the E2A is operably linked to a natural or constitutive promoter, E4 is operably linked to a natural or constitutive promoter, and E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the constitutive promoter. In some embodiments, the E2A is operably linked to a natural or constitutive promoter, E4 is operably linked to a natural or constitutive promoter, and E1A and E1B are downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and inducible promoter, wherein upon excision of the excisable element (e.g., by a recombinase), E1A and E1B are operably linked to the inducible promoter. [0413] In certain embodiments, the adenoviral helper construct provides inducible production of one or more of the helper proteins. In some embodiments, an adenoviral helper protein further comprises a protein tag. A protein tag can be a FLAG tag. In some embodiments, E2A is a FLAG tagged E2A. In some embodiments, E4 is a FLAG tagged E4. A protein tag, such as a FLAG tag, can be used to screen for or to confirm integration of the third polynucleotide construct and expression of the adenoviral helper protein from the third polynucleotide construct in a cell after induction. [0414] In some embodiments, the third integrated synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the third synthetic construct comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate integrated constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the one or more separate constructs comprises conditionally expressible recombinase and conditionally expressible adenovirus helper proteins. In some embodiments, the third integrated synthetic construct comprises conditionally expressible Cre recombinase and conditionally expressible adenovirus helper proteins. In the exemplary embodiments illustrated in FIG.1A and FIG.2A, prior to the cell being contacted with at least a third triggering agent, the third integrated construct comprises, from 5’ to 3’: an inducible promoter, a Cre coding sequence, a first polyA sequence, adenoviral helper protein coding sequences, a second polyA sequence, a constitutive promoter, a coding sequence for a protein that is responsive to the first triggering agent, and a second mammalian cell selection element. [0415] In typical embodiments, the Cre coding sequence is operatively linked to the inducible promoter. Various inducible promoters suitable for controlling expression of the Cre coding sequence and the Cap proteins coding sequences are described throughout the specification, e.g., in the section “Inducible Promoters”. In various embodiments, the inducible promoter comprises an element responsive to the third triggering agent. In some embodiments, the inducible promoter contains a regulatory sequence that allows for control of the promoter. The regulatory sequence can be operably linked to the promoter and positioned upstream of the promoter. Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. The regulatory sequence used to control expression may be endogenous or exogenous to the host cell. In some embodiments, bacterial gene control elements in combination with viral transactivator proteins are used to provide mammalian inducible expression. Examples of mammalian-compatible regulatory sequences include those capable of controlling an engineered promoter to adjust transcription in response to antibiotics including, without limitation, tetracyclines, streptogramins, and macrolides. For a description of various inducible expression systems, see, e.g., Weber et al. (2004) Methods Mol. Biol.267:451-66, Das et al. (2016) Curr. Gene Ther.16(3):156-67, Chruscicka et al. (2015) J. Biomol. Screen.20(3):350-8, Yarranton (1992) Curr. Opin. Biotechnol.3(5):506-11, Gossen & Bujard (1992) Proc. Natl.Acad. Sci. U.S.A.89(12):5547-51, Gossen et al. (1995) Science 268(5218):1766-9; herein incorporated by reference. [0416] In some embodiments, a bacterial tetracycline response element (TRE) is included in a construct to allow mammalian expression to be induced by tetracycline or a derivative thereof (e.g., doxycycline). In certain embodiments, the inducible promoter comprises a plurality of tetracycline (Tet) operator elements capable of binding to a Tet responsive activator protein in the presence of a tetracycline. In some embodiments, the plurality of tetracycline (Tet) operator elements form a Tetracycline Responsive element (TRE). In some embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence. In further embodiments, the TRE comprises seven repeats of a 19 base pair operator sequence upstream of a minimal CMV promoter sequence. [0417] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0418] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPADALDDFD LDMLPADALDDFDLDMLPG (SEQ ID NO:40). [0419] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0420] ATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCA ATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTT GAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCT GCCAATCGAGATGCTGGACAGGCATCATACCCACTTCTGCCCCCTGGAAGGCGAGT CATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATTCCGCTGTGCTCTCCTCTCAC ATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGA AACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACG CACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAAC AGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCC CCCACTTCTGAGACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCT TCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAA AGCGGCGGGCCGGCCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGA TGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGAC CTTGACATGCTCCCCGGGTAA (SEQ ID NO:69). [0421] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0422] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCACTTTACTTTAGGTTGCGTATTGGAAGAACAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCGCCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 70). [0423] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0424] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 71). [0425] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0426] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACGGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 72). [0427] In some embodiments, a Tet responsive activator protein encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0428] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACGGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT ACCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCACTTTACTTTAGGTTGCGTATTGGAAGAACAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCGCCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 73). [0429] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0430] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAGCAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGGTGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 74). [0431] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0432] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATAGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAA ACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCA TTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAA GAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCC ATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTT ATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTG GGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGC CTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCG CCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGA TGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATG CCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGT CCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTC GAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 75). [0433] In some embodiments, a Tet responsive activator protein is encoded a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0434] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGAACATTTAGGTACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCA AGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGC CATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCT TATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGT GGGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGG CCTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGC GCCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCG ATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCAT GCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGG TCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTT CGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 76). [0435] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0436] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGGGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGTGCTTTACTAAGTCATC GCGATGGAGCAAAAGTACATTTAGGTACACGGCCTACAGAAAAACAGTATGAAACT CTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGCATTA TATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCAAGAG CATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGCCATT ATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCTTATT CGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGTGGGT CCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGGCCTG CTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGCGCCT GTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCGATGT CAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCATGCCG ACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGGTCCG GGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTTCGAG TTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 77). [0437] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0438] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGAACATTTAGGTACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCA AGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGC CATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCT TATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTAAAAGT GGGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGG CCTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGC GCCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCG ATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCAT GCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGG TCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTT CGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 78). [0439] In some embodiments, a Tet responsive activator protein is encoded by a nucleic acid sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the nucleotide sequence: [0440] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACATTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAGAAGGGGAAA GCTGGCAAGATTTTTTACGTAATAACGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCGATGGAGCAAAAGAACATTTAGGTACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCA AGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGC CATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCT TATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTAAAAGT GGGTCCGCGTACAGCCGCGCGCGTACGAAAAACAATTACGGGTCTACCATCGAGGG CCTGCTCGATCTCCCGGACGACGACGCCCCCGAAGAGGCGGGGCTGGCGGCTCCGC GCCTGTCCTTTCTCCCCGCGGGACACACGCGCAGACTGTCGACGGCCCCCCCGACCG ATGTCAGCCTGGGGGACGAGCTCCACTTAGACGGCGAGGACGTGGCGATGGCGCAT GCCGACGCGCTAGACGATTTCGATCTGGACATGTTGGGGGACGGGGATTCCCCGGG TCCGGGATTTACCCCCCACGACTCCGCCCCCTACGGCGCTCTGGATATGGCCGACTT CGAGTTTGAGCAGATGTTTACCGATGCCCTTGGAATTGACGAGTACGGTGGGTAG (SEQ ID NO: 79). [0441] In some embodiments, a Tet responsive activator protein is a variant of a Tet responsive activator protein and comprises the sequence set forth in SEQ ID NO:40 but with the following amino acid substitutions:86Y A209T; V9I F86Y A209T; F67S F86Y A209T; G138D F86Y A209T; E157K F86Y A209T; R171K F86Y A209T; V9I G138D F86Y A209T; V9I E157K F86Y A209T; V9I R171K F86Y A209T; F67S R171K F86Y A209T; V9I F67S F86Y A209T; F67S G138D F86Y A209T; F67S E157K F86Y A209T; V9I F67S G138D F86Y A209T; V9I F67S E157K F86Y A209T; V9I F67S R171K F86Y A209T; V9I G138D E157K F86Y A209T; V9I G138D R171K F86Y A209T; F86Y; F86Y A209T; F67S F86Y A209T; G138d F86Y A209T; E157K F86Y A209T; R171K F86Y A209T; V9I G138D F86Y A209T; V9I E157K F86Y A209T; V9I R171K F86Y A209T; F177L F86Y A209T; F67S F177L F86Y A209T; C195S F86Y A209T; G138S F86Y A209T; C68R F86Y A209T; V9I F67S F86Y A209T; F67S G138D F86Y A209T; F67S E157K F86Y A209T; F67S R171K F86Y A209T; V9I F67S G138D F86Y A209T; V9I F67S E157K F86Y A209T; V9I F67S R171K F86Y A209T; V9I G138D E157K F86Y A209T; V9I G138D R171K F86Y A209T; S12G F67S F86Y A209T; G19M F67S F86Y A209T; E37Q F67S F86Y A209T; C68R G138D F86Y A209T; G19M G138D F86Y A209T; E37Q G138D F86Y A209T; V9I C68R G138D F86Y A209T; V9I G19M G138D F86Y A209T; V9I E37Q G138D F86Y A209T; F67S; G138D ; E157K ; R171K ; V9I G138D; V9I E157K ; V9I R171K; F177L ; F67S F177L ; C195S; G138S; C68R; V9I F67S ; F67S G138D ; F67S E157K ; F67S R171K; V9I F67S G138D; V9I F67S E157K ; V9I F67S R171K ; V9I G138D E157K ; V9I G138D R171K ; S12G F67S; G19M F67S ; E37Q F67S; V9I C68R G138D; V9I G19M G138D; V9I E37Q G138D; V9I G19M F67S G138D; V9I S12G F67S G138D; V9I F67S C68R G138D; F67S F86Y; G138D F86Y; E157K F86Y; R171K F86Y; V9I G138D F86Y; V9I E157K F86Y; V9I R171K F86Y; F177L F86Y; F67S F177L F86Y; C195S F86Y; G138S F86Y; C68R F86Y; V9I F67S F86Y; F67S G138D F86Y; F67S E157K F86Y; F67S R171K F86Y; V9I F67S G138D F86Y; V9I F67S E157K F86Y; V9I F67S R171K F86Y; V9I G138D E157K F86Y; V9I G138D R171K F86Y; S12G F67S F86Y; G19M F67S F86Y; E37Q F67S F86Y; V9I C68R G138D F86Y; V9I G19M G138D F86Y; V9I E37Q G138D F86Y; V9I G19M F67S G138D F86Y; V9I S12G F67S G138D F86Y; V9I F67S C68R G138D F86Y; F67S A209T; G138D A209T; E157K A209T; R171K A209T; V9I G138D A209T; V9I E157K A209T; V9I R171K A209T; F177L A209T; F67S F177L A209T; C195S A209T; G138S A209T; C68R A209T; V9I F67S A209T; F67S G138D A209T; F67S E157K A209T; F67S R171K A209T; V9I F67S G138D A209T; V9I F67S E157K A209T; V9I F67S R171K A209T; V9I G138D E157K A209T; V9I G138D R171K A209T; S12G F67S A209T; G19M F67S A209T; E37Q F67S A209T; V9I C68R G138D A209T; V9I G19M G138D A209T; V9I E37Q G138D A209T; V9I G19M F67S G138D A209T; V9I S12G F67S G138D A209T; V9I F67S C68R G138D A209T; G19M F67S V9I G138D F86Y A209T; S12G F67S V9I G138D F86Y A209T; or C68R F67S V9I G138D F86Y A209T;, where the numbering of the substituted amino acids is based on the numbering of amino acids in SEQ ID NO:40. [0442] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0443] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:41). [0444] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0445] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:42). [0446] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0447] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:43). [0448] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0449] MSRLDKSKVINSALELLNEVGIEGLATRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRGGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:44). [0450] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0451] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDRAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO:45). [0452] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0453] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKEHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 80). [0454] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0455] MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFSALLSHRDGAKVHLGTRPTEKQYET LENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLLR QAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 81). [0456] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0457] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKEHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCKSGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 82). [0458] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0459] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLKGESWQDFLRNNAKSFRCALLSHRNGAKVHSDTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 83). [0460] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0461] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPD DDAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFD LDMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 84). [0462] In some embodiments, a Tet responsive activator protein comprises an amino acid sequence having at least 90% identity to the amino acid sequence: [0463] MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTTDSMPPLL RQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGSAYSRARTKNNYGSTIEGLLDLPDD DAPEEAGLAAPRLSFLPAGHTRRLSTAPPTDVSLGDELHLDGEDVAMAHADALDDFDL DMLGDGDSPGPGFTPHDSAPYGALDMADFEFEQMFTDALGIDEYGG (SEQ ID NO: 85). [0464] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0465] GAATTCCTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCG AGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCT ATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGA AAAGTGAAAGTCGAGTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGA GTTTACCACTCCCTATCAGTGATAGAGAAAAGTGAAAGTCGAGTTTACCACTCCCTA TCAGTGATAGAGAAAAGTGAAAGTCGAGCTCGGTACCCGGGTCGAGTAGGCGTGTA CGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAG ACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCC GCGG (SEQ ID NO: 46). [0466] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0467] GAATTCCTCGACCCGGGTACCGAGCTCGACTTTCACTTTTCTCTATCACTGAT AGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACT CGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCT CTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGG AGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGAC TTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGAGTAGGCGTGTACGGTG GGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCC ATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGG (SEQ ID NO: 47). [0468] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0469] GAGCTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTT TCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCA CTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGT AAACTCGACTTTCACTTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCAC TTTTCTCTATCACTGATAGGGAGTGGTAAACTCGACTTTCACTTTTCTCTATCACTGA TAGGGAGTGGTAAACTCGAGATCCGGCGAATTCGAACACGCAGATGCAGTCGGGGC GGCGCGGTCCGAGGTCCACTTCGCATATTAAGGTGACGCGTGTGGCCTCGAACACC GAG (SEQ ID NO: 48). [0470] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0471] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTC CATAGAAGA (SEQ ID NO: 50). [0472] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0473] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGA (SEQ ID NO: 51). [0474] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0475] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGGTAATCAACTACCAATTC CAGCTCTCTTTTGACAACTGGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCGT AAGACAATTGCAA (SEQ ID NO: 52). [0476] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0477] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTG TCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAA (SEQ ID NO: 53). [0478] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0479] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAA AGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCTAATCAACTACCAATTC CAGCTCTCTTTTGACAACTGGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCCT AAGACAATTGCAAA (SEQ ID NO: 54). [0480] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0481] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTGCCTATGTTCTTTTGGAATCTATCCAAG TCTTATGTAAATGCTTATGTAAACCATAATATAAAAGAGTGCTGATTTTTTGAGTAA ACTTGCAACAGTCCTAACATTCTTCTCTCGTGTGTTTGTGTCTGTTCGCCATCCCGTC TCCGCTCGTCACTTATCCTTCACTTTTCAGAGGGTCCCCCCGCAGATCCCGGTCACC CTCAGGTCGG (SEQ ID NO: 55). [0482] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0483] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTCCATAATATAAAAGAGTGCTGATTTTTT GAGTAAACTTGCAACAGTCCTAACATTCTTCTCTCGTGTGTTTGTGTCTGTTCGCCAT CCCGTCTCCGCTCGTCACTTATCCTTCACTTTTCAGAGGGTCCCCCCGCAGATCCCG GTCACCCTCAGGTCGG (SEQ ID NO: 56). [0484] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0485] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTCCAGGGCGCCTATAAAAGAGTGCTGAT TTTTTGAGTAAACTTGCAACAGTCCTAACATTCTTCTCTCGTGTGTTTGTGTCTGTTC GCCATCCCGTCTCCGCTCGTCACTTATCCTTCACTTTTCAGAGGGTCCCCCCGCAGAT CCCGGTCACCCTCAGGTCGG (SEQ ID NO: 57). [0486] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0487] TTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTG ATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGT TTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGA GAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTC CCTATCAGTGATAGAGAACGTATAAGCTTTGCTTATGTAAACCAGGGCGCCTATAA AAGAGTGCTGATTTTTTGAGTAAACTTCAATTCCACAACACTTTTGTCTTATACCAA CTTTCCGTACCACTTCCTACCCTCGTAAA (SEQ ID NO: 58). [0488] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0489] GAATTCTTTACTCCCTATCAGTGATAGAGAATGTATGAAGAGTTTACTCCCTA TCAGTGATAGAGAATGTATGCAGACTTTACTCCCTATCAGTGATAGAGAATGTATA AGGAGTTTACTCCCTATCAGTGATAGAGAATGTATGACCAGTTTACTCCCTATCAGT GATAGAGAATGTATCTACAGTTTACTCCCTATCAGTGATAGAGAATGTATATCCAGT TTACTCCCTATCAGTGATAGAGAATGTATAAGCTTTAGG (SEQ ID NO: 59). [0490] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0491] CATGTACAGTGGGCACCTATAAAAGCAGAGCTCATTTAGTGAACTGTCAGAT TGCCTGGAGCAATTCCACAACACTTTTGTCTTATACCAACTTTCCATACCACTTCCTA CCCTCATAAAGTGCACACCATGG (SEQ ID NO: 60). [0492] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0493] CCATGGTGTGCACTTTATGAGGGTAGGAAGTGGTATGGAAAGTTGGTATAAG ACAAAAGTGTTGTGGAATTGCTCCAGGCAATCTGACAGTTCACTAAATGAGCTCTG CTTTTATAGGTGCCCACTGTACATGCCTAAGAATTCTTTACT (SEQ ID NO: 61). [0494] In some embodiments, a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0495] CCCTATCAGTGATAGAGAATGTATGAAGAGTTTACTCCCTATCAGTGATAGA GAATGTATGCAGACTTTACTCCCTATCAGTGATAGAGAATGTATAAGGAGTTTACTC CCTATCAGTGATAGAGAATGTATGACCAGTTTACTCCCTATCAGTGATAGAGAATGT ATCTACAGTTTACTCCCTATCAGTGATAGAGAATGTATATCCAGTTTACTCCCTATC AGTGATAGAGAATGTATAAGCTTTAGG (SEQ ID NO: 62). [0496] In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0497] GGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACC GTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGG GACCGATCCAGCCTCCGCGG (SEQ ID NO: 63). [0498] In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0499] GGTAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCG TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGG ACCGATCCAGCCTCCGCG (SEQ ID NO: 64). [0500] In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0501] TAGGCGTGTACGGTGGGCGCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTC AGATCGCCTGGAGA (SEQ ID NO: 65). [0502] In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0503] GTAATCAACTACCAATTCCAGCTCTCTTTTGACAACTGGTCTTATACCAACTT TCCGTACCACTTGCAACCCTCGTAAGACAATTGCAA (SEQ ID NO: 66). [0504] In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0505] AATTCCACAACACTTTTGTCTTATACCAACTTTCCGTACCACTTCCTACCCTC GTAAA (SEQ ID NO: 67). [0506] In some embodiments, a minimal promoter of a TRE comprises a nucleotide sequence having at least 80% identity (e.g., at least 85%, at least 90%, at least 95%, or a 100% identity) to the nucleotide sequence: [0507] GCCTATGTTCTTTTGGAATCTATCCAAGTCTTATGTAAATGCTTATGTAAACC ATAATATAAAAGAGTGCTGATTTTTTGAGTAAACTTGCAACAGTCCTAACATTCTTC TCTCGTGTGTTTGTGTCTGTTCGCCATCCCGTCTCCGCTCGTCACTTATCCTTCACTTT TCAGAGGGTCCCCCCGCAGATCCCGGTCACCCTCAGGTCGG (SEQ ID NO: 68). [0508] In some embodiments, a Tet Repressor binding protein may comprise a sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0509] MSRLDKSKVINSALELLNEVGIEGLTTRKLAQKLGVEQPTLYWHVKNKRALLD ALAIEMLDRHHTHFCPLKGESWQDFLRNKAKSFRCALLSHRNGAKVHSDTRPTEKQYE TLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEDQEHQVAKEERETPTTDSMPPLL RQAIELFDHQGAEPAFLFGLELIICGLEKQLKCESGS (SEQ ID NO: 49). [0510] In some embodiments, a Tet Repressor binding protein may comprise a sequence having at least 90% identity (e.g., at least 95% or 100% identity) to the amino acid sequence: [0511] ATGTCTAGATTAGATAAAAGTAAAGTGATTAACAGCGCATTAGAGCTGCTTA ATGAGGTCGGAATCGAAGGTTTAACAACCCGTAAACTCGCCCAGAAGCTAGGTGTA GAGCAGCCTACACTGTATTGGCATGTAAAAAATAAGCGGGCTTTGCTCGACGCCTT AGCCATTGAGATGTTAGATAGGCACCATACTCACTTTTGCCCTTTAAAAGGGGAAA GCTGGCAAGATTTTTTACGCAATAAGGCTAAAAGTTTTAGATGTGCTTTACTAAGTC ATCGCAATGGAGCAAAAGTACATTCAGATACACGGCCTACAGAAAAACAGTATGA AACTCTCGAAAATCAATTAGCCTTTTTATGCCAACAAGGTTTTTCACTAGAGAATGC ATTATATGCACTCAGCGCTGTGGGGCATTTTACTTTAGGTTGCGTATTGGAAGATCA AGAGCATCAAGTCGCTAAAGAAGAAAGGGAAACACCTACTACTGATAGTATGCCGC CATTATTACGACAAGCTATCGAATTATTTGATCACCAAGGTGCAGAGCCAGCCTTCT TATTCGGCCTTGAATTGATCATATGCGGATTAGAAAAACAACTTAAATGTGAAAGT GGGTCC (SEQ ID NO: 86). [0512] In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-86, or variants thereof. [0513] The Tet responsive activator protein or variant thereof, the Tet Repressor binding protein or variant thereof, TRE sequence, or any tetracycline-inducible promoter sequence or variant thereof can be any of those disclosed in US 7,541,446; US 8,383,364; US 6,136,954; US 5,814,618; US 6,271,348; US 5,789,156; US 7,666,668; US 6,914,124; US 5,650,298; US 5,922,927; US 5,464,758; US 5,866,755; US 5,589,362; US 5,654,168; US 6,242,667; US 5,912,411; US 6,783,756; US 5,888,981; US 6,004,941; US 6,252,136; US 5,859,310; US 6,271,341; US 6,087,166; US2003022315; US20050037335; US 9,181,556; and WO03056021, which are each herein incorporated by reference in their entirety. [0514] Any of the proteins described herein may be expressed from a nucleotide sequence that has been codon-optimized to increase expression in a host cell, e.g., a mammalian cell or a human cell line. [0515] In other embodiments, an insect gene control element is used to provide mammalian inducible expression. For example, an ecdysone-responsive element and a gene encoding the ecdysone receptor can be included in a construct to allow mammalian expression to be induced by the insect hormone ecdysone or analogs or derivatives thereof, such as ponasterone. In mammalian cells, the ecdysone receptor heterodimerizes with the retinoid X receptor (RXR). The ecdysone-responsive element comprises a binding site for the RXR-ecdysone receptor heterodimer, which is typically a synthetic recognition site for the heterodimer that preferably does not bind any endogenous transcription factors or natural nuclear hormone receptors. In the presence of ecdysone or an analog or derivative thereof, the RXR-ecdysone receptor heterodimer binds to the ecdysone-responsive element to activate transcription from the promoter. For a description of ecdysone-responsive promoters, see, e.g., No et al. (1996) Proc. Natl. Acad. Sci. USA 93(8):3346-51, Oehme et al. (2006) Cell Death and Differentiation (2006) 13:189-201; herein incorporated by reference. [0516] In some embodiments, the third construct or an additional separate construct comprises an element responsive to a fourth triggering agent. In certain embodiments, the fourth triggering agent-responsive element comprises a plurality of hormone-response elements. In particular embodiments, the hormone-response elements are estrogen responsive elements (EREs). In various embodiments, the third triggering agent is the same as the first triggering agent, and the fourth triggering agent is the same as the second triggering agent. [0517] In some embodiments, the inducible promoter comprises a plurality of Tet operator elements capable of binding to a Tet responsive activator protein in the presence of a third triggering agent. In particular embodiments, the third triggering agent is the same as the first triggering agent. [0518] In some embodiments, the recombinase coding sequence is flanked by a first recombinase site and a second recombinase site. In some embodiments, the recombinase is Cre. In some embodiments, the Cre coding sequence is flanked by a first lox site and a second lox site. In some embodiments, the first polyA sequence is positioned between the Cre coding sequence and adenoviral helper protein coding sequences that encode one or both of adenovirus E2A and E4. The strong 3’ polyadenylation signal positioned upstream (5’ to) the coding sequences for the adenovirus helper proteins prevents basal expression of the downstream adenoviral helper genes, E2A and E4. [0519] In some embodiments, helper construct does not comprise a recombinase that is self- excising. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre- ERT2 polypeptide. In some embodiments, the recombinase translocates to the nucleus in the presence of a triggering agent. In some embodiments, the triggering agent is an estrogen receptor ligand. In some embodiments, the triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the triggering agent is tamoxifen. When the recombinase is not self-excisable, the sequence or sequences encoding one or more helper proteins can be operably linked to an inducible promoter. When the recombinase is not self-excisable, the sequence or sequences encoding one or more helper proteins can be downstream of an excisable element (e.g., a sequence flanked by recombination sites and comprising a stop signal) and constitutive promoter, wherein upon excision of the excisable element (e.g., by a recombinase), the sequence or sequences encoding one or more helper proteins are operably linked to the constitutive promoter. [0520] In some embodiments, the further segment shown in FIG.1A provides for inducible production of VA-RNA from construct 3. [0521] In this embodiment, the further segment includes a Cre-inducible U6 or U7 promoter. The U6 or U7 promoter is split into 2 parts separated by a Lox flanked stuffer sequence. The U6 or U7 promoter is inactive because of the presence of the stuffer sequence. Cre mediated excision of the stuffer activates the U6 or U7 promoter. The U6 or U7 promoter drives the expression of transcriptionally dead mutants of VA-RNA1 (a preferred embodiment is a double point mutant G16A- G60A). Other embodiments provide for alternative sources of VA-RNA. [0522] In various embodiments, the coding sequence for the first triggering agent-responsive protein is operatively linked to a CMV promoter. In some embodiments, the coding sequence for the first triggering agent-responsive protein comprises a coding sequence for the Tet responsive activator protein. In particular embodiments, the Tet responsive activator protein is Tet-on 3G activator protein. [0523] In various embodiments, the second mammalian cell selection element confers antibiotic resistance. In particular embodiments, the antibiotic resistance conferring element is a blasticidin resistance gene. [0524] Multiple inducible helper polynucleotide constructs are contemplated herein. In some embodiments, the elements of the inducible helper polynucleotide constructs can be in one or more separate constructs. In some embodiments, said inducible helper polynucleotide constructs encode for one or more adenoviral helper proteins, such as VA-RNA, E2A, E4, or any combination thereof. In some embodiments, the present disclosure provides for an inducible polynucleotide construct encoding for a mutated VA-RNA gene sequence. In some embodiments, the mutations to VA-RNA render its internal promoters inactive. For example, the inducible helper polynucleotide construct may comprise from 5’ to 3’ a first part of a U6 promoter sequence, a first lox sequence, a stuffer sequence, a second lox sequence, and a second part of a U6 promoter sequence. The stuffer sequence may be any polynucleotide sequence and is excised by Cre. Cre may be exogenously provided, such as in the form of Cre gesicles. Cre may also be encoded for in the same inducible helper polynucleotide construct and expression of Cre may be conditioned on the presence of at least two triggering agents, such as doxycycline and tamoxifen. Cre may be a hormone activated Cre. [0525] In other embodiments, instead of a mutated VA-RNA gene sequence, the inducible helper constructs may comprise a constitutively expressed VA-RNA that is not mutated. [0526] In some embodiments, the inducible helper polynucleotide construct also encodes for one or more helper proteins, a self-excising element upstream of the one or more helper proteins, and an inducible promoter upstream of the self-excising element. Expression of the self-excising element may be driven by a Tet-on 3G system. For example, the construct may comprise a Tet- On 3G gene sequence, wherein expression is driven by an EF1alpha promoter. The EF1alpha promoter may be a mutated EF1alpha promoter. The mutated EF1alpha promoter can have a sequence of: ggatctgcgatcgctccggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattg aacgggtgcctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaac cgtatgtaagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctc cttcacgcgcccgccgccctacctgaggccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgc gtccgccgtctaggtaagtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctcca cgctttgcctgaccctgcttgctcaactctacgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctac (SEQ ID NO: 20). [0527] In the presence of a first triggering agent, such as doxycycline, Tet-On 3G is able to bind the Tet inducible promoter. Upon this binding event, the Tet inducible promoter drives expression of the self-excising element. In some embodiments, the self-excising element is a hormone activated Cre. In the presence of a second triggering agent, such as tamoxifen, and upon expression of Cre, Cre self-excises itself leading to expression of downstream adenoviral helper proteins. Thus, mammalian cell lines stably integrated with the inducible helper constructs disclosed herein only express adenoviral helper proteins in the presence of at least two triggering agents (e.g., doxycycline and tamoxifen). [0528] In some embodiments, an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; and b) a self-excising element upstream of the one or more helper proteins. In some embodiments, the self-excising element is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, a sequence coding for the self- excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre- ERT2 polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a triggering agent. In some embodiments, the triggering agent is an estrogen receptor ligand. In some embodiments, the triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites. In some embodiments, the self-excising element is excised upon administration of the triggering agent, thereby operably linking the constitutive promoter to the one or more helper proteins. In some embodiments, the inducible helper construct lacks sequences coding for a tetracycline inducible system (e.g., a tetracycline- responsive promoter element (TRE) and/or a reverse tetracycline-controlled transactivator (rTA)). In some embodiments, the inducible helper construct lacks sequences coding for a tetracyline-inducible system, an ecdysone-inducible system, or a cumate-inducible system. [0529] In some embodiments, an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; b) a self-excising element upstream of the one or more helper proteins; and c) an inducible promoter upstream of the self-excising element. In some embodiments, the self-excising element is operably linked to the inducible promoter. In some embodiments, expression of the self-excising element is driven by the inducible promoter. [0530] In some embodiments, an inducible helper construct is a polynucleotide construct coding for: a) one or more helper proteins; b) a recombinase; and c) an inducible promoter upstream of the one or more helper proteins; and d) an inducible promoter upstream the recombinase. In some embodiments, the recombinase is operably linked to the inducible promoter. In some embodiments, the one or more helper proteins are operably linked to the inducible promoter. In some embodiments, expression of the recombinase is driven by the inducible promoter. In some embodiments, expression of the one or more helper proteins is driven by the inducible promoter. [0531] In some embodiments, the inducible promoter is a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. In some embodiments, the minimal promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 63-68. In some embodiments, the inducible promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 22, 46-48, or 50-62. In some embodiments, transcription is activated from the inducible promoter upon binding of an activator. In some embodiments, the activator binds to the inducible promoter in the presence of a first triggering agent. In some embodiments, further comprising an activator. In some embodiments, the activator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. In some embodiments, the rTA comprises four mutations in the tetR DNA binding moiety. In some embodiments, the rTA comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NOs: 21, 40-45, or 69-86, or variants thereof. [0532] In some embodiments, the inducible promoter is bound by a repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the repressor. In some embodiments, the repressor is a tetracycline-controlled transactivator. In some embodiments, further comprising the repressor. In some embodiments, the repressor is operably linked to a constitutive promoter. In some embodiments, further comprising a tetracycline-controlled transactivator. In some embodiments, the tetracycline-controlled transactivator is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the tetracycline-controlled transactivator is unbound in the presence of a first triggering agent. In some embodiments, the tetracycline-controlled transactivator does not bind to the inducible promoter in the presence of a first triggering agent. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, transcription is activated from the inducible promoter upon binding of the first triggering agent to the repressor. In some embodiments, the repressor binds to the first triggering agent. In some embodiments, the first triggering agent is a tetracycline. In some embodiments, the tetracycline is doxycycline. [0533] In some embodiments, the inducible promoter is a cumate operator sequence. In some embodiments, the cumate operator sequence is downstream of a constitutive promoter. In some embodiments, the constitutive promoter is a human cytomegalovirus promoter. In some embodiments, wherein the inducible promoter is bound by a cymR repressor in the absence of a first triggering agent. In some embodiments, the inducible promoter is activated in the presence of a first triggering agent. In some embodiments, the first triggering agent binds to the cymR repressor. In some embodiments,, the cumate inducible system further comprises a cymR repressor. In some embodiments, the cymR repressor is operably linked to a constitutive promoter. In some embodiments, the constitutive promoter is EF1alpha promoter. In some embodiments, the EF1alpha promoter comprises at least one mutation. In some embodiments, the constitutive promoter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity with SEQ ID NO: 20. In some embodiments, the first triggering agent is a cumate. [0534] In some embodiments, a sequence coding for the self-excising element comprises a poly A sequence. In some embodiments, the self-excising element is a recombinase. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is fused to a ligand binding domain. In some embodiments, the recombinase is Cre polypeptide or flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ER2. In some embodiments, the recombinase is a ER2 Cre polypeptide. In some embodiments, the self-excising element translocates to the nucleus in the presence of a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the recombinase is flanked by recombination sites. In some embodiments, the recombination sites are lox sites or flippase recognition target (FRT) sites. In some embodiments, the lox sites are loxP sites. [0535] In some embodiments, the one or more adenoviral helper proteins comprise E2A and E4. In some embodiments, the one or more adenoviral helper proteins further comprises a protein tag. In some embodiments, the protein tag is a FLAG-tag. In some embodiments, the E2A is FLAG-tagged E2A. In some embodiments, the sequence coding for E2 and the sequence coding for E4 are separated by an internal ribosome entry site (IRES) or by P2A. [0536] In some embodiments, the inducible helper construct further comprises a sequence coding for a selectable marker. [0537] In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0538] In some embodiments, the helper construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the helper construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0539] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0540] In some embodiments, an inducible helper construct further comprises a sequence coding for VA-RNA. In some embodiments, the VA-RNA is on a separate construct from the sequences encoding one or more helper proteins. In some embodiments, the VA-RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In some embodiments, the sequence coding for VA-RNA is a transcriptionally dead sequence. In some embodiments, the sequence coding for VA-RNA comprises at least two mutations in the internal promoter. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the sequence coding for VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by the recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. [0541] In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z- Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C- terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0542] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0543] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0544] In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. [0545] In some embodiments, an inducible helper construct is in a vector. In some embodiments, an inducible helper construct is in a plasmid. In some embodiments, an inducible helper construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138, or any combination thereof. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 9 – SEQ ID NO: 19, SEQ ID 23 – SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 90 – SEQ ID NO: 99, SEQ ID NO: 101 – SEQ ID NO: 109, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138, or any combination thereof. [0546] In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA. In some embodiments, the VA-RNA is on a separate construct from the sequences encoding one or more helper proteins. In some embodiments, the VA-RNA is operably linked to a constitutive promoter or an inducible promoter. In some embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. In some embodiments, the inducible promoter is a tetracycline-inducible promoter, an ecdysone- inducible promoter, or a cumate-inducible promoter. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, a separate polynucleotide construct codes for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA-RNA comprises a sequence coding for a transcriptionally dead VA-RNA. In some embodiments, the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA-RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA- RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z- Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C- terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0547] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0548] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0549] In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct further comprising a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. In some embodiments, the construct comprising the VA-RNA as described herein further comprises a sequence coding for a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C- terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0550] In some embodiments, the construct comprising the VA-RNA further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the construct comprising the VA-RNA further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0551] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0552] In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a vector. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a plasmid. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is in a bacterial artificial chromosome or yeast artificial chromosome. In some embodiments, an inducible helper construct comprises a polynucleotide construct coding for a VA-RNA or the VA-RNA construct is a synthetic nucleic acid construct. In some embodiments, an inducible helper construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, an inducible helper construct has at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, a VA-RNA construct comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. In some embodiments, a VA-RNA construct has a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to any one of SEQ ID NO: 13 – SEQ ID NO: 19 or SEQ ID 23 – SEQ ID NO: 26. Synthetic nucleic acid constructs [0553] In typical embodiments, the nuclear genome of the cell of the stable cell line comprises a plurality of integrated synthetic nucleic acid constructs. In some embodiments, each of the plurality of synthetic nucleic acid constructs is separately integrated into the nuclear genome of the cell. In some embodiments, only a single non-auxotrophic selection is required to maintain all of the plurality of synthetic nucleic acid constructs stably within the nuclear genome of the cells. In some embodiments, antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, both a non-auxotrophic selection and antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, auxotrophic selection and antibiotic resistance is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. In some embodiments, auxotrophic selection is required to maintain the plurality of synthetic constructs stably within the nuclear genomes of the cells. [0554] In particular embodiments, the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences; the second integrated synthetic construct comprises a conditionally expressible AAV Cap coding sequence, where the AAV Cap coding sequence is operably linked to a native or an inducible promoter; the third integrated synthetic construct comprises a conditionally expressible Cre coding sequence and conditionally expressible adenoviral helper protein coding sequences; and the fourth integrated synthetic construct comprises expressible coding sequences for the payload. An example of this embodiment is depicted in FIG.1A. [0555] In particular embodiments, the first integrated synthetic construct comprises conditionally expressible AAV Rep and Cap coding sequences, where the AAV Cap coding sequence is operably linked to an inducible promoter; the second integrated synthetic construct comprises a conditionally expressible Cre coding sequence and conditionally expressible adenoviral helper protein coding sequences; and the third integrated synthetic construct comprises expressible coding sequences for the payload. An example of this embodiment is depicted in FIG.2A. Production of Single-Stranded or Self-Complementary rAAV Virion DNA [0556] In some embodiments, the region of the fourth polynucleotide construct between the two inverted terminal repeats (3’ ITR and 5’ ITR) is packaged into rAAV virions. In some embodiments, the rAAV virions comprise wild-type inverted terminal repeats, wherein the rAAV virion DNA that is generated is single-stranded (i.e., ssAAV virion). In other embodiments, a terminal resolution site in the 3' ITR is deleted, resulting in formation of an rAAV virion comprising DNA that is self-complementary (i.e., scAAV virion). The scAAV forms a single-stranded DNA molecule during replication in which two single-stranded genomes comprising a plus DNA strand and a minus DNA strand are concatenated to form a self- complementary intramolecular dsDNA genome. Unlike ssAAV virions, the scAAV virions do not need to perform second-strand DNA synthesis, which increases the efficiency of scAAV transgene expression relative to ssAAV. However, the maximum cargo capacity of scAAV (i.e., maximum length of region between the 5' ITR and 3' ITR of the fourth polynucleotide construct) that can be packaged into the rAAV virion is about half that of ssAAV because the scAAV DNA packaged into a viral particle is a concatemer of two single-stranded genomes of opposite strands. For a description of methods of producing scAAV virions, see, e.g., Raj et al. (2011) Expert Rev. Hematol.4(5):539-549, McCarty (2008) Mol. Ther.16(10):1648-1656, McCarty et al. (2003) Gene Ther.10(26):2112-2118; herein incorporated by reference in their entireties. For example, a ssAAV plasmid encoding a sequence of a payload (e.g., construct 3 of FIG.2A) can be SEQ ID NO: 159. For example, a scAAV plasmid encoding a sequence of a payload (e.g., construct 3 of FIG.2A) can be SEQ ID NO: 157. Split Auxotrophic selection [0557] In some embodiments, maintaining constructs stably in the cellular genome requires selective pressure. [0558] Typically, each integrated nucleic acid construct comprises a mammalian cell selection element. In some embodiments, the stable cell line comprises four integrated nucleic acid constructs, wherein the first nucleic acid construct comprises a first mammalian cell selection element, the second nucleic acid construct comprises a second mammalian cell selection element, the third nucleic acid construct comprises a third mammalian cell selection element and the fourth nucleic acid construct comprises a fourth mammalian cell selection element. In some embodiments, the stable cell line comprises three integrated nucleic acid constructs, wherein the first nucleic acid construct comprises a first mammalian cell selection element, the second nucleic acid construct comprises a second mammalian cell selection element, and the third nucleic acid construct comprises a third mammalian cell selection element. [0559] In some embodiments, the mammalian selection elements are components of a split auxotrophic selection system or a split antibiotic resistance gene. [0560] In some embodiments, a split auxotrophic system can be a leucine zipper based system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. In some embodiments, components of the split auxotrophic selection system described herein comprise a C-terminal fragment of the auxotrophic protein Z-Cter and an N-terminal fragment of an auxotrophic protein Z-Nter. In some embodiments, a split auxotrophic system is a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form. For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein. Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288: 6202-6211 (2013); Stevens et al, PNAS 114: 8538-8543 (2017); Jillette et al., Nat Comm 10: 4968 (2019); US 2020/0087388 A1; and US 2020/0263197 A1. In some embodiments, components of the split auxotrophic selection system described herein comprises a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein. This N-terminal fragment is enzymatically nonfunctional and this C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, the split inteins can catalyze the joining of the N-terminal fragment of the auxotrophic protein and a C-terminal fragment of the auxotrophic protein to form a functional enzyme, such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR). In some embodiments, both constructs can be stably retained in the genome of a cell by growth in a medium lacking the product produced by the enzyme. [0561] In some embodiments, a construct encoding for a component of a split auxotrophic system further encodes a helper enzyme, wherein expression of the helper enzyme facilitates growth of the host cell in conjunction with the functional enzyme upon application of the single selective pressure. [0562] In some embodiments, the first nucleic acid construct comprises a first mammalian cell selection element, and the first mammalian cell selection element is a first auxotrophic selection element. In certain embodiments, the first auxotrophic selection element encodes an active protein. In some embodiments, the first auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the first auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the first auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z- Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the first auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the first auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the first auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N- terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0563] In some embodiments, first nucleic acid construct further comprises a sequence coding for a first auxotrophic selection element and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the first nucleic acid construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0564] In some embodiments, a first auxotrophic selection element comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the first auxotrophic selection element and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0565] In various embodiments, the second nucleic acid construct comprises a second mammalian cell selection element, and the second mammalian cell selection element encodes antibiotic resistance. In particular embodiments, the antibiotic resistance gene is a blasticidin resistance gene. In certain embodiments, the second mammalian cell selection element encodes an active protein. In some embodiments, the second mammalian cell selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. [0566] In various embodiments, the third nucleic acid construct comprises a third mammalian cell selection element. In some embodiments, the third mammalian cell selection element is a second auxotrophic selection element. In certain embodiments, the second auxotrophic selection element encodes an active protein. In some embodiments, the second auxotrophic selection element is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the second auxotrophic selection element codes for an inactive protein that requires expression of a first auxotrophic selection element for activity. In some embodiments, the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the first auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the second auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the first auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the first auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the second auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the second auxotrophic selection element codes for an N- terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C- terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0567] In some embodiments, the third nucleic acid construct further comprises a sequence coding for second auxotrophic element and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the third nucleic acid construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0568] In some embodiments, a second auxotrophic selection element comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the second auxotrophic selection element and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109. [0569] In various embodiments, the selectable marker of the nucleic acid constructs is interchangeable between each other. In various embodiments, the stable mammalian cell line can be propagated in growth media lacking hypoxanthine and thymidine. [0570] In some embodiments, the average concentration of Rep protein within the cells is less than between 1-99%, 10-90%, 20-80%, 30-70%, 40-60% prior to addition of the at least first triggering agent to the cell culture medium. In some embodiments, the average concentration of Rep protein within the cells is less than about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% prior to addition of the at least first triggering agent to the cell culture medium. [0571] In some embodiments, the mammalian cell line is selected from the group consisting of a human embryonic kidney (HEK) 293 cell line, a human HeLa cell line, and a Chinese hamster ovary (CHO) cell line. In some embodiments, the mammalian cell line is a HEK293 cell line. In some embodiments, the mammalian cell line expresses adenovirus helper functions E1A and E1B. [0572] Alternative constructs as described herein can be used in a complete system. In some embodiments, the complete system can be integrated into the host cell genome to produce a stable cell line. In other embodiments, the complete system can be transfected into the host cell and then conditional production of AAV virion from the plasmids can be induced. In some embodiments, the complete system comprises episomes in the host cell and conditional production of AAV virion from the episomes is induced. [0573] In some embodiments, the cell is conditionally capable of producing rAAV virions having a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the rAAV virions have a payload encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99 prior to purification. In some embodiments, the rAAV virions have a concentration of greater than 1 × 1011 or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013 or 1 × 1014 viral genomes per milliliter prior to purification. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a titer of greater than 1 × 1011 or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013 or 1 × 1014 viral genomes per milliliter. In some embodiments, the cell is capable of producing rAAV virions comprising the payload nucleic acid sequence at a concentration of greater than 1 × 1011 or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013 or 1 × 1014 viral genomes per milliliter prior to purification. In some embodiments, this cell is expanded to produce a population of cells. In some embodiments, the population of cells produces a stable cell line as described herein. In some embodiments, this cell is passaged at least three times. In some embodiments, this cell can be passaged up to 60 times. In some embodiments, this cell can be passage more than 60 times. In some embodiments, the cell maintains the ability to be conditionally induced after each passage. 1.6. Vector/Vector System Vector [0574] In some aspects, vectors comprising the polynucleotide described in the present disclosure are provided. The first polynucleotide is described in section "First Polynucleotide encoding AAV Rep Protein" above. The second polynucleotide is described in section "AAV Cap Encoding Sequence linked to PolyA Signal Sequence" above. The third polynucleotide is described in sections "Third Polynucleotide Encoding Adenoviral Helper Proteins" or "Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA" above. The fourth polynucleotide is described in section "Fourth Polynucleotide Encoding Payload" above. The fifth polynucleotide is described in section "Fifth Polynucleotide Encoding VA-RNA" above. Vector System [0575] In some aspects, vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; b) a second vector comprising the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; c) a third vector comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; and d) a fourth vector comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above are provided. [0576] In some aspects, vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above; b) a second vector comprising the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; c) a third vector comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; d) a fourth vector comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; and d) a fifth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above are provided. [0577] In some embodiments, the first vector is a first plasmid, the second vector is a second plasmid, the third vector is a third plasmid, and the fourth vector is a fourth plasmid. In some embodiments, the fifth vector is a fifth plasmid. [0578] In some embodiments, the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 160 or 161 In certain embodiments, the first plasmid has sequence of SEQ ID NO: 32 or sequence of SEQ ID NO: 160 or 161. [0579] In some embodiments, the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149. In certain embodiments, the second plasmid has sequence of SEQ ID NO: 149. [0580] In some embodiments, the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154. In certain embodiments, the third plasmid has sequence of SEQ ID NO: 30 or sequence of SEQ ID NO: 154. [0581] In some embodiments, the fourth plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159. In certain embodiments, the fourth plasmid has sequence of SEQ ID NO: 157 or sequence of SEQ ID NO: 159. [0582] In some aspects, vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above or the first polynucleotide construct described in section “Polynucleotide Construction System” above; and one or more of: b) a second vector comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above or the second polynucleotide construct described in section “Polynucleotide Construction System” above; and c) a third vector comprising the sequence of the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above or the third polynucleotide construct described in section “Polynucleotide Construction System” above are provided. In some embodiments, the vector systems further comprise a fourth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above or the fourth polynucleotide construct described in section “Polynucleotide Construction System” above. [0583] In some aspects, vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above or the first polynucleotide construct described in section “Polynucleotide Construction System” above; and b) a second vector comprising the sequence of the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above or the second polynucleotide construct described in section “Polynucleotide Construction System” above are provided. In some embodiments, the vector systems further comprise a fourth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above or the fourth polynucleotide construct described in section “Polynucleotide Construction System” above. [0584] In some aspects, vector systems comprising: a) a first vector comprising the sequence of the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the sequence of the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above or the first polynucleotide construct described in section “Polynucleotide Construction System” above; and b) a third vector comprising the sequence of the fourth polynucleotide escribed in section “Fourth Polynucleotide Encoding Payload” above or the third polynucleotide construct described in section “Polynucleotide Construction System” above are provided. In some embodiments, the vector systems further comprise a fourth vector comprising the sequence of the fifth polynucleotide described in section “Fifth Polynucleotide Encoding VA-RNA” above or the fourth polynucleotide construct described in section “Polynucleotide Construction System” above. [0585] In some embodiments, the first vector is a first plasmid, the second vector is a second plasmid, and the third vector is a third plasmid. In some embodiments, the fourth vector is a fourth plasmid. [0586] In some embodiments, the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. In certain embodiments, the first plasmid has sequence of SEQ ID NO: 164. [0587] In some embodiments, the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154. In certain embodiments, the second plasmid has sequence of SEQ ID NO: 30 or sequence of SEQ ID NO: 154. [0588] In some embodiments, the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159. In certain embodiments, the third plasmid has sequence of SEQ ID NO: 157 or sequence of SEQ ID NO: 159. 1.7. Host production cell [0589] The present disclosure further provides host cells comprising the vector system described herein. A subject host cell can be an isolated cell, e.g., a cell in in vitro culture. A subject host cell is useful for producing rAAV virions, as described below. A subject host cell useful for producing rAAV virions can be any cell that is capable of expressing proteins from a p5 promoter. Where a subject host cell is used to produce rAAV virions, it is referred to as a “packaging cell.” In some cases, a subject host cell is stably genetically modified with the vector system. In other cases, a subject host cell is transiently genetically modified with the vector system. [0590] In some aspects, vector systems that comprise the first polynucleotide described herein; the second polynucleotide described herein; the third polynucleotide described herein; and the fourth polynucleotide described herein are provided. [0591] In some aspects, vector systems that comprise the first polynucleotide described herein; the second polynucleotide described herein; the third polynucleotide described herein; the fourth polynucleotide described herein; and the fifth polynucleotide described herein are provided. [0592] In some aspects, vector systems that comprise the first polynucleotide described herein; the second polynucleotide described herein; the third polynucleotide described herein; the fourth polynucleotide described herein; the fifth polynucleotide described herein; and the sixth polynucleotide described herein are provided. [0593] The vector system described herein can be used in a variety of host cells for rAAV virion production. For example, suitable host cells that have been transfected with the vector system are rendered capable of producing rAAV virions. The first, second, third and fourth polynucleotide constructs of the vector system can be introduced into a host cell, either simultaneously or serially, using established transfection techniques, including, but not limited to, electroporation, calcium phosphate precipitation, liposome-mediated transfection, and the like. In some embodiments, the first, second, and third polynucleotide constructs of the vector system are introduced into a host cell, and the fourth polynucleotide construct comprising the expressible payload is introduced later when production of the payload is desired. [0594] A subject host cell is generated by introducing the vector system into any of a variety of cells, e.g., mammalian cells, including, without limitation, murine cells, and primate cells (e.g., human cells). Suitable mammalian cells include, but are not limited to, primary cells and cell lines, where suitable cell lines include, but are not limited to, 293 cells, COS cells, HeLa cells, Vero cells, 3T3 mouse fibroblasts, C3H10T1/2 fibroblasts, CHO cells, and the like. Non- limiting examples of suitable host cells include, e.g., HeLa cells (e.g., American Type Culture Collection (ATCC) No. CCL-2), CHO cells (e.g., ATCC Nos. CRL9618, CCL61, CRL9096), 293 cells (e.g., ATCC No. CRL-1573), Vero cells, NIH 3T3 cells (e.g., ATCC No. CRL-1658), Huh-7 cells, BHK cells (e.g., ATCC No. CCL10), PC12 cells (ATCC No. CRL1721), COS cells, COS-7 cells (ATCC No. CRL1651), RAT1 cells, mouse L cells (ATCC No. CCLI.3), human embryonic kidney (HEK) cells (ATCC No. CRL1573), HLHepG2 cells, and the like. A subject host cell can also be made using a baculovirus to infect insect cells such as Sf9 cells, which produce AAV (see, e.g., U.S. Patent Nos.7,271,002 and 8,945,918). In some embodiments, a host cell is any cell capable of activating a p5 promoter of sequence encoding a Rep protein. [0595] In typical embodiments, the production host cell is a mammalian cell line that expresses adenovirus E1A and E1B. In particular embodiments, the cell is a human embryonic kidney (HEK) 293 cell line or derivatives thereof (HEK293T cells, HEK293F cells), a human HeLa cell line that expresses E1A and E1B, a Chinese hamster ovary (CHO) cell line that expresses E1A and E1B, or a Vero cell that expresses adenovirus E1A and E1B. In particular embodiments, the host cell is a HEK293 cell line. [0596] In certain embodiments, the host cell is or is genetically altered to be deficient in an enzyme required for production of a molecule required for cell growth, for example, an enzyme required for catalyzing production of a cofactor or nutrient. In certain embodiments, the host cell is DHFR null. In specific embodiments, the host cell is a DHFR null HEK293 cell. In some embodiments, the host cell is GS null. In some embodiments, the host cell is a GS null HEK293. [0597] In some embodiments, the host cell expresses or is genetically modified to express GTP- CH1. [0598] In some embodiments, the HEK293 cell expresses AAV E1A and E1B. In the presence of doxycycline and tamoxifen, the ER2 Cre is excised from the first integrated synthetic construct, thereby permitting expression of AAV E2A and E4. The self-excised ER2 Cre recombines by virtue of the lox sites flanking the EGFP cassette in the second integrated synthetic construct, thereby removing the EGFP segment from the second spacer element in the integrated second synthetic construct. As such, any cells comprising only the second integrated synthetic construct will be EGFP signal positive whereas cells comprising both the first and second integrated synthetic constructs will be EGFP signal negative, following the addition of the triggering agents. Absence of EGFP signal indicates successful transfection of both the first and second integrated synthetic constructs in a cell. This is further ensured by antibiotic resistance selection, e.g., blasticidin resistance. [0599] Additionally, removal of the EGFP cassette provides for the functional expression of Rep and Cap proteins, which can be linked to a first selectable marker, e.g., a first DHFR selection element, e.g., Z-Cter DHFR. The first selectable marker is capable of associating with a second selectable marker, e.g., a second DHFR selection element, e.g., a Z-Cter DHFR is capable of associating with a second DHFR selection element, e.g., Z-Nter DHFR, present in the fourth integrated synthetic construct to form an active molecule that allows the cell to survive in a selection medium, e.g., HT lacking media selection. The first selectable marker and second selectable marker can be any selectable marker as described herein, wherein expression of the first selectable marker and expression of the second selectable marker form an active molecule (e.g., a functional enzyme) that allows the cell to survive in a selection medium (e.g., a selection media deficient in the product produced by the functional enzyme). [0600] In some embodiments, the fourth integrated synthetic construct comprises a payload. The payload can be a guide RNA, an HDR homology region, or a gene of interest. [0601] In some embodiments, one or more of the synthetic nucleic acid constructs are integrated into the genome of a production host cell. In some embodiments, the integration of a construct into a chromosome is site-specific. Any method known in the art for directing integration into the genome may be used. For example, a polynucleotide construct can be cloned into a lentivirus vector that integrates into the nuclear genome of the cell. Alternatively, a transposon system, a clustered regularly interspersed short palindromic repeats (CRISPR) system, or a site-specific recombinase can be used to integrate a polynucleotide construct into the host cell genome, as described further below. [0602] In some embodiments, a polynucleotide construct is integrated into the genome using a transposon system comprising a transposase and transposon donor DNA. The transposase can be provided to a host cell with an expression vector or mRNA comprising a coding sequence encoding the transposase. The transposon donor DNA can be provided with a vector comprising transposon terminal inverted repeats (TIRs). The polynucleotide construct is cloned into the transposon donor vector between the TIRs. The host cell is cotransfected with an expression vector or mRNA encoding the transposase and the transposon donor vector containing the polynucleotide construct insert, wherein the polynucleotide construct is excised from the transposon donor vector and integrated into the genome of the host cell at a target transposon insertion site. Transposition efficiency may be improved in a host cell by codon optimization of the transposase, using engineered hyperactive transposases, and/or introduction of mutations in the transposon terminal repeats. Any suitable transposon system can be used including, without limitation, the piggyBac, Tol2, or Sleeping Beauty transposon systems. For a description of various transposon systems, see, e.g., Kawakami et al. (2007) Genome Biol.8 Suppl 1(Suppl 1):S7, Tipanee et al. (2017) Biosci Rep.37(6):BSR20160614, Yoshida et al. (2017) Sci Rep. 7:43613, Yusa et al. (2011) Proc. Natl. Acad. Sci. USA 108(4):1531-1536, Doherty et al. (2012) Hum. Gene Ther.23(3):311-320; herein incorporated by reference in their entireties. [0603] In some embodiments, a construct is integrated at a target chromosomal locus by homologous recombination using site-specific nucleases or site-specific recombinases. For example, a construct can be integrated into a double-strand DNA break at the target chromosomal site by homology-directed repair. A DNA break may be created by a site-specific nuclease, such as, but not limited to, a Cas nuclease (e.g., Cas9, Cpf1, or C2c1), an engineered RNA-guided FokI nuclease, a zinc finger nuclease (ZFN), a transcription activator-like effector- based nuclease (TALEN), a restriction endonuclease, a meganuclease, a homing endonuclease, and the like. Any site-specific nuclease that selectively cleaves a sequence at the target site for integration of the construct may be used. See, e.g., Targeted Genome Editing Using Site-Specific Nucleases: ZFNs, TALENs, and the CRISPR/Cas9 System (T. Yamamoto ed., Springer, 2015); Genome Editing: The Next Step in Gene Therapy (Advances in Experimental Medicine and Biology, T. Cathomen, M. Hirsch, and M. Porteus eds., Springer, 2016); Aachen Press Genome Editing (CreateSpace Independent Publishing Platform, 2015); herein incorporated by reference in their entireties. [0604] The construct sequence to be integrated is flanked by a pair of homology arms responsible for targeting the construct to the target chromosomal locus. A 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence can be introduced into a polynucleotide construct. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms in the polynucleotide construct. The 5' and 3' homology arms hybridize to regions within the target locus where the construct is integrated, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. [0605] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the construct and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein construct is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms. [0606] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and/or a specific site for integrating the construct. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. [0607] A homology arm can be of any length, e.g., 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more, 10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3' homology arms are substantially equal in length to one another, e.g., one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g., one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm. [0608] An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence for insertion of a polynucleotide construct) by altering its guide RNA sequence. A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed to selectively bind to the chromosomal target site where integration of the construct is desired. In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of polynucleotide constructs by the HDR mechanism can be used for selective integration at a target chromosomal site, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Cas10, Cas10d, CasF, CasG, CasH, Csy1, Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof. [0609] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. In some embodiments, Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to selectively integrate a construct at a chromosomal target site as described herein. In certain embodiments, the Cas9 need not be physically derived from an organism but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP_002989955, WP_038434062, WP_011528583); Campylobacter jejuni (WP_022552435, YP_002344900), Campylobacter coli (WP_060786116); Campylobacter fetus (WP_059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP_033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisI (NC_018721); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP_061665472); Legionella pneumophila (WP_062726656); Staphylococcus aureus (WP_001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res.42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol.198(5):797-807, Shmakov et al. (2015) Mol. Cell.60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091-6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9. [0610] In some embodiments, the CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. In certain embodiments, the bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. In certain embodiments, targeting of Cas9 typically further relies on the presence of a 5′ protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site. [0611] In some embodiments, the genomic target site may comprise a nucleotide sequence that is complementary to the gRNA and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the allele targeted by a gRNA comprises a mutation that creates a PAM within the allele, wherein the PAM promotes binding of the Cas9-gRNA complex to the allele. [0612] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 nucleotides in length. In some embodiments, the guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNA molecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules. [0613] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1) may be used. In some embodiments, Cpf1 is another class II CRISPR/Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cpf1 does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cpf1 for targeting than Cas9. Cpf1 is capable of cleaving either DNA or RNA. The PAM sites recognized by Cpf1 have the sequences 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Cpf1 cleavage of DNA produces double- stranded breaks with a sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cpf1, see, e.g., Ledford et al. (2015) Nature.526 (7571):17-17, Zetsche et al. (2015) Cell.163 (3):759-771, Murovec et al. (2017) Plant Biotechnol. J.15(8):917-926, Zhang et al. (2017) Front. Plant Sci.8:177, Fernandes et al. (2016) Postepy Biochem.62(3):315-326; herein incorporated by reference. [0614] In some embodiments, C2c1is another class II CRISPR/Cas system RNA-guided nuclease that may be used. C2c1, similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2c1, see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci.8:177; herein incorporated by reference. [0001] In yet another embodiment, an engineered RNA-guided FokI nuclease may be used. In some embodiments, RNA-guided FokI nucleases comprise fusions of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9), wherein the dCas9 portion confers guide RNA-dependent targeting on FokI. For a description of engineered RNA-guided FokI nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther.25(2):342-355, Pan et al. (2016) Sci Rep.6:35794, Tsai et al. (2014) Nat Biotechnol.32(6):569-576; herein incorporated by reference. [0615] In some embodiments, the RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA-guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector) that is introduced into the host cell. In certain embodiments, Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell or organism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a yeast cell, a bacterial cell, a human cell, a non-human cell, a mammalian cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the RNA-guided nuclease is introduced into cells, the protein can be transiently, conditionally, or constitutively expressed in the cell. [0616] In some embodiments, a polynucleotide construct is site-specifically integrated into the genome of a host cell using a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system, wherein the construct is integrated into a Cas9-induced double-strand break at the target chromosomal site. In some embodiments, a vector encoding Cas9 and a gRNA targeting the desired chromosomal site for integration is introduced into the host cell. In some embodiments, sequences with homology to the target locus are introduced into the polynucleotide construct to allow for integration by homology-directed repair. For a description of the use of a CRISPR-Cas9 systems for targeted genomic integration of AAV constructs, see, e.g., Nat. Commun. (2019) 10(1):4439; herein incorporated by reference. [0617] Alternatively, site-specific recombinases can be used to selectively integrate a polynucleotide construct at a target chromosomal site. In some embodiments, a target chromosomal site for integration of one or more polynucleotide constructs disclosed herein may include transcriptionally active chromosomal sites. Examples of transcriptionally active chromosomal sites include DNaseI hypersensitive sites (DHSs). In some embodiments, a polynucleotide construct can be site-specifically integrated into the genome of a host cell by introducing a first recombination site into the construct and expressing a site-specific recombinase in the host cell. In some embodiments, the target chromosomal site of the host cell comprises a second recombination site, wherein recombination between the first and second recombination sites mediated by the site-specific recombinase results in integration of the vector at the target chromosomal locus. In some embodiments, the target chromosomal site may comprise either a recombination site native to the genome of the host cell or an engineered recombination site recognized by the site-specific recombinase. In some embodiments, various recombinases may be used for site-specific integration of vector constructs, including, but not limited to phi C31 phage recombinase, TP901-1 phage recombinase, and R4 phage recombinase. In some cases, a recombinase engineered to improve the efficiency of genomic integration at the target chromosomal site may be used. For a description of various site-specific recombinase systems and their use in site-specific recombination and genomic integration of constructs, see, e.g., U.S. Patent No.6,632,672; Olivares et al. (2001) Gene 278:167-176; Stoll et al. (2002) J. Bacteriol.184(13):3657-3663; Thyagarajan et al. (2001) Mol. Cell Biol.21(12):3926-3934; Sclimenti et al. (2001) Nucleic Acids Res.29(24):5044-5051; Stark et al. (2011) Biochem. Soc. Trans.39(2):617-22; Olorunniji et al. (2016) Biochem. J.473(6):673-684; Birling et al. (2009) Methods Mol. Biol.561:245-63; García-Otin et al. (2006) Front. Biosci.11:1108-1136; Weasner et al. (2017) Methods Mol. Biol.1642:195-209; herein incorporated by reference in their entireties). [0618] In some embodiments, one or more of the polynucleotide constructs are not integrated into the genome of the production host cell, and instead are maintained in the cell extrachromosomally. Examples of extrachromosomal polynucleotide constructs include those that persist as stable/persistent plasmids or episomal plasmids. In some embodiments, a construct comprises Epstein-Barr virus (EBV) sequences, including the EBV origin of replication. oriP, and the EBV gene, EBNA1, to provide stable extrachromosomal maintenance and replication of the construct. For a description of methods of using EBV sequences to stably maintain vectors extrachromosomally, see, e.g., Stoll et al. (2010) Mol. Ther.4(2):122-129 and Deutsch et al. (2010) J. Virol.84(5):2533-2546; herein incorporated by reference in their entireties. In some embodiments, the polynucleotide constructs of the present disclosure may be introduced into a cell in manner similar to the currently used triple-transfection method for production of rAAV virions. [0619] In a preferred embodiment, this system requires only one antibiotic resistance marker, and two split auxotrophic constructs for selection of all three plasmids, each being transformed just once into the DHFR knockout strain- producing a master cell line for virion production which can be stored and then utilized for scaled-up production without further transformations. This approach provides inducible control over expression of the Rep/Cap products avoiding the toxicity typically associated with Rep/Cap production and also avoids selection with multiple antibiotics, which is not preferred for therapeutic products. In some embodiments, both overexpression of Rep/Cap and selection with multiple antibiotics can be toxic and result in diminished virion yield. In other embodiments, the transformed cells can be frozen for storage and thawed for subsequent applications. 1.8. Plasmids [0620] In some aspects, plasmids comprising a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 are provided. [0621] In some aspects, plasmids comprising a nucleotide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149 are provided. [0622] In some aspects, plasmids comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 are provided. [0623] In some aspects, plasmids comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 are provided. [0624] In some aspects, plasmids comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 and a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 or 165 are provided. 1.9. Cells Cells Comprising Polynucleotides [0625] In some aspects, cells comprising the polynucleotide described in the present disclosure are provided. The first polynucleotide is described in section "First Polynucleotide encoding AAV Rep Protein" above. The second polynucleotide is described in section "AAV Cap Encoding Sequence linked to PolyA Signal Sequence" above. The third polynucleotide is described in sections "Third Polynucleotide Encoding Adenoviral Helper Proteins" or "Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA" above. The fourth polynucleotide is described in section "Fourth Polynucleotide Encoding Payload" above. The fifth polynucleotide is described in section "Fifth Polynucleotide Encoding VA-RNA" above. [0626] In some embodiments, the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof. [0627] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof. In some embodiments, the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. [0628] In some embodiments, the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein. In other embodiments, the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein. [0629] In some embodiments, the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome. In other embodiments, the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. [0630] In some embodiments, the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins. In other embodiments, the third polynucleotide comprises SEQ ID NO: 30. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. [0631] In some embodiments, the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B. [0632] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient. [0633] In some embodiments, the cell expresses adenoviral helper proteins E1A and E1B. [0634] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0635] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0636] In some embodiments, after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In other embodiments, the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155. [0637] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161. [0638] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0639] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. [0640] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163. [0641] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158. [0642] In some aspects, cells that comprise a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; and d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins are provided. [0643] In other aspects, cells that comprise a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins; and e) a fifth polynucleotide comprising a fifth sequence encoding a payload are provided. [0644] In still other aspects, cells that comprise a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins; e) a fifth polynucleotide comprising a fifth sequence encoding a payload; and f) a sixth polynucleotide comprising a sixth sequence encoding a viral associated RNA (VA-RNA) are provided. In certain embodiments, the VA- RNA is a mutated VA-RNA. [0645] In some embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0646] In some embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions that encapsidates the payload of the fifth polynucleotide. [0647] In some embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide, and the sixth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions that encapsidates the payload of the fifth polynucleotide. [0648] In other embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are, in any combination, on one or more polynucleotide constructs. [0649] In still other embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and fifth polynucleotide are, in any combination, on one or more polynucleotide constructs. [0650] In further embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide, and the sixth polynucleotide are, in any combination, on one or more polynucleotide constructs. [0651] In some embodiments, cells for inducibly producing rAAVcomprise a plurality of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide, the sixth polynucleotide, or any combination thereof. [0652] In some embodiments, cells for inducibly producing rAAV comprise a single copy of each of the first polynucleotide, the second polynucleotide, and the third polynucleotide. In other embodiments, cells for inducibly producing rAAV comprise a single copy of each of the first polynucleotide construct and the third polynucleotide construct, and comprise two or more copies of the second polynucleotide construct. [0653] In some embodiments, the first polynucleotide further comprises a selectable marker operably linked to a promoter. In some embodiments, the second polynucleotide further comprises a selectable marker operably linked to a promoter. In some embodiments, the third polynucleotide further comprises a selectable marker operably linked to a promoter. In some embodiments, the fourth polynucleotide further comprises a selectable marker operably linked to a promoter. In some embodiments, the fifth polynucleotide further comprises a selectable marker operably linked to a promoter. In some embodiments, the sixth polynucleotide further comprises a selectable marker operably linked to a promoter. In some embodiments, any combination of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide comprise a selectable marker. In some embodiments, the selectable marker of any one of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide and the sixth polynucleotide are different selectable markers, the same selectable marker but as a different split portion of the selectable marker, or a combination thereof. In certain embodiments, the promoter is a constitutive promoter. [0654] In some embodiments, a) the first polynucleotide comprising the first sequence comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence comprising a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence comprising the stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, (iii) a second part of the AAV Rep proteins coding sequence; and (iv) one or more promoters operably linked to the first sequence. In certain embodiments, the coding sequence comprising the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker operably linked to the stop signaling sequence. [0655] In some embodiments, the first sequence comprises from 5’ to 3’: [0656] one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form the excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins. [0657] In some embodiments, b) the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to a promoter present in the second part of the AAV Rep proteins coding sequence or the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to an inducible promoter. [0658] In some embodiments, the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to a native AAV promoter and the third polynucleotide comprising the third sequence encoding the AAV Cap proteins is operably linked to a first inducible promoter. [0659] In some embodiments, the third polynucleotide further comprises a second constitutive promoter operably linked to a second selectable marker. [0660] In some embodiments, the fourth polynucleotide comprising the fourth sequence encoding the one or more adenoviral helper proteins further comprises a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the fourth sequence encoding the one or more AAV helper proteins; a third constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a triggering agent; and a fourth constitutive promoter operably linked to a sequence encoding a third selectable marker, wherein the cell constitutively expresses the third selectable marker, wherein in absence of activation of the first inducible promoter and second inducible promoter, the cell does not express detectable levels of the Rep proteins, the AAV Cap proteins of the second polynucleotide, the AAV Cap proteins of the third polynucleotide, the inducible recombinase, and the one or more AAV helper proteins, or wherein the fourth polynucleotide comprising the fourth sequence encoding the one or more adenoviral helper proteins further comprises a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the fourth sequence encoding the one or more AAV helper proteins; a third constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a triggering agent; and a fourth constitutive promoter operably linked to a sequence encoding a third selectable marker, wherein the cell constitutively expresses the third selectable marker, wherein in absence of activation of the first inducible promoter and second inducible promoter, the cell does not express detectable levels of the Rep proteins, the AAV Cap proteins of the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins. [0661] In some embodiments, the sequence encoding one or more adenoviral helper proteins comprises from 5’ to 3’: a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site, wherein the third recombination site and the fourth recombination site are oriented in the same direction; and a sequence encoding one or more AAV helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the second inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more AAV helper proteins and allowing expression of the one or more AAV helper proteins. [0662] In some embodiments, the fifth polynucleotide further comprises a fourth selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR); optionally, wherein the sequence encoding the payload is flanked by a 5' AAV inverted terminal repeat (5' ITR) and a 3' AAV inverted terminal repeat (3' ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146, SEQ ID NO: 156, or SEQ ID NO: 158; optionally, wherein SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159 comprises the sequence of the fifth polynucleotide. [0663] In some embodiments, the fifth polynucleotide further comprises a spacer between the 5' ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3' ITR, or a combination thereof; optionally wherein SEQ ID NO: 147 comprises the fifth polynucleotide. In certain embodiments, the spacer ranges in length from 500 base pairs to 5000 base pairs. [0664] In some embodiments, the fourth polynucleotide comprising the fourth sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins; optionally, wherein SEQ ID NO: 30 or SEQ ID NO: 154 comprises the fourth polynucleotide or the fourth polynucleotide comprises SEQ ID NO: 153. In certain embodiments, the two AAV helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein; optionally, wherein the any two proteins are E2A and E4 or E1A and E1B. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. [0665] In some embodiments, the first inducible promoter operably linked to the Cap in the third polynucleotide construct is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. In certain embodiments, the tetracycline-inducible promoter comprises a tetracycline-responsive promoter element (TRE). In certain embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In certain embodiments, the minimal promoter is a human cytomegalovirus promoter. [0666] In some embodiments, the second inducible promoter operably linked to the self- excising element in the third polynucleotide construct is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same; further optionally, wherein the first inducible promoter and the second inducible promoter are a tetracyline-inducible promoter. [0667] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0668] In some embodiments, the fifth polynucleotide comprising the fifth sequence encoding the payload comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the sequence encoding the payload a sequence encoding progranulin. [0669] In some embodiments, the fifth polynucleotide comprising the fifth sequence encoding the payload comprises a suppressor tRNA, a guide RNA, or a homology region for homology- directed repair. [0670] In some embodiments, the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. [0671] In some embodiments, the sixth polynucleotide comprising the sixth sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, wherein the fifth and sixth recombination sites are oriented in the same direction, and wherein excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. [0672] In certain embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter. [0673] In certain embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter. [0674] In certain embodiments, the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. [0675] In some embodiments, the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. [0676] In some embodiments, cells of interest for inducibly producing recombinant AAV (rAAV) virions comprise a payload. [0677] In some embodiments, transcription of the first polynucleotide and the second polynucleotide are driven by native AAV promoters. In other embodiments, transcription of the first polynucleotide is driven by native AAV promoters and transcription of the second polynucleotide is driven by a first inducible promoter. In certain embodiments, the first polynucleotide and the second polynucleotide are separated by a transcriptional blocking element. [0678] In some embodiments, transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters and transcription of the second polynucleotide is driven by the P40 native AAV promoter. In some embodiments, transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters and transcription of the second polynucleotide is driven by a first inducible promoter. In certain embodiments, the first polynucleotide and the second polynucleotide are separated by a transcriptional blocking element. [0679] In some embodiments, transcription of the third polynucleotide is driven by a first inducible promoter. [0680] In some embodiments, the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof; optionally, wherein the fourth polynucleotide comprising the fourth sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof; further optionally, wherein the third polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. [0681] In some embodiments, the cell comprises an adenovirus E1A protein and E1B protein, and the one or more AAV helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein or wherein the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the third polynucleotide construct are an adenovirus E1A protein and E1B protein. [0682] In some embodiments, the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome; optionally, wherein the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. [0683] In some embodiments, the cell is a mammalian cell. In certain embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient. Cells Comprising Polynucleotide Systems [0684] In some aspects, cells comprising the polynucleotide system described in the present disclosure are provided. The polynucleotide system is described in section “1.4. Polynucleotide System” above. Specifically, the first polynucleotide is described in section "First Polynucleotide encoding AAV Rep Protein" above; the second polynucleotide is described in section "AAV Cap Encoding Sequence linked to PolyA Signal Sequence" above; the third polynucleotide is described in sections "Third Polynucleotide Encoding Adenoviral Helper Proteins" or "Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA" above; the fourth polynucleotide is described in section "Fourth Polynucleotide Encoding Payload" above; and the fifth polynucleotide is described in section "Fifth Polynucleotide Encoding VA- RNA" above. [0685] In some embodiments, one or more of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell. [0686] In some embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0687] In some embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0688] In some embodiments, the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are, in any combination, on one or more polynucleotide constructs. The one or more polynucleotide constructs are described in section “1.5. Polynucleotide Construct System”. [0689] In some embodiments, the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof. [0690] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof. In some embodiments, the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. [0691] In some embodiments, the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein. In other embodiments, the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein. [0692] In some embodiments, the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome. In other embodiments, the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. [0693] In some embodiments, the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins. In other embodiments, the third polynucleotide comprises SEQ ID NO: 30. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. [0694] In some embodiments, the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B. [0695] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient. [0696] In some embodiments, the cell expresses adenoviral helper proteins E1A and E1B. [0697] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0698] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0699] In some embodiments, after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In other embodiments, the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155. [0700] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161. [0701] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0702] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. [0703] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163. [0704] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158. Cells Comprising Polynucleotide Construct System [0705] In some aspects, cells comprising the polynucleotide construct system described in section “1.5. Polynucleotide Construct System” above are provided. [0706] In some embodiments, one or more of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, and the fourth polynucleotide are integrated into the nuclear genome of the cell. [0707] In some embodiments, the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0708] In some embodiments, the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0709] In some embodiments, the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof. [0710] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof. In some embodiments, the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. [0711] In some embodiments, the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein. In other embodiments, the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein. [0712] In some embodiments, the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome. In other embodiments, the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. [0713] In some embodiments, the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins. In other embodiments, the third polynucleotide comprises SEQ ID NO: 30. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. [0714] In some embodiments, the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B. [0715] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient. [0716] In some embodiments, the cell expresses adenoviral helper proteins E1A and E1B. [0717] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0718] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0719] In some embodiments, after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In other embodiments, the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155. [0720] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161. [0721] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0722] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. [0723] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163. [0724] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158. [0725] In some aspects, stable mammalian cell lines that include a) a first polynucleotide construct comprising a first sequence encoding AAV Rep proteins and a second sequence encoding AAV Cap proteins; b) a second polynucleotide construct comprising a third sequence encoding the AAV Cap proteins; and c) a third polynucleotide construct comprising a fourth sequence encoding one or more adenoviral helper proteins are provided. In some embodiments, the stable mammalian cell lines further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs). [0726] In other aspects, stable mammalian cell lines that include a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins; b) a polynucleotide construct comprising a sequence encoding the AAV Cap proteins; and c) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided. In some embodiments, the stable mammalian cell lines further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs). [0727] In some aspects, stable mammalian cell lines that include a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins encoding sequence and a sequence encoding AAV Cap proteins; and b) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided. In some embodiments, the stable mammalian cell lines further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs). [0728] In some aspects, cells that comprise a) a first polynucleotide construct comprising a first sequence encoding AAV Rep proteins encoding sequence and a second sequence encoding AAV Cap proteins; b) a second polynucleotide construct comprising a third sequence encoding the AAV Cap proteins; and c) a third polynucleotide construct comprising a fourth sequence encoding one or more adenoviral helper proteins are provided. In some embodiments, the cells further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs). [0729] In other aspects, cells that comprise a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins encoding; b) a polynucleotide construct comprising a sequence encoding the AAV Cap proteins; and c) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided. In some embodiments, the cells further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs). [0730] In some aspects, cells that comprise a) a polynucleotide construct comprising a sequence encoding AAV Rep proteins encoding and a sequence encoding the AAV Cap proteins; and b) a polynucleotide construct comprising a sequence encoding one or more adenoviral helper proteins are provided. In some embodiments, the cells further include a polynucleotide construct comprising a sequence encoding a payload flanked by inverted terminal repeats (ITRs). [0731] In some embodiments, a) a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; further optionally, wherein the first polynucleotide sequence lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. In some embodiments, b) a second polynucleotide construct comprises the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, c) a third polynucleotide construct comprises the fourth polynucleotide; optionally, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. [0732] In some embodiments, a) a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, b) a second polynucleotide construct comprises the third polynucleotide; optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, c) a third polynucleotide construct comprises the fourth; optionally, wherein the third polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30. In some embodiments, d) a fourth polynucleotide construct comprises the fifth polynucleotide; optionally, wherein the fourth polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. [0733] In some embodiments, cells for inducibly producing AAV comprise a) the first polynucleotide construct comprising the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; further optionally, wherein the first polynucleotide sequence lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136; b) the second polynucleotide construct comprising the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148; and c) the third polynucleotide construct comprising the fourth polynucleotide; optionally, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. [0734] In other embodiments, cells for inducibly producing AAV comprise a) a first polynucleotide construct comprising the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32; b) a second polynucleotide construct comprising the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149; c) a third polynucleotide construct comprising the fourth, optionally, wherein the third polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; and d) a fourth polynucleotide construct comprising the fifth polynucleotide, optionally, wherein the fourth polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. [0735] In some embodiments, the first polynucleotide construct, second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0736] In some embodiments, the first polynucleotide construct, second polynucleotide construct, the third polynucleotide construct and fourth polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0737] In some embodiments, the first polynucleotide construct, second polynucleotide construct, the third polynucleotide construct, fourth polynucleotide construct and fifth polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0738] In some embodiments, cells of interest comprise a plurality of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, the fourth polynucleotide construct, or the fifth polynucleotide construct, or any combination thereof. [0739] In some embodiments, the first polynucleotide construct further comprises a first constitutive promoter operably linked to a sequence encoding a first selectable marker. [0740] In some embodiments, the third polynucleotide construct comprises a sequence encoding the activator. In certain embodiments, the sequence encoding the activator is operably linked to a constitutive promoter. In certain embodiments, the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. [0741] In some embodiments, the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. [0742] In some embodiments, the triggering agent for inducing the tetracycline-inducible promoter is tetracycline or doxycycline. [0743] In some embodiments, the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen. [0744] In some embodiments, the recombination sites in the first polynucleotide construct and the third polynucleotide construct are lox sites and the recombinase is a cre recombinase or wherein the recombination sites in the first polynucleotide construct and the third polynucleotide are flippase recognition target (FRT) sites and the recombinase is a flippase (Flp) recombinase. [0745] In some embodiments, presence of the triggering agent activates the activator for activation of the first inducible promoter to express Cap proteins from the third polynucleotide comprising the third sequence encoding the AAV Cap proteins. [0746] In some embodiments, presence of the triggering agent activates the activator for activation of the second inducible promoter to express the Rep proteins, the Cap proteins of the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins. [0747] In some aspects, the cells or stable cells may further include a fourth polynucleotide construct encoding a payload. [0748] In some embodiments, these cells provide for an increased levels of AAV capsid proteins thereby leading to increased rAAV production as compared to cells that include the first and third polynucleotide construct but do not include a second polynucleotide construct comprising a third sequence encoding the AAV Cap proteins. [0749] In some embodiments, the third polynucleotide construct permits inducible expression of a hormone-activated excising element. In some embodiments, the excising element can be a recombinase. In some embodiments, the recombinase can be a site-specific recombinase. In some embodiments, the site-specific recombinase can be a Cre polypeptide or a flippase. Triggering of Cre expression via an inducible promoter leads to recombination at lax sites, which in turn lead to expression of adenovirus helper proteins, expression of AAV Rep and Cap proteins, and production of rAAV, optionally, encapsidating a therapeutic payload (e.g., transgene, a tRNA suppressor, a guide RNA, or other oligonucleotide). [0750] In certain embodiments, expression of the Cre recombinase and the AAV capsid proteins from the second polynucleotide construct may be under the control of the same inducible promoter. Such a configuration may lead to a substantially simultaneous expression of AAV capsid proteins from the first polynucleotide construct (where the expression is activated after the action of Cre) and from the second polynucleotide construct. [0751] While the figures provided herein show specific arrangement of genes in the polynucleotide constructs, any combination that is capable of conditionally producing AAV Rep/Cap proteins and producing rAAV is also envisioned. [0752] FIG.1A depicts an exemplary system of polynucleotides for inducibly producing rAAV. In the embodiment shown, four synthetic polynucleotide constructs are separately integrated into the nuclear genome of a cell line that expresses adenovirus E1A and E1B, such as HEK 293 cells. In the pre-triggered state “off state” shown in FIG.1A, transcriptional read-through of rep on Construct 1 is blocked by an excisable element comprising a first recombination site, a coding sequence comprising a stop signaling sequence, a second recombination site. A first and second recombination sites flank the coding sequence comprising a stop signaling sequence and the first recombination site and the second recombination site are oriented in the same direction. Construct 1 comprises from 5’ to 3’: one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence. [0753] Construct 2 encodes a mammalian selectable marker under the control of a constitutive promoter and AAV capsid proteins under the control of an inducible promoter. Each of Constructs 1 and 2 include sequences that encode for the same Cap proteins. In some embodiments, each of the Constructs 1 and 2 encodes for AAV5 cap proteins, VP1, VP2, and VP3. [0754] In some embodiments, Construct 3 includes a conditionally self-excising element which permits conditional expression of ER2 Cre under the control of an inducible promoter. For example, the inducible promoter is a Tet-inducible promoter (e.g., Tet-On promoter). In certain embodiments, in the absence of a triggering agent, the inducible promoter is not active. For example, a triggering agent for Tet-inducible promoter is a tetracycline. In certain embodiments, in the absence of a tetracycline, such as doxycycline (“Dox”), Tet activator protein (TetOn3G) expressed, under the control of a constitutive promoter, cannot bind and activate the basal Tet inducible promoter. In other embodiments, in addition, the localization of Cre is under control of estrogen response elements (“ER2”) that require binding of an estrogen agonist or selective modulator, such as tamoxifen, for the translocation from the cytoplasm to the nucleus. This approach limits pre-triggering Cre expression with consequent promiscuous recombination events and toxicity. In some embodiments, the ER2 Cre element also comprises a strong 3’ polyadenylation signal, which prevents basal expression of the downstream adenoviral helper genes, E2A and E4. [0755] In some embodiments, the Cre element is split into two fragments, that can be fused in the presence of a chemical agent, such as rapamycin. In some embodiments, the Cre is a light inducible Cre. [0756] In some embodiments, the Construct 3 comprises a P2A sequence positioned between an E2A sequence and an E4 sequence. The P2A sequence encodes a self-cleaving peptide such that E2A and E4 proteins are generated after cleavage of a fusion protein comprising E4, P2A, E2A amino acid sequences. In some embodiments, the Construct 3 comprises an internal ribosomal entry site (IRES) sequence positioned between an E2A sequence and an E4 sequence. In some embodiments, an IRES can comprise at least 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to SEQ ID NO: 135. [0757] In some embodiments, the inducible promoter system of Construct 3 is a tetracycline- inducible promoter (e.g., a Tet On inducible promoter that is part of a Tet On inducible promoter system). In some embodiments, the inducible promoter system of Construct 3 is a Tet Off inducible promoter system. In some embodiments, the inducible promoter system of Construct 3 is a cumate inducible promoter system. [0758] In this embodiment, in Construct 3, the activator TetOn3G is expressed under the control of the constitutive EF1alpha promoter and a gene encoding for puromycin resistance is under the control of the constitutive CMV promoter. In some embodiments, Construct 3 includes an optional insert which includes a Cre inducible U6 promoter that drives the expression of transcriptionally dead mutants of VA RNA1 (VA RNA). Specifically, the U6 promoter is split into two parts separated by a Lox flanked stuffer sequence. The U6 promoter is inactive because of the presence of the stuffer sequence. Cre mediated excision of the stuffer sequence activates the U6 promoter, which then drives the expression of VA RNA. [0759] In some embodiments, the payload polynucleotide on Construct 4 is flanked by AAV ITRs, represented by the brackets. In other embodiments, Construct 4 also includes a constitutive promoter operably linked to a sequence encoding a selectable marker or a first portion or a second portion of a split selectable marker. In the embodiment depicted in FIG.1A, Construct 1 includes a sequence encoding a first portion or a second portion of a split selectable marker and Construct 4 includes a sequence encoding the other portion of the split selectable marker. [0760] In some embodiments, when a first triggering agent (e.g., doxycycline, which is also referred to as Dox) and a second triggering agent (e.g., tamoxifen) are added to the culture medium, Tet-on 3G and Dox bind the Tet responsive basal promoter and ER2 Cre is expressed and translocates to the nucleus. In some embodiments, ER2 Cre excises its own coding sequence from Construct 3, leaving the integrated construct shown in FIG.1B. Excision of the self-excising ER2 Cre coding sequence allows expression of E2A and E4 helper proteins under the control of the tetracycline-inducible promoter. Similarly, for the optional additional insert shown in FIG.1A, VA-RNA is expressed following Cre mediated excision of the stuffer sequence, which activates the U6 promoter which then drives the expression of VA-RNA, as shown in FIG.1B. [0761] In some embodiments, Construct 1 is designed to prevent expression of AAV Rep prior expression and translocation of ER2 Cre and permit expression of AAV Rep and Cap proteins from their endogenous promoters after translocation of ER2 Cre to the nucleus. [0762] In the embodiment shown in FIG.1A, the excisable element in Construct 1 which interrupts the rep coding sequence, blocking transcriptional read-through of the full-length rep coding sequence results in production of a pre-triggered transcript that includes the 5’ portion of AAV Rep coding sequence fused to a blue fluorescent marker protein (BFP) coding sequence. In some embodiments, the transcript contains a single intron flanked by 5’ and 3’ splice sites. Routine splicing produces a transcript that encodes a fusion protein that includes the N-terminal portion of rep fused to BFP. The fusion protein lacks the toxicity of full-length Rep protein, and presence of pre-triggered Construct 1 in the cell genome can be detected by BFP fluorescence for quality control. [0763] In some embodiments, the excisable element comprises a first spacer segment, a second spacer segment, and a third spacer segment. In some embodiments, the excisable element is inserted at an insertion site between the p19 internal promoter and the p40 internal promoter of the AAV Rep coding sequence. In some embodiments, the insertion site is CAG-G, CAG-A, AAG-G, or AAG-A, wherein the dash (-) indicates the point of insertion of the excisable spacer. In other embodiments, Construct 1 also includes a sequence encoding a selectable marker. In certain embodiments, the selectable marker may be a split selectable marker, e.g., split antibiotic resistance protein expressed under the control of a constitutive promoter. [0764] FIG.1B shows the conversion of the pre-triggered Construct 1 to a post-triggered state “on state” upon exposure to ER2 Cre within the cell nucleus. ER2 Cre excises the excisable element, which includes the BFP marker coding sequence and the upstream 3’ splice site. As rearranged, the construct now allows expression of functional Rep and Cap transcripts from their respective endogenous promoters. Loss of BFP expression indicates successful Cre-mediated genomic recombination. [0765] An exemplary embodiment of Construct 4 shown in FIG.1A and FIG.1B encodes a payload. In some embodiments, Construct 4 includes a polynucleotide encoding a payload, which can be a gene of interest flanked by ITRs, and a second portion of the selectable marker expressed from a constitutive promoter. In some embodiments, the polynucleotide payload can be any payload for which rAAV is an appropriate vehicle, including a transgene encoding a protein of interest, a homology element for homology-directed repair, or a guide RNA. In other embodiments, the polynucleotide payload is flanked by AAV ITRs, represented by the brackets. In certain embodiments, the sequence encoding the payload may be under control of a constitutive promoter. In certain embodiments, the sequence encoding the payload is under control of a tissue-specific promoter active in the tissue where the payload is to be delivered. [0766] FIG.1B depicts the post-triggered state “on state” of the constructs shown in FIG.1A following the addition of tamoxifen and doxycycline to the cell medium. Adenoviral E2A and E4 helper proteins are expressed from integrated Construct 3 under control of the inducible promoter (e.g., a Tet-On promoter activated in the presence of Dox). AAV rep and cap coding sequences are expressed from Construct 1 under control of endogenous promoters. In some embodiments, AAV cap coding sequences are expressed from Construct 2 under control of the Tet promoter. rAAV virions that encapsidate the payload are therefore produced. [0767] This approach provides numerous benefits over current AAV systems for delivery of payloads. [0768] In some embodiments, maintaining constructs stably in the cellular genome requires selective pressure. FIGS.1A and 1B depict polynucleotides for selection of cells based in antibiotics resistance. In this example, the split blasticidin is used in Constructs 1 and 4. A separate exemplary antibiotic selection approach, puromycin resistance, is used in Construct 3. A separate exemplary antibiotic selection approach, hygromycin resistance, is used in Construct 2. This results in the ability to stably maintain all four constructs in the mammalian cell line using three antibiotics. [0769] In some embodiments, to reduce the number of selective agents (and in particular, antibiotics) required to stably maintain integrated constructs within the cell line genome, other approaches that stably maintains all 4 constructs in the nuclear genome with two antibiotic selections, plus a single auxotrophic selection can also be utilized. In some embodiments, the cell line stably maintains all 4 constructs in the nuclear genome with no antibiotic selection and instead utilizes auxotrophic protein selection. [0770] In certain embodiments, a split auxotrophic selection system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure can be used. One construct encodes the N-terminal fragment of mammalian dihydrofolate reductase (DHFR) fused to a leucine zipper peptide (“Nter-DHFR”). This N-terminal fragment is enzymatically nonfunctional. The other construct encodes the C-terminal fragment of DHFR fused to a leucine zipper peptide (“Cter-DHFR”). This C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, a functional DHFR enzyme complex is formed through association of the leucine zipper peptides. Both constructs can be stably retained in the genome of a DHFR null cell by growth in a medium lacking hypoxanthine and thymidine. [0771] An exemplary deployment of this split auxotrophic selection design in the multi- construct system of FIG.1A is disclosed. In this example, the split auxotrophic selection elements are deployed on Constructs 1 and 4. A separate exemplary antibiotic selection approach, puromycin resistance, is deployed on Construct 3. A separate exemplary antibiotic selection approach, hygromycin resistance, is deployed on Construct 2. This results in the ability to stably maintain all four constructs in the mammalian cell line using two antibiotics, culturing in medium with puromycin and hygromycin, lacking thymidine and hypoxanthine. [0772] In some embodiments, the split selection system is a split intervening proteins (inteins) system that permits stable retention of two integrated nucleic acid constructs under a single selective pressure. Inteins auto catalyze a protein splicing reaction that results in excision of the intein and joining of the flanking amino acids (extein sequences) via a peptide bond. Inteins exist in nature as a single domain within a host protein or, less frequently, in a split form. For split inteins, the two separate polypeptide fragments of the intein must associate in order for protein trans-splicing to occur to excise the intein. Split intein systems are described in: Cheriyan et al, J. Biol. Chem 288: 6202-6211 (2013); Stevens et al, PNAS 114: 8538-8543 (2017); Jillette et al., Nat Comm 10: 4968 (2019); US 2020/0087388 A1; and US 2020/0263197 A1. In some embodiments, the split auxotrophic selection system described herein comprises a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein. In certain embodiments, this N-terminal fragment is enzymatically nonfunctional and this C-terminal fragment is enzymatically nonfunctional. When both fragments are concurrently expressed in the cell, the split inteins can catalyze the joining of the N-terminal fragment of the auxotrophic protein and a C-terminal fragment of the auxotrophic protein to form a functional enzyme, such as any one of the enzymes disclosed herein (e.g., PAH, GS, TYMS, DHFR). In some embodiments, both constructs can be stably retained in the genome of a cell by growth in a medium lacking the product produced by the enzyme. In some embodiments, the split auxotrophic selection elements (e.g., a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of the split intein and a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein) deployed on, for example, Constructs 1 and 4, are part of a split intein system. A separate exemplary auxotrophic selection approach, e.g., a full length auxotrophic protein, can be deployed on Construct 3. In some embodiments, a construct encoding an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of the split intein or a construct encoding the C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of the split intein further encodes a helper enzyme, wherein expression of the helper enzyme facilitates growth of the host cell in conjunction with the functional enzyme upon application of the single selective pressure. [0773] In some embodiments, following triggering and Cre-mediated genomic rearrangement, the selection elements remain unchanged, allowing continued maintenance of the four post- triggering integrated constructs using two antibiotics in medium lacking hypoxanthine and thymidine. [0774] In some embodiments, viral proteins needed for AAV virion formation are inhibited by host cell mechanisms. Inhibition of these host cell mechanisms to maximize AAV viral titers in the stable cell lines described herein include, but are not limited to: knocking out PKR (PKR KO) (pathway is responsible for inhibition of viral proteins) in the starting cell line (P0), introducing a mutant EIF2alpha (in the PKR pathway) in the starting cell line (P0), and/or manipulating or modulating virus-associated (VA) RNAs (VA-RNAs, an inhibitor of PKR). Virus-associated (VA) RNAs from adenovirus act as small-interference RNAs and are transcribed from the vector genome. These VA-RNAs can trigger the innate immune response. Moreover, VA-RNAs are processed to functional viral miRNAs and disturb the expression of numerous cellular genes. Therefore, VA-deleted adenoviral vector production constructs (AdVs) lacking VA-RNA genes, or having modified VA-RNA, would be advantageous. However, VA- deleted AdVs do not produce commercially sufficient quantities of AAV titers (e.g., resulting in fewer and poor-quality virions). Conversely, overexpressing VA-RNA also results in a low titer of AAV production that would not be commercially feasible for scale-up. Thus, developing conditional VA-RNA constructs, and combining any of those optimized constructs with the conditional helper constructs described herein, will provide commercially relevant, high-quality virions from the AAV production systems as described herein. All three of these strategies can be done in any combination. [0775] In some embodiments, VA-RNA is also an inhibitor of PKR, which is involved in a pathway responsible for inhibiting AAV viral protein synthesis. In particular, PKR phosphorylates EIF2alpha, which results in inhibition of viral protein synthesis. [0776] While the limited interactions between VA-RNA, PKR, and EIF2alpha are understood, PKR is a major kinase that may self-phosphorylate and EIF2alpha may be phosphorylated by other kinases. As such, three strategies (PKR KO, EIF2alpha mutation, manipulation of VA- RNA) are being developed for use in any combination in the AAV production systems described herein. [0777] In some aspects, the constructs of this system can also be used in a vector system, wherein the constructs do not integrate into the genome of the cell. [0778] In the embodiment shown in FIG.2A, an exemplary system of polynucleotides for inducibly producing rAAV is depicted. In absence of a first triggering agent and a second triggering agent, the system is in an off state. In Construct 1, depicted in this embodiment, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter. The coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE). The coding sequence for the Rep proteins is similar to the coding sequence for the Rep proteins described in FIG.1A. This construct may also be referred to as a Rep-Cap construct. This construct comprises from 5’ to 3’: one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence. Construct 2 is similar to Construct 4 depicted in FIGS.1A and 1B. Construct 3 is similar to Construct 3 depicted in FIGS.1A and 1B. This system does not include Construct 2 depicted in FIGS.1A and 1B. In some embodiments, the Cap proteins coding sequence may include a strong polyA signal sequence positioned 3’ to the Cap proteins coding sequence. In some embodiments, the strong polyA signal sequence may be SV40, bGH, or RBG polyA signal sequence. [0779] In the embodiment shown in FIG.2B, the system of polynucleotides for inducibly producing rAAV depicted in FIG.2A is shown in the on-state, after induction by the first triggering agent and the second triggering agent. [0780] In the embodiment shown in FIG.3A, an exemplary system of polynucleotides for inducibly producing rAAV is depicted. In some embodiments, in absence of a first triggering agent and a second triggering agent, the system is in an off state. In Construct 1, depicted in this embodiment, the sequence encoding the AAV Cap proteins is operably linked to an inducible promoter (for example, Tet-On promoter). The coding sequences and promoters for the Rep proteins are separated from the coding sequence and inducible promoter for the Cap proteins by a transcription blocking element (TBE). In some embodiments, this system includes an additional polynucleotide, depicted as Construct 4, for expressing AAV Cap proteins. In other embodiments, Construct 4 may be referred to as a Cap construct and is the same as Construct 2 shown in FIGS.1A and 1B. [0781] In the embodiment shown in FIG.3B, the system of polynucleotides for inducibly producing rAAV depicted in FIG.3A is shown in the on-state, after induction by the first triggering agent and the second triggering agent. [0782] In some embodiments, a cell comprises one construct (Rep Cap construct, inducible helper construct, and the payload construct). In some embodiments, the one construct is stably integrated into the genome of the cell. In some embodiments, the one construct is not stably integrated into the genome of the cell. In some embodiments, a plurality of the one construct is stably integrated into the genome of the cell. In some embodiments, a plurality of the one construct is not stably integrated into the genome of the cell. [0783] In some embodiments, a cell comprises two constructs (any combination of Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct). In some embodiments, the two constructs are stably integrated into the genome of the cell. In some embodiments, the two constructs are not stably integrated into the genome of the cell. In some embodiments, the two constructs are separately stably integrated into the genome of the cell. In some embodiments, the two constructs are not separately stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the two constructs are not separately stably integrated into the genome of the cell. In some embodiments, a cell comprises the Rep Cap construct and the inducible Cap construct. In some embodiments, a cell comprises the Rep Cap construct and the payload construct. In some embodiments, a cell comprises inducible Cap construct and the payload construct. In some embodiments, a cell comprises the inducible helper construct and the payload construct. In some embodiments, a cell comprises the Rep Cap construct and the inducible helper construct. In some embodiments, a cell comprises the Rep Cap construct (s) as disclosed herein, the inducible Cap construct(s) as disclosed herein, the inducible helper construct(s) as disclosed herein, payload construct(s) as disclosed herein, or any combination thereof. In some embodiments, the inducible helper construct comprises a VA-RNA construct as described herein. In some embodiments, the cell further comprises the VA-RNA construct as described herein. In some embodiments, the VA-RNA construct is stably integrated into the genome of the cell. In some embodiments, the VA-RNA construct is not stably integrated into the genome of the cell. [0784] In some embodiments, a cell comprises three constructs (any combination of Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct). For example, the constructs of FIG.2A. In some embodiments, the three constructs are stably integrated into the genome of the cell. In some embodiments, the three constructs are not stably integrated into the genome of the cell. In some embodiments, the three constructs are separately stably integrated into the genome of the cell. In some embodiments, the three constructs are not separately stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the three constructs are not separately stably integrated into the genome of the cell. In some embodiments, a cell comprises the Rep Cap construct, the inducible Cap construct, and the inducible helper construct. In some embodiments, a cell comprises the Rep Cap construct, the inducible Cap construct, and the payload construct. In some embodiments, a cell comprises the Rep Cap construct, the inducible helper construct, and the payload. In some embodiments, a cell comprises the inducible Cap construct, the inducible helper construct, and the payload. In some embodiments, a cell comprises the Rep Cap construct (s) as disclosed herein, the inducible Cap construct(s) as disclosed herein, the inducible helper construct(s) as disclosed herein, payload construct(s) as disclosed herein, or any combination thereof. In some embodiments, the inducible helper construct comprises a VA- RNA construct as described herein. In some embodiments, a cell further comprises the VA-RNA construct as described herein. In some embodiments, the VA-RNA construct is stably integrated into the genome of the cell. In some embodiments, the VA-RNA construct is not stably integrated into the genome of the cell. [0785] In some embodiments, a cell comprises three constructs (any combination of Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct). For example, the constructs of FIG.2A. In some embodiments, the one of the three constructs is stably integrated into the genome of the cell. For example, Construct 3 is stably integrated into the cell and Construct 1 and Construct 2 are transiently transfected into that cell. In some embodiments, the two of the three constructs are stably integrated into the genome of the cell. For example, Construct 3 and Construct 1 (e.g., but with different selectable markers than shown in FIG.2A) are is stably integrated into the cell and Construct 2 (e.g., but also with a different selectable marker than shown in FIG.2A) is transiently transfected into that cell. In some embodiments, the three constructs are not stably integrated into the genome of the cell, but are instead all transiently transfected into the cell. [0786] In some embodiments, a cell comprises all four constructs (Rep Cap construct, inducible Cap construct, inducible helper construct, and the payload construct). For example, the constructs of FIG.1A, FIG.3A, or FIG.7. In some embodiments, the four constructs are stably integrated into the genome of the cell. In some embodiments, the four constructs are not stably integrated into the genome of the cell. In some embodiments, the four constructs are separately stably integrated into the genome of the cell. In some embodiments, the four constructs are not separately stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are not stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are separately stably integrated into the genome of the cell. In some embodiments, a plurality of the four constructs are not separately stably integrated into the genome of the cell. In some embodiments, the cell, the inducible helper construct comprises a VA-RNA construct as described herein. In some embodiments, cell further comprises the VA-RNA construct. [0787] In some embodiments, a VA-RNA construct is a polynucleotide construct coding for a VA-RNA, wherein a sequence coding for the VA-RNA comprises at least two mutations in an internal promoter. In some embodiments, the sequence coding for the VA-RNA comprises a sequence coding for a transcriptionally dead VA-RNA. In some embodiments, the sequence coding for the VA-RNA comprises a deletion of from about 5-10 nucleotides in the promoter region. In some embodiments, the sequence coding for the VA-RNA comprises at least one mutation. In some embodiments, the at least one mutation is in the A Box promoter region. In some embodiments, the at least one mutation is in the B Box promoter region. In some embodiments, the at least one mutation is G16A and G60A. In some embodiments, the expression of the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the expression of the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the expression of the VA-RNA is under the control of an interrupted U6 or U7 promoter. In some embodiments, the polynucleotide construct comprises upstream of the VA-RNA gene sequence, from 5’ to 3’: a) a first part of a U6 or U7 promoter sequence; b) a first recombination site; c) a stuffer sequence; d) a second recombination site; e) a second part of a U6 or U7 promoter sequence. In some embodiments, the stuffer sequence is excisable by a recombinase. In some embodiments, the stuffer sequence comprises a sequence encoding a gene. In some embodiments, the stuffer sequence comprises a promoter. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is a CMV promoter. In some embodiments, the gene encodes a detectable marker or a selectable marker. In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z-Nter or DHFR Z-Cter. In some embodiments, the DHFR Z- Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein and the second auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0788] In some embodiments, the stuffer sequence further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the stuffer sequence further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0789] In some embodiments, a selectable marker comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90 – SEQ ID NO: 98, SEQ ID NO: 112 – SEQ ID NO: 131, SEQ ID NO: 137, or SEQ ID NO: 138. In some embodiments, the selectable marker and helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 101 – SEQ ID NO: 109.In some embodiments, the detectable marker comprises a luminescent marker or a fluorescent marker. In some embodiments, the fluorescent marker is GFP, EGFP, RFP, CFP, BFP, YFP, or mCherry. In some embodiments, the VA-RNA construct further comprises a sequence coding for a recombinase. In some embodiments, the recombinase is exogenously provided. In some embodiments, the recombinase is a site-specific recombinase. In some embodiments, the recombinase is a Cre polypeptide or a Flippase polypeptide. In some embodiments, the Cre polypeptide is fused to a ligand binding domain. In some embodiments, the ligand binding domain is a hormone receptor. In some embodiments, the hormone receptor is an estrogen receptor. In some embodiments, the estrogen receptor comprises a point mutation. In some embodiments, the estrogen receptor is ERT2. In some embodiments, the recombinase is a Cre-ERT2 polypeptide. In some embodiments, the first recombination site is a first lox sequence and the second recombination site is a second lox sequence. In some embodiments, the first lox sequence is a first loxP site and the second lox sequence is a second loxP site. In some embodiments, the first recombination site is a first FRT site and the second recombination site is a second FRT site. The polynucleotide construct of any one of any embodiment disclosed herein, further comprising a sequence coding for a selectable marker. [0790] In some embodiments, the selectable marker is a mammalian cell selection element. In some embodiments, the selectable marker is an auxotrophic selection element. In some embodiments, the auxotrophic selection element codes for an active protein. In some embodiments, the active protein is glutamine synthetase (GS), thymidylate synthase (TYMS), phenylalanine hydroxylase (PAH), or dihydrofolate reductase (DHFR). In some embodiments, PAH comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 90. In some embodiments, GS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, TYMS comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the auxotrophic selection element codes for an inactive protein that requires expression of a second auxotrophic selection element for activity. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein Z-Cter and the second auxotrophic selection element codes for N-terminal fragment of an auxotrophic protein Z-Nter, or vice a versa. In some embodiments, the auxotrophic selection element codes for DHFR Z-Cter or DHFR Z-Nter. In some embodiments, the selectable marker is DHFR Z- Nter or DHFR Z-Cter. In some embodiments, the DHFR Z-Nter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the DHFR Z-Cter comprises a sequence having at least 70%, 80%, 90%, 95%, 99%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein and the second auxotrophic selection element codes for an N- terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of an auxotrophic protein fused to an N-terminal intein of a split intein and the second auxotrophic selection element codes for a C-terminal fragment of the auxotrophic protein fused to a C- terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for C-terminal fragment of PAH, GS, TYMS, or DHFR fused to a C-terminal intein of a split intein. In some embodiments, the auxotrophic selection element codes for an N-terminal fragment of PAH, GS, TYMS, or DHFR fused to a N-terminal intein of a split intein. In some embodiments, the selectable marker is an antibiotic resistance protein. In some embodiments, the selectable marker is a split intein linked to an N-terminus of the antibiotic resistance protein or split intein linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the selectable marker is a leucine zipper linked to an N-terminus of the antibiotic resistance protein or leucine zipper linked to a C-terminus of the antibiotic resistance protein. In some embodiments, the antibiotic resistance protein is for puromycin resistance or blasticidin resistance. In some embodiments, the split intein is derived from the Nostoc punctiforme (Npu) DnaE intein, the Synechocystis species, strain PCC6803 (Ssp) DnaE intein, or the consensus DnaE intein (Cfa). In some embodiments, an N-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, a C-terminal intein comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 141. [0791] In some embodiments, the polynucleotide construct further comprises a sequence coding for a selectable marker and a helper enzyme, wherein expression of the helper enzyme facilitates growth of the cell in conjunction with the selectable marker. In certain embodiments, the helper enzyme is an enzyme that facilitates production of a molecule required for cell growth. For example, the helper enzyme may be required for production of a cofactor utilized by the functional enzyme to generate the molecule required for cell growth. In certain embodiments, the cell may produce the helper enzyme at low levels and the expression of the helper enzyme from the helper construct can increase helper enzyme levels thereby increasing production of the molecule required for cell growth, by, e.g., increasing levels of a co-factor required for enzyme activity. In some embodiments, the polynucleotide construct further encodes a helper enzyme involved in production of tyrosine from phenylalanine. In some embodiments, the helper enzyme facilitates PAH-mediated production of tyrosine from phenylalanine. In some embodiments, the helper enzyme catalyzes production a co-factor required by PAH for converting phenylalanine to tyrosine. In some embodiments, the helper enzyme is GTP cyclohydrolase I (GTP-CH1). In some embodiments, the helper enzyme comprises at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the GTP-CH1 produces the cofactor (6R)-5,6,7,8-tetrahydrobiopterin (BH4) that is required for conversion of phenylalanine to tyrosine. In some embodiments, expression of GTP-CH1 facilitates growth of the host cell in conjunction with functional PAH upon application of the single selective pressure. [0792] In some embodiments, a stable cell line is produced from the cell or the population of cells as described herein. In some embodiments, the stable cell line is derived from a single cell and is monoclonal. In certain embodiments, the stable cell line can be a mammalian stable cell line. The stable cell line can be produced by expanding or passaging a cell as described herein. In some embodiments, a stable cell line comprises the population of cells as disclosed herein. In some embodiments, the population of cells are derived from a single cell. In some embodiments, at least 70%, 80%, 90%, 95%, 99%, or 100% of the cells of the stable cell line are the population of cells as disclosed herein. Cells Comprising Vector/Vector System [0793] In some aspects, cells comprising vector system described in section “1.6. Vector/Vector System” above are provided. [0794] In some embodiments, one or more of the first vector, the second vector, the third vector, and the fourth vector are integrated into the nuclear genome of the cell. [0795] In some embodiments, wherein the first vector, the second vector, and the third vector are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0796] In some embodiments, wherein the first vector, the second vector, and the third vector, are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. [0797] In some embodiments, the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof. [0798] In some embodiments, the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof. In some embodiments, the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. [0799] In some embodiments, the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein. In other embodiments, the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein. [0800] In some embodiments, the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome. In other embodiments, the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. [0801] In some embodiments, the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins. In other embodiments, the third polynucleotide comprises SEQ ID NO: 30. In certain embodiments, the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. [0802] In some embodiments, the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein. In other embodiments, the any two proteins are E2A and E4 or E1A and E1B. [0803] In some embodiments, the cell is a mammalian cell. In some embodiments, the mammalian cell is a HEK293 cell. In certain embodiments, the HEK293 cell is DHFR-deficient or GS-deficient. [0804] In some embodiments, the cell expresses adenoviral helper proteins E1A and E1B. [0805] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0806] In some embodiments, upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0807] In some embodiments, after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In other embodiments, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In other embodiments, the cell comprises has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155. [0808] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161. [0809] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. [0810] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. [0811] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163. [0812] In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158. 1.10. Conditional expression [0813] In a first aspect, stable cell lines, e.g., mammalian stable cell lines, capable of conditionally producing recombinant AAV (rAAV) virions are provided. The rAAV virions package an expressible payload, such as, a sequence encoding a therapeutic nucleic acid or protein or a complex therefore. In certain embodiments, production of virions is not conditioned on the presence of an episome or independent plasmid within the cell. [0814] In another aspect, the plasmids are provided comprising the constructs as disclosed herein. In some embodiments, the plasmids further comprise Epstein-Barr virus (EBV) sequences to stably maintain the constructs extrachromosomally. Rep Protein and Cap Protein Inducible Expression [0815] As discussed herein, expression of AAV Rep and Cap proteins is conditional. In certain embodiments, expression of AAV Cap proteins is conditioned on addition of at least a first triggering agent to the cell culture medium. In certain embodiments, expression of AAV Rep proteins is conditioned on addition of a first triggering agent and a second triggering agent to the cell culture medium. [0816] In some embodiments, the first triggering agent is . In certain embodiments, doxycycline is used to the control a Tet inducible promoter. In certain embodiments, the expression of the AAV cap proteins is under the control of a Tet inducible promoter. [0817] In some embodiments, expression of AAV Rep proteins is conditional. In certain embodiments, expression of AAV Rep proteins is conditioned on addition of at least a first triggering agent to the cell culture medium. In certain embodiments, expression of AAV Rep proteins is conditioned on addition of a first triggering agent and a second triggering agent to the cell culture medium. In certain embodiments, a first triggering agent is used to the control a Tet inducible promoter. In certain embodiments, the expression of the AAV cap proteins is under the control of a Tet inducible promoter. In some embodiments, a first triggering agent is doxycycline. [0818] In some embodiments, alternatively, other inducible promoters can be utilized instead of a Tet inducible promoter, such as, but not limited to, a cumate inducible promoter system, which is under the control of cumate as the triggering agent or an ecdysone-inducible promoter, which is under the control of ecdysone or ponasterone as the triggering agent. Site-Specific Recombinase System [0819] In some embodiments, any suitable inducible excising agent (e.g., recombinase) can be utilized. In certain embodiments, an excising agent can be a recombinase. In certain cases, an excising agent can be a site-specific recombinase. Exemplary site-specific recombinase systems include, without limitation, Cre-loxP, Flp-FRT, PhiC31-att, Dre-rox, and Tre-loxLTR site- specific recombinase systems. In some embodiments, the Cre-loxP system uses a Cre recombinase to catalyze site-specific recombination between two loxP sites. In some embodiments, the Flp-FRT system uses a flippase (FLP) recombinase to catalyze site-specific recombination between two flippase recognition target (FRT) sites. In some embodiments, the PhiC31-att system uses a phiC31 recombinase to catalyze site-specific recombination between two attachment (att) sites referred to as attB and attP. In some embodiments, the Dre-rox system uses a DreO recombinase to catalyze site-specific recombination between two rox sites. In some embodiments, the Tre-loxLTR system uses a Tre recombinase to catalyze site-specific recombination between two loxP sites that are modified with HIV long terminal repeats (loxLTR). For a description of various site-specific recombinase systems, see, e.g., Stark et al. (2011) Biochem. Soc. Trans.39(2):617-22; Olorunniji et al. (2016) Biochem. J.473(6):673-684; Birling et al. (2009) Methods Mol. Biol.561:245-63; García-Otin et al. (2006) Front. Biosci. 11:1108-1136; Weasner et al. (2017) Methods Mol. Biol.1642:195-209; herein incorporated by reference in their entireties. [0820] In some embodiments, an excising agent can target a recombination site. Examples of suitable inducible excising agents include Cre and a flippase. In certain embodiments, the Cre element can be hormone activated Cre, or light inducible Cre. In some embodiments, a recombination site can be a lox site. In certain embodiments, a lox site can be a loxP site. In other embodiments, a recombination site can be an FRT site. [0821] In some embodiments, the Flippase recombinase system is based on Flp-FRT recombination, a site-directed recombination technology used to manipulate DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase (Flp) derived from the 2 µ plasmid of baker's yeast Saccharomyces cerevisiae. The Flp protein, much like Cre, is a tyrosine family site-specific recombinase. [0822] In typical embodiments, the cells do not express cytotoxic levels of Rep protein prior to addition of both the first expression and second triggering agents to the cell culture medium. In certain embodiments, the cells do not express cytostatic levels of Rep protein prior to addition of both the first and second triggering agents to the cell culture medium. In certain embodiments, the average concentration of Rep protein within the cells is less than the amount prior to addition of both of the first and second triggering agents to the cell culture medium. In some embodiments, expression of Rep and Cap proteins becomes constitutive after addition of all of the at least first triggering agents to the cell culture medium. [0823] In some embodiments, expression of at least one adenoviral helper protein is conditional. In certain embodiments, expression of the at least one adenoviral helper protein is conditioned on addition of at least a third triggering agent to the cell culture medium. In particular embodiments, the third triggering agent is the same as the first triggering agent. In certain embodiments, expression of adenoviral helper proteins is conditioned on addition of a third triggering agent and a fourth triggering agent to the cell culture medium. In particular embodiments, the fourth triggering agent is the same as the second triggering agent. In particular embodiments, the third triggering agent is the same as the first triggering agent and the fourth triggering agent is the same as the second triggering agent. For example, the first triggering agent and the third triggering agent are both doxycycline. For example, the second triggering agent and the fourth triggering agent are both tamoxifen. [0824] In some embodiments, continued expression of adenoviral helper proteins following triggering of expression by contact of the cell with the at least third triggering agent requires the presence of only the third triggering agent in the cell culture medium. In certain embodiments, the third triggering agent is the same as the first triggering agent. For example, the first triggering agent and the third triggering agent are both doxycycline. [0825] In some embodiments, expression of at least one adenoviral helper RNA is conditional. In certain embodiments, the adenoviral helper proteins comprise Ad E2A. In certain embodiments, the adenoviral helper proteins comprise Ad E4. In some embodiments, the adenoviral helper protein is tagged. A tag can be a protein tag. A protein tag can be a FLAG tag. In some embodiments, E2A is FLAG-tagged. In some embodiments, E4 is FLAG-tagged. [0826] In particular embodiments, the adenoviral helper RNA is a VA-RNA. In particular embodiments, the adenoviral helper RNA is expressed from an inducible VA-RNA construct. In some embodiments, the VA-RNA is a mutant VA-RNA. In some embodiments, the VA-RNA is a transcriptionally dead VA-RNA. In some embodiments, the VA-RNA is under the control of an RNA polymerase III promoter. In some embodiments, the VA-RNA is under the control of an interrupted RNA polymerase III promoter. In some embodiments, the VA-RNA is under the control of a U6 or U7 promoter. In some embodiments, the VA-RNA is under the control of an interrupted U6 or U7 promoter. [0827] In some embodiments, the third triggering agent is a tetracycline. In certain embodiments, the tetracycline is doxycycline (“Dox”). In some embodiments, the fourth triggering agent is an estrogen receptor ligand. In certain embodiments, the estrogen receptor ligand is a selective estrogen receptor modulator (SERM). In particular embodiments, the estrogen receptor ligand is tamoxifen. [0828] In some embodiments of the stable cell line, expression of the payload is conditioned on addition of at least a fifth triggering agent to the cell culture medium. In some embodiments, expression of the payload is not conditioned on addition of an triggering agent to the cell culture medium. [0829] In some embodiments, expression of Rep and Cap proteins, Cap proteins, adenoviral helper proteins, and the payload becomes constitutive after addition of only one triggering agent to the cell culture medium. In certain embodiments, expression of Rep and Cap proteins, Cap proteins, and the adenoviral helper proteins becomes constitutive after addition of only one triggering agent to the cell culture medium. [0830] In certain embodiments, the one triggering agent is the first triggering agent. In certain embodiments, the first triggering agent is a tetracycline. In particular embodiments, the first triggering agent is doxycycline. 1.11. Cell Cultures and Bioreactors [0831] In some embodiments, a cell, population of cells, or stable cell line as disclosed herein is in a cell culture. In some embodiments, a cell culture composition comprises: a) suspension- adapted cells, b) serum-free cell culture media, and c) recombinant AAV (rAAV) virions, wherein the cell culture composition is free of herpes simplex virus, baculovirus, and adenovirus, and wherein the cell culture composition is free of plasmid and transfection agent. In some embodiments, the cell culture composition is free of polyethylenimine (PEI). In some embodiments, the suspension-adapted cells are suspension-adapted mammalian cells. In some embodiments, the suspension-adapted cells are suspension-adapted HEK293 cells or derivatives thereof. In some embodiments, the suspension-adapted mammalian cells are cells from the stable cell line of as disclosed herein, the population of cells as disclosed herein, or comprise a cell as disclosed herein. In some embodiments, the cell culture composition has a prepurification rAAV concentration of no less than 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, or 5×1015 viral genome (vg)/L. In some embodiments, the cell culture composition has a prepurification rAAV encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. [0832] In some embodiments, rAAV virion from the stable cells as disclosed herein is produced in a bioreactor. In some embodiments, a bioreactor comprises the stable cell line as disclosed herein. In some embodiments, a bioreactor comprising the population of cells of as disclosed herein. In some embodiments, a bioreactor comprising the cell as disclosed herein. In some embodiments, a bioreactor contains the cell culture as disclosed herein. In some embodiments, the bioreactor is a 1L bioreactor. In some embodiments, the 1L bioreactor has a total rAAV yield of greater than 1×1014 viral genome (vg). In some embodiments, the bioreactor is a 5L bioreactor. In some embodiments, the 5L bioreactor has a total rAAV yield of greater than 5×1014 viral genome (vg). In some embodiments, the bioreactor is a 50L bioreactor. In some embodiments, the 50L bioreactor has a total rAAV yield of greater than 5×1015 viral genome (vg). In some embodiments, the bioreactor is a 100L bioreactor. In some embodiments, the 100L bioreactor has a total rAAV yield of greater than 1×1016 viral genome (vg). In some embodiments, the bioreactor is a 500L bioreactor. In some embodiments, the 500L bioreactor has a total rAAV yield of greater than 5×1016 viral genome (vg). In some embodiments, the bioreactor is a 2000L bioreactor. In some embodiments, the 2000L bioreactor has a total rAAV yield of greater than 2×1017 viral genome (vg). In some embodiments, a bioreactor comprises a plurality of rAAV virions having a concentration of greater than 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, or 5×1015 viral genome (vg)/L. In some embodiments, a bioreactor comprises a plurality of rAAV virions having a prepurification concentration of greater than 1×1014, 2×1014, 3×1014, 4×1014, 5×1014, 6×1014, 7×1014, 8×1014, 9×1014, 1×1015, or 5×1015 viral genome (vg)/L. In some embodiments, the bioreactor is a 1L, 5L, 50L, 100L, 500L, or 2000L bioreactor. In some embodiments, the bioreactor is a single use bioreactor. 1.12. Compositions of rAAV [0833] In some embodiments, the cell, population of cells, or stable cell line as disclosed herein is induced (as disclosed herein, e.g., after administration of a first and a second triggering agent in a bioreactor) to produce a plurality of rAAV virions. In some embodiments, a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification concentration of greater than 1 × 1011 or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013 or 1 × 1014 viral genomes per milliliter. In some embodiments, a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, a composition comprises a plurality of rAAV virions encapsidating a viral genome, wherein the composition has a prepurification F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, a composition comprises an rAAV virion encapsidating a viral genome, wherein the composition has an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99% at an MOI of 1 × 105 vg/target cell or less. In some embodiments, the rAAV virion has an increased infectivity compared an rAAV virion produced by an otherwise comparable cell capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virion has at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared an rAAV virion produced by an otherwise comparable cell capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the rAAV virion has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared to an AAV virion produced by a cell having wildtype AAV at the same MOI. In some embodiments, the rAAV virion has at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared an AAV virion produced by a cell having wildtype AAV at the same MOI. In some embodiments, the compositions further comprise a plurality of the rAAV virion. In some embodiments, the plurality of rAAV virions have a prepurification concentration of greater than 1 × 1011 or no less than 5 × 1011, 1 × 1012, 5 × 1012, 1 × 1013 or 1 × 1014 viral genomes per milliliter. In some embodiments, the plurality of rAAV virions have a prepurification encapsidation ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the plurality of rAAV virions have a prepurification F:E ratio of no less than 0.5, 0.6, 0.7, 0.8, 0.9, 0.95, 0.97, or 0.99. In some embodiments, the plurality of rAAV virions have an infectivity of no less than 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the plurality of rAAV virions have an increased infectivity compared to a plurality of rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the plurality of rAAV virions have at least 1%, 5%, 10%, 15%, 20%, 30%, 40%, or 50% greater infectivity compared a plurality of rAAV virions produced by an otherwise comparable the population of cells capable of producing rAAV virions upon transient transfection at the same MOI. In some embodiments, the plurality of rAAV virions have at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 98%, 99%, or 100% infectivity as compared a plurality of AAV virions produced by a cell having wildtype AAV at the same MOI. In some embodiments, the MOI is 1 × 101, 1 × 102, 2 × 103, 5 × 104, or 1 × 105 vg/target cell. In some embodiments, the MOI is selected from a range of 1 × 101 to 1 × 105 vg/target cell. In some embodiments, the viral genome comprises a sequence coding for a payload. In some embodiments, expression of the sequence of the payload is driven by a constitutive promoter. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a gene. In some embodiments, the gene codes for a selectable marker or detectable marker. In some embodiments, the gene codes for a therapeutic polypeptide or transgene. In some embodiments, the sequence of the payload comprises a polynucleotide sequence coding for a therapeutic polynucleotide. In some embodiments, the therapeutic polynucleotide is a tRNA suppressor or a guide RNA. In some embodiments, the guide RNA is a polyribonucleotide capable of binding to a protein. In some embodiments, the protein is nuclease. In some embodiments, the protein is a Cas protein, an ADAR protein, or an ADAT protein. In some embodiments, the Cas protein is catalytically inactive Cas protein. In some embodiments, the rAAV virion comprises a Cap polypeptide. In some embodiments, the Cap polypeptide is an AAV capsid protein. In some embodiments, the AAV capsid protein is VP1, VP2, or VP3. In some embodiments, a serotype of the AAV capsid protein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11, AAV 12, AAV13, AAV 14, AAV 15 and AAV 16, AAV.rh8, AAV.rh10, AAV.rh20, AAV.rh39, AAV.Rh74, AAV.RHM4-1, AAV.hu37, AAV.Anc80, AAV.Anc80L65, AAV.7m8, AAV.PHP.B, AAV2.5, AAV2tYF, AAV3B, AAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, AAV.HSC16, and AAVhu68. In some embodiments, rAAV virions as disclosed herein are in a first composition and a second composition. In some embodiments, the first composition and the second composition have an encapsidation ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition and the second composition have an F:E ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition and the second composition have a concentration of viral genomes per milliliter that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition and the second composition have an infectivity that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition is a first dose and the second composition is a second dose. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days before a plurality of rAAV virions of the second composition is produced. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months before the second composition is produced. In some embodiments, the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the second composition is produced. In some embodiments, a plurality of rAAV virions of the first composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years before the second composition is produced. In some embodiments, the first composition is produced from a plurality of virions from a first bioreactor and the second composition is produced from a plurality of virions from a second bioreactor. In some embodiments, a third composition or more compositions are produced from the rAAV as disclosed herein. In some embodiments, the first composition, the second composition, and the third composition have an encapsidation ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition, the second composition, and the third composition have an F:E ratio that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition, the second composition, and the third composition have a concentration of viral genomes per milliliter that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the first composition, the second composition, and the third composition have an infectivity that varies by no more than 20%, 10%, 5%, 4%, 3%, 2%, or 1%. In some embodiments, the third composition is a third dose. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, or 7 days after a plurality of rAAV virions of the second composition is produced. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months after the second composition is produced. In some embodiments, the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after the second composition is produced. In some embodiments, a plurality of rAAV virions of the third composition is produced at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 years after the second composition is produced. In some embodiments, the third composition is produced from a plurality of virions from a third bioreactor. 1.13. Pharmaceutical Compositions [0834] The present disclosure provides pharmaceutical compositions comprising the polynucleotides, or vector system described herein or an rAAV virion encapsidating a polynucleotide payload (e.g., for encoding a therapeutic protein, such as an antibody or any fragment or derivative thereof), produced from such a vector system, and a pharmaceutically acceptable carrier, diluent, excipient, or buffer. In some cases, the pharmaceutically acceptable carrier, diluent, excipient, or buffer is suitable for use in a human. Such excipients, carriers, diluents, and buffers include any pharmaceutical agent that may be administered without undue toxicity. [0835] Pharmaceutically acceptable excipients include, but are not limited to, liquids such as water, saline, glycerol, polyethylene glycol, hyaluronic acid, and ethanol. Pharmaceutically acceptable salts may be included therein, for example, mineral acid salts such as hydrochlorides, hydrobromides, phosphates, sulfates, and the like; and the salts of organic acids such as acetates, propionates, malonates, benzoates, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles. A wide variety of pharmaceutically acceptable excipients are known in the art and need not be discussed in detail herein. Pharmaceutically acceptable excipients have been amply described in a variety of publications, including, for example, A. Gennaro (2000) “Remington: The Science and Practice of Pharmacy,” 20th edition, Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; and Handbook of Pharmaceutical Excipients (2000) A.H. Kibbe et al., eds., 3rd ed. Amer. Pharmaceutical Assoc. Certain facilitators of nucleic acid uptake and/or expression may also be included in the compositions or coadministered. [0836] In some embodiments, a pharmaceutical composition comprises the plurality of rAAV virions of any one of the embodiments as disclosed herein as disclosed herein and a pharmaceutically acceptable carrier. In some embodiment, a plurality of pharmaceutical doses each independently comprise the plurality of rAAV virions of any one of the embodiments as disclosed herein as disclosed herein and a pharmaceutically acceptable carrier. In some embodiments, the encapsidation ratio has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the F:E ratio has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the concentration of viral genomes has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the concentration of vector genomes has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. In some embodiments, the rAAV virion infectivity has a difference of not more than 20%, 10%, 5%, 4%, 3%, 2%, or 1% between a first dose and a second dose of a plurality of pharmaceutical doses. 1.14. Methods Methods of Generating a Cell Line – Polynucleotides [0837] In some aspects, methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: (i) introducing into a cell the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” or “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; (ii) selecting for a cell expressing a selectable marker of the third polynucleotide; (iii) introducing into the cell expressing the selectable marker of the third polynucleotide, the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above, the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above, and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above; (iv) selecting for a cell expressing the selectable marker of the third polynucleotide, a selectable marker of the first polynucleotide, a selectable marker of the second polynucleotide, and a selectable marker of the fourth polynucleotide; (v) expanding the cell expressing the selectable marker of the third polynucleotide, the selectable marker of the first polynucleotide, the selectable marker of the second polynucleotide, and the selectable marker of the fourth polynucleotide into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions are provided. [0838] In some embodiments, the methods further comprise contacting a cell of the cell line to a first triggering agent and a second triggering agent. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase. [0839] In some embodiments, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase. [0840] In some embodiments, the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In certain embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein. [0841] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0842] In some embodiments, in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide. [0843] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating a Cell Line – Polynucleotide Constructs [0844] In some aspects, A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: (i) introducing into a cell the second polynucleotide construct described in section “1.5. Polynucleotide Construct System” above; (ii) selecting for a cell expressing a selectable marker of the second polynucleotide construct; (iii) introducing into the cell expressing the selectable marker of the second polynucleotide construct, the first polynucleotide construct described in section “1.5. Polynucleotide Construct System” above, and the third polynucleotide construct described in section “1.5. Polynucleotide Construct System” above; (iv) selecting for a cell expressing the selectable marker of the second polynucleotide construct, a selectable marker of the first polynucleotide construct, and a selectable marker of the third polynucleotide construct; (v) expanding the cell expressing the selectable marker of the second polynucleotide construct, the selectable marker of the first polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions are provided. [0845] In some embodiments, the method further comprises contacting a cell of the cell line to a first triggering agent and a second triggering agent. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase. [0846] In some embodiments, recombination between the first recombination site and the second recombination site in the second polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase. [0847] In some embodiments, the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In certain embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein. [0848] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0849] In some embodiments, in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide construct. [0850] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating a Cell Line – Vectors [0851] In some aspects, methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: (i) introducing into a cell the third vector described in section “1.6. Vector/Vector System”; (ii) selecting for a cell expressing a selectable marker of the third vector; (iii) introducing into the cell expressing the selectable marker of the third vector, the first vector described in section “1.6. Vector/Vector System”, the second vector described in section “1.6. Vector/Vector System”, and the fourth vector described in section “1.6. Vector/Vector System”; (iv) selecting for a cell expressing the selectable marker of the third vector, a selectable marker of the first vector, a selectable marker of the vector polynucleotide, and a selectable marker of the fourth vector; (v) expanding the cell expressing the selectable marker of the third vector, the selectable marker of the first vector, the selectable marker of the second vector, and the selectable marker of the fourth vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions are provided. [0852] In some embodiments, the methods further comprise contacting a cell of the cell line to a first triggering agent and a second triggering agent. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase. In some embodiments, recombination between the first recombination site and the second recombination site in the first vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the third vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase. [0853] In some embodiments, the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In some embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein. [0854] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0855] In some embodiments, in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector. [0856] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. [0857] In some aspects, methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: (i) introducing into a cell the second vector described in section “1.6. Vector/Vector System”; (ii) selecting for a cell expressing a selectable marker of the second vector; (iii) introducing into the cell expressing the selectable marker of the second vector, the first vector described in section “1.6. Vector/Vector System”, and the third vector described in section “1.6. Vector/Vector System”; (iv) selecting for a cell expressing the selectable marker of the second vector, a selectable marker of the first vector, and a selectable marker of the third vector; (v) expanding the cell expressing the selectable marker of the second vector, the selectable marker of the first vector, and the selectable marker of the third vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions are provided. [0858] In some embodiments, the methods further comprise contacting a cell of the cell line to a first triggering agent and a second triggering agent. In some embodiments, in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase. [0859] In some embodiments, recombination between the first recombination site and the second recombination site in the second vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence; and recombination between the third recombination site and the fourth recombination site in the second vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase. [0860] In some embodiments, the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein. In some embodiments, the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein. [0861] In some embodiments, the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. [0862] In some embodiments, in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector. [0863] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Method of producing rAAV [0864] In some aspect, methods of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload are provided and the methods comprise introducing into a cell the first polynucleotide construct as described herein; selecting for a cell expressing the selectable marker of the first polynucleotide construct; introducing into the cell expressing the selectable marker of the first polynucleotide construct, the second polynucleotide as described herein and the fourth polynucleotide construct as described herein; selecting for a cell expressing the selectable marker of the first polynucleotide construct, a selectable marker of the second polynucleotide construct, and a selectable marker of the fourth polynucleotide construct; introducing into the cell expressing the selectable marker of the first polynucleotide construct, the selectable marker of the second polynucleotide construct, and the selectable marker of the fourth polynucleotide construct, the third polynucleotide construct as described herein; selecting for a cell expressing the selectable marker of the first polynucleotide construct, the selectable marker of the second polynucleotide construct, the selectable marker of the fourth polynucleotide construct, and a selectable marker of the third polynucleotide construct; and expanding the cell expressing the selectable marker of the first polynucleotide construct, the selectable marker of the second polynucleotide construct, the selectable marker of the fourth polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions. In certain embodiments, the methods produce the rAAV virion. [0865] In some embodiments, the methods further comprise contacting a cell of the cell line with the triggering agent, wherein in the presence of the triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein (i) recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, or (ii) recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in inversion of the inversible element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap proteins, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap proteins; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins. In certain embodiments, the methods produce the rAAV virion. [0866] In some aspects, methods for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload of interest are provided, and the method comprises: contacting the cell as described herein with the triggering agent, wherein in the presence of the triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide construct by the recombinase results in excision of the excisable element or inversion of the inversible element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap proteins, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein in the presence of the triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins; wherein the expression of the one or more AAV helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload. In certain embodiments, the methods produce the rAAV virion. [0867] In some embodiments, the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the third polynucleotide or the third polynucleotide construct. [0868] In another aspect, methods of producing rAAV from stable cell lines are provided. The methods comprise adding the at least first and at least second triggering agents to the medium within which the stable mammalian cell lines described above are being cultured. [0869] In another aspect, methods of producing rAAV from a cell or population of cells as described herein are provided. The methods comprise adding the at least first and at least second triggering agents to the medium within which the cell or population of cells described above are being cultured. [0870] In particular embodiments, the first triggering agent is a tetracycline. In specific embodiments, the first triggering agent is Dox. In particular embodiments, the second triggering agent is an estrogen agonist or selective estrogen receptor modulator. In specific embodiments, the second triggering agent is tamoxifen. [0871] In some embodiments, the method further comprises a later step of culturing the stable mammalian cell line only in the presence of the first triggering agent. In some embodiments, the method further comprises a later step of culturing the cell or population of cells only in the presence of the first triggering agent. [0872] In some embodiments, the method further comprises purifying rAAV from culture medium. In some embodiments, the purifying comprises performing chromatographic purification. In some embodiments, the chromatographic purification comprises using a positively charged anion exchange resin, using a negatively charged anion exchange resin, using cation exchange chromatography, using affinity chromatography, using size exclusion chromatography, or a combination thereof. In some embodiments, the chromatographic purification comprises using column chromatographic fractionation. [0873] In some embodiments, rAAV is produced in a bioreactor as described herein. [0874] In some embodiments, a method of inducing the cell as described herein, the population of cells as described herein, or the stable cell line as described herein comprises administering a first triggering agent to the cell, population of cells, or the stable cell line, thereby inducing expression of the Rep polypeptides, Cap polypeptides, and one or more adenoviral helper proteins, in the cell, population of cells, or stable cell line. In some embodiments, the first triggering agent binds to an activator or a repressor. In some embodiments, activation of an inducible promoter is induced. In some embodiments, the activated inducible promoter transcribes a recombinase. In some embodiments, the first triggering agent is tetracycline or cumate. In some embodiments, the tetracycline is doxycycline. The methods described herein further comprise culturing the cell, population of cells, or the stable cell line with a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the second triggering agent binds to the recombinase. In some embodiments, the second triggering agent induces the recombinase to translocate to a nucleus of the cell, of a cell of the population of cells, of a cell of the stable cell lines. [0875] In some embodiments, a method of producing rAAV virion comprises administering a first triggering agent to the cell, population of cells, or the stable cell line, administering a second triggering agent to the cell, population of cells, or stable cell line, thereby producing the rAAV virion in the cell, population of cells, or stable cell line. In some embodiments, the first triggering agent binds to an activator or a repressor. In some embodiments, activation of an inducible promoter is induced. In some embodiments, the activated inducible promoter transcribes a recombinase. In some embodiments, the activated inducible promoter transcribes a Cap protein or Cap proteins. In some embodiments, a first activated inducible promoter transcribes a Cap protein or Cap proteins and a second activated inducible promoter transcribes a recombinase. In some embodiments, the first triggering agent is tetracycline or cumate. In some embodiments, the tetracycline is doxycycline. In some embodiments, the method further comprises culturing the cell, population of cells, or the stable cell line with a second triggering agent. In some embodiments, the second triggering agent is an estrogen receptor ligand. In some embodiments, the second triggering agent is a selective estrogen receptor modulator (SERM). In some embodiments, the second triggering agent is tamoxifen. In some embodiments, the second triggering agent binds to the recombinase. In some embodiments, the second triggering agent induces the recombinase to translocate to a nucleus of the cell, of a cell of the population of cells, of a cell of the stable cell lines. In some embodiments, the recombinase cuts at recombinase sites. In some embodiments, the at least one adenoviral help proteins, the Rep polypeptides, and the Cap polypeptides are expressed. In some embodiments, the Rep polypeptides and the Cap polypeptides assemble into an rAAV virion. In some embodiments, the rAAV virion encapsidates a sequence of a payload. In some embodiments, the cell, population of cells, or stable cell line do not express cytotoxic levels of Rep polypeptides prior to administration of both the first triggering agent and the second triggering agent. In some embodiments, the cell, population of cells, or stable cell line do not express cytotoxic levels of Cap polypeptides prior to administration of both the first triggering agent and the second triggering agent. In some embodiments, the cell, population of cells, or stable cell line do not express cytostatic levels of Rep polypeptides prior to administration of both the first triggering agent and the second triggering agent. In some embodiments, the average concentration of Rep polypeptides within the cell, population of cells, or stable cell line is less than the amount prior to administration of both of the first triggering agent and second triggering agent. In some embodiments, expression of Rep polypeptides and Cap polypeptides becomes constitutive after administration of both the first triggering agent and the second triggering agent. In some embodiments, the method further comprises performing at least a portion of the method in a bioreactor. In some embodiments, the bioreactor is not less than 20 L, 30 L, 40 L, 50 L, 100 L, 250 L, 300 L, or 500 L. [0876] In some embodiments, the method further comprises producing the rAAV virions in a plurality of batches. In some embodiments, the method further comprises producing the rAAV virions having a difference in the encapsidation ratio of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in the F:E ratio of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in the concentration of viral genomes of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in the concentration of vector genomes of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises producing the rAAV virions having a difference in infectivity of not more than 20%, 15%, 10%, 5%, 3%, 2%, or 1% between a first batch and a second batch. In some embodiments, the method further comprises performing the method according to good manufacturing practice (GMP) standards. In some embodiments, the method further comprises performing the method in a GMP facility. In some embodiments, the method further comprises culturing the cells in a culture medium and collecting a portion of the plurality of rAAV virions from the culture medium. In some embodiments, the method further comprises purifying at least some of the plurality of rAAV virions collected from the culture medium to obtain a purified rAAV population. In some embodiments, the purifying comprises performing chromatographic purification. In some embodiments, the chromatographic purification comprises using a positively charged anion exchange resin, using a negatively charged anion exchange resin, using cation exchange chromatography, using affinity chromatography, using size exclusion chromatography, or a combination thereof. In some embodiments, the chromatographic purification comprises using column chromatographic fractionation. [0877] In some embodiments, an rAAV virion is made by the methods as disclosed herein. In some embodiments, a composition comprising a plurality of rAAV virions is made by the methods as disclosed herein. In some embodiments, the rAAV virion produced as disclosed herein has increased infectivity compared to an rAAV virion produced by comparable transient transfection methods. Methods of Generating rAAV – Polynucleotides [0878] In some aspects, methods for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell described in section “1.9. Cells” above to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second polynucleotide; wherein the expression of the one or more AAV helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload, are provided. [0879] In some embodiments, the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first polynucleotide construct. [0880] In some embodiments, the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first polynucleotide construct. [0881] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating rAAV – Vectors [0882] In some aspects, methods for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell described in section “1.9. Cells” above to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload, are provided. [0883] In some embodiments, the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second vector. [0884] In some embodiments, the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second vector. [0885] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. [0886] In some aspects, methods for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell described in section “1.9. Cells” above to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload, are provided. [0887] In some embodiments, the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first vector. [0888] In some embodiments, the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first vector. [0889] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Producing rAAV via Transfection [0890] In some aspects, methods for producing recombinant AAV, the methods comprising performing transfection of a cell with the polynucleotides of the system described in section “1.4. Polynucleotide System” above, the polynucleotide constructs of the system described in section “1.5. Polynucleotide Construction System” above, or the vectors of the system described in section “1.6. Vector/Vector System” above and contacting the cell to a first triggering agent and a second triggering agent are provided. [0891] In some embodiments, the polynucleotides of the system described in section “1.4. Polynucleotide System” above, the polynucleotide constructs of the system described in section “1.5. Polynucleotide Construction System” above, or the vectors of the system described in section “1.6. Vector/Vector System” above are integrated into a nuclear genome of the cell. [0892] In some embodiments, the polynucleotides of the system described in section “1.4. Polynucleotide System” above, the polynucleotide constructs of the system described in section “1.5. Polynucleotide Construction System” above, or the vectors of the system described in section “1.6. Vector/Vector System” above are not integrated into a nuclear genome of the cell. [0893] In some embodiments, one or more of the polynucleotides of the system described in section “1.4. Polynucleotide System” above, of the polynucleotide constructs of the system described in section “1.5. Polynucleotide Construction System” above, or of the vectors of the system described in section “1.6. Vector/Vector System” above are integrated into a nuclear genome of the cell and one or more of the polynucleotides of the system described in section “1.4. Polynucleotide System” above, of the polynucleotide constructs of the system of described in section “1.5. Polynucleotide Construction System” above, or of the vectors of the system described in section “1.6. Vector/Vector System” above are not integrated into the nuclear genome of the cell. [0894] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Inducibly Producing rAAV – Polynucleotides [0895] In some aspects, methods for inducibly producing recombinant AAV, the methods comprising: culturing a cell comprising the polynucleotides of the system described in section “1.4. Polynucleotide System” above integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload, are provided. [0896] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Inducibly Producing rAAV – Polynucleotides Constructs [0897] In some aspects, methods for inducibly producing recombinant AAV, the methods comprising: culturing a cell comprising the polynucleotide constructs of the system described in section “1.5. Polynucleotide Construction System” above integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide construct resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload. [0898] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Inducibly Producing rAAV – Vectors [0899] In some aspects, methods for inducibly producing recombinant AAV, the methods comprising: culturing a cell comprising the vectors of the system described in section “1.6. Vector/Vector System” above integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload, are provided. [0900] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. [0901] In some aspects, methods for inducibly producing recombinant AAV, the methods comprising: culturing a cell comprising the vectors of the system described in section “1.6. Vector/Vector System” above integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload, are provided. [0902] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating a Cell for Inducibly Producing rAAV – Polynucleotides [0903] In some aspects, methods of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the methods comprising: (i) introducing into cells the third polynucleotide described in sections “Third Polynucleotide Encoding Adenoviral Helper Proteins” and “Third Polynucleotide Encoding Adenoviral Helper Proteins and VA-RNA” above; (ii) selecting for cells expressing a selectable marker encoded by third polynucleotide; (iii) introducing into the cells selected in (ii) the first polynucleotide described in section “First Polynucleotide encoding AAV Rep Protein” above and the fourth polynucleotide described in section “Fourth Polynucleotide Encoding Payload” above, wherein the first polynucleotide encodes a first part of a split selectable marker and the fourth polynucleotide encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third polynucleotide and the selectable marker encoded by the first polynucleotide and the fourth polynucleotide, thereby generating the cells for inducibly producing the rAAV virions, are provided. [0904] In some embodiments, the methods further comprise: (iii) introducing into the cells selected in (iv) or the cells expanded in (v), the second polynucleotide described in section “1.3. Polynucleotides Encoding AAV Capsid Proteins” above; and (iv) selecting for cells expressing a selectable marker encoded by the second polynucleotide. [0905] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating a Cell for Inducibly Producing rAAV – Polynucleotides Constructs [0906] In some aspects, methods of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the methods comprising: (i) introducing into cells the second polynucleotide construct described in section “1.5. Polynucleotide Construct System” above; (ii) selecting for cells expressing a selectable marker encoded by second polynucleotide construct; (iii) introducing into the cells selected in (ii) the first polynucleotide construct and the third polynucleotide construct described in section “1.5. Polynucleotide Construct System” above, wherein the first polynucleotide construct encodes a first part of a split selectable marker and the third polynucleotide construct encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second polynucleotide construct and the selectable marker encoded by the first polynucleotide construct and the third polynucleotide construct, thereby generating the cells for inducibly producing the rAAV virions, are provided. [0907] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating a Cell for Inducibly Producing rAAV – Vectors [0908] In some aspects, methods of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the methods comprising: (i) introducing into cells the third vector described in section “1.6. Vector/Vector System” above; (ii) selecting for cells expressing a selectable marker encoded by third vector; (iii) introducing into the cells selected in (ii) the first vector and the fourth vector described in section “1.6. Vector/Vector System” above, wherein the first vector encodes a first part of a split selectable marker and the fourth vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third vector and the selectable marker encoded by the first vector and the fourth vector, thereby generating the cells for inducibly producing the rAAV virions, are provided. [0909] In some embodiments, the methods further comprise: (vi) introducing into the cells selected in (iv) or the cells expanded in (v), the second vector described in section “1.6. Vector/Vector System” above; and (vii) selecting for cells expressing a selectable marker encoded by the second vector. [0910] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. [0911] In some aspects, methods of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second vector described in section “1.6. Vector/Vector System” above; (ii) selecting for cells expressing a selectable marker encoded by second vector; (iii) introducing into the cells selected in (ii) the first vector and the third vector described in section “1.6. Vector/Vector System” above, wherein the first vector encodes a first part of a split selectable marker and the vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second vector and the selectable marker encoded by the first vector and the third vector, thereby generating the cells for inducibly producing the rAAV virions, are provided. [0912] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of Generating a Cell for Inducibly Producing rAAV – Polynucleotides [0913] In some aspects, methods of generating a cell for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell a first polynucleotide comprising: a first sequence comprising from 5' to 3': a first inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a first recombination site, the sequence encoding the inducible recombinase, and a second recombination site, wherein the first recombination site and the second recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the first inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the first recombination site and the second recombination site resulting in excision of the self-excising element, thereby operably linking the first inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins; and a third sequence comprising a second constitutive promoter operably linked to a sequence encoding a first selectable marker, wherein the cell constitutively expresses the first selectable marker, selecting for a cell expressing the first selectable marker; introducing a second polynucleotide and a third polynucleotide into the cell expressing the first selectable marker, the second polynucleotide comprising: a first sequence encoding AAV Cap proteins operably linked to a second inducible promoter; and from 5' to 3': one or more promoters operably linked to a second sequence comprising a first part of an AAV Rep coding sequence, a 5' splice site, a first part of an intron, a third recombination site, a first 3' splice site, a coding sequence comprising a stop signaling sequence, a fourth recombination site, a second part of the intron, a second 3' splice site, and a third sequence comprising a second part of the AAV Rep coding sequence, wherein the third recombination site, the first 3' splice site, the coding sequence comprising the stop signaling sequence, and the fourth recombination site form an excisable element, wherein the third recombination site and the fourth recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the third sequence comprising the second part of the AAV Rep coding sequence, wherein the third and fourth recombination sites are recombined by the inducible recombinase in the presence of the first triggering agent and the second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins; and a third constitutive promoter operably linked to a sequence encoding a first portion of a second selectable marker, the third polynucleotide comprising a sequence encoding the payload and a fourth constitutive promoter operably linked to a second portion of the second selectable marker, wherein the sequence encoding the payload is flanked by AAV inverted terminal repeats (ITRs); selecting for a cell expressing the first selectable marker and the second selectable marker, thereby generating the cell for inducibly producing recombinant AAV (rAAV) virions comprising the payload, are provided. [0914] In some embodiments, the methods further comprise contacting the cell with the first triggering agent and the second triggering agent for inducibly producing recombinant AAV (rAAV) virions comprising the payload. [0915] In some embodiments, the coding sequence encoding the stop signaling sequence of the second polynucleotide encodes for from 5' to 3': an exon and the stop signaling sequence. [0916] In some embodiments, the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide further comprises a polyadenylation signal. In some embodiments, the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. In certain embodiments, the polyadenylation signal sequence is a 3' of the sequence encoding AAV Cap proteins. [0917] In some embodiments, the polyadenylation signal sequence is a SV40 polyadenylation signal sequence or a bovine growth hormone polyadenylation signal sequence. [0918] In some embodiments, the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151. [0919] In some embodiments, the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162. [0920] In some embodiments, the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide is not flanked by inverted terminal repeat sequences. [0921] In some embodiments, the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof. [0922] In some embodiments, the first inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter and second inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same. [0923] In some embodiments, the first inducible promoter comprises a tetracycline-responsive promoter element (TRE) and the second inducible promoter comprises a tetracycline-responsive promoter element (TRE). In some embodiments, the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. In some embodiments, the minimal promoter is a human cytomegalovirus promoter. [0924] In some embodiments, the first inducible promoter is a Tet-On promoter and the second inducible promoter is a Tet-On promoter. [0925] In some embodiments, the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163. [0926] In some embodiments, the AAV Cap proteins comprise VP1, VP2, and VP3. [0927] In some embodiments, the first triggering agent is tetracycline and the second triggering agent is tamoxifen. In other embodiments, the first triggering agent is doxycycline and the second triggering agent is tamoxifen. Methods of treatment [0928] In another aspect, methods of treatment are provided. In various embodiments, the method comprises administering rAAV produced by the process described above to a patient in need thereof. In some embodiments, the administering is by intravenous administration, intramuscular administration, intrathecal administration, intracisternal administration, or administration via brain surgery. [0929] In some embodiments, a method of treating a condition or disorder comprises administering a therapeutically effective amount of the pharmaceutical composition of as disclosed herein to a patient in need thereof. In some embodiments, the disorder is a monogenic disorder. In some embodiments, the treatment results in at least one undesirable side effect and wherein the undesirable side effect is reduced relative to administering a daily dose that deviates more than 50%, 40%, 30%, 30%, 15%, 10%, 5%, or 2% from an expected dose. In some embodiments, the administering is by injection. In some embodiments, the injection is an infusion. In some embodiments, the daily dose is administered to the patient once. In some embodiments, the daily dose is administered to the patient two or more times. In some embodiments, the treatment results in at least one undesirable side effect and wherein the undesirable side effect is reduced relative to administering a plurality of rAAV virions produced from a triple transfection method. [0930] In some embodiments, the methods reduce the immunogenicity of a dose of rAAV having a predetermined number of viral genomes (VG) as compared to the same rAAV VG dose prepared by transient triple transfection. In some embodiments, the immunogenicity is measured by the titer or concentration of neutralizing antibodies in a subject. In some embodiments, a concentration of rAAV virion neutralizing antibody in the blood serum of the patient is reduced relative to a concentration of rAAV virion neutralizing antibody in the blood serum of a patient after administering a plurality of rAAV virions produced from a triple transfection method. In some embodiments, the concentration of rAAV virion neutralizing antibodies is measured by an ELISA assay. [0931] In some embodiments, the methods reduce the number or intensity of adverse effects caused by administering a dose of rAAV having a predetermined number of viral genomes (VG) as compared to the same rAAV VG dose prepared by transient triple transfection. In some embodiments, the methods reduce the number of adverse effects. In some embodiments, the predetermined number of VG in a dose is no greater than 3x1014 vg/kg. In some embodiments, the predetermined number of VG in a dose is no greater than 1x1014 vg/kg. In some embodiments, the predetermined number of VG in a dose is no greater than 5x1013 vg/kg. In some embodiments, the methods reduce the intensity of adverse effects. In some embodiments, the methods reduce both the number and the intensity of adverse events. [0932] In some embodiments, a method of administering a dose of rAAV virions having a predetermined number of viral genomes (VG) to a subject with reduced number or intensity of adverse effects as compared to administration of the same rAAV VG dose prepared by transient triple transfection comprises: administering a dose of rAAV produced in the cell as disclosed herein, the population of cells disclosed herein, or the stable cells as disclosed herein. In some embodiments, the adverse effect is selected from the group consisting of: liver dysfunction, liver inflammation, gastrointestinal infection, vomiting, bacterial infection, sepsis, increases in troponin levels, decreases in red blood cell counts, decreases in platelet counts, activation of the complement immune system response, acute kidney injury, cardio-pulmonary insufficiency, and death. In some embodiments, the adverse effect is an increase in serum levels of one or more proinflammatory cytokines. In some embodiments, the adverse effect is an increase in serum levels of one or more of interferon gamma (IFNγ), interleukin 1β (IL-1β), and interleukin 6 (IL- 6). [0933] In another aspects, a method of repeatedly administering a dose of rAAV to a subject in need thereof is provided. In some embodiments, the method comprises administering a first dose of rAAV produced by the cell lines and the processes described above, and then administering at least a second dose of rAAV produced by the cell lines and the processes described above. In some embodiments, the method comprises administering a first dose and a second dose of rAAV produced by the cell lines and the processes described above. In some embodiments, the method comprises administering a first dose, a second dose, and a third dose of rAAV produced by the cell lines and the processes described above. In some embodiments, the method comprises administering more than three doses of rAAV produced by the cell lines and the processes described above. In some embodiments, the first dose of rAAV and the at least second dose of rAAV are administered through the same route of administration. In some embodiments, the first dose of rAAV and the at least second dose of rAAV are administered through different routes of administration. In some embodiments, the route of administration is intravenous administration, intramuscular administration, intrathecal administration, intracisternal administration, or administration via brain surgery. [0934] In some embodiments, a method of treating a condition or disorder comprises administering a first therapeutically effective amount of the pharmaceutical composition of as disclosed herein having a predetermined number of viral genomes to a patient in need thereof and a second therapeutically effective amount of the pharmaceutical composition as disclosed herein having the predetermined number of viral genomes to the patient in need thereof. In some embodiments, the first therapeutically effective amount and the second therapeutically effective amount vary by no more than 1%, 5%, 10%, or 15%. Methods of Delivering a Payload or Protein [0935] Once formulated, compositions comprising an rAAV virion or protein (e.g., therapeutic protein such as an antibody or any fragment or derivative thereof) may be administered directly to a subject or, alternatively, delivered ex vivo, to cells derived from the subject. For example, methods for the ex vivo delivery and reimplantation of transformed cells into a subject are known in the art and may include, e.g., dextran-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, lipofectamine and LT-1 mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei. Direct delivery of a vector system comprising an expressible sequence encoding a payload of interest in vivo will generally be accomplished by injection using either a conventional syringe, needless devices such as Bioject or a gene gun, such as the Accell gene delivery system (PowderMed Ltd, Oxford, England). [0936] In certain embodiments, rAAV of the present disclosure or compositions comprising the rAAV may be administered to a subject in need thereof by any suitable route, such as, intravenous, intramuscular, intracranial, intracerebroventicular, intrathecal, intracisternal, or via brain surgery. [0937] In some embodiments, the rAAV virions comprising an expressible sequence encoding a payload of interest are used in gene therapy applications to treat a disease. The payload may be, for example, a polypeptide, a protein, or an RNA. A polypeptide or a protein may be, for example, an enzyme, an antibody, a hormone, an aptamer, or an endonuclease (e.g., a site- specific endonuclease such an RNA-guided endonuclease), a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, or any combination thereof, as described above. The payload may be progranulin. An RNA may be, for example, a guide RNA, a tRNA, a suppressor tRNA, a siRNA, a miRNA, an mRNA, a shRNA, a circular RNA, an antisense oligonucleotide (ASO), a ribozyme, a DNAzyme, an aptamer, or any combination thereof. In some embodiments, the rAAV virions used in gene therapy applications to treat a disease comprise one or more expressible sequences encoding one or more payloads of interest. For example, the rAAV virions comprise two expressible sequences, wherein a first expressible sequence encodes for a first gRNA and a second expressible sequence encodes for a second gRNA. In some embodiments, the first gRNA and the second gRNA are different. In some embodiments, the first gRNA and the second gRNA are the same. [0938] In some embodiments, the protein (e.g., therapeutic protein such as an antibody or any fragment or derivative thereof) is used in gene therapy applications to treat a disease. The protein may be, for example, a polypeptide. A polypeptide or a protein may be, for example, an enzyme, an antibody, a hormone, an aptamer, or an endonuclease (e.g., a site-specific endonuclease such an RNA-guided endonuclease), a component of a CRISPR/Cas system, an adenosine deaminase acting on RNA (ADAR) enzyme, a transcriptional activator, a transcriptional repressor, or any combination thereof, as described above. [0939] The rAAV virions or protein (e.g., therapeutic protein) may be formulated into compositions for delivery to a vertebrate subject (e.g., mammalian subject, preferably human). These compositions may either be prophylactic (to prevent a disease or condition) or therapeutic (to treat a disease or condition). The compositions will comprise a “therapeutically effective amount” of the rAAV virions such that amounts of the payload of interest may be produced in vivo sufficient to have a therapeutic benefit in the individual to which it is administered. The compositions will comprise a “therapeutically effective amount” of the protein (e.g., therapeutic protein) such that amounts have a therapeutic benefit in the individual to which it is administered. The exact amounts necessary will vary depending on the subject being treated; the age and general condition of the subject to be treated; the degree of protection desired; the severity of the condition being treated; the particular therapeutic agent produced, and the mode of administration, among other factors. An appropriate effective amount may be readily determined by one of skill in the art. Thus, a “therapeutically effective amount” will fall in a relatively broad range that may be determined through routine trials. [0940] A “therapeutically effective amount” of virion comprising an expressible sequence encoding a payload of interest will fall in a relatively broad range that may be determined through experimentation and/or clinical trials. For example, for in vivo injection, a therapeutically effective dose of rAAV virions will be on the order of from about 106 to about 1015 of the rAAV virions, e.g., from about 108 to 1012 rAAV virions. For in vitro transduction, an effective amount of rAAV virions to be delivered to cells will be on the order of from about 108 to about 1013 of the rAAV virions. Other effective dosages may be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. [0941] In some cases, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve the desired level of gene expression. In some cases, more than one administration is administered at various intervals, e.g., daily, weekly, twice monthly, monthly, every 3 months, every 6 months, yearly, etc. In some cases, multiple administrations are administered over a period of time from 1 month to 2 months, from 2 months to 4 months, from 4 months to 8 months, from 8 months to 12 months, from 1 year to 2 years, from 2 years to 5 years, or more than 5 years. 1.15. rAAV Virion Produced by Methods of the Present Disclosure [0942] In some aspects, the rAAV virion produced by the methods described in section “1.14. Methods”. 1.16. Kits [0943] In another aspect, components or embodiments described herein for the system are provided in a kit. For example, any of the plasmids, as well as the mammalian cells, related buffers, media, triggering agents, or other components related to cell culture and virion production can be provided, with optional components frozen and packaged as a kit, alone or along with separate containers of any of the other agents and optional instructions for use. In some embodiments, the kit may comprise culture vessels, vials, tubes, or the like. [0944] The methods for producing and packaging recombinant vectors in desired AAV capsids to produce the rAAVs are not meant to be limiting and other suitable methods will be apparent to the skilled artisan. ASPECTS OF THE INVENTION [0945] The below items disclose various aspects of the invention. Each of the aspects described below can be combined with other aspects and embodiments disclosed elsewhere herein, including the claims, where the combinations are clearly compatible. Certain aspects include: 1. A polynucleotide comprising: (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter; and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins. 2. The polynucleotide of aspect 1, wherein the polyadenylation signal sequence is a SV40 polyadenylation signal sequence, a bovine growth hormone polyadenylation signal sequence, or a Rabbit Beta Globin polyadenylation signal sequence. 3. The polynucleotide of aspect 1 or 2, wherein the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152, has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 170. 4. The polynucleotide construct of aspect 1, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162. 5. The polynucleotide of any one of aspects 1-4, wherein the polynucleotide is not flanked by inverted terminal repeat sequences. 6. The polynucleotide of aspect 1, wherein the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof. 7. The polynucleotide of aspect 1, wherein the inducible promoter is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. 8. The polynucleotide of aspect 1, wherein the inducible promoter comprises a tetracycline- responsive promoter element (TRE). 9. The polynucleotide of aspect 8, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. 10. The polynucleotide of aspect 9, wherein the minimal promoter is a human cytomegalovirus promoter. 11. The polynucleotide of aspect 1, wherein the inducible promoter is a Tet-On promoter. 12. The polynucleotide of aspect 1, wherein the inducible promoter comprises a first inducible promoter. 13. The polynucleotide of aspect 1, wherein the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163. 14. The polynucleotide of aspect 1, wherein the AAV Cap proteins comprise VP1, VP2, and VP3. 15. The polynucleotide of aspect 1, wherein the AAV Cap proteins encode for AAV5 Cap proteins, AAV9 Cap proteins, PhP.EB Cap proteins, AAV8 Cap proteins, AAV2 proteins, or AAV6 Cap proteins. 16. A polynucleotide comprising: (i) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and (ii) a second sequence encoding the polynucleotide of any one of aspects 1-15. 17. The polynucleotide of aspect 16, wherein transcription of the first sequence is driven by native AAV promoters; optionally, wherein transcription of the first sequence is driven by the P5 and P19 native AAV promoters. 18. The polynucleotide of aspect 16, wherein the one or more promoters comprise P5 and P19 native promoters. 19. The polynucleotide of aspect 16, wherein the first sequence has: a. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161; b. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or c. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. 20. The polynucleotide of any one of aspects 16-19, wherein the first sequence is separated from the second sequence by an intervening sequence. 21. The polynucleotide of aspect 20, wherein the intervening sequence comprises a transcriptional blocking element (TBE); optionally, wherein a sequence of the TBE has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 169. 22. The polynucleotide of any one of aspects 16-21, wherein the first sequence encoding AAV Rep proteins comprises: a. a first part of an AAV Rep proteins coding sequence, b. an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and c. a second part of the AAV Rep proteins coding sequence. 23. The polynucleotide of any one of aspects 16-22, the first sequence encoding AAV Rep proteins comprises from 5’ to 3’: one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins. 24. The system of polynucleotides of aspect 23, wherein the coding sequence encoding the stop signaling sequence of the first sequence encodes for from 5’ to 3’: an exon and the stop signaling sequence. 25. The polynucleotide of aspect 23, wherein the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence. 26. The polynucleotide of any one of aspects 16-25, further comprising a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker. 27. The polynucleotide of any one of aspects 16-26, wherein the polynucleotide is a polynucleotide construct. 28. The polynucleotide of any one of aspects 16-27, wherein the polynucleotide further comprises a selectable marker operably linked to a promoter; optionally wherein the promoter is a constitutive promoter. 29. A system of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising the sequence of the polynucleotide of any one of aspects 1-15; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload. 30. The system of polynucleotides of aspect 29, wherein transcription of the first polynucleotide is driven by native AAV promoters; optionally, wherein transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters. 31. The system of polynucleotides of aspect 29, wherein the one or more promoters comprise a P5 native AAV promoter and a P19 native AAV promoter. 32. The system of polynucleotides of any one of aspects 29-31, wherein the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises: a. a first part of an AAV Rep proteins coding sequence, b. an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and c. a second part of the AAV Rep proteins coding sequence. 33. The system of polynucleotides of any one of aspects 29-32, wherein the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises from 5’ to 3’: one or more promoters operably linked to a first sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a second sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the second sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins. 34. The system of polynucleotides of aspect 32 or 33, wherein the coding sequence encoding the stop signaling sequence of the first polynucleotide encodes for from 5’ to 3’: an exon and the stop signaling sequence. 35. The system of polynucleotides of any one of aspects 32-34, wherein the coding sequence encoding the stop signaling sequence of the first polynucleotide further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence. 36. The system of polynucleotides of any one of aspects 29-35, wherein the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161. 37. The system of polynucleotides of any one of aspects 29-36, the first polynucleotide further comprises a sequence encoding AAV Cap proteins. 38. The system of polynucleotides of aspect 37, wherein the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide. 39. The system of polynucleotides of aspect 37, wherein transcription of the sequence encoding the AAV Cap proteins on the first polynucleotide is driven by a native AAV Cap proteins promoter; optionally, wherein the native AAV Cap proteins promoter is a P40 native AAV promoter. 40. The system of polynucleotides of any one of aspects 29-39, wherein the first polynucleotide has: a. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or b. at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. 41. The system of polynucleotides of any one of aspects 29-40, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a first constitutive promoter operably linked to a sequence encoding an activator, wherein the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a first triggering agent; wherein in absence of activation of the first inducible promoter and the second inducible promoter, detectable levels of the Rep proteins from the first polynucleotide or if present the Cap proteins from the first polynucleotide, the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins are not expressed, and wherein the inducible recombinase is activated in the presence of a second triggering agent. 42. The system of polynucleotides of any one of aspects 29-41, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a first sequence comprising from 5’ to 3’: a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site, wherein the third recombination site and the fourth recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the second inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins 43. The system of polynucleotides of aspect 41 or 42, wherein the one or more adenoviral helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein. 44. The system of polynucleotides of any one of aspects 29-43, wherein the third polynucleotide comprising the sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins; optionally, wherein the SEQ ID NO: 30 comprises the third polynucleotide. 45. The system of polynucleotides of any one of aspects 41-44, wherein the one or more adenoviral helper proteins are separated by a bicistronic open reading frame; optionally, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. 46. The system of polynucleotides of any one of aspects 41-45, wherein the second inducible promoter operably linked to the self-excising element in the third polynucleotide is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same; further optionally, wherein the first inducible promoter and the second inducible promoter are a tetracycline-inducible promoter. 47. The system of polynucleotides of aspect 46, wherein the tetracycline-inducible promoter comprises a tetracycline-responsive promoter element (TRE). 48. The system of polynucleotides of aspect 47, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. 49. The system of polynucleotides of aspect 48, wherein the minimal promoter is a human cytomegalovirus promoter. 50. The system of polynucleotides of any one of aspects 41-49, wherein the first constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. 51. The system of polynucleotides of any one of aspects 41-50, wherein the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. 52. The system of polynucleotides of aspect 47, wherein a triggering agent for inducing the tetracycline-inducible promoter is tetracycline or doxycycline. 53. The system of polynucleotides of any one of aspects 41-53, wherein the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen. 54. The system of polynucleotides of any one of aspects 41-53, wherein the recombination sites in the first polynucleotide and the third polynucleotide are lox sites and the inducible recombinase is a cre recombinase or wherein the recombination sites in the first polynucleotide and the third polynucleotide are flippase recognition target (FRT) sites and the inducible recombinase is a flippase (Flp) recombinase. 55. The system of polynucleotides of any one of aspects 41-54, wherein presence of the triggering agent activates the activator for activation of the first inducible promoter to express AAV Cap proteins from the second polynucleotide encoding the AAV Cap proteins. 56. The system of polynucleotides of any one of aspects 41-55, wherein presence of the triggering agent activates the activator for activation of the second inducible promoter to express the AAV Rep proteins of the first polynucleotide, if present the AAV Cap proteins of the first polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins. 57. The system of polynucleotides of any one of aspects 41-56, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide and if present the AAV Cap Proteins of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins. 58. The system of polynucleotides of any one of aspects 29-57, wherein the third polynucleotide further comprises a third selectable marker operably linked to a third promoter. 59. The system of polynucleotides of any one of aspects 29-58, wherein the third polynucleotide further comprises a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA. 60. The system of polynucleotides of aspect 59, wherein the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. 61. The system of polynucleotides of aspect 59 or 60, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a second constitutive promoter and a second part of the second constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete second constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. 62. The system of polynucleotides of aspect 61, wherein the first part of the second constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. 63. The system of polynucleotides of any one of aspects 59-62, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. 64. The system of polynucleotides of any one of aspects 59-63, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 153. 65. The system of polynucleotides of any one of aspects 29-64, wherein the payload of the fourth polynucleotide comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the payload of the fourth polynucleotide is progranulin. 66. The system of polynucleotides of any one of aspects 29-65, wherein the sequence encoding the payload of the fourth polynucleotide comprises a sequence encoding a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the sequence encoding the payload of the fourth polynucleotide is a sequence encoding progranulin; further optionally, wherein the sequence encoding progranulin has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 166. 67. The system of polynucleotides of any one of aspects 29-66, wherein the sequence encoding the payload comprises a sequence encoding a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair. 68. The system of polynucleotides of any one of aspects 29-67, wherein the fourth polynucleotide comprising the sequence encoding the payload comprises the sequence encoding the payload flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR). 69. The system of polynucleotides of any one of aspects 29-68, wherein the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158; optionally, wherein a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159 comprises the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR). 70. The system of polynucleotides of any one of aspects 29-69 further comprising a fifth polynucleotide comprising a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA. 71. The system of polynucleotides of aspect 70, wherein the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. 72. The system of polynucleotides of aspect 70 or 71, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. 73. The system of polynucleotides of aspect 72, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. 74. The system of polynucleotides of any one of aspects 70-73, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. 75. The system of polynucleotides of any one of aspects 70-74, wherein: a) the first polynucleotide further comprises a sequence encoding a first selectable marker operably linked to a first promoter, b) the second polynucleotide further comprises a sequence encoding a second selectable marker operably linked to a second promoter, c) the third polynucleotide further comprises a sequence encoding a third selectable marker operably linked to a third promoter, d) the fourth polynucleotide further comprises a sequence encoding a fourth selectable marker operably linked to a fourth promoter, e) the fifth polynucleotide further comprises a sequence encoding a fifth selectable marker operably linked to a fifth promoter, or g) any combination of thereof; optionally, wherein any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker are different selectable markers; the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, the fifth selectable marker, or any combinations thereof are the same selectable marker but as a different split portion of the selectable marker; or any combinations thereof; further optionally, wherein any combinations of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are the same constitutive promoter or different constitutive promoters. 76. The system of polynucleotides of aspect 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is an antibiotic resistance gene; optionally, wherein the antibiotic resistance gene is a blasticidin resistance gene, a hygromycin resistance gene, or a puromycin resistance gene. 77. The system of polynucleotides of aspect 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a first split portion of an antibiotic resistance gene; optionally, wherein the first split portion of the antibiotic resistance gene is a first split portion of the blasticidin resistance gene. 78. The system of polynucleotides of aspect 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a second split portion of an antibiotic resistance gene; optionally, wherein the second split portion of the antibiotic resistance gene is a second split portion of the blasticidin resistance gene. 79. The system of polynucleotides of aspect 75, wherein the first promoter, the second promoter, the third promoter, the fourth promoter, the fifth promoter, or any combination thereof, is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutation in the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter. 80. The system of polynucleotides of any one of aspects 29-79, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5’ ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof. 81. The system of polynucleotides of any one of aspects 29-80, wherein: (i) the first polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the split selectable marker is a split antibiotic resistance protein; (ii) the second polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally wherein the constitutive promoter is an EF1 alpha promoter and/or the selectable marker is a first antibiotic resistance protein; (iii) the third polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the fourth polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter. 82. The system of polynucleotides of any one of aspects 29-81, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5’ ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof. 83. The system of polynucleotides of aspect 82, wherein the spacer ranges in length from 500 base pairs to 5000 base pairs. 84. The system of polynucleotides of any one of aspects 29-83, comprising: a) the first polynucleotide of any one of aspects 16-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and c) the third polynucleotide of any one of aspects 41-64 and 75-83. 85. The system of polynucleotides of any one of aspects 29-83, comprising: a) the first polynucleotide of any one of aspects 16-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and c) the fourth polynucleotide of any one of aspects 65-69 and 75-83. 86. The system of polynucleotides of any one of aspects 29-83, comprising: a) the first polynucleotide of any one of aspects 6-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; c) the third polynucleotide of any one of aspects 41-64 and 75-83; d) the fourth polynucleotide of any one of aspects 65-69 and 75-83. 87. The system of polynucleotides of any one of aspects 29-83, comprising: a) the first polynucleotide of any one of aspects 16-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; c) the third polynucleotide of any one of aspects 41-64 and 75-83; and d) the fifth polynucleotide of any one of aspects 70-83. 88. The system of polynucleotides of any one of aspects, comprising: a) the first polynucleotide of any one of aspects 16-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; c) the fourth polynucleotide of any one of aspects 65-69 and 75-83; and d) the fifth polynucleotide of any one of aspects 70-83. 89. The system of polynucleotides of any one of aspects 16-40 and 75-83, comprising: a) the first polynucleotide of any one of aspects 16-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and c) the fifth polynucleotide of any one of aspects 70-83. 90. The system of polynucleotides of any one of aspects 16-40 and 75-83, comprising: a) the first polynucleotide of any one of aspects 16-40 and 75-83; b) the second polynucleotide of any one of aspects 1-15, 29 and 75-83; c) the third polynucleotide of any one of aspects 41-64 and 75-83; d) the fourth polynucleotide of any one of aspects 65-69 and 75-83; and e) the fifth polynucleotide of any one of aspects 70-83. 91. A system of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and one or more of: b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of aspects 41-64 and 75-83; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of aspects 65-69 and 75-83. 92. The system of polynucleotide constructs of aspect 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of aspects 41-64 and 75-83. 93. The system of polynucleotide constructs of aspect 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and b) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of aspects 65-69 and 75-83. 94. The system of polynucleotide constructs of aspect 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1-15, 29 and 75-83; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of aspects 41-64 and 75-83; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of aspects 65-69 and 75-83. 95. The system of polynucleotide constructs of any one of aspects 91-94, further comprising d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide of any one of aspects 70-83. 96. The system of polynucleotide constructs of any one of aspects 91-95, wherein the sequence of the first polynucleotide is separated from the sequence of the second polynucleotide by an intervening sequence. 97. The system of polynucleotide constructs of aspect 96, wherein the intervening sequence comprises a transcriptional blocking element (TBE). 98. The system of polynucleotide constructs of any one of aspects 91-97, wherein the first polynucleotide construct comprises a sequence encoding a single selectable marker. 99. The system of polynucleotide constructs of any one of aspects 91-98, wherein: (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein; (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein. 100. The system of polynucleotide constructs of aspect 99, wherein the first antibiotic resistance protein is a blasticidin resistance protein and the second antibiotic resistance protein is a puromycin resistance protein. 101. A vector comprising the polynucleotide of any one of aspects 1-100. 102. A vector system comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83; b) a second vector comprising the sequence of the second polynucleotide of any one of aspects 1-15, 29 and 75-83; c) a third vector comprising the sequence of the third polynucleotide of any one of aspects 41-64 and 75-83; and d) a fourth vector comprising the sequence of the fourth polynucleotide of any one of aspects 65-69 and 75-83. 103. The vector system of aspect 102, further comprising d) a fifth vector comprising the sequence of the fifth polynucleotide of any one of aspects 70-83. 104. The vector system of any one of aspects 102 or 103, wherein the first vector is a first plasmid, the second vector is a second plasmid, the third vector is a third plasmid, and the fourth vector is a fourth plasmid. 105. The vector system of any one of aspects 103, wherein the fifth vector is a fifth plasmid. 106. The vector system of any one of aspects 102-105, wherein the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 160 or 161. 107. The vector system of any one of aspects 102-105, wherein the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149. 108. The vector system of any one of aspects 102-105, wherein the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154. 109. The vector system of any one of aspects 102-105, wherein the fourth plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159. 110. A vector system comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of aspects 91-100; and one or more of: b) a second vector comprising the sequence of the third polynucleotide of any one of aspects 41-64 and 75-83 or the second polynucleotide construct of any one of aspects 1- 15, 29 and 75-83; and c) a third vector comprising the sequence of the fourth polynucleotide of any one of aspects 65-69 and 75-83 or the third polynucleotide construct of any one of aspects 41- 64 and 75-83. 111. The vector system of aspect 110, comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of aspects 91-100; and b) a second vector comprising the sequence of the third polynucleotide of any one of aspects 41-64 and 75-83 or the second polynucleotide construct of any one of aspects 91- 100. 112. The vector system of aspect 110 or 111, comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of aspects 16-40 and 75-83 and the sequence of the second polynucleotide of any one of aspects 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of aspects 91-100; and b) a third vector comprising the sequence of the fourth polynucleotide of any one of aspects 65-69 and 75-83 or the third polynucleotide construct of any one of aspects 91- 100. 113. The vector system of any one of aspects 110-112, further comprising a fourth vector comprising the sequence of the fifth polynucleotide of any one of aspects 70-83 or the fourth polynucleotide construct of any one of aspects 91-100. 114. The vector system of any one of aspects 110-113, wherein the first vector is a first plasmid, the second vector is a second plasmid, and the third vector is a third plasmid. 115. The vector system of any one of aspects 113, wherein the fourth vector is a fourth plasmid. 116. The vector system of aspect 114, wherein the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160. 117. The vector system of aspect 114, wherein the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154. 118. The vector system of aspect 114, wherein the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159. 119. A plasmid comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160. 120. A plasmid comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149. 121. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148. 122. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. 123. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 and a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 or 165. 124. A cell comprising the polynucleotide of any one of aspects 1-28. 125. A cell comprising the polynucleotide system of any one of aspects 29-100. 126. The cell of aspect 125, wherein one or more of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell. 127. The cell of aspect 126, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 128. The cell of aspect 126, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 129. The cell of any one of aspects 125-128, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are, in any combination, on one or more polynucleotide constructs. 130. A cell comprising the polynucleotide construct system of any one of aspects 91-100. 131. The cell of aspect 130, wherein one or more of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, and the fourth polynucleotide are integrated into the nuclear genome of the cell. 132. The cell of aspect 131, wherein the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 133. The cell of aspect 131, wherein the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 134. A cell comprising the vector system of any of aspects 102-118. 135. The cell of aspect 134, wherein one or more of the first vector, the second vector, the third vector, and the fourth vector are integrated into the nuclear genome of the cell. 136. The cell of aspect 135, wherein the first vector, the second vector, and the third vector are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 137. The cell of aspect 135, wherein the first vector, the second vector, and the third vector, are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 138. The cell of any one of aspects 124-137, wherein the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof. 139. The cell of any one of aspects 124-138, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof; optionally, wherein the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. 140. The cell of any one of aspects 124-139, wherein the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein or wherein the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein. 141. The cell of any one of aspects 124-140, wherein the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome; optionally, wherein the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. 142. The cell of any one of aspects 124-141, wherein the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins; optionally, wherein the third polynucleotide comprises SEQ ID NO: 30. 143. The cell of any one of aspects 126-142, wherein the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein; optionally, wherein the any two proteins are E2A and E4 or E1A and E1B. 144. The cell of aspect 142, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. 145. The cell of any one of aspects 126-144, wherein the cell is a mammalian cell. 146. The cell of aspect 145, wherein the mammalian cell is a HEK293 cell. 147. The cell of aspect 146, wherein the HEK293 cell is DHFR-deficient or GS-deficient. 148. The cell of any one of aspects 126-147, wherein the cell expresses adenoviral helper proteins E1A and E1B. 149. The cell of any one of aspects 126-148, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. 150. The cell of any one of aspects 126-149, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. 151. The cell of any one of aspects 126-150, wherein after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155. 152. The cell of any one of aspects 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. 153. The cell of any one of aspects 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises a nucleic acid having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165. 154. The cell of any one of aspects 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164. 155. The cell of any one of aspects 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163. 156. The cell of any one of aspects 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158. 157. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the third polynucleotide of any one of aspects 41-64 and 75-83; selecting for a cell expressing a selectable marker of the third polynucleotide; introducing into the cell expressing the selectable marker of the third polynucleotide, the first polynucleotide of any one of aspects 16-40 and 75-83, the second polynucleotide of any one of aspects 1-15, 29 and 75-83, and the fourth polynucleotide of any one of aspects 65-69 and 75-83; selecting for a cell expressing the selectable marker of the third polynucleotide, a selectable marker of the first polynucleotide, a selectable marker of the second polynucleotide, and a selectable marker of the fourth polynucleotide; expanding the cell expressing the selectable marker of the third polynucleotide, the selectable marker of the first polynucleotide, the selectable marker of the second polynucleotide, and the selectable marker of the fourth polynucleotide into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions. 158. The method of aspect 157, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide. 159. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the second polynucleotide construct of any one of aspects 91-100; selecting for a cell expressing a selectable marker of the second polynucleotide construct; introducing into the cell expressing the selectable marker of the second polynucleotide construct, the first polynucleotide construct of any one of aspects 91-100, and the third polynucleotide construct of any one of aspects 91-100; selecting for a cell expressing the selectable marker of the second polynucleotide construct, a selectable marker of the first polynucleotide construct, and a selectable marker of the third polynucleotide construct; expanding the cell expressing the selectable marker of the second polynucleotide construct, the selectable marker of the first polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions. 160. The method of aspect 159, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide construct. 161. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the third vector of any one of aspects 102-118; selecting for a cell expressing a selectable marker of the third vector; introducing into the cell expressing the selectable marker of the third vector, the first vector of any one of aspects 102-118, the second vector of any of any one of aspects 102-118, and the fourth vector of any one of aspects 102-118; selecting for a cell expressing the selectable marker of the third vector, a selectable marker of the first vector, a selectable marker of the vector polynucleotide, and a selectable marker of the fourth vector; expanding the cell expressing the selectable marker of the third vector, the selectable marker of the first vector, the selectable marker of the second vector, and the selectable marker of the fourth vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions. 162. The method of aspect 161, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the third vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector. 163. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the second vector of any one of aspects 102-118; selecting for a cell expressing a selectable marker of the second vector; introducing into the cell expressing the selectable marker of the second vector, the first vector of any one of aspects 102-118, and the third vector of any one of aspects 102-118; selecting for a cell expressing the selectable marker of the second vector, a selectable marker of the first vector, and a selectable marker of the third vector; expanding the cell expressing the selectable marker of the second vector, the selectable marker of the first vector, and the selectable marker of the third vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions. 164. The method of aspect 163, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the second vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector. 165. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of aspects 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second polynucleotide; wherein the expression of the one or more AAV helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload. 166. The method of aspect 165, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide. 167. The method of aspect 165, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide. 168. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of aspects 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide construct by the recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first polynucleotide construct; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload. 169. The method of aspect 168, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first polynucleotide construct. 170. The method of aspect 168, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first polynucleotide construct. 171. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of aspects 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload. 172. The method of aspect 171, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second vector. 173. The method of aspect 171, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second vector. 174. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of aspects 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload. 175. The method of aspect 174, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first vector. 176. The method of aspect 174, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first vector. 177. A method for producing recombinant AAV, the method comprising performing transfection of a cell with the polynucleotides of the system of any one of aspects 29-100, the polynucleotide constructs of the system of any one of aspects 91-100, or the vectors of the system of any one of aspects 102-118 and contacting the cell to a first triggering agent and a second triggering agent. 178. The method of aspect 177, wherein the polynucleotides of the system of any one of aspects 29-100, the polynucleotide constructs of the system of any one of aspects 91-100, or the vectors of the system of any one of aspects 102-118 are integrated into a nuclear genome of the cell. 179. The method of aspect 177, wherein the polynucleotides of the system of any one of aspects 29-100, the polynucleotide constructs of the system of any one of aspects 91-100, or the vectors of the system of any one of aspects 102-118 are not integrated into a nuclear genome of the cell. 180. The method of any one of aspects 177-179, wherein one or more of the polynucleotides of the system of any one of aspects 29-100, of the polynucleotide constructs of the system of any one of aspects 91-100, or of the vectors of the system of any one of aspects 102-118 are integrated into a nuclear genome of the cell and one or more of the polynucleotides of the system of any one of aspects 29-100, of the polynucleotide constructs of the system of any one of aspects 91-100, or of the vectors of the system of any one of aspects 102-118 are not integrated into the nuclear genome of the cell. 181. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotides of the system of any one of aspects 29- 100 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload. 182. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotide constructs of the system of any one of aspects 91-100 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide construct resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload. 183. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the vectors of the system of any one of aspects 102-118 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload. 184. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the vectors of the system of any one of aspects 102-118 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload. 185. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third polynucleotide of any one of aspects 41-64 and 75-83; (ii) selecting for cells expressing a selectable marker encoded by third polynucleotide; (iii) introducing into the cells selected in (ii) the first polynucleotide and the fourth polynucleotide of any one of aspects 65-69 and 75-83, wherein the first polynucleotide encodes a first part of a split selectable marker and the fourth polynucleotide encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third polynucleotide and the selectable marker encoded by the first polynucleotide and the fourth polynucleotide, thereby generating the cells for inducibly producing the rAAV virions. 186. The method of aspect 185, further comprising: (iii) introducing into the cells selected in (iv) or the cells expanded in (v), the second polynucleotide of any one of aspects 1-15 and 75-83; and (iv) selecting for cells expressing a selectable marker encoded by the second polynucleotide. 187. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second polynucleotide construct of any one of aspects 91- 100; (ii) selecting for cells expressing a selectable marker encoded by second polynucleotide construct; (iii) introducing into the cells selected in (ii) the first polynucleotide construct and the third polynucleotide construct of any one of aspects 91-100, wherein the first polynucleotide construct encodes a first part of a split selectable marker and the third polynucleotide construct encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second polynucleotide construct and the selectable marker encoded by the first polynucleotide construct and the third polynucleotide construct, thereby generating the cells for inducibly producing the rAAV virions. 188. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third vector of any one of aspects 102-118; (ii) selecting for cells expressing a selectable marker encoded by third vector; (iii) introducing into the cells selected in (ii) the first vector and the fourth vector of any one of aspects 102-118, wherein the first vector encodes a first part of a split selectable marker and the fourth vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third vector and the selectable marker encoded by the first vector and the fourth vector, thereby generating the cells for inducibly producing the rAAV virions. 189. The method of aspect 188, further comprising: (vi) introducing into the cells selected in (iv) or the cells expanded in (v), the second vector of any one of aspects 102-118; and (vii) selecting for cells expressing a selectable marker encoded by the second vector. 190. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second vector of any one of aspects 102-118; (ii) selecting for cells expressing a selectable marker encoded by second vector; (iii) introducing into the cells selected in (ii) the first vector and the third vector of any one of aspects 102-118, wherein the first vector encodes a first part of a split selectable marker and the vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second vector and the selectable marker encoded by the first vector and the third vector, thereby generating the cells for inducibly producing the rAAV virions. 191. A method of generating a cell for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell a first polynucleotide comprising: a first sequence comprising from 5’ to 3’: a first inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a first recombination site, the sequence encoding the inducible recombinase, and a second recombination site, wherein the first recombination site and the second recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the first inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the first recombination site and the second recombination site resulting in excision of the self-excising element, thereby operably linking the first inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins; and a third sequence comprising a second constitutive promoter operably linked to a sequence encoding a first selectable marker, wherein the cell constitutively expresses the first selectable marker, selecting for a cell expressing the first selectable marker; introducing a second polynucleotide and a third polynucleotide into the cell expressing the first selectable marker, the second polynucleotide comprising: a first sequence encoding AAV Cap proteins operably linked to a second inducible promoter; and from 5’ to 3’: one or more promoters operably linked to a second sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a third recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a fourth recombination site, a second part of the intron, a second 3’ splice site, and a third sequence comprising a second part of the AAV Rep coding sequence, wherein the third recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the fourth recombination site form an excisable element, wherein the third recombination site and the fourth recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the third sequence comprising the second part of the AAV Rep coding sequence, wherein the third and fourth recombination sites are recombined by the inducible recombinase in the presence of the first triggering agent and the second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins; and a third constitutive promoter operably linked to a sequence encoding a first portion of a second selectable marker, the third polynucleotide comprising a sequence encoding the payload and a fourth constitutive promoter operably linked to a second portion of the second selectable marker, wherein the sequence encoding the payload is flanked by AAV inverted terminal repeats (ITRs); selecting for a cell expressing the first selectable marker and the second selectable marker, thereby generating the cell for inducibly producing recombinant AAV (rAAV) virions comprising the payload. 192. The method of aspect 191, further comprising contacting the cell with the first triggering agent and the second triggering agent for inducibly producing recombinant AAV (rAAV) virions comprising the payload. 193. The method of aspect 191 or 192, wherein the coding sequence encoding the stop signaling sequence of the second polynucleotide encodes for from 5’ to 3’: an exon and the stop signaling sequence. 194. The method of any one of aspects 191-193, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide further comprises a polyadenylation signal. 195. The method of aspect 194, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence. 196. The polynucleotide of aspect 194 or 195, wherein the polyadenylation signal sequence is a 3’ of the sequence encoding AAV Cap proteins. 197. The method of any one of aspects 194-196, wherein the polyadenylation signal sequence is a SV40 polyadenylation signal sequence or a bovine growth hormone polyadenylation signal sequence. 198. The method of any one of aspects 194-197, wherein the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151. 199. The method of aspect 195, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162. 200. The method of any one of aspects 191-199, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide is not flanked by inverted terminal repeat sequences. 201. The method of aspect 195, wherein the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof. 202. The method of any one of aspects 191-201, wherein the first inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter and second inducible promoter is a tetracycline-inducible promoter, an ecdysone- inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same. 203. The method of any one of aspects 191-202, wherein the first inducible promoter comprises a tetracycline-responsive promoter element (TRE) and the second inducible promoter comprises a tetracycline-responsive promoter element (TRE). 204. The method of aspect 203, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. 205. The method of aspect 204, wherein the minimal promoter is a human cytomegalovirus promoter. 206. The method of any one of aspects 191-203, wherein the first inducible promoter is a Tet- On promoter and the second inducible promoter is a Tet-On promoter. 207. The method of any one of aspects 191-206, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163. 208. The method of any one of aspects 191-207, wherein the AAV Cap proteins comprise VP1, VP2, and VP3. 209. The method of any one of aspects 157-208, wherein the first triggering agent is tetracycline and the second triggering agent is tamoxifen. 210. The method of any one of aspects 157-208, wherein the first triggering agent is doxycycline and the second triggering agent is tamoxifen. 211. The rAAV virion produced by the method of any one of aspects 157-210. [0946] The below items disclose various additional aspects of the invention. Each of the aspects described below can be combined with other aspects and embodiments disclosed elsewhere herein, including the aspects, where the combinations are clearly compatible. Certain additional aspects include: 1. A cell comprising: a) a first polynucleotide comprising a first sequence encoding AAV Rep proteins; b) a second polynucleotide comprising a second sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a third sequence encoding the AAV Cap proteins; and d) a fourth polynucleotide comprising a fourth sequence encoding one or more adenoviral helper proteins. 2. The cell of aspect 2, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 3. The cell of aspect 1 or 2, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are, in any combination, on one or more polynucleotide constructs. 4. The cell of any one of aspects 1-3, wherein: a) a first polynucleotide construct comprises the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; further optionally, wherein the first polynucleotide sequence lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136. b) a second polynucleotide construct comprises the third polynucleotide, optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148; and c) a third polynucleotide construct comprises the fourth polynucleotide; optionally, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 11. 5. The cell of aspect 4, wherein the first polynucleotide construct, second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions. 6. The cell of any one of aspects 1-5, further comprising a fifth polynucleotide comprising a fifth sequence encoding a payload. 7. The cell of aspect 6, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are, in any combination, on one or more polynucleotide constructs. 8. The cell of aspect 6 or 7, wherein: a) the first polynucleotide construct comprises the first polynucleotide and the second polynucleotide; optionally, wherein the first polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 137, b) the second polynucleotide construct comprises the third polynucleotide; optionally, wherein the second polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149; c) the third polynucleotide construct comprises the fourth; optionally, wherein the third polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30; and d) the fourth polynucleotide construct comprises the fifth polynucleotide; optionally, wherein the fourth polynucleotide construct has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. 9. The cell of any one of aspects 6-8, wherein the fifth polynucleotide is integrated in the nuclear genome of the cell. 10. The cell of aspect 8, wherein the fourth polynucleotide construct is integrated in the nuclear genome of the cell. 11. The cell of any one of aspects 1-10, further comprising a sixth polynucleotide comprising a sixth sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA. 12. The cell of aspect 11, wherein the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, the fourth polynucleotide construct, or the fifth polynucleotide construct comprises the sixth polynucleotide. 13. The cell of aspects 11 or 12, wherein the sixth polynucleotide is integrated in the nuclear genome of the cell. 14. The cell of aspect 12, wherein the fifth polynucleotide construct is integrated in the nuclear genome of the cell. 15. The cell of any one of aspects 1-14, wherein the cell comprises a single copy of each of the first polynucleotide, the second polynucleotide, and the third polynucleotide. 16. The cell of any one of aspects 3-15, wherein the cell comprises a single copy of each of the first polynucleotide construct and the third polynucleotide construct, and comprises two or more copies of the second polynucleotide construct. 17. The cell of any one of aspects 1-14, wherein the cell comprises a plurality of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, the fifth polynucleotide, the sixth polynucleotide, or any combination thereof. 18. The cell of any one of aspects 12-14 or 17, wherein the cell comprises a plurality of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, the fourth polynucleotide construct, or the fifth polynucleotide construct, or any combination thereof. 19. The cell of any one of aspects 1-18, wherein: a) the first polynucleotide further comprises a selectable marker operably linked to a promoter, b) the second polynucleotide further comprises a selectable marker operably linked to a promoter, c) the third polynucleotide further comprises a selectable marker operably linked to a promoter, d) the fourth polynucleotide further comprises a selectable marker operably linked to a promoter, e) the fifth polynucleotide further comprises a selectable marker operably linked to a promoter, f) the sixth polynucleotide further comprises a selectable marker operably linked to a promoter, or g) any combination of polynucleotides of a)-f) comprise a selectable marker; optionally, wherein the selectable marker of any one of a)-g) are different selectable markers, the same selectable marker but as a different split portion of the selectable marker, or a combination thereof; further optionally, wherein the promoter is a constitutive promoter. 20. The cell of any one of aspects 1-19, wherein a) the first polynucleotide comprising the first sequence comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, (iii) a second part of the AAV Rep proteins coding sequence; and (iv) one or more promoters operably linked to the first sequence; and b) the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to a promoter present in the second part of the AAV Rep proteins coding sequence or the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to an inducible promoter. 21. The cell of aspect 20, wherein the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker and comprising the stop signaling sequence. 22. The cell of any one of aspects 3-21, wherein the first polynucleotide construct further comprises a first constitutive promoter operably linked to a sequence encoding a first selectable marker. 23. The cell of any one of aspects 1-22, wherein transcription of the first polynucleotide and the second polynucleotide are driven by native AAV promoters or wherein transcription of the first polynucleotide is driven by native AAV promoters and transcription of the second polynucleotide is driven by a first inducible promoter; optionally, wherein the first polynucleotide and the second polynucleotide are separated by a transcriptional blocking element. 24. The cell of aspect 23, wherein transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters, and transcription of the second polynucleotide is driven by the P40 native AAV promoter or wherein transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters, and transcription of the second polynucleotide is driven by a first inducible promoter; optionally, wherein the first polynucleotide and the second polynucleotide are separated by a transcriptional blocking element. 25. The cell of any one of aspects 1-24, wherein transcription of the third polynucleotide is driven by a first inducible promoter. 26. The cell of any one of aspects 1-25, wherein the second polynucleotide comprising the second sequence encoding the AAV Cap proteins is operably linked to a native AAV promoter and the third polynucleotide comprising the third sequence encoding the AAV Cap proteins is operably linked to a first inducible promoter. 27. The cell of any one of aspects 1-26, wherein the AAV capsid proteins comprise VP1, VP2, and VP3. 28. The cell of any one of aspects 1-27, wherein the third polynucleotide further comprises a second constitutive promoter operably linked to a second selectable marker. 29. The cell of any one of aspects 1-28, wherein the fourth polynucleotide comprising the fourth sequence encoding the one or more adenoviral helper proteins further comprises a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the fourth sequence encoding the one or more AAV helper proteins; a third constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a triggering agent; and a fourth constitutive promoter operably linked to a sequence encoding a third selectable marker, wherein the cell constitutively expresses the third selectable marker, wherein in absence of activation of the first inducible promoter and second inducible promoter, the cell does not express detectable levels of the Rep proteins, the Cap proteins of the second polynucleotide, the Cap proteins of the third polynucleotide, the inducible recombinase, and the one or more AAV helper proteins, or wherein the fourth polynucleotide comprising the fourth sequence encoding the one or more adenoviral helper proteins further comprises a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the fourth sequence encoding the one or more AAV helper proteins; a third constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a triggering agent; and a fourth constitutive promoter operably linked to a sequence encoding a third selectable marker, wherein the cell constitutively expresses the third selectable marker, wherein in absence of activation of the first inducible promoter and second inducible promoter, the cell does not express detectable levels of the Rep proteins, the Cap proteins of the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins. 30. The cell of any one of aspects 6-29, wherein the fifth polynucleotide further comprises a fourth selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR); optionally, wherein the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. 31. The cell of aspect 30, wherein the fifth polynucleotide further comprises a spacer between the 5’ ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof; optionally wherein SEQ ID NO: 147 comprises the fifth polynucleotide. 32. The cell of aspect 31, wherein the spacer ranges in length from 500 base pairs to 5000 base pairs. 33. The cell of any one of aspects 1-32, wherein the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof; optionally, wherein the fourth polynucleotide comprising the fourth sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof; further optionally, wherein the third polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof. 34. The cell of any one of aspects 1-33, wherein the cell comprises an adenovirus E1A protein and E1B protein, and the one or more AAV helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein or wherein the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the third polynucleotide construct are an adenovirus E1A protein and E1B protein. 35. The cell of any one of aspects 1-34, wherein the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome; optionally, wherein the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein. 36. The cell of any one of aspects 1-35, wherein the fourth polynucleotide comprising the fourth sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins; optionally, wherein SEQ ID NO: 30 comprises the fourth polynucleotide. 37. The cell of aspect 36, wherein the two AAV helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein; optionally, wherein the any two proteins are E2A and E4 or E1A and E1B. 38. The cell of aspect 36 or 37, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence. 39. The cell of any one of aspects 1-38, wherein the cell is a mammalian cell. 40. The cell of aspect 39, wherein the mammalian cell is a HEK293 cell. 41. The cell of aspect 40, wherein the HEK293 cell is DHFR-deficient or GS-deficient. 42. The cell of any one of aspects 29-41, wherein the first inducible promoter operably linked to the Cap in the third polynucleotide construct is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter. 43. The cell of any one of aspects 29-42, wherein the second inducible promoter operably linked to the self-excising element in the third polynucleotide construct is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same; further optionally, wherein the first inducible promoter and the second inducible promoter are a tetracyline-inducible promoter. 44. The cell of any aspect 42, wherein the tetracycline-inducible promoter comprises a tetracycline-responsive promoter element (TRE). 45. The cell of aspect 44, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. 46. The cell of aspect 45, wherein the minimal promoter is a human cytomegalovirus promoter. 47. The cell of any one of aspects 29-46, wherein the third polynucleotide construct comprises a sequence encoding the activator. 48. The cell of aspect 47, wherein the sequence encoding the activator is operably linked to a constitutive promoter. 49. The cell of aspect 48, wherein the constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter. 50. The cell of any one of aspects 29-49, wherein the activator is reverse tetracycline- controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain. 51. The cell of any one of aspects 29-50, wherein the triggering agent for inducing the tetracycline-inducible promoter is tetracycline or doxycycline. 52. The cell of any one of aspects 29-51, wherein the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen. 53. The cell of any one of aspects 20-52, wherein the recombination sites in the first polynucleotide construct and the third polynucleotide construct are lox sites and the recombinase is a cre recombinase or wherein the recombination sites in the first polynucleotide construct and the third polynucleotide are flippase recognition target (FRT) sites and the recombinase is a flippase (Flp) recombinase. 54. The cell of any one of aspects 29-53, wherein presence of the triggering agent activates the activator for activation of the first inducible promoter to express Cap proteins from the third polynucleotide comprising the third sequence encoding the AAV Cap proteins. 55. The cell of any one of aspects 29-54, wherein presence of the triggering agent activates the activator for activation of the second inducible promoter to express the Rep proteins, the Cap proteins of the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins. 56. The cell of any one of aspects 29-55, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins. 57. The cell of any one of aspects 6-56, wherein the fifth polynucleotide comprising the fifth sequence encoding the payload comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the sequence encoding the payload a sequence encoding progranulin. 58. The cell of any one of aspects 6-57, wherein the fifth polynucleotide comprising the fifth sequence encoding the payload comprises a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair. 59. The cell of any one of aspects 11-58, wherein the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. 60. The cell of any one of aspects 1-59, wherein the sixth polynucleotide comprising the sixth sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, wherein the fifth and sixth recombination sites are oriented in the same direction, and wherein excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA- RNA coding sequence to allow expression of the VA-RNA. 61. The cell of aspect 60, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter. 62. The cell of aspect 60, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter. 63. The cell of aspect 60, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. 64. The cell of any one of aspects 11-63, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. 65. The cell of any one of aspects 1-64 for inducibly producing recombinant AAV (rAAV) virions comprising a payload. 66. A vector system comprising: a) the first polynucleotide of any one of aspects 1-65; b) the second polynucleotide of any one of aspects 1-65; c) the third polynucleotide of any one of aspects 1-65; and d) the fourth polynucleotide of any one of aspects 1-65. 67. The vector system of aspect 66, further comprising the fifth polynucleotide of any one of aspects 6-65. 68. The vector system of aspect 66 or 67 further comprising the sixth polynucleotide of any one of aspects 11-65. 69. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the first polynucleotide construct of any one of aspects 19-65; selecting for a cell expressing the selectable marker of the first polynucleotide construct; introducing into the cell expressing the selectable marker of the first polynucleotide construct, the second polynucleotide of any of any one of aspects 19-65 and the fourth polynucleotide construct of any one of aspects 19-65; selecting for a cell expressing the selectable marker of the first polynucleotide construct, a selectable marker of the second polynucleotide construct, and a selectable marker of the fourth polynucleotide construct; introducing into the cell expressing the selectable marker of the first polynucleotide construct, the selectable marker of the second polynucleotide construct, and the selectable marker of the fourth polynucleotide construct, the third polynucleotide construct of any one of aspects 19-65; selecting for a cell expressing the selectable marker of the first polynucleotide construct, the selectable marker of the second polynucleotide construct, the selectable marker of the fourth polynucleotide construct, and a selectable marker of the third polynucleotide construct; and expanding the cell expressing the selectable marker of the first polynucleotide construct, the selectable marker of the second polynucleotide construct, the selectable marker of the fourth polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions. 70. The method of aspect 69, further comprising contacting a cell of the cell line with the triggering agent, wherein in the presence of the triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein (i) recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, or (ii) recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in inversion of the inversible element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap proteins, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap proteins; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins. 71. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload of interest, the method comprising contacting the cell according to any one of aspects 6-65 with the triggering agent, [0947] wherein in the presence of the triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide construct by the recombinase results in excision of the excisable element or inversion of the inversible element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and an AAV Cap proteins, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein in the presence of the triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins; [0948] wherein the expression of the one or more AAV helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload. 72. The method of aspect 71, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the third polynucleotide or the third polynucleotide construct. 73. The rAAV virion produced by the method of any one of aspects 70-72. 74. A polynucleotide comprising: a first sequence encoding AAV Rep proteins and a second sequence encoding AAV Cap proteins, wherein the first sequence is operably linked to one or more promoters and the second sequence is operably linked to an inducible promoter. 75. The polynucleotide of aspect 74, wherein the one or more promoters comprise P5 and P19 native promoters and the inducible promoter comprises a first inducible promoter. 76. The polynucleotide of aspect 75, wherein the first inducible promoter comprises a tetracycline-responsive promoter element (TRE). 77. The polynucleotide of aspect 76, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter. 78. The polynucleotide of aspect 77, wherein the minimal promoter is a human cytomegalovirus promoter. 79. The polynucleotide of any one of aspects 74-78, wherein the first sequence and the one or more promoters operably linked to the first sequence are separated from the second sequence and the inducible promoter operably linked to the second sequence by an intervening sequence. 80. The polynucleotide of aspect 79, wherein the intervening sequence comprises a transcriptional blocking element (TBE). 81. The polynucleotide of aspect 74, wherein the inducible promoter is a Tet-On promoter. 82. The polynucleotide of any one of aspects 74-81, wherein the first sequence encoding AAV Rep proteins comprises: (i) a first part of an AAV Rep proteins coding sequence, (ii) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and (iii) a second part of the AAV Rep proteins coding sequence. 83. The polynucleotide of aspect 82, wherein the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence. 84. The polynucleotide of any one of aspects 74-83, further comprising a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker. 85. A system of polynucleotides comprising: (a) the polynucleotide of any one of aspects 74-84, wherein the polynucleotide is a first polynucleotide; and one or more of: b) a second polynucleotide comprising a sequence encoding AAV Cap proteins; c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload. 86. The system of polynucleotides of aspect 85, wherein the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide. 87. The system of polynucleotides of aspect 85 or 86, wherein the second polynucleotide comprises an inducible promoter operably linked to the sequence encoding the AAV Cap proteins. 88. The system of polynucleotides of aspect 87, wherein the inducible promoter is a second inducible promoter. 89. The system of polynucleotides of any one of aspects 85-88, wherein the second polynucleotide further comprises a selectable marker operably linked to a promoter. 90. The system of polynucleotides of aspect 89, wherein the selectable marker is a second selectable marker and the promoter is a constitutive promoter, optionally wherein the constitutive promoter is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutation in the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter. 91. The system of polynucleotides of any one of aspects 85-90, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: an inducible promoter operably linked to a self-excising element, wherein the inducible promoter is a third inducible promoter; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the third inducible promoter is not operably linked to the sequence encoding the one or more AAV helper proteins; a constitutive promoter operably linked to a sequence encoding an activator, wherein a cell comprising the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter, if present the second inducible promoter, or the third inducible promoter in absence of a first triggering agent; wherein in absence of activation of the first inducible promoter, if present the second inducible promoter, and the third inducible promoter, the cell does not express detectable levels of the Rep proteins or the Cap proteins from the first polynucleotide, if present the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more AAV helper proteins, and wherein the inducible recombinase is activated in the presence of a second triggering agent. 92. The system of polynucleotides of aspect 91, wherein the one or more helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein. 93. The system of polynucleotides of any one of aspects 85-92, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises: a selectable marker or a second part or a first part of the split selectable marker operably linked to a constitutive promoter and the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR); optionally, wherein the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147. 94. The system of polynucleotides of any one of aspects 85-93, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises further comprises a spacer between the 5’ ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the selectable marker and the 3’ ITR, or a combination thereof; optionally wherein SEQ ID NO: 147 comprises the fourth polynucleotide. 95. The system of polynucleotides of aspect 94, wherein the spacer ranges in length from 500 base pairs to 5000 base pairs. 96. The system of polynucleotides of any one of aspects 85-95, wherein the third polynucleotide comprises a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA. 97. The system of polynucleotides of aspect 96, wherein the VA-RNA is wild-type VA-RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter. 98. The system of polynucleotides of aspect 96 or 97, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA. 99. The system of polynucleotides of aspect 98, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter. 100. The system of polynucleotides of any one of aspects 96-99, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof. 101. The system of polynucleotides of any one of aspects 85-100, wherein: (i) the first polynucleotide comprising the sequence encoding the first sequence encoding AAV Rep proteins and the second sequence encoding AAV Cap proteins comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1alpha promoter and/or the split selectable marker is a split antibiotic resistance protein; (ii) if present, the second polynucleotide comprising the sequence encoding the AAV Cap proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally wherein the constitutive promoter is an EF1alpha promoter and/or the selectable marker is a first antibiotic resistance protein; (iii) the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the fourth polynucleotide comprising the sequence encoding the payload comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1alpha promoter. 102. A vector comprising the polynucleotide of any one of aspects 74-84. 103. A cell comprising the vector of aspect 102. 104. A cell comprising one or more polynucleotides of the system of any one of aspects 85-101. 105. The cell of aspect 103 or 104, wherein the cell is a mammalian cell, optionally wherein the mammalian cell is a HEK293 cell, further optionally, wherein the HEK293 cell expresses AAV helper proteins E1A and E1B. 106. The cell of any one of aspects 103-105, wherein the polynucleotides are integrated into a genome of the cell. 107. A method for producing recombinant AAV, the method comprising performing transfection of a cell with the polynucleotides of the system of any one of aspects 85-101. 108. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotides of the system of any one of aspects 85- 101, integrated into the nuclear genome in the presence of the first triggering agent and the second triggering agent, wherein in the presence of the first triggering agent, the activator activates the third inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide results resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide construct by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the third inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more AAV helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload. 109. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third polynucleotide of any one of aspects 85-101; (ii) selecting for cells expressing a selectable marker encoded by the third polynucleotide; (iii) introducing into the cells selected in (ii) the first polynucleotide and the fourth polynucleotide of any one of aspects 85-101, wherein the first polynucleotide encodes a first part of a split selectable marker and the fourth polynucleotide encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first and second parts of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third polynucleotide construct and the selectable marker encoded by the first and fourth polynucleotide constructs, thereby generating the cells for inducibly producing the rAAV virions. 110. The method of aspect 109, further comprising: (vi) introducing into the cells selected in (iv) or the cells expanded in (v), the second polynucleotide of any one of aspects 85-101; and (vii) selecting for cells expressing a selectable marker encoded by the second polynucleotide. EXAMPLES [0949] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for. [0950] The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. Example 1 - Mammalian cell line with three stably integrated plasmids [0951] A stable mammalian cell line v1.0 capable of inducible expression of rAAV encapsidating a payload was constructed by integrating three nucleic acid constructs (e.g., see FIG.16) via plasmids into the nuclear genome of a cell line that expresses adenovirus E1A and E1B. The plasmids were a plasmid having the sequence of SEQ ID NO: 30, a plasmid having the sequence of SEQ ID NO: 32, and either a plasmid having the sequence of SEQ ID NO: 33 or a plasmid having the sequence of SEQ ID NO: 147. Cells that successfully integrated all three v1.0 constructs are selected and maintained by growth in media in the presence of puromycin and blasticidin. See FIG.4. SCC refers to single cell clones. Example 2 – Mammalian cell line with four stably integrated plasmids [0952] v1.0 cells that successfully integrated all three v1.0 constructs are transfected with a plasmid encoding AAV5 capsid proteins under control of an inducible promoter and resistance to hygromycin and selected for hygromycin resistance to produce v1.2 cells, referred to as T231 (P3) or T231 in subsequent figures. The plasmid encoding AAV5 capsid proteins under control of an inducible promoter and resistance to hygromycin had a sequence of SEQ ID NO: 149. Control cells were produced by transfecting v1.0 cells with a stuffer control plasmid, referred to as T232 (P3) or T232 in subsequent figures. The plasmid stuffer control plasmid had a sequence of SEQ ID NO: 150. See FIG.5. [0953] FIG.6 provides data for the titer of AAV Capsid proteins and the titer of AAV Capsids proteins encapsidating the gene encoding progranulin (PGRN) produced in T231 (P3) and T232 (P3) cells. The table shows percent packaging of the gene encoding PGRN. The T231 (P3) cells or the T232 (P3) cells are pooled cells (also referred to as T231 stable cell pool or T232 stable cell pool), in which a pool comprises more than one single cell clone that can later be isolated and expanded to produce a monoclonal cell population. [0954] FIG.7 depicts v1.2 system that in addition to the polynucleotides of the v1.0 system (Contruct 1, Construct 3, and Construct 4) includes an additional polynucleotide (Construct 2) for encoding AAV Cap proteins to achieve an overall increase in AAV Cap proteins expression as compared to the v1.0 system. The payload (“GOI”) of polynucleotide (Construct 4) may encode for either progranulin or GFP. [0955] FIG.8 provides an overview schematic summarizing induction of cells that include the v1.2 system of polynucleotide constructs “v1.2 cells” and characterization of the Capsid levels, excision kinetics for production of AAV Rep and Cap, GOI levels, and levels of encapsidated GOI. [0956] FIG.9 provides a timeline from the point of induction of cells when tamoxifen and doxycycline are added (T=0) through 96 hours post-induction (T=96 hours). Expression of a protein marker, blue fluorescent protein, encoded by Construct 1 of FIG.7 prior to the induction in v1.2 cells (pooled) was measured and compared to the an expanded single cell clone that has only integrated the helper construct (T177CL9; see FIG.4 schematic). Presence of the additional Construct 2 of FIG.7 was shown to not impact excision of the gene encoding BFP such that AAV Rep and Cap proteins were expressed. [0957] FIG.10 provides data for characterization of rAAV produced by v1.2 cells (wherein the encapsidated payload is progranulin and the induced cells are pooled cells) and v1.0 cells (wherein the encapsidated payload is progranulin and the induced cells are pooled cells) (left two graphs). [0958] Encapsidated gene of interest (GOI) was measured using droplet digital PCR (ddPCR). Crude lysate was treated with Benzonase to remove unencapsidated DNA. Benzonase was then inactivated and viral particles are denatured to expose encapsidated GOI. This DNA was quantified by ddPCR using GOI specific primer and probe set. Capsids levels were measured by ELISA. [0959] Total gene of interest was measured using ddPCR. Viral particles in crude cell lysate are denatured to expose encapsidated GOI. Both unencapsidated and encapsidated DNA are quantified by ddPCR using GOI specific primer and probe set. [0960] Capsids titer was measured by ELISA. [0961] FIG.11 shows a timeline for production of viral particles over a 7 day period after induction of v1.2 cells (wherein the encapsidated payload is progranulin and the induced cells are pooled cells) and v1.0 cells (wherein the encapsidated payload is GFP and the induced cells are from an expanded single cell clone) with tamoxifen and doxycycline. In FIG.11, expression of total capsid production was measured. Capsids levels were measured by ELISA. [0962] FIG.12 shows a timeline for production of viral particles over a 7-day period after induction of v1.2 cells (wherein the encapsidated payload is progranulin and the induced cells are pooled cells) and v1.0 cells (wherein the encapsidated payload is GFP and the induced cells are from an expanded single cell clone) with tamoxifen and doxycycline. In FIG.12, expression of capsid encapsidated vector genomes (encapsidated payload) was measured. [0963] FIG.13 shows high titer produced after induction of v1.2 cells from the T231 stable cell pool wherein the payload is progranulin (“v1.2 Pool (PGRN)”). This v1.2 Pool (PGRN) titer is higher than the titer produced via transient transfection wherein the payload is eGFP (Transient Transfection (eGFP)), titer produced by a v1.0 stable cell pool wherein the payload is progranulin (v1.0 Pool (PGRN)), and titer produce by a v1.0 stable cell pool wherein the payload is eGFP (v1.0 Pool (eGFP)), indicating the v1.2 stable cell pool may comprise single cell clones, that when isolated and expanded to monoclonal populations, may be capable of producing higher titer than titer produced by single cell clones from v1.0 stable cell pools isolated and expanded to monoclonal populations, and higher titer than titer produced by transient transfection. [0964] FIG.14A shows high capsid titer produced after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)). The v1.2 Pool (PGRN) capsid titer and v1.2 Clones (PGRN) capsid titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) capsid titer, v1.0 single stable cell clones wherein the payload is eGFP (v1.0 eGFP clones) capsid titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) capsid titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones)capsid titer, indicating the v1.2 stable cells have successfully increased capsid titer >2 log. [0965] FIG.14B shows high vector genome production after induction of v1.2 pool cells wherein the payload is progranulin (v1.2 Pool (PGRN)) and after induction of v1.2 single cell clones wherein the payload is progranulin (v1.2 Clones (PGRN)). The v1.2 Pool (PGRN) vector genome titer and v1.2 Clones (PGRN) vector genome titer are higher than the v1.0 stable pool cells wherein the payload is eGFP (v1.0 eGFP Pool) vector genome titer, v1.0 single stable cell clones wherein the payload iseGFP (v1.0 eGFP clones) vector genome titer, v1.0 stable pool cells wherein the payload is progranulin (v1.0 PGRN Pool) vector genome titer, and v1.0 single stable cell clones wherein the payload is progranulin (v1.0 PGRN Clones) vector genome titer, indicating the v1.2 stable cells have successfully increased vector genome titer. [0966] FIG.15 shows high encapsidated vector genome (vg) production and high capsid (vp) titer produced after induction of a v1.2 pooled cell line (T318). T318 was generated from T205 CL23 clone. T205 CL23 clone was the top clone selected on the basis of highest vg/ml and vp/ml production from pool of T205 cells that have stably integrated Construct 1, Construct 3, and Construct 4 (see FIG.7). T318 cells were generated by then introducing Construct 2 into T205 CL23 clone and selecting for cells resistant to puromycin, blasticidin, and hygromycin. Example 3 – VA-RNA constructs [0967] The tables below show sequences of various elements of the VA-RNA/Inducible VA- RNA constructs as described herein. Construct Sequence of VA-RNA C T C T A C T C T
Figure imgf000304_0001
Construct Sequence of 5’ U6 DSE to 3’ VA-RNA A G
Figure imgf000304_0002
SEQ ID TAAACACAAAGATATTAGTACAAAATAATAACTTCGTATAATG NO:17 TATGCTATACGAAGTTATTTTGCAGTTTTAAAATTATGTTTTAA G C A G G A G C T T C A A A G A
Figure imgf000305_0001
Example 4 - Rep Cap construct Integration Into a Stable Cell Line [0968] This example describes integration of a construct 1 encoding Rep and Cap polypeptides (SEQ ID NO: 7) into a stable cell line and describes inducible expression of Rep and Cap polypeptides in said stable cell line. An AAV2 genome without the ITRs and with the polynucleotide construct was cloned into a piggybac vector with a Blasticidin resistance gene (SEQ ID NO: 8). The excisable element interrupting Rep was inserted downstream of the p19 promoter, as shown in FIG.1A. [0969] Suspension HEK293 cells (viral production cells, VPCs; also referred to as the parental cells or parental VPC pool) were transfected using a PEI pro transfection reagent. The ratio of transposon to transposase ratio used was 2:1. Cells were allowed to recover in non-selective media for 72 hours and passaged into selective media (10 µg/ml Blasticidin). Example 5 - Inducible Helper Constructs [0970] This example describes inducible helper constructs of the present disclosure. A tetracycline/ doxycycline (“Dox”) inducible promoter (TRE3G) drives the expression of estrogen inducible cre (ER2 cre). The estrogen inducible cre has a strong polyadenylation signal (stop signal) at its 3’ end. The cre gene and the polyadenylation signal are flanked by lox sites. Following this is a bicistronic E2A E4, orf6 cassette. The plasmid also has a constitutive promoter (mutant EF1alpha), which drives the expression of Tet-on 3G (Tet responsive activator protein). [0971] Mechanism of action: In the off state (in the absence of Dox), Tet-on 3G is unable to bind the Tet operator elements in the TRE3G promoter and, thus, the TRE3G promoter is not active. In an embodiment of the system, an estrogen responsive Cre is used instead of simple Cre to counteract any basal (or “leaky”) expression of the TRE3G promoter. Thus, even if the system yields leaky expression of the Cre gene, the expressed Cre protein will be held inactive in the cytoplasm. The strong polyadenylation stop signal positioned 3’ of the Cre gene will prevent basal expression of adenoviral helper genes (E2A and E4). [0972] To induce expression, Dox and Tamoxifen are added to the cell culture. Dox binds to the Tet-on 3G protein and promotes binding of Tet-on 3G to the Tet operator elements in the TRE3G promoter. This triggers activation of the promoter. ER2 Cre is expressed at high levels and Tamoxifen brings Cre to the nucleus. Cre recombines the lox sites, causing excision of the Cre-polyadenylation cassette. This brings the bicistronic E2A and E4 cassette next to the tetracycline-inducible promoter triggering their expression. Self-excision of Cre will limit the duration of Cre expression in the cells thus limiting Cre related toxicity and promiscuous recombination events. [0973] A first version of an inducible helper construct (STXC0123) is shown in FIG.1A. The construct also has a mutant of VA-RNA (G16A and G60A, which disable the internal PolIII promoter) driven by a U6 promoter. The proximal sequence element (PSE) and distal sequence element (DSE) of the U6 promoter are separated by a Lox sequence-flanked stuffer sequence (PGK driven Fusion Red-PuroR), thus, disabling the promoter. The promoter is reconstituted by Cre mediated excision of the stuffer sequence, resulting in VA-RNA expression conditional upon Cre expression. The inducible helper construct can have a sequence of SEQ ID NO: 153. The inducible helper construct can be in a plasmid having a sequence of SEQ ID NO: 30 or SEQ ID NO: 154. [0974] A second version of an inducible helper construct (STXC0133) can include a constitutively expressed VA-RNA element driven by its internal native promoters. The inducible helper construct can be in a plasmid having a sequence of SEQ ID NO: 31. [0975] Multiple variations of the inducible helper construct are envisioned. Fusion Red-PuroR can be replaced with PuromycinR and the PGK promoter can be replaced with a CMV promoter in two different orientations. [0976] The Rep Cap construct can be a STXC0137 plasmid (SEQ ID NO: 32) and payload construct can be a STXC0136 plasmid (SEQ ID NO: 33). The payload construct (STXC0136) comprises a sequence that encodes GFP flanked by ITRs (STX650 (sc GFP AAV) (SEQ ID NO: 34)) and the other half of a split blasticidin resistance gene, both under control of a constitutive promoter. Example 6 – Stable Cell Line for Production of Virions Encapsidating a Sequence Coding for Progranulin [0977] A stable mammalian cell line (“Progranulin Stable Cell line”) capable of inducible expression of rAAV encapsidating a sequence coding for progranulin is constructed by integrating three nucleic acid constructs (STXC-0123 (SEQ ID NO: 30), STXC0137 (SEQ ID NO: 32), and STXC0136 (SEQ ID NO: 33), in which the GFP of STXC0136 is replaced with a sequence coding for progranulin (e.g., SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159)) into the nuclear genome of a cell line that expresses adenovirus E1A and E1B. Cells that successfully integrated all 3 constructs are selected and maintained by growth in media in the presence of puromyocin and blasticidin to produce a stable cell line. [0978] The stable cell line is cultured, and then a construct similar to construct 2 (e.g., SEQ ID NO: 149) is transfected into the stable cell line. After selection, the v1.2 stable cell line is therefore producede and these v1.2 stable cells are induced to produce rAAV by adding Dox and Tamoxifen to the Progranulin Stable Cell line. Example 7 – AAV production in GS KO cells that integrated a helper construct comprising a GS selectable marker, a Rep Cap construct comprising a C-term of a split Blasticidin resistance protein selectable marker, a payload construct comprising a N-term of a split Blasticidin resistance protein selectable marker, and a Cap construct [0979] Glutamine Synthetase KO cells were transfected with a vector system comprising the helper construct comprising Glutamine Synthetase as a selectable marker, a Rep Cap construct comprising a C-term of a split Blasticidin resistance protein as a selectable marker, a payload construct comprising a N-term of a split Blasticidin resistance protein as a selectable marker, and a Cap construct with a third selectable marker. Example 8 – Exemplary V1.2 System [0980] This example describes another V1.2 system in which Cap proteins are expressed under the control of an inducible promoter that is stronger than native AAV p40 promoter. See, for example, Construct 1 of FIG.2A. The Rep proteins coding sequence is oriented in a direction opposite to the Cap proteins coding sequence. A TBE is positioned between the two sequences. The Cap proteins coding sequence includes a SV40 polyA signal sequence such that the SV40 polyA signal sequence is 3’ to the Cap coding sequence. [0981] In one method for generating cells with the V1.2 system, Construct 3 as shown in FIG. 2A is introduced in the cells and cells resistant to puromycin are selected. Constructs 1 and 2 as shown in FIG.2A are introduced into cells resistant to puromycin and cells resistant to puromycin and blasticidin are selected to generate cells harboring all three constructs. [0982] In another method, Constructs 1 and 2 as shown in FIG.2A are introduced into cells and cells resistant to blasticidin are selected. Construct 3 as shown in FIG.2A is introduced in the cells resistant to blasticidin and cells resistant to puromycin and blasticidin are selected to generate cells harboring all three constructs. [0983] Cells harboring all three constructs are separated into single cells which are expanded to generate clonal cell lines. Alternatively, a pool of cells harboring all three constructs are expanded. [0984] Clonal cell lines or pooled cells harboring all three constructs are induced by addition of doxycycline and tamoxifen. Addition of doxycycline results in expression of Cap proteins and ER2 Cre. In the presence of tamoxifen, ER2 Cre translocates to the nucleus and excises the excisable element positioned between the first part and the second part of the Rep coding sequence, resulting in expression of Rep. After induction, the Construct 3 of FIG.2A having a sequence of SEQ ID NO: 153 is induced to a Construct 3 of FIG.2B having a sequence of SEQ ID NO: 155. After induction, the Rep construct of Construct 1 of FIG.2A having a sequence of SEQ ID NO: 160 is induced to a Rep construct of Construct 1 of FIG.2B having a sequence of SEQ ID NO: 161. After induction, the Construct 1 of FIG.2A having a sequence of SEQ ID NO: 164 is induced to a Construct 1 of FIG.2B having a sequence of SEQ ID NO: 165. After induction, the VA-RNA construct of Construct 3 of FIG.2A having a sequence of SEQ ID NO: 167 is induced to a VA-RNA construct of Construct 3 of FIG.2B having a sequence of SEQ ID NO: 168. Since Cap is expressed under the control of Tet-On promoter which is stronger than the AAV p40 promoter used for expressing Cap proteins in the V1.0 system, these cells lines will produce higher levels of Cap proteins and higher levels of rAAV as compared to V1.0 system. Example 9 – AAV Cap encoding sequence comprising bGH or RBG poly A signal sequence [0985] This example describes the V1.2 system described in Example 8. The Cap proteins are expressed under the control of Tet-On promoter. The Cap proteins coding sequence includes a bGH or RBG poly A signal sequence located 3’ to the Cap proteins coding sequence. [0986] In one method for generating cells with the V1.2 system, Construct 3 as shown in FIG. 2A is introduced in the cells and cells resistant to puromycin are selected. Constructs 1 and 2 as shown in FIG.2A are introduced into cells resistant to puromycin and cells resistant to puromycin and blasticidin are selected to generate cells harboring all three constructs. [0987] In another method, Constructs 1 and 2 as shown in FIG.2A are introduced into cells and cells resistant to blasticidin are selected. Construct 3 as shown in FIG.2A is introduced in the cells resistant to blasticidin and cells resistant to puromycin and blasticidin are selected to generate cells harboring all three constructs. [0988] Cells harboring all three constructs are separated into single cells which are expanded to generate clonal cell lines. Alternatively, a pool of cells harboring all three constructs are expanded. [0989] Clonal cell lines or pooled cells harboring all three constructs are induced by addition of doxycycline and tamoxifen. Addition of doxycycline results in expression of Cap proteins and ER2 Cre. In the presence of tamoxifen, ER2 Cre translocates to the nucleus and excises the excisable element positioned between the first part and the second part of the Rep coding sequence, resulting in expression of Rep. Since Cap is expressed under the control of Tet-On promoter which is stronger than the AAV p40 promoter used for expressing Cap proteins in the V1.0 system, these cells lines will produce higher levels of Cap proteins and higher levels of rAAV as compared to V1.0 system. Example 10 – Exemplary V1.2 System [0990] This example describes another V1.2 system which includes two separate nucleic acids for inducibly expressing Cap proteins. Such a system is depicted in FIGS.3A and 3B. In this V1.2 system, Cap proteins are expressed under the control of Tet-On promoter. The constructs shown in FIG.3A may be introduced into cells in any suitable order. Cells harboring all four constructs are selected by growing the transduced cells in the presence of puromycin, blasticidin and hygromycin. The Cap proteins coding sequence present on the same polynucleotide as the Rep coding sequence may include a native polyA signal sequence and the Cap proteins coding sequence present on the separate polynucleotide may include a SV40 polyA signal sequence positioned 3’ to the Cap proteins coding sequence. [0991] Addition of doxycycline results in production of Cap proteins. Addition of doxycycline and tamoxifen results in production of rAAV. As compared to the V1.0 system which has a high level of unencapsidated payload, the increased level of Capsid proteins is expected to increase the levels of rAAV as observed in Fig.10 when using the V1.2 system depicted in FIG.1. Example 11 – Exemplary V1.2 System [0992] This example describes another V1.2 system which includes two separate nucleic acids for inducibly expressing Cap proteins. Such a system is depicted in FIGS.3A and 3B. In this V1.2 system, Cap proteins are expressed under the control of Tet-On promoter. The constructs shown in FIG.3A may be introduced into cells in any suitable order. Cells harboring all four constructs are selected by growing the transduced cells in the presence of puromycin, blasticidin and hygromycin. The Cap proteins coding sequence present on the same polynucleotide as the Rep coding sequence may include a native polyA signal sequence and the Cap proteins coding sequence present on the separate polynucleotide may include a bGH or RBG polyA signal sequence positioned 3’ to the Cap proteins coding sequence. Addition of doxycycline results in production of Cap proteins. Addition of doxycycline and tamoxifen results in production of rAAV. As compared to the V1.0 system which has a high level of unencapsidated payload, the increased level of Capsid proteins is expected to increase the levels of rAAV as observed in Fig. 10 when using the V1.2 system depicted in FIG.1. Example 12 – Viral production in cells selected to comprise a high construct copy number using an attenuated promoter [0993] This example describes viral production in cells that are selected to comprise a high copy number of a construct integrated into a cell using an attenuated promoter. [0994] A plasmid encoding helper proteins and a puromycin resistance gene (helper construct, e.g., a polynucleotide construct comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, or SEQ ID NO: 153) is produced. [0995] A glutamine synthetase (GS) protein is split at a Cys residue within the GS protein, in which an attenuated promoter operably linked to a C-Term GS/C-Term intein (referred to as the split-GS C-term module) is integrated into a construct encoding Rep (Rep2) and Cap (Cap5) proteins (e.g., SEQ ID NO: 164) to produce the C-term GS Rep/Cap plasmid, and an attenuated promoter operably linked to an N-Term intein/N-Term GS (referred to as the split-GS N-term module) is integrated into a construct encoding a GFP AAV (e.g., SEQ ID NO: 139) to produce the N-term GS payload plasmid. C-term GS Rep/Cap plasmids and N-term GS payload plasmids comprising the split-GS C-term module or the split-GS N-term module are generated, in which the GS split is at Cys53, Cys183, Cys229, or Cys252. The attenuated promoter is an attenuated EF1alpha promoter having a sequence of SEQ ID NO: 132. [0996] VPCs knocked out for GS are transfected with the helper construct and grown in media having puromycin. Surviving cells containing the helper construct are further transfected (independently for each GS split pair) with C-term GS Rep/Cap plasmids and N-term GS payload plasmids, and then are cultured in media deficient in glutamine to select for cells comprising high copy number integration of C-term GS Rep Cap constructs and N-term GS payload constructs, are expanded, and the copy number integration of C-term GS Rep Cap constructs and N-term GS payload constructs are assessed. The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. Titer is measured by qPCR. Example 13 – Viral production in cells selected to comprise a high construct copy number using a selectable marker having weak activity [0997] This example describes viral production in cells that are selected to comprise a high copy number of a construct integrated into a cell using a selectable marker having weak activity, such as a selectable marker mutated to have decreased activity. [0998] A plasmid encoding helper proteins and a puromycin resistance gene (helper construct, e.g., a polynucleotide construct comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, or SEQ ID NO: 153) is produced. [0999] A glutamine synthetase (GS) protein is split at a Cys residue within the GS protein, in which a promoter operably linked to a C-Term GS/C-Term intein (referred to as the split-GS C- term module) is integrated into a construct encoding Rep (Rep2) and Cap (Cap5) proteins (e.g., SEQ ID NO: 164) to produce the C-term GS Rep/Cap plasmid and a promoter operably linked to an N-Term intein/N-Term GS (referred to as the split-GS N-term module) is integrated into a construct encoding a GFP AAV (e.g., SEQ ID NO: 139) to produce the N-term GS GOI plasmid. C-term GS Rep/Cap plasmids and N-term GS GOI plasmids comprising the split-GS C-term module or the split-GS N-term module are generated, in which the GS split is at Cys53, Cys183, Cys229, or Cys252, and wherein the GS is a mutated GS having a R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112. The promoter is an EF1alpha promoter having a sequence of SEQ ID NO: 132. [1000] VPCs knocked out for GS are transfected with the helper construct and grown in media having puromycin. Surviving cells containing the helper construct are further transfected (independently for each GS split pair) with C-term GS Rep/Cap plasmids and N-term GS payload plasmids, and then are cultured in media deficient in glutamine to select for cells comprising high copy number integration of C-term GS Rep Cap constructs and N-term GS payload constructs, are expanded, and the copy number integration of C-term GS Rep Cap constructs and N-term GS payload constructs are assessed. The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. Titer is measured by qPCR. Example 14 – Viral production in cells selected to comprise a high construct copy number by culturing the cells with an inhibitor of the selectable marker [1001] This example describes viral production in cells that are selected to comprise a high copy number of a construct integrated into a cell by culturing the cells with an inhibitor of the selectable marker. [1002] A plasmid encoding helper proteins and a puromycin resistance gene (helper construct, e.g., a polynucleotide construct comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, or SEQ ID NO: 153) is produced. [1003] A glutamine synthetase (GS) protein is split at a Cys residue within the GS protein, in which a promoter operably linked to a C-Term GS/C-Term intein (referred to as the split-GS C- term module) is integrated into a construct encoding Rep (Rep2) and Cap (Cap5) proteins (e.g., SEQ ID NO: 164) to produce the C-term GS Rep/Cap plasmid and a promoter operably linked to an N-Term intein/N-Term GS (referred to as the split-GS N-term module) is integrated into a construct encoding a GFP AAV (e.g., SEQ ID NO: 139) to produce the N-term GS payload plasmid. C-term GS Rep/Cap plasmids and N-term GS payload plasmids comprising the split- GS C-term module or the split-GS N-term module are generated, in which the GS split is at Cys53, Cys183, Cys229, or Cys252. The promoter is an EF1alpha promoter having a sequence of SEQ ID NO: 132. [1004] VPCs knocked out for GS are transfected with the helper construct and grown in media having puromycin. Surviving cells containing the helper construct are further transfected (independently for each GS split pair) with C-term GS Rep/Cap plasmids and N-term GS payload plasmids, and then are cultured in media deficient in glutamine and comprising 0 uM, 50 uM, 125 uM, 250 uM , or 500 uM MSX to select for cells comprising high copy number integration of C-term GS Rep Cap constructs and N-term GS payload constructs, are expanded, and the copy number integration of C-term GS Rep Cap constructs and N-term GS payload constructs are assessed. The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. Titer is measured by qPCR. Example 15 – Viral production in cells selected to comprise a high Rep Cap construct copy number using an attenuated promoter [1005] This example describes viral production in cells that are selected to comprise a high copy number of a Rep Cap construct integrated into a cell using an attenuated promoter. [1006] A helper plasmid encoding helper proteins and a puromycin resistance gene (helper construct, e.g., a polynucleotide construct comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, or SEQ ID NO: 153) is produced. A payload plasmid comprising a construct encoding a GFP AAV (e.g., SEQ ID NO: 139) and a blasticidin resistance protein is produced. A Rep/Cap plasmid comprising a construct encoding Rep (Rep2) and Cap (Cap5) proteins (e.g., SEQ ID NO: 164) and an attenuated promoter operably linked to a GS protein (e.g., SEQ ID NO: 112) is produced. The attenuated promoter is an attenuated EF1alpha promoter having a sequence of SEQ ID NO: 132. [1007] VPCs knocked out for GS are transfected with the helper plasmid and grown in media having puromycin. Surviving cells containing the helper construct are further transfected with the payload plasmid and then cells containing the helper construct and the payload construct are selected in media having blasticidin. Surviving cells containing the helper construct and the payload construct are further transfected with the Rep/Cap plasmid and then cells containing the helper construct, the payload construct, and the Rep Cap construct are selected in media deficient in glutamine, are expanded, and the copy number integration the Rep Cap constructs are assessed. The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. Titer is measured by qPCR. Example 16 – Viral production in cells selected to comprise a high Rep Cap construct copy number using a selectable marker having weak activity [1008] This example describes viral production in cells that are selected to comprise a high copy number of a Rep Cap construct integrated into a cell using a selectable marker having weak activity, such as a selectable marker mutated to have decreased activity. [1009] A helper plasmid encoding helper proteins and a puromycin resistance gene (helper construct, e.g., a polynucleotide construct comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, or SEQ ID NO: 153) is produced. A payload plasmid comprising a construct encoding a GFP AAV (e.g., SEQ ID NO: 139) and a blasticidin resistance protein is produced. A Rep/Cap plasmid comprising a construct encoding Rep (Rep2) and Cap (Cap5) proteins (e.g., SEQ ID NO: 164) and a promoter operably linked to a mutated GS having a R324C, R324S, or R341C mutation as compared to SEQ ID NO: 112. The promoter is an EF1alpha promoter having a sequence of SEQ ID NO: 132. [1010] VPCs knocked out for GS are transfected with the helper plasmid and grown in media having puromycin. Surviving cells containing the helper construct are further transfected with the payload plasmid and then cells containing the helper construct and the payload construct are selected in media having blasticidin. Surviving cells containing the helper construct and the payload construct are further transfected with the Rep/Cap plasmid and then cells containing the helper construct, the payload construct, and the Rep Cap construct are selected in media deficient in glutamine, are expanded, and the copy number integration the Rep Cap constructs are assessed. The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. Titer is measured by qPCR. Example 17 – Viral production in cells selected to comprise a high Rep Cap construct copy number by culturing the cells with an inhibitor of the selectable marker [1011] This example describes viral production in cells that are selected to comprise a high copy number of a Rep Cap construct integrated into a cell by culturing the cells with an inhibitor of the selectable marker. [1012] A helper plasmid encoding helper proteins and a puromycin resistance gene (helper construct, e.g., a polynucleotide construct comprising SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 30, or SEQ ID NO: 153) is produced. A payload plasmid comprising a construct encoding a GFP AAV (e.g., SEQ ID NO: 139) and a blasticidin resistance protein is produced. A Rep/Cap plasmid comprising a construct encoding Rep (Rep2) and Cap (Cap5) proteins (e.g., SEQ ID NO: 164) and a promoter operably linked to a GS protein (e.g., SEQ ID NO: 112) is produced. The promoter is an EF1alpha promoter having a sequence of SEQ ID NO: 132. [1013] VPCs knocked out for GS are transfected with the helper plasmid and grown in media having puromycin. Surviving cells containing the helper construct are further transfected with the payload plasmid and then cells containing the helper construct and the payload construct are selected in media having blasticidin. Surviving cells containing the helper construct and the payload construct are further transfected with the Rep/Cap plasmid and then cells containing the helper construct, the payload construct, and the Rep Cap construct are selected in media deficient in glutamine and comprising 0 uM, 50 uM, 125 uM, 250 uM, or 500 uM MSX, are expanded, and the copy number integration the Rep Cap constructs are assessed. The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. Titer is measured by qPCR. Example 18 – Viral production in cells selected to comprise a high Rep Cap construct copy number [1014] This example describes several metabolic selection schemes for increasing amplification of a construct (by, e.g., increased expression and/or higher DNA copy number), such as a Rep Cap construct that encodes for a metabolic selectable marker and AAV Rep/Cap proteins (e.g., SEQ ID NO: 164) is tested to identify conditions that lead to amplification of the construct when stably integrated into the genome of a cell. Three conditions are tested: promoter mutagenesis, enzymatic inhibition, and mutation of the metabolic marker expressing gene (e.g., an attenuated version of the metabolic marker). The metabolic marker that are tested are glutamine synthetase (GS). For promoter mutagenesis, the TATA box in the EF1alpha promoter is mutated (SEQ ID NO: 132) and operably linked to full length GS (SEQ ID NO: 112). For GS inhibition, methionine sulfoximine (MSX), which is a direct inhibitor of GS activity, is tested at various concentrations with full length GS (SEQ ID NO: 23) driven by an EF1alpha promoter (SEQ ID NO: 133). For mutation of GS, three different mutations in GS are tested: GS R324C (SEQ ID NO: 142), R324S (SEQ ID NO: 143), and R341C (SEQ ID NO: 144). [1015] The host cell is a HEK293 cell lacking GS (GS-knock out “GS-KO”). The first generation of stable cell lines, P1, comprises cells selected for integration of a construct that expresses AAV helper proteins upon induction and the selectable marker, puromycin, after puromycin selection. The P1 cell line is then transfected with a construct that comprises the payload flanked by ITRs and the selectable marker, blasticidin, to produce P2 cell line after selection in blasticidin. P2 cell line is independently transfected with a different construct that express Rep/Cap upon induction and variations of GS to produce P3 cell line after GS selection. For Path 1, the GS is a full-length GS driven by an attenuated EF1alpha promoter (comprising a TATGTA mutation). For Path 2, the GS was GS R324C, GS R324S, or GS R341C. For Path 3, the GS is a full-length GS that is further selected in media comprising different concentrations of MSX for GS inhibition. [1016] A GS-KO P2 cell line is independently transfected with the various Rep/Cap GS constructs. P3 cells are selected in cell culture media lacking glutamine (-Gln) to select for cells expressing a higher level of GS. For Path 3, P3 cells are cultured in media lacking Gln and in the presence of a GS inhibitor, MSX, at various concentrations, to select for cells expressing a higher level of GS. [1017] The cells are then transfected with a construct encoding an AAV cap protein and another selectable marker. The selected cells are then induced with doxycycline and tamoxifen to produce virions. [1018] Exemplary sequences of constructs and plasmids described herein may be as follows. [1019] A construct as described herein (e.g., Construct 3 in FIGs.1A, 2A, and 3A) that inducibly encodes the adenoviral helper proteins E2A and E4 and mutant VA RNA1 and the inducible recombinase may have a sequence prior to induction that is at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% identical to the sequence set forth in SEQ ID NO:153. [1020] A plasmid that includes the construct as described herein (e.g., Construct 3 in FIGs.1A, 2A, and 3A) that encodes the adenoviral helper proteins E2A and E4 and mutant VA RNA1 and the inducible recombinase may have a sequence having at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% sequence identity or 100% sequence identity to the sequence set forth in SEQ ID NO: 154. [1021] A construct as described herein (e.g., Construct 3 in FIGs.1A, 2A, and 3A) that encodes the adenoviral helper proteins E2A and E4 and mutant VA RNA1 and the inducible recombinase may have a sequence after induction that has at least 70%, 75%, 80% 85%, 90%, 95%, 96%, 97%, 98%, 99% or a 100% sequence identity to the sequence set forth in SEQ ID NO:155. [1022] The “payload” construct, e.g., Construct 4 in FIGs.1A-1B, Construct 2 in FIGs.2A-2B and FIGs.3A-3B may include a sequence of a gene of interest. In some specific embodiments, the gene of interest is progranulin. As will be understood to one of skill in the art, the payload can be a single stranded sequence of a gene of interest or a self-complementary sequence of the gene of interest. [1023] In one embodiment, the payload construct may comprise a self-complementary sequence of the gene of interest, progranulin. In such an embodiment, the payload construct may have a sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or a 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 156. [1024] In one embodiment, the payload construct may comprise a self-complementary sequence of progranulin and is included in a plasmid. In such an embodiment, the plasmid may have a sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or a 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO: 157. [1025] In one embodiment, the payload construct may comprise a single stranded sequence of the gene of interest, progranulin. In such an embodiment, the payload construct may have a sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or a 100% sequence identity to SEQ ID NO: 158. [1026] In one embodiment, the payload construct may comprise a single stranded sequence of progranulin and is included in a plasmid. In such an embodiment, the plasmid may have a sequence having at least 70%, at least 75%, at least 80% at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity or a 100% sequence identity to SEQ ID NO: 159.
INFORMAL SEQUENCE LISTING [1028] First spacer segment- SEQ ID NO: 1: [1029] GTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATG TTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTG TATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAAC CATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTG CATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTC TCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTT TTAGAGAAC [1030] Second spacer segment - SEQ ID NO: 2: [1031] ataacttcgtataatgtatgctatacgaagttatCGGGCCCCTCTGCTAACCATGTTCATGCCTTCT TCTTTTTCCTACAGatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacg taaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccgg caagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagc acgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccg aggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaa gctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccaca acatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaacc actacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgg gatcactctcggcatggacgagctgtacaagtaaCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGC TGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAA AAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGA AATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGAC ATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGG CAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCAT CAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACT TGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAA ATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTC ATAGCTGTCCCTCTTCTCTTATGGAGATCataacttcgtataatgtatgctatacgaagttat [1032] Third spacer segment - SEQ ID NO: 3: [1033] AATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCT GGCGTGGAAATATTCTTATTGGTAGAAACAACTACACCCTGGTCATCATCCTGCCTT TCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGT CCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTtTTTCCTACAG [1034] DHFR Z-Nter - SEQ ID NO: 4: [1035] ATGAGAGGGTCAGATCCGGCAGCGTTGAAGCGGGCTAGGAACACTGAGGCA GCAAGGCGCTCTCGAGCAAGGAAGTTGCAACGGATGAAACAATTGGAAGACAAAG TTGAAGAGCTGCTTTCAAAAAACTACCACCTTGAAAATGAAGTCGCGAGGCTGAAG AAATTGGTCGGATCTGCTGGCAGCGCAGCGGGGAGCGGTGAGTTTATGGTCAGACC TCTCAACTGTATTGTCGCTGTCTCACAGAACATGGGTATCGGAAAGAACGGTGACTT GCCGTGGCCGCCACTGCGGAATGAGTTCAAATACTTTCAGCGCATGACGACCACCA GCAGTGTGGAGGGTAAGCAAAATCTTGTCATAATGGGTCGCAAGACTTGGTTTTCT ATTCCAGAGAAAAACAGACCGCTTAAAGATAGGATTAACATCGTGTTGAGCCGGGA ACTGAAAGAGCCACCAAGGGGAGCACATTTTTTGGCTAAGTCCTTGGATGACGCCC TGCGACTGATAGAGCAACCAGAACTTGCTTAGTAA [1036] DHFR Z-Cter - SEQ ID NO: 5: [1037] ATGCGCGGTTCCGACCCAGCAGCTTTGAAACGAGCACGAAACACGGAAGCA GCCCGCAGGAGTCGAGCGAGAAAACTTCAGCGGATGAAGCAGCTTGAAGATAAAG TCGAGGAATTGCTTAGCAAGAATTATCACCTCGAGAATGAAGTGGCGCGACTGAAA AAACTTGTAGGTTCTGCTGGGAGCGCAGCCGGAAGCGGCGAGTTCTCAAAAGTTGA CATGGTGTGGATCGTGGGTGGAAGTTCTGTCTATCAAGAGGCGATGAATCAGCCTG GCCACCTCAGACTGTTTGTTACAAGGATCATGCAGGAGTTCGAGTCTGACACGTTTT TTCCAGAGATCGACCTGGGGAAATATAAACTCCTCCCAGAGTACCCAGGAGTGCTT AGTGAGGTCCAAGAAGAGAAGGGAATCAAATATAAATTTGAAGTTTACGAAAAGA AGGATTAGTAA [1038] ITR deleted AAV2 genome with Construct 1 (cloned in pCRII Topo vector) - SEQ ID NO: 6 [1039] ggaggggtggagtcgtgacgtgaattacgtcatagggttagggaggtcctgtattagaggtcacgtgagtgttttgcgacattttg cgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccATTTTGAAGCGGGAGGTTTGAA CGCGCAGCCGCCatgccggggttttacgagattgtgattaaggtccccagcgaccttgacgagcatctgcccggcatttctgaca gctttgtgaactgggtggccgagaaggaatgggagttgccgccagattctgacatggatctgaatctgattgagcaggcacccctgaccgt ggccgagaagctgcagcgcgactttctgacggaatggcgccgtgtgagtaaggccccggaggcccttttctttgtgcaatttgagaaggg agagagctacttccacatgcacgtgctcgtggaaaccaccggggtgaaatccatggttttgggacgtttcctgagtcagattcgcgaaaaac tgattcagagaatttaccgcgggatcgagccgactttgccaaactggttcgcggtcacaaagaccagaaatggcgccggaggcgggaac aaggtggtggatgagtgctacatccccaattacttgctccccaaaacccagcctgagctccagtgggcgtggactaatatggaacagtattt aagcgcctgtttgaatctcacggagcgtaaacggttggtggcgcagcatctgacgcacgtgtcgcagacgcaggagcagaacaaagaga atcagaatcccaattctgatgcgccggtgatcagatcaaaaacttcagccaggtacatggagctggtcgggtggctcgtggacaaGGT GAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATAT GGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCA CCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATTG TCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTT GTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAA TCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGA GAACataacttcgtataatgtatgctatacgaagttatCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTT CTTTTTCCTACAGatggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgta aacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggca agctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcac gacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccga ggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaag ctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaac atcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccac tacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggat cactctcggcatggacgagctgtacaagtaaCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTG GTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAA ATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAA TTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACAT ATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCA ACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCA GTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTG AGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAAT TTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCAT AGCTGTCCCTCTTCTCTTATGGAGATCataacttcgtataatgtatgctatacgaagttatAATTGTTATAA TTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTC TTATTGGTAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACA ATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCT CTGCTAACCATGTTCATGCCTTCTTCTtTTTCCTACAGgggattacctcggagaagcagtggatccagga ggaccaggcctcatacatctccttcaatgcggcctccaactcgcggtcccaaatcaaggctgccttggacaatgcgggaaagattatgagc ctgactaaaaccgcccccgactacctggtgggccagcagcccgtggaggacatttccagcaatcggatttataaaattttggaactaaacg ggtacgatccccaatatgcggcttccgtctttctgggatgggccacgaaaaagttcggcaagaggaacaccatctggctgtttgggcctgc aactaccgggaagaccaacatcgcggaggccatagcccacactgtgcccttctacgggtgcgtaaactggaccaatgagaactttcccttc aacgactgtgtcgacaagatggtgatctggtgggaggaggggaagatgaccgccaaggtcgtggagtcggccaaagccattctcggag gaagcaaggtgcgcgtggaccagaaatgcaagtcctcggcccagatagacccgactcccgtgatcgtcacctccaacaccaacatgtgc gccgtgattgacgggaactcaacgaccttcgaacaccagcagccgttgcaagaccggatgttcaaatttgaactcacccgccgtctggatc atgactttgggaaggtcaccaagcaggaagtcaaagactttttccggtgggcaaaggatcacgtggttgaggtggagcatgaattctacgtc aaaaagggtggagccaagaaaagacccgcccccagtgacgcagatataagtgagcccaaacgggtgcgcgagtcAGTTGCGC agccatcgacgtcagacgcggaagcttcgatcaactacgcagacagGTACCAAAACAAATGTTCTCGTCACGT GGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATT CAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAG AATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATC ATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGAT TTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGG TTATCTTCCAGattggctcgaggacactctctctgaaggaataagacagtggtggaagctcaaacctggcccaccaccaccaaa gcccgcagagcggcataaggacgacagcaggggtcttgtgcttcctgggtacaagtacctcggacccttcaacggactcgacaagggag agccggtcaacgaggcagacgccgcggccctcgagcacgacaaagcctacgaccggcagctcgacagcggagacaacccgtacctc aagtacaaccacgccgacgcggagtttcaggagcgccttaaagaagatacgtcttttgggggcaacctcggacgagcagtcttccaggcg aaaaagagggttcttgaacctctgggcctggttgaggaacctgttaagacggctccgggaaaaaagaggccggtagagcactctcctgtg gagccagactcctcctcgggaaccggaaaggcgggccagcagcctgcaagaaaaagattgaattttggtcagactggagacgcagact cagtacctgacccccagcctctcggacagccaccagcagccccctctggtctgggaactaatacgatggctacaggcagtggcgcacca atggcagacaataacgagggcgccgacggagtgggtaattcctcgggaaattggcattgcgattccacatggatgggcgacagagtcatc accaccagcacccgaacctgggccctgcccacctacaacaaccacctctacaaacaaatttccagccaatcaggagcctcgaacgacaat cactactttggctacagcaccccttgggggtattttgacttcaacagattccactgccacttttcaccacgtgactggcaaagactcatcaaca acaactggggattccgacccaagagactcaacttcaagctctttaacattcaagtcaaagaggtcacgcagaatgacggtacgacgacgat tgccaataaccttaccagcacggttcaggtgtttactgactcggagtaccagctcccgtacgtcctcggctcggcgcatcaaggatgcctcc cgccgttcccagcagacgtcttcatggtgccacagtatggatacctcaccctgaacaacgggagtcaggcagtaggacgctcttcattttac tgcctggagtactttccttctcagatgctgcgtaccggaaacaactttaccttcagctacacttttgaggacgttcctttccacagcagctacgct cacagccagagtctggaccgtctcatgaatcctctcatcgaccagtacctgtattacttgagcagaacaaacactccaagtggaaccaccac gcagtcaaggcttcagttttctcaggccggagcgagtgacattcgggaccagtctaggaactggcttcctggaccctgttaccgccagcag cgagtatcaaagacatctgcggataacaacaacagtgaatactcgtggactggagctaccaagtaccacctcaatggcagagactctctgg tgaatccgggcccggccatggcaagccacaaggacgatgaagaaaagttttttcctcagagcggggttctcatctttgggaagcaaggctc agagaaaacaaatgtggacattgaaaaggtcatgattacagacgaagaggaaatcaggacaaccaatcccgtggctacggagcagtatg gttctgtatctaccaacctccagagaggcaacagacaagcagctaccgcagatgtcaacacacaaggcgttcttccaggcatggtctggca ggacagagatgtgtaccttcaggggcccatctgggcaaagattccacacacggacggacattttcacccctctcccctcatgggtggattc ggacttaaacaccctcctccacagattctcatcaagaacaccccggtacctgcgaatccttcgaccaccttcagtgcggcaaagtttgcttcc ttcatcacacagtactccacgggacaggtcagcgtggagatcgagtgggagctgcagaaggaaaacagcaaacgctggaatcccgaaat tcagtacacttccaactacaacaagtctgttaatgtggactttactgtggacactaatggcgtgtattcagagcctcgccccattggcaccaga tacctgactcgtaatctgtaattgcttgttaatcaataaaccgtttaattcgtttcagttgaactttggtctctgcgtatttctttcttatctagtttccat ggctacgtagataagtagcatggcgggttaatcattaactacaTAAGGGCGAATTCTGCAGATATCCATCACA CTGGCGGCCGCTCGAGCATGCATCTAGAGGGCCCAATTCGCCCTATAGTGAGTCGT ATTACAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTA CCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAG AGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTATACGTACGGCAGTTTA AGGTTTACACCTATAAAAGAGAGAGCCGTTATCGTCTGTTTGTGGATGTACAGAGT GATATTATTGACACGCCGGGGCGACGGATGGTGATCCCCCTGGCCAGTGCACGTCT GCTGTCAGATAAAGTCTCCCGTGAACTTTACCCGGTGGTGCATATCGGGGATGAAA GCTGGCGCATGATGACCACCGATATGGCCAGTGTGCCGGTCTCCGTTATCGGGGAA GAAGTGGCTGATCTCAGCCACCGCGAAAATGACATCAAAAACGCCATTAACCTGAT GTTCTGGGGAATATAAATGTCAGGCATGAGATTATCAAAAAGGATCTTCACCTAGA TCCTTTTCACGTAGAAAGCCAGTCCGCAGAAACGGTGCTGACCCCGGATGAATGTC AGCTACTGGGCTATCTGGACAAGGGAAAACGCAAGCGCAAAGAGAAAGCAGGTAG CTTGCAGTGGGCTTACATGGCGATAGCTAGACTGGGCGGTTTTATGGACAGCAAGC GAACCGGAATTGCCAGCTGGGGCGCCCTCTGGTAAGGTTGGGAAGCCCTGCAAAGT AAACTGGATGGCTTTCTCGCCGCCAAGGATCTGATGGCGCAGGGGATCAAGCTCTG ATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCA GGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGAC AATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCT TTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCAGCGC GGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCA CTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTG TCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGG CTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATC GAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGA AGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGC CCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATG GTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGA CCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCG AATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCA TCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAATTATTAACGCTTACAATTTCCT GATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATACAGGTGGC ACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCA AATATGTATCCGCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGT GAGGAGGGCCACCATGGCCAAGTTGACCAGTGCCGTTCCGGTGCTCACCGCGCGCG ACGTCGCCGGAGCGGTCGAGTTCTGGACCGACCGGCTCGGGTTCTCCCGGGACTTC GTGGAGGACGACTTCGCCGGTGTGGTCCGGGACGACGTGACCCTGTTCATCAGCGC GGTCCAGGACCAGGTGGTGCCGGACAACACCCTGGCCTGGGTGTGGGTGCGCGGCC TGGACGAGCTGTACGCCGAGTGGTCGGAGGTCGTGTCCACGAACTTCCGGGACGCC TCCGGGCCGGCCATGACCGAGATCGGCGAGCAGCCGTGGGGGCGGGAGTTCGCCCT GCGCGACCCGGCCGGCAACTGCGTGCACTTCGTGGCCGAGGAGCAGGACTGACACG TGCTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATC TCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAG AAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGC AAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCA ACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTT CTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATAC CTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTT ACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAAC GGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGAT ACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGA CAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCA GGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAG CGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAA CGCGGCCTTTTTACGGTTCCTGGGCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCT GCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCT GGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTG AGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATG TTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGA TTACGCCAAGCTATTTAGGTGACACTATAGAATACTCAAGCTATGCATCAAGCTTGG TACCGAGCTCGGATCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCT [1040] SEQ ID NO: 7 (AAV2 Rep AAV2 Cap construct) [1041] GGCCTCCACGGCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCTGGA GGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTAGAG GTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAA GCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCC GCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCA TCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGT TGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTG GCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCC GGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGT GCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGA TTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAAC TGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGG ATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGG CGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAA CGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAG AGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGG TACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGTGAGTTTGGGGACCCTTGATTG TTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGG GTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTT TGTTTCTTTCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATT TTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACT TTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATC AGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACATAACTTCGTATAATGT ATGCTATACGAAGTTATCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTC CTACAGATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGT CGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAG GGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCT GCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAG CCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAG GCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGC GCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCAT CGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAAC AGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTT CAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGC AGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGC ACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCT GGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAAC CTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCT CACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAG CCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTG TGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTA AAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCT GCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGC TGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTT TGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGC CAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGG AGATCATAACTTCGTATAATGTATGCTATACGAAGTTATAATTGTTATAATTAAATG ATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGG TAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATA TACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTA ACCATGTTCATGCCTTCTTCTTTTTCCTACAGGGGATTACCTCGGAGAAGCAGTGGA TCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCC AAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCC CCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTA TAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGG ATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAA CTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGG TGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGT GATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCC ATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGAT AGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACG GGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTT GAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAA AGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACG TCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTT CGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAAT CTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTG CTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGG AAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACT GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCA GATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACC TGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTT GTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCC GGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAG CTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCA GGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCC AGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACG GCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTC GGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAG ACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGC CCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAG ACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGAT TCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCC CACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACG ACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCC ACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTC CGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCA GAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTA CTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCC CGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACA ACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTC AGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTT TCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCG ACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAG TCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAA CTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATA ACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGA GACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAA AGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACA AATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCA ATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAAC AGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTG GCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGG ACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTC CACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTG CGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAG ATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGT ACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCG TGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATTGC TTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTC TTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAA CTACA [1042] SEQ ID NO: 8 (STXC0068) [1043] ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGT TAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCC CTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT GTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAG GACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGAT GCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTG TGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTGT ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTA CATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAA AACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCC CCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGC GAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCA GCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAA CGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCC TCCACGGCCACTAGTAACGGCCGCCAGTGTGCTGGAATTCGCCCTGGAGGGGTGGA GTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTG AGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGT GAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGCC GGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCG GCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCA GATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAA GCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCC TTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGG AAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAA AAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGC GGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGC TACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACT AATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGT GGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAG AATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGA GCTGGTCGGGTGGCTCGTGGACAAGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTT TTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTT TAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTT TCACTTTCTACTCTGTTGACAACCATTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAA CTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTAT TTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATAT TATATTGTACTTCAGCACAGTTTTAGAGAACATAACTTCGTATAATGTATGCTATAC GAAGTTATCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGATG GTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGA CGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC CTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCC CGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCC AGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTG AAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAA GGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAC GTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCC CCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC GCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGT GACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACCTCAGGTGC AGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATAC CACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAG CATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAAT TTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAG AATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAAC AAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTC CTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTG TTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTT CCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCATAA CTTCGTATAATGTATGCTATACGAAGTTATAATTGTTATAATTAAATGATAAGGTAG AATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAA CTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTT GAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCA TGCCTTCTTCTTTTTCCTACAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGA CCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGC TGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACC TGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTG GAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCAC GAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGA AGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAAC TGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTG GGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGA GGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGA CTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCA ACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCAC CCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTT TCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAG GGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAAC GGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAAC TACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCT GTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCA CGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGT CGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGC CAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTG AACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCAGATTGGCTC GAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACC ACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTG GGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAG GCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCG GAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTT AAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAA GAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAA AAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGA AAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACG CAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTC TGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGA GGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGA TGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAAC AACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTA CTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTT TTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGA GACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGT ACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGA GTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCC AGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTC AGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGC GTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCA GCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACC TGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTT CAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCT GGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAG TGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGG TGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCT CAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACAT TGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCT ACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGC TACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAG ATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTT CACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTC ATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTT GCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGA GCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACT ACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGC CTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAATTGCTTGTTAATCAAT AAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTTCTTATCTAG TTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACATAAGGG CGAATTCTGCAGATATCCATCACACTGGCGGCCGCTCGAGCATGCATCTAGAGCTA GCGAATTCGAATTTAAATCGGATCCGCGGCCGCAAGGATCTGCGATCGCTCCGGTG CCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGG GGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGT GATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT GCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTG AAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGC CATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACT GCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGC GCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCT TGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTG TGACCGGCGCCTACGATATCGCCACCATGAAAACATTTAACATTTCTCAACAGGATC TAGAATTAGTAGAAGTAGCGACAGAGAAGATTACAATGCTTTATGAGGATAATAAA CATCATGTGGGAGCGGCAATTCGTACGAAAACAGGAGAAATCATTTCGGCAGTACA TATTGAAGCGTATATAGGACGAGTAACTGTTTGTGCAGAAGCCATTGCGATTGGTA GTGCAGTTTCGAATGGACAAAAGGATTTTGACACGATTGTAGCTGTTAGACACCCTT ATTCTGACGAAGTAGATAGAAGTATTCGAGTGGTAAGTCCTTGTGGTATGTGTAGG GAGTTGATTTCAGACTATGCACCAGATTGTTTTGTGTTAATAGAAATGAATGGCAAG TTAGTCAAAACTACGATTGAAGAACTCATTCCACTCAAATATACCCGAAATTAAGG TACCTCGACAACCTTCCAAACTGAGTGCATGACCCGCAAGCCCGGTGCCTGAAATC AACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTC CTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCGTTAACTAAACT TGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAA ATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATC TTATCATGTCTGGAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTATCTGG TCTCCCTTCCGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGTTCCCAA ACTGGGTTCTTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGTCCTCAC AGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAATTGACTG GATTCCTTTTTTAGGGCCCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTG CAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCC CCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGG ACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTA ATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAA TATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGT ATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGC GGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGG GAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATT TAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCT TTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCA GTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTT TCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCAT TGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGC ACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGC TTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAAT ACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGC GCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGA CATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGC TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAA GCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC CTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGG TATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTAC TTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAG GGAATAAGGGCGACACGGAAATGTTGAATACTCAT [1044] SEQ ID NO: 9 (STXC0090) [1045] ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGT TAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCC CTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT GTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAG GACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGAT GCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTG TGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTGT ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTA CATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAA AACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCC CCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGC GAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCA GCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAA CGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCC TCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTT CCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATAGAATGACACC TACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTG GCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGC AACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGC GGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAG GAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCAGCATGAG ACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCAGCACAGTA ACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATC CAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAG GTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGG CATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCC ATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTATGCACTGCA GGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAACCATGGAT CATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTGCATACACTT CCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGGAACAACCCATT CCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTG TGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTATGGTAGC GCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGAGTGCGCCGAG ACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGACGTAGTC ATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTT CGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACC GAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCCTTCAGCCCCT TGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAA CGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGC CGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACA GACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAAC AGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAA GACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAA ATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACG TTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCTGCCGCGCATC ACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAA CTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCA CCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGG CCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTAT CAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGT CCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCA AAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAGAAGG TGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTGATCTG CTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGC CGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGCGTCGGTG TTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTA GACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGT GCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGC GCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGTTACCT CTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTC TTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGGTCTTG CATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCTTTAGA TCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCTTCTCC CACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTTCCGCT TCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGGGTCGTCTT CATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGCACCGGTG GGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCAC GATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTTTTTCTT TTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGGTGTGC GCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCC TCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGTCGGGAGGCG GCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACGTCGCGCCGCA CCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATGGTG GCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCAGACATGATAA GATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTT ATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAA CAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGG GAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCT CTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCTCTGCCTCCCC CGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCAGTGGCCAAGT GGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGCGCATGTAGGC GGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCACGTTCTTGCAC TTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATGTGGGAGAGG ATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCAGGCCTGCCTT GGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGTGGATATGGTC CTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTCCAGAATTAAG CAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTCATCATGCGGA ACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCCCTCTACACAT TTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTTCACTGGGTGC TCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGGCACATTCTAG AAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACCCTCTTCGCCC AGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAGCCCATCATCG AAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTATGGGGGGCTCA GCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGGCCAGGCTGTT CTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAGCTCTCATGTC TCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTC ACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGC TTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGAC ACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTAAAATA CACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAATTTTATATTTA CCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGA GTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCA AAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGA CCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCG TTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTT AACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAG AAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGGAC TTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATC AACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGG TCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGA TAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCGCGAACATCTT CAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACC GGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTG AACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCA GGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGTAATAACTTCGT ATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGGAAGTGGTACG GAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGACGGTTC ACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTATACGTT CTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGGGAGTA AACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTCTCTAT CACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAAAGTCT GCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATCACTGA TAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCACTTGCACTGGG GACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTT AGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAAGATAT TTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCA CAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAG GTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGA GGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCA ACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCAC GCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGC CTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCA GGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGC TCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTT CTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGCGGATCCGCCGCC ACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAA TGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTG AGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTG CCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTC ATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTCTCTCACA TCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAA ACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGC ACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACA GGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCC CCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTT CCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAA GCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGAT GCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGAC CTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGTGCCTTCTAGT TGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA GAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCTATTTCCCAT GATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAA TTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTATAATGTATGCT ATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACT AGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTT GTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTTTTCGCGTCA TGCACCACGTCCGTGGGCCCTCGGGTACTTCAACGTCAGCAGTAACTGTAAATCCG AGCCGTTCATAGAAGGGCAAATTCCTTGGCGCTGACGTTTCAAGAAAGGCTGGCAC TCCGGCTCGTTCTGCGGCTTCTACTCCGGGCAATACCACCGCGGAACCAAGGCCCTT TCCCTGATGATCGGGGCTAACGCCCACAGTAGCGAGGAACCAAGCTGGTTCTTTAG GGCGGTGAGGGGCGAGGAGTCCTTCCATTTGTTGCTGAGCCGCGAGACGAGAGCCA CTAAGCTCAGCCATTCGGGGACCAATTTCTGCAAATACAGCCCCGGCCTCAACGCTC TCCGGAGTCGTCCACACTGCCACTGCAGCCCCGTCGTCGGCGACCCAAACTTTACCG ATGTCCAATCCTACCCTGGTCAAAAAAAGTTCTTGCAATTCTGTAACCCGTTCAATA TGTCTATCAGGATCAACTGTGTGGCGTGTAGCGGGATAATCCGCGAAAGCGGCAGC CAATGTTCTCACGGCCCTAGGGACGTCGTCTCGAGTTGCCAGTCTGACAGTAGGTTT ATATTCTGTCATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGGCT GAAGTTAGTAGCTCCGGATCCTTTACCTCCATCACCAGCGCCACCAGTAGAGTATCT GGCCACAGCCACCTCGTGCTGCTCGACGTAGGTCTCATCGTCGGCCTCCTTGATTCT TTCCAGTCTGTGGTCCACGTTGTAGACGCCGGGCATCTTGAGGTTCGTAGCGGGTTT CTTGGATCTGTATGTGGTCTCAAGGTTGCAGATCAGGTGGCCCCCGCCCACGAGCTT CAGGGCCATGTCACATGCGCCTTCCAGGCCGCCGTCAGCGGGGTACATCGTCTCGG TGGAGGCCTCCCAGCCGAGTGTTTTCTTCTGCATCACAGGGCCGTTGGCTGGGAAGT TCACCCCTCTAACCTTGACGTTGTAGATGAGGCAGCCGTCCTGGAGGCTGGTGTCCT GGGTAGCGGTCAGCACGCCCCCGTCTTCGTATGTGGTGACTCTCTCCCATGTGAAGC CCTCAGGGAAGGACTGCTTAAAGAAGTCGGGGATGCCCGGAGGGTGCTTGATGAAG GTTCTGCTGCCGTACATGAAGCTGGTAGCCAGGATGTCGAAGGCGAAGGGGAGAGG GCCGCCCTCGACGACCTTGATTCTCATGGTCTGGGTGCCCTCGTAGGGCTTGCCTTC GCCCTCGGATGTGCACTTGAAGTGGTGGTTGTTCACGGTGCCCTCCATGTACAGCTT CATGGGCATGTTCTCCTTAATCAGCTCGCTCACGGTGGCGGCGAATTCCGAAAGGCC CGGAGATGAGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCAG AATGCCGGGCCTCCGGAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCCT GAGCCCGCCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAGC AAAGCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTGC TGTCCATCTGCACGAGACTAGTGAGACGTGCTACTTCCATTTGTCACGTCCTGCACG ACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGGGAGGAGTAGAAGGTGG CGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTGGATGTGGAA TGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCTAA AGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATAA TGTATGCTATACGAAGTTATTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATC ATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAA AGGACGAAACACCGGGCACTCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATC ATGGCGGACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCG GTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTC CTTTTTGGGCCCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTC AAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCC CGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGA GCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACAT CCCTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATAT ATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGT TAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGT TATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTC CAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAA GAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGG GTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCG CCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCG AGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTT GAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTT TAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCG GACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCG ACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGA TATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGAT ACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCT GTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA GTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGA CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAA CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCAC CGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATC GTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGG CGACACGGAAATGTTGAATACTCAT [1046] SEQ ID NO: 10 (STXC0110) [1047] ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGT TAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCC CTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT GTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAG GACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGAT GCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTG TGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTGT ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTA CATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAA AACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCC CCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGC GAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCA GCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAA CGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCC TCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTT CCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATAGAATGACACC TACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTG GCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGC AACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGC GGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAG GAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCAGCATGAG ACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCAGCACAGTA ACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATC CAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAG GTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGG CATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCC ATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTATGCACTGCA GGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAACCATGGAT CATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTGCATACACTT CCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGGAACAACCCATT CCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTG TGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTATGGTAGC GCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGAGTGCGCCGAG ACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGACGTAGTC ATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTT CGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACC GAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCCTTCAGCCCCT TGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAA CGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGC CGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACA GACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAAC AGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAA GACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAA ATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACG TTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCTGCCGCGCATC ACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAA CTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCA CCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGG CCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAACACTAT CAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGT CCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCA AAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAGAAGG TGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTGATCTG CTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGC CGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGCGTCGGTG TTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTA GACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGT GCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGC GCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGTTACCT CTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTC TTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGGTCTTG CATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCTTTAGA TCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCTTCTCC CACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTTCCGCT TCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGGGTCGTCTT CATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGCACCGGTG GGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCAC GATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTTTTTCTT TTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGGTGTGC GCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCC TCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGTCGGGAGGCG GCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACGTCGCGCCGCA CCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATGGTG GCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCAGACATGATAA GATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTT ATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAA CAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGG GAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATGATCCT CTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCTCTGCCTCCCC CGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCAGTGGCCAAGT GGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGCGCATGTAGGC GGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCACGTTCTTGCAC TTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATGTGGGAGAGG ATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCAGGCCTGCCTT GGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGTGGATATGGTC CTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTCCAGAATTAAG CAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTCATCATGCGGA ACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCCCTCTACACAT TTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTTCACTGGGTGC TCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGGCACATTCTAG AAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACCCTCTTCGCCC AGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAGCCCATCATCG AAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTATGGGGGGCTCA GCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGGCCAGGCTGTT CTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAGCTCTCATGTC TCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTC ACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGC TTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGAC ACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTAAAATA CACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAATTTTATATTTA CCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGA GTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCA AAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGA CCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCAGCG TTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATATCTTT AACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCCCAG AAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGGAC TTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATC AACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCACTTGG TCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGCTGGA TAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCGCGAACATCTT CAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACC GGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTG AACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCA GGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGTAATAACTTCGT ATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGGAAGTGGTACG GAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGACGGTTC ACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTATACGTT CTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGGGAGTA AACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTCTCTAT CACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAAAGTCT GCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATCACTGA TAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCACTTGCACTGGG GACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTT AGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAAGATAT TTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCA CAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAG GTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGA GGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCA ACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCAC GCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGC CTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCA GGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGC TCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTT CTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGCGGATCCGCCGCC ACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAA TGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTG AGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTG CCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTC ATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTCTCTCACA TCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAA ACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGC ACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACA GGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCC CCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTT CCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAA GCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGAT GCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGAC CTTGACATGCTCCCCGGGGGATCCGGAGCTACTAACTTCAGCCTGCTGAAGCAGGC TGGAGACGTGGAGGAGAACCCTGGACCTATGACAGAATATAAACCTACTGTCAGAC TGGCAACTCGAGACGACGTCCCTAGGGCCGTGAGAACATTGGCTGCCGCTTTCGCG GATTATCCCGCTACACGCCACACAGTTGATCCTGATAGACATATTGAACGGGTTACA GAATTGCAAGAACTTTTTTTGACCAGGGTAGGATTGGACATCGGTAAAGTTTGGGTC GCCGACGACGGGGCTGCAGTGGCAGTGTGGACGACTCCGGAGAGCGTTGAGGCCG GGGCTGTATTTGCAGAAATTGGTCCCCGAATGGCTGAGCTTAGTGGCTCTCGTCTCG CGGCTCAGCAACAAATGGAAGGACTCCTCGCCCCTCACCGCCCTAAAGAACCAGCT TGGTTCCTCGCTACTGTGGGCGTTAGCCCCGATCATCAGGGAAAGGGCCTTGGTTCC GCGGTGGTATTGCCCGGAGTAGAAGCCGCAGAACGAGCCGGAGTGCCAGCCTTTCT TGAAACGTCAGCGCCAAGGAATTTGCCCTTCTATGAACGGCTCGGATTTACAGTTAC TGCTGACGTTGAAGTACCCGAGGGCCCACGGACGTGGTGCATGACGCGAAAACCCG GCGCTTGAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATC TGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTC CTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATT CTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCA GGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGG GGCTCGACTAGAGCTTGCGGAACCCTTAGTTTAAACGGGCCCTTAATTAATCGATGT AGGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGTCTGC CGCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGG ATGCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTGCAA GCGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCC GAGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTAC AGGGAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCG GCCAGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCAAAA GGAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGGTAA CCGCATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCGCCA TGATACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCT CCTTTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGCGTC CCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTT CCGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGG ATTACGGGGCCCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCT CAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGC CCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGA GCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACAT CCCTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATAT ATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGT TAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGT TATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTC CAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAA GAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGG GTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCG CCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCG AGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTT GAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTT TAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCG GACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCG ACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGA TATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGAT ACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCT GTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAA GTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTC ACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCA ACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGA CTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGG TAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAA AGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGA AACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCG CTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGA AGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTT CGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTA TCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTT CTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCG CTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAA CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGG AACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTA AACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTG TCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACG GGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCAC CGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGT GGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGA GTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATC GTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCT CCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCA CTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGT ACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGG CGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATT GGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAG TTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGG CGACACGGAAATGTTGAATACTCAT [1048] SEQ ID NO: 11 (Helper construct, V1) [1049] GCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCTGCT ATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATAGA ATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGT GGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGAT GGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATCAGG ATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGT CCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCA GCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCA GCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGC GCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACA AGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACC TCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAAC ATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTAT GCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAA CCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTG CATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGGAAC AACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAAC TCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTA TGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGAGTG CGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGA CGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCC CGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATG TGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCCTTCA GCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATCCAAC TCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCT TTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTC GCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACA TTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGT CAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGG TGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCA GTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCTGCCG CGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCA CTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCT GCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGT TGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAAC ACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATC CGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCT TCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCA GAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTG ATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGA CTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGCGT CGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCT TGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAAT CACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCGATC TCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGT TACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAA AGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGG TCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCT TTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCT TCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTT CCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGGGTC GTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGCAC CGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTG TCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTT TTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGG TGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACG CCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGTCGGG AGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACGTCGCG CCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCA TGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCAGACAT GATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAAT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAA TAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGT GTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATG ATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCTCTGCC TCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCAGTGGC CAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGCGCATG TAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCACGTTCT TGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATGTGGG AGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCAGGCCT GCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGTGGATA TGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTCCAGAA TTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTCATCATG CGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCCCTCTAC ACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTTCACTGG GTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGGCACATT CTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACCCTCTTC GCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAGCCCATC ATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTATGGGGGG CTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGGCCAGGC TGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAGCTCTCA TGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTG TTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCAC CAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTC CGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTAA AATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAATTTTATA TTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCG CGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCG GCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCT CGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCA GCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATAT CTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCC CCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAAC GGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGG CATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCAC TTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGC TGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCGCGAAC ATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCA CGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGAT CCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTA ATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGTAATAA CTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGGAAGTG GTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGAC GGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTAT ACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGG GAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTC TCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAA AGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATC ACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCACTTGCA CTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGT ATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAA GATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATC GCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAG AGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTT TCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTT TTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCT CCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTT CTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTA AAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTC AGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGT TTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGCGGATC CGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAAT TACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTG GGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGA TGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAG GCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTC TCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAG TACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAG AACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAG GAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTA TGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCT GCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGC GAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGC CGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTT GACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGTGCCTTCT AGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGT GCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGT AGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTG GGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGG AAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCTATTTCC CATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAAT TAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTATAATGTAT GCTATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACA ACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTA TTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTTTTCGCGT CATGCACCACGTCCGTGGGCCCTCGGGTACTTCAACGTCAGCAGTAACTGTAAATCC GAGCCGTTCATAGAAGGGCAAATTCCTTGGCGCTGACGTTTCAAGAAAGGCTGGCA CTCCGGCTCGTTCTGCGGCTTCTACTCCGGGCAATACCACCGCGGAACCAAGGCCCT TTCCCTGATGATCGGGGCTAACGCCCACAGTAGCGAGGAACCAAGCTGGTTCTTTA GGGCGGTGAGGGGCGAGGAGTCCTTCCATTTGTTGCTGAGCCGCGAGACGAGAGCC ACTAAGCTCAGCCATTCGGGGACCAATTTCTGCAAATACAGCCCCGGCCTCAACGC TCTCCGGAGTCGTCCACACTGCCACTGCAGCCCCGTCGTCGGCGACCCAAACTTTAC CGATGTCCAATCCTACCCTGGTCAAAAAAAGTTCTTGCAATTCTGTAACCCGTTCAA TATGTCTATCAGGATCAACTGTGTGGCGTGTAGCGGGATAATCCGCGAAAGCGGCA GCCAATGTTCTCACGGCCCTAGGGACGTCGTCTCGAGTTGCCAGTCTGACAGTAGGT TTATATTCTGTCATAGGTCCAGGGTTCTCCTCCACGTCTCCAGCCTGCTTCAGCAGG CTGAAGTTAGTAGCTCCGGATCCTTTACCTCCATCACCAGCGCCACCAGTAGAGTAT CTGGCCACAGCCACCTCGTGCTGCTCGACGTAGGTCTCATCGTCGGCCTCCTTGATT CTTTCCAGTCTGTGGTCCACGTTGTAGACGCCGGGCATCTTGAGGTTCGTAGCGGGT TTCTTGGATCTGTATGTGGTCTCAAGGTTGCAGATCAGGTGGCCCCCGCCCACGAGC TTCAGGGCCATGTCACATGCGCCTTCCAGGCCGCCGTCAGCGGGGTACATCGTCTCG GTGGAGGCCTCCCAGCCGAGTGTTTTCTTCTGCATCACAGGGCCGTTGGCTGGGAAG TTCACCCCTCTAACCTTGACGTTGTAGATGAGGCAGCCGTCCTGGAGGCTGGTGTCC TGGGTAGCGGTCAGCACGCCCCCGTCTTCGTATGTGGTGACTCTCTCCCATGTGAAG CCCTCAGGGAAGGACTGCTTAAAGAAGTCGGGGATGCCCGGAGGGTGCTTGATGAA GGTTCTGCTGCCGTACATGAAGCTGGTAGCCAGGATGTCGAAGGCGAAGGGGAGAG GGCCGCCCTCGACGACCTTGATTCTCATGGTCTGGGTGCCCTCGTAGGGCTTGCCTT CGCCCTCGGATGTGCACTTGAAGTGGTGGTTGTTCACGGTGCCCTCCATGTACAGCT TCATGGGCATGTTCTCCTTAATCAGCTCGCTCACGGTGGCGGCGAATTCCGAAAGGC CCGGAGATGAGGAAGAGGAGAACAGCGCGGCAGACGTGCGCTTTTGAAGCGTGCA GAATGCCGGGCCTCCGGAGGACCTTCGGGCGCCCGCCCCGCCCCTGAGCCCGCCCC TGAGCCCGCCCCCGGACCCACCCCTTCCCAGCCTCTGAGCCCAGAAAGCGAAGGAG CAAAGCTGCTATTGGCCGCTGCCCCAAAGGCCTACCCGCTTCCATTGCTCAGCGGTG CTGTCCATCTGCACGAGACTAGTGAGACGTGCTACTTCCATTTGTCACGTCCTGCAC GACGCGAGCTGCGGGGCGGGGGGGAACTTCCTGACTAGGGGAGGAGTAGAAGGTG GCGCGAAGGGGCCACCAAAGAACGGAGCCGGTTGGCGCCTACCGGTGGATGTGGA ATGTGTGCGAGGCCAGAGGCCACTTGTGTAGCGCCAAGTGCCCAGCGGGGCTGCTA AAGCGCATGCTCCAGACTGCCTTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATA ATGTATGCTATACGAAGTTATTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTAT CATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGA AAGGACGAAACACCGGGCACTCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTAT CATGGCGGACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGC GGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCT CC [1050] SEQ ID NO: 12 (Helper construct, v2) [1051] GCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCTGCT ATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATAGA ATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGACAGT GGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGAT GGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATCAGG ATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGT CCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCA GCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAATCA GCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAGGC GCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACA AGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACC TCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTAAAC ATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGCTAT GCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCGTAA CCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTG CATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGGAAC AACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGTAAC TCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCAGTA TGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGAGTG CGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGA CGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCC CGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATG TGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCCTTCA GCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATCCAAC TCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGTGGCT TTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTC GCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACA TTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGT CAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAATATGG TGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCGGCCA GTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCTGCCG CGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCA CTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCT GCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGT TGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGGAAC ACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAGATC CGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCT TCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCA GAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTG ATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAAGA CTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGCGT CGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGCCT TGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAAT CACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCGATC TCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGT TACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCACAA AGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGG TCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCT TTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCT TCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTT CCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGGGTC GTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGCAC CGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTG TCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTT TTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGG TGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACG CCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGTCGGG AGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACGTCGCG CCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCA TGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCAGACAT GATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAAT GCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAA TAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGT GTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGATTATG ATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCTCTGCC TCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCAGTGGC CAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGCGCATG TAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCACGTTCT TGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATGTGGG AGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCAGGCCT GCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGTGGATA TGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTCCAGAA TTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTCATCATG CGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCCCTCTAC ACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTTCACTGG GTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGGCACATT CTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACCCTCTTC GCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAGCCCATC ATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTATGGGGGG CTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGGCCAGGC TGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAGCTCTCA TGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTG TTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCAC CAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCGCGGCTC CGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCATCCTAA AATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAATTTTATA TTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCG CGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCG GCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCCATCGCT CGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGCTAACCA GCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTGAGATAT CTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCC CCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAAC GGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGG CATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGCTGTCAC TTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAATGTTGC TGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCGCGAAC ATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCA CGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGAT CCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGACCGGTA ATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGTAATAA CTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGGAAGTG GTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGAC GGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTAT ACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGG GAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTC TCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAA AGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATC ACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCACTTGCA CTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGT ATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAA GATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATC GCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAG AGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTT TCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTT TTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCT CCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTT CTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTA AAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTC AGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGT TTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGCGGATC CGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAAT TACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTG GGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGA TGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAG GCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTC TCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAG TACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAG AACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAG GAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTA TGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCT GCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGC GAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGC CGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTT GACCTTGACATGCTCCCCGGGGGATCCGGAGCTACTAACTTCAGCCTGCTGAAGCA GGCTGGAGACGTGGAGGAGAACCCTGGACCTATGACAGAATATAAACCTACTGTCA GACTGGCAACTCGAGACGACGTCCCTAGGGCCGTGAGAACATTGGCTGCCGCTTTC GCGGATTATCCCGCTACACGCCACACAGTTGATCCTGATAGACATATTGAACGGGTT ACAGAATTGCAAGAACTTTTTTTGACCAGGGTAGGATTGGACATCGGTAAAGTTTG GGTCGCCGACGACGGGGCTGCAGTGGCAGTGTGGACGACTCCGGAGAGCGTTGAG GCCGGGGCTGTATTTGCAGAAATTGGTCCCCGAATGGCTGAGCTTAGTGGCTCTCGT CTCGCGGCTCAGCAACAAATGGAAGGACTCCTCGCCCCTCACCGCCCTAAAGAACC AGCTTGGTTCCTCGCTACTGTGGGCGTTAGCCCCGATCATCAGGGAAAGGGCCTTGG TTCCGCGGTGGTATTGCCCGGAGTAGAAGCCGCAGAACGAGCCGGAGTGCCAGCCT TTCTTGAAACGTCAGCGCCAAGGAATTTGCCCTTCTATGAACGGCTCGGATTTACAG TTACTGCTGACGTTGAAGTACCCGAGGGCCCACGGACGTGGTGCATGACGCGAAAA CCCGGCGCTTGAGTTTAAACCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGC CATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCAC TGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTC TATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAAT AGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAG CTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGTTTAAACGGGCCCTTAATTAATCG ATGTAGGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGT CTGCCGCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCAC CGGATGCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTG CAAGCGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGG CCCGAGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGC TACAGGGAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGC GCGGCCAGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCA AAAGGAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGG TAACCGCATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCG CCATGATACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGG CTCTCCTTTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGC GTCCCGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGAC TTTCCGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTAC GGATTACGGGGCCCATTGGTATG [1052] SEQ ID NO: 21 (reverse tetracycline-controlled transactivator mutant) [1053] ATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCA ATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTT GAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCT GCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGT CATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTCTCTCAC ATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGA AACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACG CACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAAC AGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCC CCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCT TCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAA AGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGA TGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGAC CTTGACATGCTCCCCGGG [1054] SEQ ID NO: 22 (tetracycline-inducible promoter sequence) [1055] GAGTTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATC AGTGATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAG GAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGA TAGAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTT ACTCCCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTA TAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACAC TTTTGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAA [1056] SEQ ID NO: 23 (STXC0034) [1057] GGTACCCAACTCCATGCTTAACAGTCCCCAGGTACAGCCCACCCTGCGTCGC AACCAGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCA CAGTGCGCAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACATGTAAAAATA ATGTACTAGGAGACACTTTCAATAAAGGCAAATGTTTTTATTTGTACACTCTCGGGT GATTATTTACCCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGC CGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTC CACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAG GCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGC AGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGG AACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAG ATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTG CCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCAT CAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCT TGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAA GACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGC GTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGC CTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCA ATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCG ATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTA GGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCA CAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCC AGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTG CCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGC CCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCAC TTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGG GTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGC ACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGC TGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTC TTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTG GGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGA CGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGGGGAGGCGGCGGCGACGGCGACGG GGACGACACGTCCTCCATGGTTGGTGGACGTCGCGCCGCACCGCGTCCGCGCTCGG GGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAGGCAGA AAAAGATCATGGAGTCAGTCGAGAAGGAGGACAGCCTAACCGCCCCCTTTGAGTTC GCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGC ACCCCCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCG AAGACGACGAGGATCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACGA CGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGGACCAAAGGCATGGCGACTAC CTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTAT CTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCC TTGCCTACGAACGCCACCTGTTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAAC GGCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAG GTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGT GCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACC TGATATCGCCTCGCTCGACGAAGTGCCAAAAATCTTTGAGGGTCTTGGACGCGACG AGAAACGCGCGGCAAACGCTCTGCAACAAGAAAACAGCGAAAATGAAAGTCACTG TGGAGTGCTGGTGGAACTTGAGGGTGACAACGCGCGCCTAGCCGTGCTGAAACGCA GCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTTATGA GCACAGTCATGAGCGAGCTGATCGTGCGCCGTGCACGACCCCTGGAGAGGGATGCA AACTTGCAAGAACAAACCGAGGAGGGCCTACCCGCAGTTGGCGATGAGCAGCTGG CGCGCTGGCTTGAGACGCGCGAGCCTGCCGACTTGGAGGAGCGACGCAAGCTAATG ATGGCCGCAGTGCTTGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGAC CCGGAGATGCAGCGCAAGCTAGAGGAAACGTTGCACTACACCTTTCGCCAGGGCTA CGTGCGCCAGGCCTGCAAAATTTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCT TGGAATTTTGCACGAAAACCGCCTCGGGCAAAACGTGCTTCATTCCACGCTCAAGG GCGAGGCGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTGTGCTACACCT GGCAAACGGCCATGGGCGTGTGGCAGCAATGCCTGGAGGAGCGCAACCTAAAGGA GCTGCAGAAGCTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAGC GCTCCGTGGCCGCGCACCTGGCGGACATTATCTTCCCCGAACGCCTGCTTAAAACCC TGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAAAACTTTAGGAAC TTTATCCTAGAGCGTTCAGGAATTCTGCCCGCCACCTGCTGTGCGCTTCCTAGCGAC TTTGTGCCCATTAAGTACCGTGAATGCCCTCCGCCGCTTTGGGGTCACTGCTACCTT CTGCAGCTAGCCAACTACCTTGCCTACCACTCCGACATCATGGAAGACGTGAGCGG TGACGGCCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCT GGTCTGCAATTCGCAACTGCTTAGCGAAAGTCAAATTATCGGTACCTTTGAGCTGCA GGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGC TGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGCCCACGAGA TTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGCTTACCGCCTGCGTC ATTACCCAGGGCCACATCCTTGGCCAATTGCAAGCCATCAACAAAGCCCGCCAAGA GTTTCTGCTACGAAAGGGACGGGGGGTTTACCTGGACCCCCAGTCCGGCGAGGAGC TCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCCGCGGGCCCTTGCTTCCC AGGATGGCACCCAAAAAGAAGCTGCAGCTGCCGCCGCCGCCACCCACGGACGAGG AGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGGAGGAGATGAT GGAAGACTGGGACAGCCTAGACGAAGCTTCCGAGGCCGAAGAGGTGTCAGACGAA ACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATTGGCAACCGTT CCCAGCATCGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCTGTTCGCCGA CCCAACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCTAAGCAGCCGCC GCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGCTCGTGGCGCGGGCACA AGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGGCAACATCTCCTTCGCCCGCC GCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCG TCATCTCTACAGCCCCTACTGCACCGGCGGCAGCGGCAGCGGCAGCAACAGCAGCG GTCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAAT CCACAGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACC CGTATCGACCCGCGAGCTTAGAAATAGGATTTTTCCCACTCTGTATGCTATATTTCA ACAAAGCAGGGGCCAAGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGCTCC CTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGA AGACGCGGAGGCTCTCTTCAGCAAATACTGCGCGCTGACTCTTAAGGACTAGTTTCG CGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGC GCCAGCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGT GGAGTTACCAGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACC CGAATAAACTACATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGAATCCG CGCCCACCGAAACCGAATTCTCCTCGAACAGGCGGCTATTACCACCACACCTCGTA ATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTC CCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCA GGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCGTTTTAG GGCGGAGTAACTTGCATGTATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGT ATCGTGGGAAAACGGAAGTGAAGATTTGAGGAAGTTGTGGGTTTTTTGGCTTTCGTT TCTGGGCGTAGGTTCGCGTGCGGTTTTCTGGGTGTTTTTTGTGGACTTTAACCGTTAC GTCATTTTTTAGTCCTATATATACTCGCTCTGTACTTGGCCCTTTTTACACTGTGACT GATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTTTTTACTGGTAAGG CTGACTGTTATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGT GCTATTTTGCCTAGGCAGGAGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTA ATTTTGTTATACCTCCTATGGGGGCTGTAATGTTGTCTCTACGCCTGCGGGTATGTAT TCCCCCGGGCTATTTCGGTCGCTTTTTAGCACTGACCGATGTTAACCAACCTGATGT GTTTACCGAGTCTTACATTATGACTCCGGACATGACCGAGGAACTGTCGGTGGTGCT TTTTAATCACGGTGACCAGTTTTTTTACGGTCACGCCGGCATGGCCGTAGTCCGTCT TATGCTTATAAGGGTTGTTTTTCCTGTTGTAAGACAGGCTTCTAATGTTTAAATGTTT TTTTTTTTGTTATTTTATTTTGTGTTTAATGCAGGAACCCGCAGACATGTTTGAGAGA AAAATGGTGTCTTTTTCTGTGGTGGTTCCGGAACTTACCTGCCTTTATCTGCATGAGC ATGACTACGATGTGCTTGCTTTTTTGCGCGAGGCTTTGCCTGATTTTTTGAGCAGCAC CTTGCATTTTATATCGCCGCCCATGCAACAAGCTTACATAGGGGCTACGCTGGTTAG CATAGCTCCGAGTATGCGTGTCATAATCAGTGTGGGTTCTTTTGTCATGGTTCCTGG CGGGGAAGTGGCCGCGCTGGTCCGTGCAGACCTGCACGATTATGTTCAGCTGGCCC TGCGAAGGGACCTACGGGATCGCGGTATTTTTGTTAATGTTCCGCTTTTGAATCTTA TACAGGTCTGTGAGGAACCTGAATTTTTGCAATCATGATTCGCTGCTTGAGGCTGAA GGTGGAGGGCGCTCTGGAGCAGATTTTTACAATGGCCGGACTTAATATTCGGGATTT GCTTAGAGACATATTGATAAGGTGGCGAGATGAAAATTATTTGGGCATGGTTGAAG GTGCTGGAATGTTTATAGAGGAGATTCACCCTGAAGGGTTTAGCCTTTACGTCCACT TGGACGTGAGGGCAGTTTGCCTTTTGGAAGCCATTGTGCAACATCTTACAAATGCCA TTATCTGTTCTTTGGCTGTAGAGTTTGACCACGCCACCGGAGGGGAGCGCGTTCACT TAATAGATCTTCATTTTGAGGTTTTGGATAATCTTTTGGAATAAAAAAAAAAAAACA TGGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGACTCGCAGAACGAATGTGTAG GTTGGCTGGGTGTGGCTTATTCTGCGGTGGTGGATGTTATCAGGGCAGCGGCGCATG AAGGAGTTTACATAGAACCCGAAGCCAGGGGGCGCCTGGATGCTTTGAGAGAGTGG ATATACTACAACTACTACACAGAGCGAGCTAAGCGACGAGACCGGAGACGCAGAT CTGTTTGTCACGCCCGCACCTGGTTTTGCTTCAGGAAATATGACTACGTCCGGCGTT CCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTAC AGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGA TCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCG AGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGA TATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCC TGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCT GGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGC AGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGT TTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATG TCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGC CACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGG ATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATC AGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAAT CATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGC AGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATG ACTCTACCCCCATGTAGGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAG TTGGACAGCAGCCTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTG CCCGGGGAGTTTATTAATATCACTGATGAGCGTTTGGCTCGACAGGAAACCGTGTG GAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATGCTTTTTAAGGCCAG CCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGAATA CTAGGGTTCTGTGAGTTTGATTAAGGTACGGTGATCAATATAAGCTATGTGGTGGTG GGGCTATACTACTGAATGAAAAATGACTTGAAATTTTCTGCAATTGAAAAATAAAC ACGTTGAAACATAACATGCAACAGGTTCACGATTCTTTATTCCTGGGCAATGTAGGA GAAGGTGTAAGAGTTGGTAGCAAAAGTTTCAGTGGTGTATTTTCCACTTTCCCAGGA CCATGTAAAAGACATAGAGTAAGTGCTTACCTCGCTAGTTTCTGTGGATTCACTAGA ATCGATGTCGACGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAAGGGTATCAT GGCGGACGAACCGGTACCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGT TACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCT TTTTCATATGATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCT TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT GCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAA CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAA ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT CCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAA ATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAG AGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGT GCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGC GCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGC GCTTAATGCGCCGCTACAGGGCGCGATGGATCC [1058] SEQ ID NO: 24 (STXC0036) [1059] GGTACCCAACTCCATGCTTAACAGTCCCCAGGTACAGCCCACCCTGCGTCGC AACCAGGAACAGCTCTACAGCTTCCTGGAGCGCCACTCGCCCTACTTCCGCAGCCA CAGTGCGCAGATTAGGAGCGCCACTTCTTTTTGTCACTTGAAAAACATGTAAAAATA ATGTACTAGGAGACACTTTCAATAAAGGCAAATGTTTTTATTTGTACACTCTCGGGT GATTATTTACCCCCCACCCTTGCCGTCTGCGCCGTTTAAAAATCAAAGGGGTTCTGC CGCGCATCGCTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTC CACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAG GCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTCGC AGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACTGG AACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATCAG ATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTG CCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCAT CAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCT TGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGCAA GACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTTGC GTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTGGC CTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCA ATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCG ATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGTA GGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTCA CAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGCC AGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTG CCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGC CCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCAC TTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTGG GTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTAGC ACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCGC TGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTC TTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTG GGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGA CGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGGGGAGGCGGCGGCGACGGCGACGG GGACGACACGTCCTCCATGGTTGGTGGACGTCGCGCCGCACCGCGTCCGCGCTCGG GGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATTTCCTTCTCCTATAGGCAGA AAAAGATCATGGAGTCAGTCGAGAAGGAGGACAGCCTAACCGCCCCCTTTGAGTTC GCCACCACCGCCTCCACCGATGCCGCCAACGCGCCTACCACCTTCCCCGTCGAGGC ACCCCCGCTTGAGGAGGAGGAAGTGATTATCGAGCAGGACCCAGGTTTTGTAAGCG AAGACGACGAGGATCGCTCAGTACCAACAGAGGATAAAAAGCAAGACCAGGACGA CGCAGAGGCAAACGAGGAACAAGTCGGGCGGGGGGACCAAAGGCATGGCGACTAC CTAGATGTGGGAGACGACGTGCTGTTGAAGCATCTGCAGCGCCAGTGCGCCATTAT CTGCGACGCGTTGCAAGAGCGCAGCGATGTGCCCCTCGCCATAGCGGATGTCAGCC TTGCCTACGAACGCCACCTGTTCTCACCGCGCGTACCCCCCAAACGCCAAGAAAAC GGCACATGCGAGCCCAACCCGCGCCTCAACTTCTACCCCGTATTTGCCGTGCCAGAG GTGCTTGCCACCTATCACATCTTTTTCCAAAACTGCAAGATACCCCTATCCTGCCGT GCCAACCGCAGCCGAGCGGACAAGCAGCTGGCCTTGCGGCAGGGCGCTGTCATACC TGATATCGCCTCGCTCGACGAAGTGCCAAAAATCTTTGAGGGTCTTGGACGCGACG AGAAACGCGCGGCAAACGCTCTGCAACAAGAAAACAGCGAAAATGAAAGTCACTG TGGAGTGCTGGTGGAACTTGAGGGTGACAACGCGCGCCTAGCCGTGCTGAAACGCA GCATCGAGGTCACCCACTTTGCCTACCCGGCACTTAACCTACCCCCCAAGGTTATGA GCACAGTCATGAGCGAGCTGATCGTGCGCCGTGCACGACCCCTGGAGAGGGATGCA AACTTGCAAGAACAAACCGAGGAGGGCCTACCCGCAGTTGGCGATGAGCAGCTGG CGCGCTGGCTTGAGACGCGCGAGCCTGCCGACTTGGAGGAGCGACGCAAGCTAATG ATGGCCGCAGTGCTTGTTACCGTGGAGCTTGAGTGCATGCAGCGGTTCTTTGCTGAC CCGGAGATGCAGCGCAAGCTAGAGGAAACGTTGCACTACACCTTTCGCCAGGGCTA CGTGCGCCAGGCCTGCAAAATTTCCAACGTGGAGCTCTGCAACCTGGTCTCCTACCT TGGAATTTTGCACGAAAACCGCCTCGGGCAAAACGTGCTTCATTCCACGCTCAAGG GCGAGGCGCGCCGCGACTACGTCCGCGACTGCGTTTACTTATTTCTGTGCTACACCT GGCAAACGGCCATGGGCGTGTGGCAGCAATGCCTGGAGGAGCGCAACCTAAAGGA GCTGCAGAAGCTGCTAAAGCAAAACTTGAAGGACCTATGGACGGCCTTCAACGAGC GCTCCGTGGCCGCGCACCTGGCGGACATTATCTTCCCCGAACGCCTGCTTAAAACCC TGCAACAGGGTCTGCCAGACTTCACCAGTCAAAGCATGTTGCAAAACTTTAGGAAC TTTATCCTAGAGCGTTCAGGAATTCTGCCCGCCACCTGCTGTGCGCTTCCTAGCGAC TTTGTGCCCATTAAGTACCGTGAATGCCCTCCGCCGCTTTGGGGTCACTGCTACCTT CTGCAGCTAGCCAACTACCTTGCCTACCACTCCGACATCATGGAAGACGTGAGCGG TGACGGCCTACTGGAGTGTCACTGTCGCTGCAACCTATGCACCCCGCACCGCTCCCT GGTCTGCAATTCGCAACTGCTTAGCGAAAGTCAAATTATCGGTACCTTTGAGCTGCA GGGTCCCTCGCCTGACGAAAAGTCCGCGGCTCCGGGGTTGAAACTCACTCCGGGGC TGTGGACGTCGGCTTACCTTCGCAAATTTGTACCTGAGGACTACCACGCCCACGAGA TTAGGTTCTACGAAGACCAATCCCGCCCGCCAAATGCGGAGCTTACCGCCTGCGTC ATTACCCAGGGCCACATCCTTGGCCAATTGCAAGCCATCAACAAAGCCCGCCAAGA GTTTCTGCTACGAAAGGGACGGGGGGTTTACCTGGACCCCCAGTCCGGCGAGGAGC TCAACCCAATCCCCCCGCCGCCGCAGCCCTATCAGCAGCCGCGGGCCCTTGCTTCCC AGGATGGCACCCAAAAAGAAGCTGCAGCTGCCGCCGCCGCCACCCACGGACGAGG AGGAATACTGGGACAGTCAGGCAGAGGAGGTTTTGGACGAGGAGGAGGAGATGAT GGAAGACTGGGACAGCCTAGACGAAGCTTCCGAGGCCGAAGAGGTGTCAGACGAA ACACCGTCACCCTCGGTCGCATTCCCCTCGCCGGCGCCCCAGAAATTGGCAACCGTT CCCAGCATCGCTACAACCTCCGCTCCTCAGGCGCCGCCGGCACTGCCTGTTCGCCGA CCCAACCGTAGATGGGACACCACTGGAACCAGGGCCGGTAAGTCTAAGCAGCCGCC GCCGTTAGCCCAAGAGCAACAACAGCGCCAAGGCTACCGCTCGTGGCGCGGGCACA AGAACGCCATAGTTGCTTGCTTGCAAGACTGTGGGGGCAACATCTCCTTCGCCCGCC GCTTTCTTCTCTACCATCACGGCGTGGCCTTCCCCCGTAACATCCTGCATTACTACCG TCATCTCTACAGCCCCTACTGCACCGGCGGCAGCGGCAGCGGCAGCAACAGCAGCG GTCACACAGAAGCAAAGGCGACCGGATAGCAAGACTCTGACAAAGCCCAAGAAAT CCACAGCGGCGGCAGCAGCAGGAGGAGGAGCGCTGCGTCTGGCGCCCAACGAACC CGTATCGACCCGCGAGCTTAGAAATAGGATTTTTCCCACTCTGTATGCTATATTTCA ACAAAGCAGGGGCCAAGAACAAGAGCTGAAAATAAAAAACAGGTCTCTGCGCTCC CTCACCCGCAGCTGCCTGTATCACAAAAGCGAAGATCAGCTTCGGCGCACGCTGGA AGACGCGGAGGCTCTCTTCAGCAAATACTGCGCGCTGACTCTTAAGGACTAGTTTCG CGCCCTTTCTCAAATTTAAGCGCGAAAACTACGTCATCTCCAGCGGCCACACCCGGC GCCAGCACCTGTCGTCAGCGCCATTATGAGCAAGGAAATTCCCACGCCCTACATGT GGAGTTACCAGCCACAAATGGGACTTGCGGCTGGAGCTGCCCAAGACTACTCAACC CGAATAAACTACATGAGCGCGGGACCCCACATGATATCCCGGGTCAACGGAATCCG CGCCCACCGAAACCGAATTCTCCTCGAACAGGCGGCTATTACCACCACACCTCGTA ATAACCTTAATCCCCGTAGTTGGCCCGCTGCCCTGGTGTACCAGGAAAGTCCCGCTC CCACCACTGTGGTACTTCCCAGAGACGCCCAGGCCGAAGTTCAGATGACTAACTCA GGGGCGCAGCTTGCGGGCGGCTTTCGTCACAGGGTGCGGTCGCCCGGGCGTTTTAG GGCGGAGTAACTTGCATGTATTGGGAATTGTAGTTTTTTTAAAATGGGAAGTGACGT ATCGTGGGAAAACGGAAGTGAAGATTTGAGGAAGTTGTGGGTTTTTTGGCTTTCGTT TCTGGGCGTAGGTTCGCGTGCGGTTTTCTGGGTGTTTTTTGTGGACTTTAACCGTTAC GTCATTTTTTAGTCCTATATATACTCGCTCTGTACTTGGCCCTTTTTACACTGTGACT GATTGAGCTGGTGCCGTGTCGAGTGGTGTTTTTTAATAGGTTTTTTTACTGGTAAGG CTGACTGTTATGGCTGCCGCTGTGGAAGCGCTGTATGTTGTTCTGGAGCGGGAGGGT GCTATTTTGCCTAGGCAGGAGGGTTTTTCAGGTGTTTATGTGTTTTTCTCTCCTATTA ATTTTGTTATACCTCCTATGGGGGCTGTAATGTTGTCTCTACGCCTGCGGGTATGTAT TCCCCCGGGCTATTTCGGTCGCTTTTTAGCACTGACCGATGTTAACCAACCTGATGT GTTTACCGAGTCTTACATTATGACTCCGGACATGACCGAGGAACTGTCGGTGGTGCT TTTTAATCACGGTGACCAGTTTTTTTACGGTCACGCCGGCATGGCCGTAGTCCGTCT TATGCTTATAAGGGTTGTTTTTCCTGTTGTAAGACAGGCTTCTAATGTTTAAATGTTT TTTTTTTTGTTATTTTATTTTGTGTTTAATGCAGGAACCCGCAGACATGTTTGAGAGA AAAATGGTGTCTTTTTCTGTGGTGGTTCCGGAACTTACCTGCCTTTATCTGCATGAGC ATGACTACGATGTGCTTGCTTTTTTGCGCGAGGCTTTGCCTGATTTTTTGAGCAGCAC CTTGCATTTTATATCGCCGCCCATGCAACAAGCTTACATAGGGGCTACGCTGGTTAG CATAGCTCCGAGTATGCGTGTCATAATCAGTGTGGGTTCTTTTGTCATGGTTCCTGG CGGGGAAGTGGCCGCGCTGGTCCGTGCAGACCTGCACGATTATGTTCAGCTGGCCC TGCGAAGGGACCTACGGGATCGCGGTATTTTTGTTAATGTTCCGCTTTTGAATCTTA TACAGGTCTGTGAGGAACCTGAATTTTTGCAATCATGATTCGCTGCTTGAGGCTGAA GGTGGAGGGCGCTCTGGAGCAGATTTTTACAATGGCCGGACTTAATATTCGGGATTT GCTTAGAGACATATTGATAAGGTGGCGAGATGAAAATTATTTGGGCATGGTTGAAG GTGCTGGAATGTTTATAGAGGAGATTCACCCTGAAGGGTTTAGCCTTTACGTCCACT TGGACGTGAGGGCAGTTTGCCTTTTGGAAGCCATTGTGCAACATCTTACAAATGCCA TTATCTGTTCTTTGGCTGTAGAGTTTGACCACGCCACCGGAGGGGAGCGCGTTCACT TAATAGATCTTCATTTTGAGGTTTTGGATAATCTTTTGGAATAAAAAAAAAAAAACA TGGTTCTTCCAGCTCTTCCCGCTCCTCCCGTGTGTGACTCGCAGAACGAATGTGTAG GTTGGCTGGGTGTGGCTTATTCTGCGGTGGTGGATGTTATCAGGGCAGCGGCGCATG AAGGAGTTTACATAGAACCCGAAGCCAGGGGGCGCCTGGATGCTTTGAGAGAGTGG ATATACTACAACTACTACACAGAGCGAGCTAAGCGACGAGACCGGAGACGCAGAT CTGTTTGTCACGCCCGCACCTGGTTTTGCTTCAGGAAATATGACTACGTCCGGCGTT CCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTAC AGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGA TCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCG AGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGA TATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCC TGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCT GGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGC AGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGT TTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATG TCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGC CACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGG ATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATC AGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAAT CATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGC AGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATG ACTCTACCCCCATGTAGGCGTGGACTTCCCCTTCGCCGCCCGTTGAGCAACCGCAAG TTGGACAGCAGCCTGTGGCTCAGCAGCTGGACAGCGACATGAACTTAAGCGAGCTG CCCGGGGAGTTTATTAATATCACTGATGAGCGTTTGGCTCGACAGGAAACCGTGTG GAATATAACACCTAAGAATATGTCTGTTACCCATGATATGATGCTTTTTAAGGCCAG CCGGGGAGAAAGGACTGTGTACTCTGTGTGTTGGGAGGGAGGTGGCAGGTTGAATA CTAGGGTTCTGTGAGTTTGATTAAGGTACGGTGATCAATATAAGCTATGTGGTGGTG GGGCTATACTACTGAATGAAAAATGACTTGAAATTTTCTGCAATTGAAAAATAAAC ACGTTGAAACATAACATGCAACAGGTTCACGATTCTTTATTCCTGGGCAATGTAGGA GAAGGTGTAAGAGTTGGTAGCAAAAGTTTCAGTGGTGTATTTTCCACTTTCCCAGGA CCATGTAAAAGACATAGAGTAAGTGCTTACCTCGCTAGTTTCTGTGGATTCACTAGA ATCGATGTCGACGGGCACTCTTCCGTGGTCTGGTGGATAAATTCGCAAGGGTATCAT GGCGGACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGT TACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCT TTTTCATATGATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGG GATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTA AAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACA AAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCA GGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACC GGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGC TGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAA CCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAAC CCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAG AGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCT ACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAA AAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTT TTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTT GATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTT GGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAA GTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCT TAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT GCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAA CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAA ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT CCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAA ATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTT ATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAG AGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCA GGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGT GCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGG GGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGC GCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGC GCTTAATGCGCCGCTACAGGGCGCGATGGATCC [1060] SEQ ID NO: 25 (STXC0030) [1061] CCGCGGCCGCCAACTTTGTATAGAAAAGTTGTAGTTATTAATAGTAATCAAT TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGT AAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGC CCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGG GATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGT AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGAT CCAAGTTTGTACAAAAAAGCAGGCTGCCACCATGGCCAGTCGGGAAGAGGAGCAG CGCGAAACCACCCCCGAGCGCGGACGCGGTGCGGCGCGACGTCCACCAACCATGG AGGACGTGTCGTCCCCGTCGCCGTCGCCGCCGCCTCCCCGCGCGCCCCCAAAAAAG CGGCTGAGGCGGCGTCTCGAGTCCGAGGACGAAGAAGACTCGTCACAAGATGCGCT GGTGCCGCGCACACCCAGCCCGCGGCCATCGACCTCGACGGCGGATTTGGCCATTG CGTCCAAAAAGAAAAAGAAGCGCCCCTCTCCCAAGCCCGAGCGCCCGCCATCCCCA GAGGTGATCGTGGACAGCGAGGAAGAAAGAGAAGATGTGGCGCTACAAATGGTGG GTTTCAGCAACCCACCGGTGCTAATCAAGCACGGCAAGGGAGGTAAGCGCACGGTG CGGCGGCTGAATGAAGACGACCCAGTGGCGCGGGGTATGCGGACGCAAGAGGAAA AGGAAGAGTCCAGTGAAGCGGAAAGTGAAAGCACGGTGATAAACCCGCTGAGCCT GCCGATCGTGTCTGCGTGGGAGAAGGGCATGGAGGCTGCGCGCGCGTTGATGGACA AGTACCACGTGGATAACGATCTAAAGGCAAACTTCAAGCTACTGCCTGACCAAGTG GAAGCTCTGGCGGCCGTATGCAAGACCTGGCTAAACGAGGAGCACCGCGGGTTGCA GCTGACCTTCACCAGCAACAAGACCTTTGTGACGATGATGGGGCGATTCCTGCAGG CGTACCTGCAGTCGTTTGCAGAGGTAACCTACAAGCACCACGAGCCCACGGGCTGC GCGTTGTGGCTGCACCGCTGCGCTGAGATCGAAGGCGAGCTTAAGTGTCTACACGG GAGCATTATGATAAATAAGGAGCACGTGATTGAAATGGATGTGACGAGCGAAAAC GGGCAGCGCGCGCTGAAGGAGCAGTCTAGCAAGGCCAAGATCGTGAAGAACCGGT GGGGCCGAAATGTGGTGCAGATCTCCAACACCGACGCAAGGTGCTGCGTGCATGAC GCGGCCTGTCCGGCCAATCAGTTTTCCGGCAAGTCTTGCGGCATGTTCTTCTCTGAA GGCGCAAAGGCTCAGGTGGCTTTTAAGCAGATCAAGGCTTTCATGCAGGCGCTGTA TCCTAACGCCCAGACCGGGCACGGTCACCTTCTGATGCCACTACGGTGCGAGTGCA ACTCAAAGCCTGGGCATGCACCCTTTTTGGGAAGGCAGCTACCAAAGTTGACTCCG TTCGCCCTGAGCAACGCGGAGGACCTGGACGCGGATCTGATCTCCGACAAGAGCGT GCTGGCCAGCGTGCACCACCCGGCGCTGATAGTGTTCCAGTGCTGCAACCCTGTGTA TCGCAACTCGCGCGCGCAGGGCGGAGGCCCCAACTGCGACTTCAAGATATCGGCGC CCGACCTGCTAAACGCGTTGGTGATGGTGCGCAGCCTGTGGAGTGAAAACTTCACC GAGCTGCCGCGGATGGTTGTGCCTGAGTTTAAGTGGAGCACTAAACACCAGTATCG CAACGTGTCCCTGCCAGTGGCGCATAGCGATGCGCGGCAGAACCCCTTTGATTTTTA AACCCAGCTTTCTTGTACAAAGTGGGCCCCTCTCCCTCCCCCCCCCCTAACGTTACT GGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACC ATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACG AGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTC GTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGAC CCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGC CACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGT TGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCT GAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCA CATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACG GGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGACTAC GTCCGGCGTTCCATTTGGCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCG CACTCCGTACAGTAGGGATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCAT ACTGGAGGATCATCCGCTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGA GTTACGTGCGAGGTCTTCCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTG TTCCCTGGGATATGGTTCTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATG CACGTGTGCCTGTGTTGTGCCAACATTGATATCATGACGAGCATGATGATCCATGGT TACGAGTCCTGGGCTCTCCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATA GCCGGCGGGCAGGTTTTGGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTT TAATCAGAGGTTTATATGGTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGG TAATGTTTATGTCCAGCGTGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGT GGTATGATGGCCACGTGGGTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCT TGCACTGTGGGATTTTGAACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATT TAAGTGAGATCAGGGTGCGCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGG GCGGTGCGAATCATCGCTGAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGA GCGGCGGCGGCAGCAGTTTATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGAT GCACGATTATGACTCTACCCCCATGTAGCAACTTTATTATACATAGTTGATGGCCGG CCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAG AATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGT AACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTT TCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAAT GTGGTAGCGGCCGCGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGT CGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCA CAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGC CAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGA CGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTA TAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACC CTGCCGCTTACCGGATACCTGTCCGCCTTTCTCTCTTCGGGAAGCGTGGCGCTTTCTC ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCT GTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAAC AGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCC TAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGT TACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAA GAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGT TAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAAT TAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGT TACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCC ATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCT GGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATC AGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTAT CCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAG TTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGT CGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGAT CCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAA GTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTA CTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCAT TCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGAT AATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTC GGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCA CTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGC AAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATG TTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGT CTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCC GCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGAC ATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCGGCGCGCCTCGATGTA GGATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGTCTGCC GCTGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGGA TGCATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTGCAAG CGGGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCCG AGACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTACA GGGAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCGG CCAGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCAAAAG GAGCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGGTAAC CGCATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCGCCAT GATACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCTC CTTTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGCGTCC CGACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTTC CGCGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGGA TTACGGGCGCG [1062] SEQ ID NO: 26 (STXC0031) [1063] CCGCGGCCGCCAACTTTGTATAGAAAAGTTGTAGTTATTAATAGTAATCAAT TACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGT AAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGT ATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGC CCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGA CCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCAT GGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGG GATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATC AACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGT AGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGAT CCAAGTTTGTACAAAAAAGCAGGCTGCCACCATGACTACGTCCGGCGTTCCATTTG GCATGACACTACGACCAACACGATCTCGGTTGTCTCGGCGCACTCCGTACAGTAGG GATCGCCTACCTCCTTTTGAGACAGAGACCCGCGCTACCATACTGGAGGATCATCCG CTGCTGCCCGAATGTAACACTTTGACAATGCACAACGTGAGTTACGTGCGAGGTCTT CCCTGCAGTGTGGGATTTACGCTGATTCAGGAATGGGTTGTTCCCTGGGATATGGTT CTGACGCGGGAGGAGCTTGTAATCCTGAGGAAGTGTATGCACGTGTGCCTGTGTTG TGCCAACATTGATATCATGACGAGCATGATGATCCATGGTTACGAGTCCTGGGCTCT CCACTGTCATTGTTCCAGTCCCGGTTCCCTGCAGTGCATAGCCGGCGGGCAGGTTTT GGCCAGCTGGTTTAGGATGGTGGTGGATGGCGCCATGTTTAATCAGAGGTTTATATG GTACCGGGAGGTGGTGAATTACAACATGCCAAAAGAGGTAATGTTTATGTCCAGCG TGTTTATGAGGGGTCGCCACTTAATCTACCTGCGCTTGTGGTATGATGGCCACGTGG GTTCTGTGGTCCCCGCCATGAGCTTTGGATACAGCGCCTTGCACTGTGGGATTTTGA ACAATATTGTGGTGCTGTGCTGCAGTTACTGTGCTGATTTAAGTGAGATCAGGGTGC GCTGCTGTGCCCGGAGGACAAGGCGTCTCATGCTGCGGGCGGTGCGAATCATCGCT GAGGAGACCACTGCCATGTTGTATTCCTGCAGGACGGAGCGGCGGCGGCAGCAGTT TATTCGCGCGCTGCTGCAGCACCACCGCCCTATCCTGATGCACGATTATGACTCTAC CCCCATGTAGACCCAGCTTTCTTGTACAAAGTGGGCCCCTCTCCCTCCCCCCCCCCT AACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTA TTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTC TTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTG TTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTC TGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCG GCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCAC GTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAAC AAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCC TCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCC GAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACC ATGGCCAGTCGGGAAGAGGAGCAGCGCGAAACCACCCCCGAGCGCGGACGCGGTG CGGCGCGACGTCCACCAACCATGGAGGACGTGTCGTCCCCGTCGCCGTCGCCGCCG CCTCCCCGCGCGCCCCCAAAAAAGCGGCTGAGGCGGCGTCTCGAGTCCGAGGACGA AGAAGACTCGTCACAAGATGCGCTGGTGCCGCGCACACCCAGCCCGCGGCCATCGA CCTCGACGGCGGATTTGGCCATTGCGTCCAAAAAGAAAAAGAAGCGCCCCTCTCCC AAGCCCGAGCGCCCGCCATCCCCAGAGGTGATCGTGGACAGCGAGGAAGAAAGAG AAGATGTGGCGCTACAAATGGTGGGTTTCAGCAACCCACCGGTGCTAATCAAGCAC GGCAAGGGAGGTAAGCGCACGGTGCGGCGGCTGAATGAAGACGACCCAGTGGCGC GGGGTATGCGGACGCAAGAGGAAAAGGAAGAGTCCAGTGAAGCGGAAAGTGAAA GCACGGTGATAAACCCGCTGAGCCTGCCGATCGTGTCTGCGTGGGAGAAGGGCATG GAGGCTGCGCGCGCGTTGATGGACAAGTACCACGTGGATAACGATCTAAAGGCAAA CTTCAAGCTACTGCCTGACCAAGTGGAAGCTCTGGCGGCCGTATGCAAGACCTGGC TAAACGAGGAGCACCGCGGGTTGCAGCTGACCTTCACCAGCAACAAGACCTTTGTG ACGATGATGGGGCGATTCCTGCAGGCGTACCTGCAGTCGTTTGCAGAGGTAACCTA CAAGCACCACGAGCCCACGGGCTGCGCGTTGTGGCTGCACCGCTGCGCTGAGATCG AAGGCGAGCTTAAGTGTCTACACGGGAGCATTATGATAAATAAGGAGCACGTGATT GAAATGGATGTGACGAGCGAAAACGGGCAGCGCGCGCTGAAGGAGCAGTCTAGCA AGGCCAAGATCGTGAAGAACCGGTGGGGCCGAAATGTGGTGCAGATCTCCAACACC GACGCAAGGTGCTGCGTGCATGACGCGGCCTGTCCGGCCAATCAGTTTTCCGGCAA GTCTTGCGGCATGTTCTTCTCTGAAGGCGCAAAGGCTCAGGTGGCTTTTAAGCAGAT CAAGGCTTTCATGCAGGCGCTGTATCCTAACGCCCAGACCGGGCACGGTCACCTTCT GATGCCACTACGGTGCGAGTGCAACTCAAAGCCTGGGCATGCACCCTTTTTGGGAA GGCAGCTACCAAAGTTGACTCCGTTCGCCCTGAGCAACGCGGAGGACCTGGACGCG GATCTGATCTCCGACAAGAGCGTGCTGGCCAGCGTGCACCACCCGGCGCTGATAGT GTTCCAGTGCTGCAACCCTGTGTATCGCAACTCGCGCGCGCAGGGCGGAGGCCCCA ACTGCGACTTCAAGATATCGGCGCCCGACCTGCTAAACGCGTTGGTGATGGTGCGC AGCCTGTGGAGTGAAAACTTCACCGAGCTGCCGCGGATGGTTGTGCCTGAGTTTAA GTGGAGCACTAAACACCAGTATCGCAACGTGTCCCTGCCAGTGGCGCATAGCGATG CGCGGCAGAACCCCTTTGATTTTTAACAACTTTATTATACATAGTTGATGGCCGGCC GCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAA TGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAA CCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTC AGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGT GGTAGCGGCCGCGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACA GAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCA GGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATA AAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCT GCCGCTTACCGGATACCTGTCCGCCTTTCTCTCTTCGGGAAGCGTGGCGCTTTCTCAT AGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGT GTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAG GATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTA ACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTA CCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGC GGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGA AGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTA AGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTA AAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTA CCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGG CCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAG CAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCC GCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCG TTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCC CCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGT AAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACT GTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTC TGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAA TACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGG GGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTC GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAA AAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTG AATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCG CACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATT AACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCGGCGCGCCTCGATGTAGG ATGTTGCCCCTCCTGACGCGGTAGGAGAAGGGGAGGGTGCCCTGCATGTCTGCCGC TGCTCTTGCTCTTGCCGCTGCTGAGGAGGGGGGCGCATCTGCCGCAGCACCGGATG CATCTGGGAAAAGCAAAAAAGGGGCTCGTCCCTGTTTCCGGAGGAATTTGCAAGCG GGGTCTTGCATGACGGGGAGGCAAACCCCCGTTCGCCGCAGTCCGGCCGGCCCGAG ACTCGAACCGGGGGTCCTGCGACTCAACCCTTGGAAAATAACCCTCCGGCTACAGG GAGCGAGCCACTTAATGCTTTCGCTTTCCAGCCTAACCGCTTACGCCGCGCGCGGCC AGTGGCCAAAAAAGCTAGCGCAGCAGCCGCCGCGCCTGGAAGGAAGCCAAAAGGA GCGCTCCCCCGTTGTCTGACGTCGCACACCTGGGTTCGACACGCGGGCGGTAACCG CATGGATCACGGCGGACGGCCGGATCCGGGGTTCGAACCCCGGTCGTCCGCCATGA TACCCTTGCGAATTTATCCACCAGACCACGGAAGAGTGCCCGCTTACAGGCTCTCCT TTTGCACGGTCTAGAGCGTCAACGACTGCGCACGCCTCACCGGCCAGAGCGTCCCG ACCATGGAGCACTTTTTGCCGCTGCGCAACATCTGGAACCGCGTCCGCGACTTTCCG CGCGCCTCCACCACCGCCGCCGGCATCACCTGGATGTCCAGGTACATCTACGGATTA CGGGCGCG [1064] SEQ ID NO: 27 (STXC00124) [1065] TGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCA GCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGT CCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCC CCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGG GCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAG TTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTA AAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCA AAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCG ACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCA ATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTA GCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTG GCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAG CGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAA CGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGA ACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTT CCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGG AACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGT CAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTG AGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTG CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA GGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGT TAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAA TCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCG AAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTT TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAG CGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAAC CACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC CAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTT GAACCCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGA GCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAG ATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTT TTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTA TATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTAT TTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGA GGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGG GGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCC CGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGC TTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGG GGAAAAGGCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCT GCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATA GAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGAC AGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACA GATGGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATC AGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTC CGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCC GCAGCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAA TCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAA GGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACC ACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATT ACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTA AACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGC TATGCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCG TAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACAC GTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGG AACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGT AACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCA GTATGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGA GTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCC GGACGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGT CCCCGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGC ATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCC TTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATC CAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGT GGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAG GGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCAC GACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGC TCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAAT ATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCG GCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCT GCCGCGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTG CTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCAC AGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTC GCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACT GGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATC AGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAG CTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGG CATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAG CCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGC AAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTT GCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTG GCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTT CAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTC GATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGT AGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTC ACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGC CAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTT GCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATG CCCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCA CTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTG GGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTA GCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTC GCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCT TCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGC TGGGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGA GACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGT CGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACG TCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACT GGCCATGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAA AAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGC TGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGA TTATGATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCT CTGCCTCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCA GTGGCCAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGC GCATGTAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCAC GTTCTTGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATG TGGGAGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCA GGCCTGCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGT GGATATGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTC CAGAATTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTC ATCATGCGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCC CTCTACACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTT CACTGGGTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGG CACATTCTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACC CTCTTCGCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAG CCCATCATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTAT GGGGGGCTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGG CCAGGCTGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAG CTCTCATGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTG CCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCC AGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCG CGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCA TCCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAA TTTTATATTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCC AGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCT GACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCC ATCGCTCGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGC TAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTG AGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAG CAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGA GTGAACGGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTT CACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGC TGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAA TGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCG CGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCC GCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTG GCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGAC CGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGT AATAACTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGG AAGTGGTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGAT CTGACGGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAG CTTATACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTG ATAGGGAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCAT ACGTTCTCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGG GAGTAAAGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTC TCTATCACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCA CTTGCACTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGA GAGCGTATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAA AATAAAGATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGC GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGT GCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG CCTTTTTCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCA TCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTC GCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAA GTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTA GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGT TTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC GGATCCGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCT GGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAA AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTG CTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTG GAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGC TCTTCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGA AACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCC CTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTA TTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCC GAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTA AAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCT CCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGA CGATTTTGACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTG AGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTAT AATGTATGCTATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACA AACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTAT TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTT TTCGCGTCATGCACCACGTCCGTGGTAGAACTAGTATTATGCCCAGTACATGACCTT ATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT GATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAAC GGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGG CGTGTACGGTGGGAGGTTTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGC CTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGAGAATTCGCCGCCACCAT GACAGAATATAAACCTACTGTCAGACTGGCAACTCGAGACGACGTCCCTAGGGCCG TGAGAACATTGGCTGCCGCTTTCGCGGATTATCCCGCTACACGCCACACAGTTGATC CTGATAGACATATTGAACGGGTTACAGAATTGCAAGAACTTTTTTTGACCAGGGTA GGATTGGACATCGGTAAAGTTTGGGTCGCCGACGACGGGGCTGCAGTGGCAGTGTG GACGACTCCGGAGAGCGTTGAGGCCGGGGCTGTATTTGCAGAAATTGGTCCCCGAA TGGCTGAGCTTAGTGGCTCTCGTCTCGCGGCTCAGCAACAAATGGAAGGACTCCTC GCCCCTCACCGCCCTAAAGAACCAGCTTGGTTCCTCGCTACTGTGGGCGTTAGCCCC GATCATCAGGGAAAGGGCCTTGGTTCCGCGGTGGTATTGCCCGGAGTAGAAGCCGC AGAACGAGCCGGAGTGCCAGCCTTTCTTGAAACGTCAGCGCCAAGGAATTTGCCCT TCTATGAACGGCTCGGATTTACAGTTACTGCTGACGTTGAAGTACCCGAGGGCCCAC GGACGTGGTGCATGACGCGAAAACCCGGCGCTTGAACCGGTCGCTGATCAGCCTCG ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGA CCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGC ATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAG GGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG CTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGG TTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATAATGTATGCTATACGAAGTTAT TTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGA AAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGCAC TCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGATT CGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGCGTGTCGA ACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTGGGCCCAT [1066] SEQ ID NO: 28 (STXC0125) [1067] TGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCA GCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGT CCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCC CCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGG GCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAG TTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTA AAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCA AAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCG ACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCA ATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTA GCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTG GCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAG CGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAA CGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGA ACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTT CCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGG AACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGT CAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTG AGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTG CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA GGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGT TAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAA TCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCG AAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTT TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAG CGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAAC CACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC CAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTT GAACCCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGA GCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAG ATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTT TTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTA TATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTAT TTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGA GGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGG GGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCC CGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGC TTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGG GGAAAAGGCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCT GCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATA GAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGAC AGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACA GATGGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATC AGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTC CGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCC GCAGCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAA TCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAA GGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACC ACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATT ACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTA AACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGC TATGCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCG TAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACAC GTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGG AACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGT AACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCA GTATGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGA GTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCC GGACGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGT CCCCGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGC ATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCC TTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATC CAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGT GGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAG GGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCAC GACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGC TCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAAT ATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCG GCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCT GCCGCGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTG CTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCAC AGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTC GCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACT GGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATC AGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAG CTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGG CATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAG CCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGC AAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTT GCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTG GCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTT CAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTC GATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGT AGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTC ACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGC CAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTT GCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATG CCCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCA CTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTG GGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTA GCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTC GCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCT TCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGC TGGGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGA GACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGT CGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACG TCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACT GGCCATGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAA AAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGC TGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGA TTATGATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCT CTGCCTCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCA GTGGCCAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGC GCATGTAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCAC GTTCTTGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATG TGGGAGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCA GGCCTGCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGT GGATATGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTC CAGAATTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTC ATCATGCGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCC CTCTACACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTT CACTGGGTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGG CACATTCTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACC CTCTTCGCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAG CCCATCATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTAT GGGGGGCTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGG CCAGGCTGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAG CTCTCATGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTG CCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCC AGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCG CGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCA TCCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAA TTTTATATTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCC AGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCT GACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCC ATCGCTCGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGC TAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTG AGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAG CAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGA GTGAACGGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTT CACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGC TGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAA TGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCG CGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCC GCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTG GCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGAC CGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGT AATAACTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGG AAGTGGTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGAT CTGACGGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAG CTTATACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTG ATAGGGAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCAT ACGTTCTCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGG GAGTAAAGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTC TCTATCACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCA CTTGCACTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGA GAGCGTATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAA AATAAAGATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGC GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGT GCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG CCTTTTTCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCA TCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTC GCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAA GTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTA GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGT TTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC GGATCCGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCT GGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAA AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTG CTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTG GAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGC TCTTCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGA AACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCC CTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTA TTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCC GAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTA AAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCT CCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGA CGATTTTGACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTG AGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTAT AATGTATGCTATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACA AACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTAT TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTT TTCGCGTCATGCACCACGTCCGTGGTCAACCCTCCCACACGTAACCAGATGGGAGA AGCTCTCGTATTCCAACAGCTGTAGGTTGACCGTCTGAATCCTTCACTATGGCCTTT ATACCTGGATGCAGATCCAACAGGACTTGTCTACATCTACCACAAGGTGACAAGAT CCCGCGATTCTCGTTACCAATAGCAACGATACAAGTCAGATTCCCTGCGGCGGCGG CAGCGGCGGTTCCCAGTACTACGAGTTCTGCGCAGGGGCCACCCGTAAAATGATAC ACATTTACCCCAGTAAAAATCCGCCCATCGGATGACAAGGCTGCACTAGCCACGGA ATAGTCGTCAGATATAGGTATGGAATTGATTGTGGCGGTAGCCCTCTCTATCAGCGT GGACTCCTCCTGAGAGAGGGGTTTTGCCATGGTGGCGGCTTAAGGGTTCGATCCTCT AGAGTCCGGAGGCTGGATCGGTCCCGGTGTCTTCTATGGAGGTCAAAACAGCGTGG ATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAGACCTC CCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTT TGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGT GGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGC CAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGT AGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGT CATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGT GGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCG TTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGTTGGGCGGT CAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATATATGGGCT ATGAACTAATGACCCCGTAATTGATTACTATTAATAACTAGTCAATAATCAATGTCA TTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATAATGTATGCTATACGAAGTTAT TTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGA AAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGCAC TCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGATT CGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGCGTGTCGA ACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTGGGCCCAT [1068] SEQ ID NO: 29 (STXC0126) [1069] TGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCA GCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGT CCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCC CCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGG GCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAG TTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTA AAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCA AAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCG ACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCA ATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTA GCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTG GCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAG CGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAA CGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGA ACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTT CCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGG AACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGT CAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTG AGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTG CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA GGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGT TAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAA TCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCG AAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTT TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAG CGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAAC CACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC CAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTT GAACCCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGA GCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAG ATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTT TTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTA TATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTAT TTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGA GGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGG GGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCC CGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGC TTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGG GGAAAAGGCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCT GCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATA GAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGAC AGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACA GATGGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATC AGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTC CGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCC GCAGCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAA TCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAA GGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACC ACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATT ACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTA AACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGC TATGCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCG TAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACAC GTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGG AACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGT AACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCA GTATGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGA GTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCC GGACGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGT CCCCGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGC ATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCC TTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATC CAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGT GGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAG GGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCAC GACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGC TCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAAT ATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCG GCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCT GCCGCGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTG CTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCAC AGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTC GCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACT GGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATC AGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAG CTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGG CATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAG CCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGC AAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTT GCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTG GCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTT CAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTC GATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGT AGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTC ACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGC CAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTT GCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATG CCCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCA CTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTG GGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTA GCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTC GCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCT TCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGC TGGGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGA GACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGT CGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACG TCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACT GGCCATGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAA AAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGC TGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGA TTATGATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCT CTGCCTCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCA GTGGCCAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGC GCATGTAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCAC GTTCTTGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATG TGGGAGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCA GGCCTGCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGT GGATATGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTC CAGAATTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTC ATCATGCGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCC CTCTACACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTT CACTGGGTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGG CACATTCTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACC CTCTTCGCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAG CCCATCATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTAT GGGGGGCTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGG CCAGGCTGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAG CTCTCATGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTG CCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCC AGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCG CGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCA TCCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAA TTTTATATTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCC AGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCT GACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCC ATCGCTCGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGC TAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTG AGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAG CAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGA GTGAACGGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTT CACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGC TGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAA TGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCG CGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCC GCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTG GCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGAC CGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGT AATAACTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGG AAGTGGTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGAT CTGACGGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAG CTTATACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTG ATAGGGAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCAT ACGTTCTCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGG GAGTAAAGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTC TCTATCACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCA CTTGCACTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGA GAGCGTATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAA AATAAAGATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGC GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGT GCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG CCTTTTTCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCA TCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTC GCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAA GTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTA GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGT TTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC GGATCCGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCT GGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAA AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTG CTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTG GAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGC TCTTCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGA AACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCC CTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTA TTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCC GAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTA AAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCT CCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGA CGATTTTGACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTG AGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTAT AATGTATGCTATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACA AACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTAT TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTT TTCGCGTCATGCACCACGTCCGTGGTCTAGAACTAGTATTATGCCCAGTACATGACC TTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATG GTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGG ATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCA ACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTA GGCGTGTACGGTGGGAGGTTTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC GCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGAGAATTCGCCGCCAC CATGAAAACATTTAACATTTCTCAACAGGATCTAGAATTAGTAGAAGTAGCGACAG AGAAGATTACAATGCTTTATGAGGATAATAAACATCATGTGGGAGCGGCAATTCGT ACGAAAACAGGAGAAATCATTTCGGCAGTACATATTGAAGCGTATATAGGACGAGT AACTGTTTGTGCAGAAGCCATTGCGATTGGTAGTGCAGTTTCGAATGGACAAAAGG ATTTTGACACGATTGTAGCTGTTAGACACCCTTATTCTGACGAAGTAGATAGAAGTA TTCGAGTGGTAAGTCCTTGTGGTATGTGTAGGGAGTTGATTTCAGACTATGCACCAG ATTGTTTTGTGTTAATAGAAATGAATGGCAAGTTAGTCAAAACTACGATTGAAGAA CTCATTCCACTCAAATATACCCGAAATTAAACCGGTCGCTGATCAGCCTCGACTGTG CCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGG AAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTC TGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAG GATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGA GGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGGGCCCATT GGTATGGCTTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATAATGTATGCTATAC GAAGTTATTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGT AACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACAC CGGGCACTCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGAC CGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGC GTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTGGGCCC AT [1070] SEQ ID NO: 30 (STXC0123) [1071] TGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCA GCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGT CCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCC CCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGG GCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAG TTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTA AAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCA AAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCG ACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCA ATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTA GCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTG GCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAG CGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAA CGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGA ACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTT CCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGG AACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGT CAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTG AGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTG CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA GGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT ATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGT TAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAA TCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTT CCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCG AAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTT TTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGA TTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAG CGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAAC CACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAG GCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGC TGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCC CAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTT GAACCCGTGGAGGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGA GCAGATGAAGATGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAG ATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTT TTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTA TATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTAT TTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGA GGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGCTAGG GGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCC CGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGC TTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGG GGAAAAGGCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCATGCCT GCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAGAATA GAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAGGAC AGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACA GATGGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATC AGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTC CGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGCCC GCAGCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTAAA TCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAA GGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACC ACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATT ACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATTA AACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGGC TATGCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTCG TAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACAC GTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGG AACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACGT AACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCCA GTATGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGGA GTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCC GGACGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGT CCCCGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGC ATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATCC TTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTATC CAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACGT GGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAG GGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCAC GACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGC TCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAAT ATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTCG GCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCT GCCGCGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTG CTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCAC AGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTC GCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACT GGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATC AGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAG CTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGG CATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAG CCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGC AAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTT GCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTG GCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTT CAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTC GATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGT AGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTC ACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGC CAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTT GCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATG CCCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCA CTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTG GGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTA GCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTC GCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCT TCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGC TGGGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGA GACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGT CGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACG TCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACT GGCCATGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAA AAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGC TGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGA TTATGATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCT CTGCCTCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCA GTGGCCAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGC GCATGTAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCAC GTTCTTGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATG TGGGAGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCA GGCCTGCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGT GGATATGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTC CAGAATTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTC ATCATGCGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCC CTCTACACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTT CACTGGGTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGG CACATTCTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACC CTCTTCGCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAG CCCATCATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTAT GGGGGGCTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGG CCAGGCTGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAG CTCTCATGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTG CCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCC AGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCG CGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCA TCCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAA TTTTATATTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCC AGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCT GACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCC ATCGCTCGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGC TAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTG AGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAG CAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGA GTGAACGGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTT CACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGC TGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAA TGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCG CGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCC GCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTG GCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGAC CGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGT AATAACTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGG AAGTGGTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGAT CTGACGGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAG CTTATACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTG ATAGGGAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCAT ACGTTCTCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGG GAGTAAAGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTC TCTATCACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCA CTTGCACTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGA GAGCGTATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAA AATAAAGATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGC GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGT GCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG CCTTTTTCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCA TCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTC GCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAA GTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTA GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGT TTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC GGATCCGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCT GGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAA AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTG CTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTG GAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGC TCTTCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGA AACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCC CTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTA TTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCC GAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTA AAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCT CCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGA CGATTTTGACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTG AGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTAT AATGTATGCTATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACA AACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTAT TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTT TTCGCGTCATGCACCACGTCCGTGGTTCAAGCGCCGGGTTTTCGCGTCATGCACCAC GTCCGTGGGCCCTCGGGTACTTCAACGTCAGCAGTAACTGTAAATCCGAGCCGTTCA TAGAAGGGCAAATTCCTTGGCGCTGACGTTTCAAGAAAGGCTGGCACTCCGGCTCG TTCTGCGGCTTCTACTCCGGGCAATACCACCGCGGAACCAAGGCCCTTTCCCTGATG ATCGGGGCTAACGCCCACAGTAGCGAGGAACCAAGCTGGTTCTTTAGGGCGGTGAG GGGCGAGGAGTCCTTCCATTTGTTGCTGAGCCGCGAGACGAGAGCCACTAAGCTCA GCCATTCGGGGACCAATTTCTGCAAATACAGCCCCGGCCTCAACGCTCTCCGGAGTC GTCCACACTGCCACTGCAGCCCCGTCGTCGGCGACCCAAACTTTACCGATGTCCAAT CCTACCCTGGTCAAAAAAAGTTCTTGCAATTCTGTAACCCGTTCAATATGTCTATCA GGATCAACTGTGTGGCGTGTAGCGGGATAATCCGCGAAAGCGGCAGCCAATGTTCT CACGGCCCTAGGGACGTCGTCTCGAGTTGCCAGTCTGACAGTAGGTTTATATTCTGT CATGGTGGCGGCGAATTCTCTTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCC AGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATATAAACCTCCCACCGTACAC GCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTTTGGAAAGTCCC GTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGA AATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATC ACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCA TAAGGTCATGTACTGGGCATAATACTAGTTCTTGGGAAAAGCGCTCCCCTACCCATA ACTTCGTATAATGTATGCTATACGAAGTTATTTTGCAGTTTTAAAATTATGTTTTAAA ATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATA TCTTGTGGAAAGGACGAAACACCGGGCACTCTTCCGTGATCTGGTGGATAAATTCG CAAGGGTATCATGGCGGACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGCCGT GATCCATGCGGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGG GGGAGCGCTCCTTTTTGGGCCCAT [1072] SEQ ID NO: 31 (STXC0133) [1073] actcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaa taggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcat tttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaaga gtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatc aagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaa cgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccacca cacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcc tcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaa aacgacggccagtgagcgcgcctcgttcattcacgtttttgaacccgtggaggacgggcagactcgcggtgcaaatgtgttttacagcgtg atggagcagatgaagatgctcgacacgctgcagaacacgcagctagattaaccctagaaagataatcatattgtgacgtacgttaaagata atcatgtgtaaaattgacgcatgtgttttatcggtctgtatatcgaggtttatttattaatttgaatagatattaagttttattatatttacacttacatact aataataaattcaacaaacaatttatttatgtttatttatttattaaaaaaaacaaaaactcaaaatttcttctataaagtaacaaaacttttatgaggg acagcccccccccaaagcccccagggatgtaattacgtccctcccccgctagggggcagcagcgagccgcccggggctccgctccggt ccggcgctccccccgcatccccgagccggcagcgtgcggggacagcccgggcacggggaaggtggcacgggatcgctttcctctgaa cgcttctcgctgctctttgagcctgcagacacctggggggatacggggaaaaggcctccacggccactagtccatagagcccaccgcatc cccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccctagaatagaatgacacctactcagacaatg cgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacag atggctggcaactagaaggcacagctacatgggggtagagtcataatcgtgcatcaggatagggcggtggtgctgcagcagcgcgcga ataaactgctgccgccgccgctccgtcctgcaggaatacaacatggcagtggtctcctcagcgatgattcgcaccgcccgcagcatgaga cgccttgtcctccgggcacagcagcgcaccctgatctcacttaaatcagcacagtaactgcagcacagcaccacaatattgttcaaaatccc acagtgcaaggcgctgtatccaaagctcatggcggggaccacagaacccacgtggccatcataccacaagcgcaggtagattaagtggc gacccctcataaacacgctggacataaacattacctcttttggcatgttgtaattcaccacctcccggtaccatataaacctctgattaaacatg gcgccatccaccaccatcctaaaccagctggccaaaacctgcccgccggctatgcactgcagggaaccgggactggaacaatgacagt ggagagcccaggactcgtaaccatggatcatcatgctcgtcatgatatcaatgttggcacaacacaggcacacgtgcatacacttcctcag gattacaagctcctcccgcgtcagaaccatatcccagggaacaacccattcctgaatcagcgtaaatcccacactgcagggaagacctcg cacgtaactcacgttgtgcattgtcaaagtgttacattcgggcagcagcggatgatcctccagtatggtagcgcgggtctctgtctcaaaagg aggtaggcgatccctactgtacggagtgcgccgagacaaccgagatcgtgttggtcgtagtgtcatgccaaatggaacgccggacgtagt catggttgtggccatattatcatcgtgtttttcaaaggaaaaccacgtccccgtggttcggggggcctagacgtttttttaacctcgactaaaca catgtaaagcatgtgcaccgaggccccagatcagatcccatacaatggggtaccttctgggcatccttcagccccttgttgaatacgcttga ggagagccatttgactctttccacaactatccaactcacaacgtggcactggggttgtgccgcctttgcaggtgtatcttatacacgtggctttt ggccgcagaggcacctgtcgccaggtggggggttccgctgcctgcaaagggtcgctacagacgttgtttgtcttcaagaagcttccagag gaactgcttccttcacgacattcaacagaccttgcattcctttggcgagaggggaaagacccctaggaatgctcgtcaagaagacagggcc aggtttccgggccctcacattgccaaaagacggcaatatggtggaaaataacatatagacaaacgcacaccggccttattccaagcggctt cggccagtaacgttaggggggggggagggagaggggcttaaaaatcaaaggggttctgccgcgcatcactatgcgccactggcaggg acacgttgcgatactggtgtttagtgctccacttaaactcaggcacaaccatccgcggcagctcggtgaagttttcactccacaggctgcgc accatcaccaacgcgtttagcaggtcgggcgccgatatcttgaagtcgcagttggggcctccgccctgcgcgcgcgagttgcgatacaca gggttgcagcactggaacactatcagcgccgggtggtgcacgctggccagcacgctcttgtcggagatcagatccgcgtccaggtcctcc gcgttgctcagggcgaacggagtcaactttggtagctgccttcccaaaaagggtgcatgcccaggctttgagttgcactcgcaccgtagtg gcatcagaaggtgaccgtgcccggtctgggcgttaggatacagcgcctgcatgaaagccttgatctgcttaaaagccacctgagcctttgc gccttcagagaagaacatgccgcaagacttgccggaaaactgattggccggacaggccgcgtcatgcacgcagcaccttgcgtcggtgtt ggagatctgcaccacatttcggccccaccggttcttcacgatcttggccttgctagactgctccttcagcgcgcgctgcccgttttcgctcgtc acatccatttcaatcacgtgctccttatttatcataatgctcccgtgtagacacttaagctcgccttcgatctcagcgcagcggtgcagccacaa cgcgcagcccgtgggctcgtggtgcttgtaggttacctctgcaaacgactgcaggtacgcctgcaggaatcgccccatcatcgtcacaaa ggtcttgttgctggtgaaggtcagctgcaacccgcggtgctcctcgtttagccaggtcttgcatacggccgccagagcttccacttggtcag gcagtagcttgaagtttgcctttagatcgttatccacgtggtacttgtccatcaacgcgcgcgcagcctccatgcccttctcccacgcagaca cgatcggcaggctcagcgggtttatcaccgtgctttcactttccgcttcactggactcttccttttcctcttgcgtccgcataccccgcgccact gggtcgtcttcattcagccgccgcaccgtgcgcttacctcccttgccgtgcttgattagcaccggtgggttgctgaaacccaccatttgtagc gccacatcttctctttcttcctcgctgtccacgatcacctctggggatggcgggcgctcgggcttgggagaggggcgcttctttttctttttgga cgcaatggccaaatccgccgtcgaggtcgatggccgcgggctgggtgtgcgcggcaccagcgcatcttgtgacgagtcttcttcgtcctc ggactcgagacgccgcctcagccgcttttttgggggcgcgcgcttgtcgtcatcgtctttgtagtcgggaggcggcggcgacggcgacgg ggacgacacgtcctccatggttggtggacgtcgcgccgcaccgcgtccgcgctcgggggtggtttcgcgctgctcctcttcccgactggc catggtggccgaggataacttcgtatatggtttcttatacgaagttatgatccagacatgataagatacattgatgagtttggacaaaccacaa ctagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgtaaccattataagctgcaataaacaagttaacaacaa caattgcattcattttatgtttcaggttcagggggaggtgtgggaggttttttaaagcaagtaaaacctctacaaatgtggtatggctgattatga tcctctagagtcgcagatctgctacgtatcaagctgtggcagggaaaccctctgcctcccccgtgatgtaatacttttgcaaggaatgcgatg aagtagagcccgcagtggccaagtggctttggtccgtctcctccacggatgcccctccacggctagtgggcgcatgtaggcggtgggcgt ccgccgcctccagcagcaggtcatagaggggcaccacgttcttgcacttcatgctgtacagatgctccatgcctttgttactcatgtgtcgga tgtgggagaggatgaggaggagctgggccagccgctggtgctgctgctgcagggtcaggcctgccttggccatcaggtggatcaaagtg tctgtgatcttgtccaggactcggtggatatggtccttctcttccagagacttcagggtgctggacagaaatgtgtacactccagaattaagca aaataatagatttgaggcacacaaactcctctccctgcagattcatcatgcggaaccgagatgatgtagccagcagcatgtcgaagatctcc accatgccctctacacattttccctggttcctgtccaagagcaagttaggagcaaacagtagcttcactgggtgctccatggagcgccagac gagaccaatcatcaggatctctagccaggcacattctagaaggtggacctgatcatggagggtcaaatccacaaagcctggcaccctcttc gcccagttgatcatgtgaaccagctccctgtctgccaggttggtcagtaagcccatcatcgaagcttcactgaagggtctggtaggatcatac tcggaatagagtatggggggctcagcatccaacaaggcactgaccatctggtcggccgtcagggacaaggccaggctgttcttcttagag cgtttgatcatgagcgggcttggccaaaggttggcagctctcatgtctccagcagatggctcgagatcgccatcttccagcaggcgcaccat tgcccctgtttcactatccaggttacggatatagttcatgacaatatttacattggtccagccaccagcttgcatgatctccggtattgaaactcc agcgcgggccatatctcgcgcggctccgacacgggcactgtgtccagaccaggccaggtatctctgaccagagtcatcctaaaatacaca aacaattagaatcagtagtttaacacattatacacttaaaaattttatatttaccttagcgccgtaaatcaatcgatgagttgcttcaaaaatccctt ccagggcgcgagttgatagctggctggtggcagatggcgcggcaacaccattttttctgacccggcaaaacaggtagttattcggatcatc agctacaccagagacggaaatccatcgctcgaccagtttagtgactcccaggctaagtgccttctctacacctgcggtgctaaccagcgtttt cgttctgccaatatggattaacattctcccaccgtcagtacgtgagatatctttaaccctgatcctggcaatttcggctatacgtaacagggtgtt ataagcaatccccagaaatgccagattacgtatatcctggcagcgatcgctattttccatgagtgaacggacttggtcgaaatcagtgcgttc gaacgctagagcctgttttgcacgttcaccggcatcaacgttttcttttcggatccgccgcataaccagtgaaacagcattgctgtcacttggt cgtggcagcccggaccgacgatgaagcatgtttagctggcccaaatgttgctggatagtttttactgccagaccgcgcgcttgaagatatag aagataatcgcgaacatcttcaggttctgcgggaaaccatttccggttattcaacttgcaccatgccgcccacgaccggcaaacggacaga agcattttccaggtatgctcagaaaacgcctggcgatccctgaacatgtccatcaggttcttgcgaacctcatcactcgttgcatcgaccggt aatgcaggcaaattttggtgtacggtcagtaaattggacatggtggctacgtaataacttcgtatatggtttcttatacgaagttatgcggccgc tttacgagggtaggaagtggtacggaaagttggtataagacaaaagtgttgtggaattgctccaggcgatctgacggttcactaaacgagct ctgcttttataggcgcccaccgtacacgcctaaagcttatacgttctctatcactgatagggagtaaactggatatacgttctctatcactgatag ggagtaaactgtagatacgttctctatcactgatagggagtaaactggtcatacgttctctatcactgatagggagtaaactccttatacgttctc tatcactgatagggagtaaagtctgcatacgttctctatcactgatagggagtaaactcttcatacgttctctatcactgatagggagtaaactc gcggccgcagagaaatgttctggcacctgcacttgcactggggacagcctattttgctagtttgttttgtttcgttttgttttgatggagagcgtat gttagtactatcgattcacacaaaaaaccaacacacagatgtaatgaaaataaagatattttattggatctgcgatcgctccggtgcccgtcag tgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaacgggtgcctagagaaggtggcgcgg ggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatgtaagtgcagtagtcgccgtgaac gttctttttcgcaacgggtttgccgccagaacacagctgaagcttcgaggggctcgcatctctccttcacgcgcccgccgccctacctgagg ccgccatccacgccggttgagtcgcgttctgccgcctcccgcctgtggtgcctcctgaactgcgtccgccgtctaggtaagtttaaagctca ggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactcagccggctctccacgctttgcctgaccctgcttgctcaactct acgtctttgtttcgttttctgttctgcgccgttacagatccaagctgtgaccggcgcctacgctagcggatccgccgccaccatgtctagactg gacaagagcaaagtcataaactctgctctggaattactcaatggagtcggtatcgaaggcctgacgacaaggaaactcgctcaaaagctg ggagttgagcagcctaccctgtactggcacgtgaagaacaagcgggccctgctcgatgccctgccaatcgagatgctggacaggcatcat acccactcctgccccctggaaggcgagtcatggcaagactttctgcggaacaacgccaagtcataccgctgtgctcttctctcacatcgcga cggggctaaagtgcatctcggcacccgcccaacagagaaacagtacgaaaccctggaaaatcagctcgcgttcctgtgtcagcaaggctt ctccctggagaacgcactgtacgctctgtccgccgtgggccactttacactgggctgcgtattggaggaacaggagcatcaagtagcaaa agaggaaagagagacacctaccaccgattctatgcccccacttctgaaacaagcaattgagctgttcgaccggcagggagccgaacctg ccttccttttcggcctggaactaatcatatgtggcctggagaaacagctaaagtgcgaaagcggcgggccgaccgacgcccttgacgatttt gacttagacatgctcccagccgatgcccttgacgactttgaccttgatatgctgcctgctgacgctcttgacgattttgaccttgacatgctccc cgggtgaaccggtcgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaa ggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggc aggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcggaaagaacca gctggggctcgactagagcttgcggaacccttagagggcctatttcccatggtggtagaactagtattatgcccagtacatgaccttatggga ctttcctacttggcagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttga ctcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaac tccgccccattgacgcaaatgggcggtaggcgtgtacggtgggaggtttatataagcagagctcgtttagtgaaccgtcagatcgcctgga gacgccatccacgctgttttgacctccatagaagagaattcgccgccaccatgacagaatataaacctactgtcagactggcaactcgaga cgacgtccctagggccgtgagaacattggctgccgctttcgcggattatcccgctacacgccacacagttgatcctgatagacatattgaac gggttacagaattgcaagaactttttttgaccagggtaggattggacatcggtaaagtttgggtcgccgacgacggggctgcagtggcagt gtggacgactccggagagcgttgaggccggggctgtatttgcagaaattggtccccgaatggctgagcttagtggctctcgtctcgcggct cagcaacaaatggaaggactcctcgcccctcaccgccctaaagaaccagcttggttcctcgctactgtgggcgttagccccgatcatcagg gaaagggccttggttccgcggtggtattgcccggagtagaagccgcagaacgagccggagtgccagcctttcttgaaacgtcagcgcca aggaatttgcccttctatgaacggctcggatttacagttactgctgacgttgaagtacccgagggcccacggacgtggtgcatgacgcgaa aacccggcgcttgagtttaaaccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttga ccctggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtgg ggtggggcaggacagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggcttctgaggcgga aagaaccagctggggctcgactagagcttgcggaacccttagtttaaacgggcccttaattaatcgatgtaggatgttgcccctcctgacgc ggtaggagaaggggagggtgccctgcatgtctgccgctgctcttgctcttgccgctgctgaggaggggggcgcatctgccgcagcaccg gatgcatctgggaaaagcaaaaaaggggctcgtccctgtttccggaggaatttgcaagcggggtcttgcatgacggggaggcaaacccc cgttcgccgcagtccggccggcccgagactcgaaccgggggtcctgcgactcaacccttggaaaataaccctccggctacagggagcg agccacttaatgctttcgctttccagcctaaccgcttacgccgcgcgcggccagtggccaaaaaagctagcgcagcagccgccgcgcctg gaaggaagccaaaaggagcgctcccccgttgtctgacgtcgcacacctgggttcgacacgcgggcggtaaccgcatggatcacggcgg acggccggatccggggttcgaaccccggtcgtccgccatgatacccttgcgaatttatccaccagaccacggaagagtgcccgcttacag gctctccttttgcacggtctagagcgtcaacgactgcgcacgcctcaccggccagagcgtcccgaccatggagcactttttgccgctgcgc aacatctggaaccgcgtccgcgactttccgcgcgcctccaccaccgccgccggcatcacctggatgtccaggtacatctacggattacgg ggcccattggtatggctttttccccgtatccccccaggtgtctgcaggctcaaagagcagcgagaagcgttcagaggaaagcgatcccgtg ccaccttccccgtgcccgggctgtccccgcacgctgccggctcggggatgcggggggagcgccggaccggagcggagccccgggc ggctcgctgctgccccctagcgggggagggacgtaattacatccctgggggctttgggggggggctgtccctgatatctataacaagaaa atatatatataataagttatcacgtaagtagaacatgaaataacaatataattatcgtatgagttaaatcttaaaagtcacgtaaaagataatcatg cgtcattttgactcacgcggtcgttatagttcaaaatcagtgacacttaccgcattgacaagcacgcctcacgggagctccaagcggcgact gagatgtcctaaatgcacagcgacggattcgcgctatttagaaagagagagcaatatttcaagaatgcatgcgtcaattttacgcagactatc tttctagggttaatctagctgcatcaggatcatatcgtcgggtcttttttccggctcagtcatcgcccaagctggcgctatctgggcatcgggga ggaagaagcccgtgccttttcccgcgaggttgaagcggcatggaaagagtttgccgaggatgactgctgctgcattgacgttgagcgaaa acgcacgtttaccatgatgattcgggaaggtgtggccatgcacgcctttaacggtgaactgttcgttcaggccacctgggataccagttcgtc gcggcttttccggacacagttccggatggtcagcccgaagcgcatcagcaacccgaacaataccggcgacagccggaactgccgtgcc ggtgtgcagattaatgacagcggtgcggcgctgggatattacgtcagcgaggacgggtatcctggctggatgccgcagaaatggacatg gataccccgtgagttacccggcgggcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacaca acatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttc cagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcct cgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcagg ggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccatagg ctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttcc ccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttct catagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctg cgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcaga gcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctg aagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcag attacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggatt ttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtc tgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataac tacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaac cagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaa gtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccg gttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagtt ggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactc aaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactt taaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgc acccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagg gcgacacggaaatgttgaatactcat [1074] SEQ ID NO: 32 (STXC0137) [1075] ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGT TAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCC CTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT GTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAG GACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGAT GCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTG TGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTGT ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTA CATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAA AACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATGGAGGGGTGGAG TCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTGA GTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGCCCGAGTG AGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGCCG GGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGC ATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGA TTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGC TGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTT TTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAA ACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTGAGTCAGATTCGCGAAAA ACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTTGCCAAACTGGTTCGCGG TCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAGGTGGTGGATGAGTGCTA CATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCCAGTGGGCGTGGACTAA TATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAAACGGTTGGTGG CGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAATCAGAA TCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAGC TGGTCGGGTGGCTCGTGGACAAGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTT TCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTA GAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTC ACTTTCTACTCTGTTGACAACCGTTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACT TTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTT GTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGGCAATCAGGGTATATTA TATTGTACTTCAGCACAGTTTTAGAGAACATAACTTCGTATAAAGTATACTATACGA AGTTATCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGATGTC AGAACTCATTAAAGAGAATATGCACATGAAGCTGTATATGGAAGGTACTGTAGACA ACCACCATTTCAAATGCACGTCCGAAGGTGAGGGGAAGCCATACGAGGGTACCCAA ACTATGCGCATCAAAGTGGTTGAGGGTGGCCCCCTGCCATTCGCATTCGACATCCTG GCAACTAGCTTTCTTTACGGTTCCAAGACATTCATAAATCATACCCAGGGTATTCCC GATTTCTTCAAACAATCCTTCCCGGAAGGGTTTACTTGGGAGCGGGTCACGACATAT GAAGACGGGGGTGTTCTTACAGCCACACAGGATACGAGTTTGCAAGACGGTTGTCT TATCTATAACGTGAAGATTCGGGGTGTGAATTTCACATCCAATGGCCCGGTGATGCA GAAAAAAACACTGGGCTGGGAAGCATTTACGGAGACGTTGTATCCCGCCGATGGAG GTCTCGAGGGCCGAAACGATATGGCCCTCAAGTTGGTAGGTGGTTCTCACCTTATAG CAAACATTAAGACCACGTATCGATCAAAAAAACCCGCTAAGAATCTGAAAATGCCA GGCGTGTATTATGTTGATTACAGACTGGAGCGAATAAAAGAGGCTAACAATGAGAC CTACGTCGAACAGCATGAAGTCGCTGTAGCTAGATATTGCGACCTCCCGTCAAAGTT GGGCCATAAATTGAATTAACCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTG GTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAA ATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAA TTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACAT ATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCA ACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCA GTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTG AGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAAT TTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCAT AGCTGTCCCTCTTCTCTTATGGAGATCATAACTTCGTATAAAGTATACTATACGAAG TTATAATTGTTATAATTAAATGATAAGGTAGAATATTTCTGCATATAAATTCTGGCT GGCGTGGAAATATTCTTATTGGTAGAAACAACTACACCCTGGTCATCATCCTGCCTT TCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATAAAATACTCTGAGT CCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGGGGAT TACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATG CGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATT ATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGA CATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATA TGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCA TCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCC CACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAAC GACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGG TCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAA ATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGATCGTCACCTCCAACACCA ACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACACCAGCAGCCGTTG CAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTTTGGGAA GGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTG AGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCC CAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCAT CGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGT TCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATG AATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTT CCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGC TACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTC AATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTGTAAATAAATTTAG TAGTCATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTC TTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAG CATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGA AACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGC ACGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTAC AACCACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGG AAACCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCC TGGTTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTT CCAAAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAG ACGCCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCC TCAAGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAA TAACCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCA CGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGC TACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACG CCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCC ACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTC AGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGT GCAGGACTCCACCACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTAC GGACGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGC CGGCCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACC GCGACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTT CCCAGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGA GGTGCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTGTTCAAGCTGGCCAACCC GCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCC AGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCG GGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAG TGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGG TGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCC CTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCAC GTACCTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACC GCGTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCCACCACT GCCCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGAT GGAGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGG GCGCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCC ATGATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGT GCCCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGG AGTGGGAGCTCAAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACAC AAACAACTACAACGACCCCCAGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAAT ACAGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAATTGCTTG TTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTTT CTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTA CAGCCCGGGCGTTTAAACAGCGGGCGGAGGGGTGGAGTCGTGACGTGAATTACGTC ATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCG ACACCATGTGGTCCGCGGCCGCAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGG GCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATT GAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTA CTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCG CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAG GGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCC GGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCG TCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGA GCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCT ACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCC TACGATATCGCCACCATGATTAAGATCGCTACGCGGAAGTACCTGGGGAAACAGAA CGTCTACGACATAGGTGTGGAGCGCGATCACAACTTTGCTCTGAAAAATGGATTTAT CGCCAGCAACTGTAGGGAGTTGATTTCAGACTATGCACCAGATTGTTTTGTGTTAAT AGAAATGAATGGCAAGTTAGTCAAAACTACGATTGAAGAACTCATTCCACTCAAAT ATACCCGAAATTAAGTGCATGACCCGCAAGCCCGGTGCCTGAAATCAACCTCTGGA TTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTA TGTGGATACGCTGCTTTAATGCCTTTGTATCATGCGTTAACTAAACTTGTTTATTGCA GCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATT TTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTC TGGAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTCATCTGGTCTCCCTTC CGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGTTCCCAAACTGGGTTC TTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGTCCTCACAGGAACGA AGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTT TTTTAGGGCCCATTGGTATGGCGATATCTATAACAAGAAAATATATATATAATAAGT TATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTA AAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCA AAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCG ACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCA ATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTA GCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTG GCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAG CGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAA CGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGA ACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTT CCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGG AACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGT CAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTG AGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAG CCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCG CTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCG GGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTG CGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACG GTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAG CAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCG CCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGA CAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTG TTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGG CGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCA AGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCC ACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAA GTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCT GAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAA GGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAA AACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATC CTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGG TCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTT CGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGC TTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCA GATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGC AACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAG TTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTC ACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGT TACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAA TTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACC AAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATA CGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACG TTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTA ACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGG TGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG AAATGTTGAATACTCAT [1076] SEQ ID NO: 33 (STXC0136) [1077] ACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGA GCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACA TTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGT TAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCC CTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAAC AAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTA TCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAG GTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGAC GGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGG GCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCC GCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGT TGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGG ATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTT GTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAG GACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGAT GCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATTG TGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTGT ATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTTA CATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAA AACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATTTCTTTCCTGCGT TATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTC GCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCG CCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCA CGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTT AGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGT GTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGACCATGATTAC GCCAAGCTTTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGA CCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGC GCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCTGCAGCCGCGACCG GCCAAGGTTTAATGATAGGCTGCAACGGGATGTTGGGAATATGTTGCACTGGTCCG TGAGGGTACCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCAT CACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAA ACTCATCAATGTATCTTATCATGTCTGACCGGTTCACTTGAGCTCGAGATCTGAGTA CTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGGCGGTCACGAACTCCAGCA GGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCGGACTGGGTGCTCA GGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGTTCTGCTGG TAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAGTTC ACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGT TGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCA GCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGT AGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATG GCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTG CACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCG GTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCG CCGGACACGCTGAACTTGTGGCCGTTTACGTCGCCGTCCAGCTCGACCAGGATGGG CACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTGGCGGCTTAAGGGTT CGATCCTCTAGAGTCCGGAGGCTGGATCGGTCCCGGTGTCTACTATGGAGGTCAAA ACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTAT ATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTT ACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTC AATGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGA TGTACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTAC TGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCA TTTACCGTCATTGACGTCAATAGGGGGCGTACTTGGCATATGATACACTTGATGTAC TGCCAAGTGGGCAGTTTACCGTAAATACTCCACCCATTGACGTCAATGGAAAGTCC CTATTGGCGTTACTATGGGAACATACGTCATTATTGACGTCAATGGGCGGGGGTCGT TGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGTTATGTAACGCGGAACTCCATA TATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTAATAACTAGTCAATAAT CAATGTCAACGCGTATGGTACCTGCGGAGGATGCCGAGGATAACCTTGTTACTAGC CTCCGCCTGGCCGTTGGACTGTGGATAATATGGCGTAGAGGATCCTCTGCGCGCTCG CTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCC CGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAATTCACTGGCCGTCGTTTTACAACG TCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCC TTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGT TGCGCAGCCTGAATGGCGAATGGTCTAGAGCTAGCGAATTCGAATTTAAATCGGAT CCGCGGCCGCAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTA GAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTT TTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTT TTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCT CCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTT CTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTA AAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTC AGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGT TTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGATATCGCCAC CATGAAAACATTTAACATTTCTCAACAGGATCTAGAATTAGTAGAAGTAGCGACAG AGAAGATTACAATGCTTTATGAGGATAATAAACATCATGTGGGAGCGGCAATTCGT ACGAAAACAGGAGAAATCATTTCGGCAGTACATATTGAAGCGTATATAGGACGAGT AACTGTTTGTGCAGAAGCCATTGCGATTGGTAGTGCAGTTTCGAATGGACAAAAGG ATTTTGACACGATTGTAGCTGTTAGACACCCTTATTCTGACGAAGTAGATAGAAGTA TTCGAGTGGTAAGTCCTTGTGGTATGTGCCTTTCATACGAGACCGAGATCCTGACTG TCGAGTACGGATTGCTTCCTATCGGCAAAATCGTGGAGAAGAGGATTGAATGTACC GTCTATTCAGTCGATAATAATGGGAACATCTACACACAGCCCGTGGCTCAATGGCA CGACAGAGGAGAGCAGGAAGTTTTTGAATACTGTCTCGAGGACGGATCCCTCATCC GCGCTACTAAAGATCATAAGTTTATGACCGTGGACGGCCAGATGCTGCCAATTGAC GAAATTTTTGAACGAGAGCTGGATCTGATGAGAGTCGACAACCTTCCAAACTGAGT GCATGACCCGCAAGCCCGGTGCCTGAAATCAACCTCTGGATTACAAAATTTGTGAA AGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTT TAATGCCTTTGTATCATGCGTTAACTAAACTTGTTTATTGCAGCTTATAATGGTTACA AATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTA GTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGAATTGACTCAAAT GATGTCAATTAGTCTATCAGAAGCTATCTGGTCTCCCTTCCGGGGGACAAGACATCC CTGTTTAATATTTAAACAGCAGTGTTCCCAAACTGGGTTCTTATATCCCTTGCTCTGG TCAACCAGGTTGCAGGGTTTCCTGTCCTCACAGGAACGAAGTCCCTAAAGAAACAG TGGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATTGGTA TGGCTGATATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAA CATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGA TAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTA CCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAA TGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCA TGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCAT ATCGTCGGGTCTTTTTTCCGGCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATC GGGGAGGAAGAAGCCCGTGCCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTT TGCCGAGGATGACTGCTGCTGCATTGACGTTGAGCGAAAACGCACGTTTACCATGA TGATTCGGGAAGGTGTGGCCATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCA CCTGGGATACCAGTTCGTCGCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGA AGCGCATCAGCAACCCGAACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTG CAGATTAATGACAGCGGTGCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCC TGGCTGGATGCCGCAGAAATGGACATGGATACCCCGTGAGTTACCCGGCGGGCGCG CTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATT CCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGT GAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCT GTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTA TTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCG GCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAA AAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAA AAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCT GTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAAC CCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACC CGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGA GCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTA CACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAA AAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTT TGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGG TCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAA TCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACT CCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAG CCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCA GTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCG CAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGC TTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTG CAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGC AGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATC CGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTG TATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCAC ATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTC TCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAAC TGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGG CAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCAT [1078] SEQ ID NO: 34 (STX650) [1079] TCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGA GACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCG CGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAG ATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAG AAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGC GATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCA AGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAC GGCCAGTGAATTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGG ATCCTCTACGCCATATTATCCACAGTCCAACGGCCAGGCGGAGGCTAGTAACAAGG TTATCCTCGGCATCCTCCGCAGGTACCATACGCGTTGACATTGATTATTGACTAGTT ATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCAT TGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGAC GTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATC ATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATT ATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAG TCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGC GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGT TTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGA CGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTA GTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGTAGA CACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCGAACCCTTAAGCCGCCACC ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCT GGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGAT GCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGT GCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTA CCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACG TCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAG GTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTT CAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAC AACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGAT CCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACA CCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAG TCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTT CGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTACTCAGATC TCGAGCTCAAGTGAACCGGTCAGACATGATAAGATACATTGATGAGTTTGGACAAA CCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTG CTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTGGTACCCTCACGGACCAGT GCAACATATTCCCAACATCCCGTTGCAGCCTATCATTAAACCTTGGCCGGTCGCGGC TGCAGAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCT CACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCT CAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAAAGCTTGGCGTAATCATG GTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACG AGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACAT TAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGC ATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCC GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCA GCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAA GAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTG CTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGG AAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGC CTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCC CGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGA CTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAG GCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACA GTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAG CAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGG GTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTAT CAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCT AAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCA CCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGT AGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCG CGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAG GGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTG TTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGC CATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTC CGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGG TTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCAC TCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCT TTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGAC CGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACT TTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTA CCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCA TCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGC AAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTC AATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAAT GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCA CCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATC ACGAGGCCCTTTCGTC [1080] SEQ ID NO: 35 (STXC002) [1081] ggtacccaactccatgcttaacagtccccaggtacagcccaccctgcgtcgcaaccaggaacagctctacagcttcctggagc gccactcgccctacttccgcagccacagtgcgcagattaggagcgccacttctttttgtcacttgaaaaacatgtaaaaataatgtactagga gacactttcaataaaggcaaatgtttttatttgtacactctcgggtgattatttaccccccacccttgccgtctgcgccgtttaaaaatcaaaggg gttctgccgcgcatcgctatgcgccactggcagggacacgttgcgatactggtgtttagtgctccacttaaactcaggcacaaccatccgcg gcagctcggtgaagttttcactccacaggctgcgcaccatcaccaacgcgtttagcaggtcgggcgccgatatcttgaagtcgcagttggg gcctccgccctgcgcgcgcgagttgcgatacacagggttgcagcactggaacactatcagcgccgggtggtgcacgctggccagcacg ctcttgtcggagatcagatccgcgtccaggtcctccgcgttgctcagggcgaacggagtcaactttggtagctgccttcccaaaaagggtg catgcccaggctttgagttgcactcgcaccgtagtggcatcagaaggtgaccgtgcccggtctgggcgttaggatacagcgcctgcatga aagccttgatctgcttaaaagccacctgagcctttgcgccttcagagaagaacatgccgcaagacttgccggaaaactgattggccggaca ggccgcgtcatgcacgcagcaccttgcgtcggtgttggagatctgcaccacatttcggccccaccggttcttcacgatcttggccttgctag actgctccttcagcgcgcgctgcccgttttcgctcgtcacatccatttcaatcacgtgctccttatttatcataatgctcccgtgtagacacttaag ctcgccttcgatctcagcgcagcggtgcagccacaacgcgcagcccgtgggctcgtggtgcttgtaggttacctctgcaaacgactgcag gtacgcctgcaggaatcgccccatcatcgtcacaaaggtcttgttgctggtgaaggtcagctgcaacccgcggtgctcctcgtttagccagg tcttgcatacggccgccagagcttccacttggtcaggcagtagcttgaagtttgcctttagatcgttatccacgtggtacttgtccatcaacgcg cgcgcagcctccatgcccttctcccacgcagacacgatcggcaggctcagcgggtttatcaccgtgctttcactttccgcttcactggactct tccttttcctcttgcgtccgcataccccgcgccactgggtcgtcttcattcagccgccgcaccgtgcgcttacctcccttgccgtgcttgattag caccggtgggttgctgaaacccaccatttgtagcgccacatcttctctttcttcctcgctgtccacgatcacctctggggatggcgggcgctc gggcttgggagaggggcgcttctttttctttttggacgcaatggccaaatccgccgtcgaggtcgatggccgcgggctgggtgtgcgcggc accagcgcatcttgtgacgagtcttcttcgtcctcggactcgagacgccgcctcagccgcttttttgggggcgcgcggggaggcggcggc gacggcgacggggacgacacgtcctccatggttggtggacgtcgcgccgcaccgcgtccgcgctcgggggtggtttcgcgctgctcctc ttcccgactggccatttccttctcctataggcagaaaaagatcatggagtcagtcgagaaggaggacagcctaaccgccccctttgagttcg ccaccaccgcctccaccgatgccgccaacgcgcctaccaccttccccgtcgaggcacccccgcttgaggaggaggaagtgattatcgag caggacccaggttttgtaagcgaagacgacgaggatcgctcagtaccaacagaggataaaaagcaagaccaggacgacgcagaggca aacgaggaacaagtcgggcggggggaccaaaggcatggcgactacctagatgtgggagacgacgtgctgttgaagcatctgcagcgc cagtgcgccattatctgcgacgcgttgcaagagcgcagcgatgtgcccctcgccatagcggatgtcagccttgcctacgaacgccacctgt tctcaccgcgcgtaccccccaaacgccaagaaaacggcacatgcgagcccaacccgcgcctcaacttctaccccgtatttgccgtgccag aggtgcttgccacctatcacatctttttccaaaactgcaagatacccctatcctgccgtgccaaccgcagccgagcggacaagcagctggc cttgcggcagggcgctgtcatacctgatatcgcctcgctcgacgaagtgccaaaaatctttgagggtcttggacgcgacgagaaacgcgc ggcaaacgctctgcaacaagaaaacagcgaaaatgaaagtcactgtggagtgctggtggaacttgagggtgacaacgcgcgcctagcc gtgctgaaacgcagcatcgaggtcacccactttgcctacccggcacttaacctaccccccaaggttatgagcacagtcatgagcgagctga tcgtgcgccgtgcacgacccctggagagggatgcaaacttgcaagaacaaaccgaggagggcctacccgcagttggcgatgagcagct ggcgcgctggcttgagacgcgcgagcctgccgacttggaggagcgacgcaagctaatgatggccgcagtgcttgttaccgtggagcttg agtgcatgcagcggttctttgctgacccggagatgcagcgcaagctagaggaaacgttgcactacacctttcgccagggctacgtgcgcc aggcctgcaaaatttccaacgtggagctctgcaacctggtctcctaccttggaattttgcacgaaaaccgcctcgggcaaaacgtgcttcatt ccacgctcaagggcgaggcgcgccgcgactacgtccgcgactgcgtttacttatttctgtgctacacctggcaaacggccatgggcgtgtg gcagcaatgcctggaggagcgcaacctaaaggagctgcagaagctgctaaagcaaaacttgaaggacctatggacggccttcaacgag cgctccgtggccgcgcacctggcggacattatcttccccgaacgcctgcttaaaaccctgcaacagggtctgccagacttcaccagtcaaa gcatgttgcaaaactttaggaactttatcctagagcgttcaggaattctgcccgccacctgctgtgcgcttcctagcgactttgtgcccattaag taccgtgaatgccctccgccgctttggggtcactgctaccttctgcagctagccaactaccttgcctaccactccgacatcatggaagacgtg agcggtgacggcctactggagtgtcactgtcgctgcaacctatgcaccccgcaccgctccctggtctgcaattcgcaactgcttagcgaaa gtcaaattatcggtacctttgagctgcagggtccctcgcctgacgaaaagtccgcggctccggggttgaaactcactccggggctgtggac gtcggcttaccttcgcaaatttgtacctgaggactaccacgcccacgagattaggttctacgaagaccaatcccgcccgccaaatgcggag cttaccgcctgcgtcattacccagggccacatccttggccaattgcaagccatcaacaaagcccgccaagagtttctgctacgaaagggac ggggggtttacctggacccccagtccggcgaggagctcaacccaatccccccgccgccgcagccctatcagcagccgcgggcccttgc ttcccaggatggcacccaaaaagaagctgcagctgccgccgccgccacccacggacgaggaggaatactgggacagtcaggcagag gaggttttggacgaggaggaggagatgatggaagactgggacagcctagacgaagcttccgaggccgaagaggtgtcagacgaaaca ccgtcaccctcggtcgcattcccctcgccggcgccccagaaattggcaaccgttcccagcatcgctacaacctccgctcctcaggcgccg ccggcactgcctgttcgccgacccaaccgtagatgggacaccactggaaccagggccggtaagtctaagcagccgccgccgttagccc aagagcaacaacagcgccaaggctaccgctcgtggcgcgggcacaagaacgccatagttgcttgcttgcaagactgtgggggcaacat ctccttcgcccgccgctttcttctctaccatcacggcgtggccttcccccgtaacatcctgcattactaccgtcatctctacagcccctactgca ccggcggcagcggcagcggcagcaacagcagcggtcacacagaagcaaaggcgaccggatagcaagactctgacaaagcccaaga aatccacagcggcggcagcagcaggaggaggagcgctgcgtctggcgcccaacgaacccgtatcgacccgcgagcttagaaatagga tttttcccactctgtatgctatatttcaacaaagcaggggccaagaacaagagctgaaaataaaaaacaggtctctgcgctccctcacccgca gctgcctgtatcacaaaagcgaagatcagcttcggcgcacgctggaagacgcggaggctctcttcagcaaatactgcgcgctgactcttaa ggactagtttcgcgccctttctcaaatttaagcgcgaaaactacgtcatctccagcggccacacccggcgccagcacctgtcgtcagcgcc attatgagcaaggaaattcccacgccctacatgtggagttaccagccacaaatgggacttgcggctggagctgcccaagactactcaaccc gaataaactacatgagcgcgggaccccacatgatatcccgggtcaacggaatccgcgcccaccgaaaccgaattctcctcgaacaggcg gctattaccaccacacctcgtaataaccttaatccccgtagttggcccgctgccctggtgtaccaggaaagtcccgctcccaccactgtggta cttcccagagacgcccaggccgaagttcagatgactaactcaggggcgcagcttgcgggcggctttcgtcacagggtgcggtcgcccgg gcgttttagggcggagtaacttgcatgtattgggaattgtagtttttttaaaatgggaagtgacgtatcgtgggaaaacggaagtgaagatttg aggaagttgtgggttttttggctttcgtttctgggcgtaggttcgcgtgcggttttctgggtgttttttgtggactttaaccgttacgtcattttttagt cctatatatactcgctctgtacttggccctttttacactgtgactgattgagctggtgccgtgtcgagtggtgttttttaataggtttttttactggtaa ggctgactgttatggctgccgctgtggaagcgctgtatgttgttctggagcgggagggtgctattttgcctaggcaggagggtttttcaggtgt ttatgtgtttttctctcctattaattttgttatacctcctatgggggctgtaatgttgtctctacgcctgcgggtatgtattcccccgggctatttcggt cgctttttagcactgaccgatgttaaccaacctgatgtgtttaccgagtcttacattatgactccggacatgaccgaggaactgtcggtggtgct ttttaatcacggtgaccagtttttttacggtcacgccggcatggccgtagtccgtcttatgcttataagggttgtttttcctgttgtaagacaggctt ctaatgtttaaatgtttttttttttgttattttattttgtgtttaatgcaggaacccgcagacatgtttgagagaaaaatggtgtctttttctgtggtggttc cggaacttacctgcctttatctgcatgagcatgactacgatgtgcttgcttttttgcgcgaggctttgcctgattttttgagcagcaccttgcatttt atatcgccgcccatgcaacaagcttacataggggctacgctggttagcatagctccgagtatgcgtgtcataatcagtgtgggttcttttgtca tggttcctggcggggaagtggccgcgctggtccgtgcagacctgcacgattatgttcagctggccctgcgaagggacctacgggatcgc ggtatttttgttaatgttccgcttttgaatcttatacaggtctgtgaggaacctgaatttttgcaatcatgattcgctgcttgaggctgaaggtggag ggcgctctggagcagatttttacaatggccggacttaatattcgggatttgcttagagacatattgataaggtggcgagatgaaaattatttgg gcatggttgaaggtgctggaatgtttatagaggagattcaccctgaagggtttagcctttacgtccacttggacgtgagggcagtttgcctttt ggaagccattgtgcaacatcttacaaatgccattatctgttctttggctgtagagtttgaccacgccaccggaggggagcgcgttcacttaata gatcttcattttgaggttttggataatcttttggaataaaaaaaaaaaaacatggttcttccagctcttcccgctcctcccgtgtgtgactcgcaga acgaatgtgtaggttggctgggtgtggcttattctgcggtggtggatgttatcagggcagcggcgcatgaaggagtttacatagaacccgaa gccagggggcgcctggatgctttgagagagtggatatactacaactactacacagagcgagctaagcgacgagaccggagacgcagat ctgtttgtcacgcccgcacctggttttgcttcaggaaatatgactacgtccggcgttccatttggcatgacactacgaccaacacgatctcggt tgtctcggcgcactccgtacagtagggatcgcctacctccttttgagacagagacccgcgctaccatactggaggatcatccgctgctgccc gaatgtaacactttgacaatgcacaacgtgagttacgtgcgaggtcttccctgcagtgtgggatttacgctgattcaggaatgggttgttccct gggatatggttctgacgcgggaggagcttgtaatcctgaggaagtgtatgcacgtgtgcctgtgttgtgccaacattgatatcatgacgagc atgatgatccatggttacgagtcctgggctctccactgtcattgttccagtcccggttccctgcagtgcatagccggcgggcaggttttggcc agctggtttaggatggtggtggatggcgccatgtttaatcagaggtttatatggtaccgggaggtggtgaattacaacatgccaaaagaggt aatgtttatgtccagcgtgtttatgaggggtcgccacttaatctacctgcgcttgtggtatgatggccacgtgggttctgtggtccccgccatga gctttggatacagcgccttgcactgtgggattttgaacaatattgtggtgctgtgctgcagttactgtgctgatttaagtgagatcagggtgcgc tgctgtgcccggaggacaaggcgtctcatgctgcgggcggtgcgaatcatcgctgaggagaccactgccatgttgtattcctgcaggacg gagcggcggcggcagcagtttattcgcgcgctgctgcagcaccaccgccctatcctgatgcacgattatgactctacccccatgtaggcgt ggacttccccttcgccgcccgttgagcaaccgcaagttggacagcagcctgtggctcagcagctggacagcgacatgaacttaagcgag ctgcccggggagtttattaatatcactgatgagcgtttggctcgacaggaaaccgtgtggaatataacacctaagaatatgtctgttacccatg atatgatgctttttaaggccagccggggagaaaggactgtgtactctgtgtgttgggagggaggtggcaggttgaatactagggttctgtga gtttgattaaggtacggtgatcaatataagctatgtggtggtggggctatactactgaatgaaaaatgacttgaaattttctgcaattgaaaaata aacacgttgaaacataacatgcaacaggttcacgattctttattcctgggcaatgtaggagaaggtgtaagagttggtagcaaaagtttcagt ggtgtattttccactttcccaggaccatgtaaaagacatagagtaagtgcttacctcgctagtttctgtggattcactagaatcgatgtcgacgtt taaaccatatgatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggc cagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacg ctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgacc ctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggt cgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccgg taagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtg gtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttg atccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctt tgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatc cttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctc agcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgct gcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtc ctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgcca ttgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccat gttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactg cataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccg agttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcg aaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgt ttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttc aatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatt tccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccga aatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgt ggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgagg tgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaa gggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaat gcgccgctacagggcgcgatggatcc [1082] Full-length Phenylalanine Hydroxylase (PAH) (SEQ ID NO: 90) [1083] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKL ATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLY YVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK [1084] N-terminal PAH fragment C237/N-terminal NpuDnaE intein (SEQ ID NO: 91) [1085] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCLSYETEILTVEYGLLPIG KIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDG QMLPIDEIFERELDLMRVDNLPN [1086] C-terminal NpuDnaE intein/C-terminal PAH fragment C237 (SEQ ID NO: 92) [1087] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTGFRLRPVAGLLSSRDFLGGL AFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIY WFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAE SFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIK [1088] N-terminal PAH fragment C265/N-terminal NpuDnaE intein (SEQ ID NO: 93) [1089] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQ EVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN [1090] C-terminal NpuDnaE intein/C-terminal PAH fragment C265 (SEQ ID NO: 94) [1091] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTQYIRHGSKPMYTPEPDICHE LLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFG ELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPY TQRIEVLDNTQQLKILADSINSEIGILCSALQKIK [1092] N-terminal PAH fragment C284/N-terminal NpuDnaE intein (SEQ ID NO: 95) [1093] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCTQYIRHGSKPMYTPEPDICLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNN GNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMR VDNLPN [1094] C-terminal NpuDnaE intein/C-terminal PAH fragment C284 (SEQ ID NO: 96) [1095] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHELLGHVPLFSDRSFAQFSQE IGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEK TAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSI NSEIGILCSALQKIK [1096] N-terminal PAH fragment C334/N-terminal NpuDnaE intein (SEQ ID NO: 97) [1097] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKL ATIYWFTVEFGLCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN [1098] C-terminal NpuDnaE intein/C-terminal PAH fragment C334 (SEQ ID NO: 98) [1099] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCKQGDSIKAYGAGLLSSFGEL QYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQ RIEVLDNTQQLKILADSINSEIGILCSALQKIK [1100] GTP-CH1 (SEQ ID NO: 99) [1101] MEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPR SEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDE DHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQI AVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS [1102] P2A (SEQ ID NO: 100) [1103] GSGATNFSLLKQAGDVEENPGP [1104] Full-length PAH-P2A-(GTP-CH1) (SEQ ID NO: 101) [1105] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKL ATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLY YVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIKG SGATNFSLLKQAGDVEENPGPMEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPR PEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASA MQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQ VLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQK MNSKTVTSTMLGVFREDPKTREEFLTLIRS [1106] N-terminal PAH fragment C237/N-terminal NpuDnaE intein-P2A-(GTP-CH1) (SEQ ID NO: 102) [1107] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCLSYETEILTVEYGLLPIG KIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDG QMLPIDEIFERELDLMRVDNLPNGSGATNFSLLKQAGDVEENPGPMEKGPVRAPAEKPRGAR CSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSL GENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHL VPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATH MCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS [1108] C-terminal NpuDnaE intein/C-terminal PAH fragment C237-P2A-(GTP-CH1) (SEQ ID NO: 103) [1109] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTGFRLRPVAGLLSSRDFLGGL AFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIY WFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAE SFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSINSEIGILCSALQKIKGSGAT NFSLLKQAGDVEENPGPMEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSA QPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQ ETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIY SRRLQVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDP KTREEFLTLIRS [1110] N-terminal PAH fragment C265/N-terminal NpuDnaE intein-P2A-(GTP-CH1) SEQ ID NO: 104 [1111] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQ EVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNGSGATNFSLLKQ AGDVEENPGPMEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGW KGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLN DAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQ ERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFL TLIRS [1112] C-terminal NpuDnaE intein/C-terminal PAH fragment C265-P2A-(GTP-CH1) SEQ ID NO: 105 [1113] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCTQYIRHGSKPMYTPEPDICHE LLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFG ELQYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPY TQRIEVLDNTQQLKILADSINSEIGILCSALQKIKGSGATNFSLLKQAGDVEENPGPMEKGPVRAP AEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAA AYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDM FSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGV GVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS [1114] N-terminal PAH fragment C284/N-terminal NpuDnaE intein-P2A-(GTP-CH1) SEQ ID NO: 106 [1115] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCTQYIRHGSKPMYTPEPDICLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNN GNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMR VDNLPNGSGATNFSLLKQAGDVEENPGPMEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRP AEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAA SAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQV LGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTV TSTMLGVFREDPKTREEFLTLIRS [1116] C-terminal NpuDnaE intein/C-terminal PAH fragment C284-P2A-(GTP-CH1) SEQ ID NO: 107 [1117] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHELLGHVPLFSDRSFAQFSQE IGLASLGAPDEYIEKLATIYWFTVEFGLCKQGDSIKAYGAGLLSSFGELQYCLSEKPKLLPLELEK TAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQRIEVLDNTQQLKILADSI NSEIGILCSALQKIKGSGATNFSLLKQAGDVEENPGPMEKGPVRAPAEKPRGARCSNGFPERDPP RPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLK TPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGY LPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQK MNSKTVTSTMLGVFREDPKTREEFLTLIRS [1118] N-terminal PAH fragment C334/N-terminal NpuDnaE intein-P2A-(GTP-CH1) SEQ ID NO: 108 [1119] MSTAVLENPGLGRKLSDFGQETSYIEDNCNQNGAISLIFSLKEEVGALAKVLRLFEENDV NLTHIESRPSRLKKDEYEFFTHLDKRSLPALTNIIKILRHDIGATVHELSRDKKKDTVPWFPRTIQE LDRFANQILSYGAELDADHPGFKDPVYRARRKQFADIAYNYRHGQPIPRVEYMEEEKKTWGTV FKTLKSLYKTHACYEYNHIFPLLEKYCGFHEDNIPQLEDVSQFLQTCTGFRLRPVAGLLSSRDFL GGLAFRVFHCTQYIRHGSKPMYTPEPDICHELLGHVPLFSDRSFAQFSQEIGLASLGAPDEYIEKL ATIYWFTVEFGLCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDR GEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNGSGATNFSL LKQAGDVEENPGPMEKGPVRAPAEKPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPAD GWKGERPRSEEDNELNLPNLAAAYSSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISD VLNDAIFDEDHDEMVIVKDIDMFSMCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRL QVQERLTKQIAVAITEALRPAGVGVVVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTRE EFLTLIRS [1120] C-terminal NpuDnaE intein/C-terminal PAH fragment C334-P2A-(GTP-CH1) SEQ ID NO: 109 [1121] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCKQGDSIKAYGAGLLSSFGEL QYCLSEKPKLLPLELEKTAIQNYTVTEFQPLYYVAESFNDAKEKVRNFAATIPRPFSVRYDPYTQ RIEVLDNTQQLKILADSINSEIGILCSALQKIKGSGATNFSLLKQAGDVEENPGPMEKGPVRAPAE KPRGARCSNGFPERDPPRPGPSRPAEKPPRPEAKSAQPADGWKGERPRSEEDNELNLPNLAAAY SSILSSLGENPQRQGLLKTPWRAASAMQFFTKGYQETISDVLNDAIFDEDHDEMVIVKDIDMFS MCEHHLVPFVGKVHIGYLPNKQVLGLSKLARIVEIYSRRLQVQERLTKQIAVAITEALRPAGVGV VVEATHMCMVMRGVQKMNSKTVTSTMLGVFREDPKTREEFLTLIRS [1122] EGFP SEQ ID NO: 110 [1123] MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWP TLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVN RIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQN TPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKYSDLELK [1124] mCherry SEQ ID NO: 111 [1125] MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGP LPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDG EFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVK TTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYK [1126] Full-length glutamine synthetase (GS) SEQ ID NO: 112 [1127] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNW NGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNIND FSAGVANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1128] N-terminal GS fragment C53/N-terminal NpuDnaE intein SEQ ID NO: 113 [1129] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCLSYETE ILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDH KFMTVDGQMLPIDEIFERELDLMRVDNLPN [1130] C-terminal NpuDnaE intein/C-terminal GS fragment C53 SEQ ID NO: 114 [1131] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCVEELPEWNFDGSSTLQSEGSN SDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMDMVSNQHPWFGMEQE YTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVM PAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMR EENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRT VGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1132] N-terminal GS fragment C117/N-terminal NpuDnaE intein SEQ ID NO: 115 [1133] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCLSYET EILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRA TKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN [1134] C-terminal NpuDnaE intein/C-terminal GS fragment C117 SEQ ID NO: 116 [1135] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCKRIMDMVSNQHPWFGMEQE YTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRACLYAGVKIAGTNAEVM PAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMR EENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRT VGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1136] N-terminal GS fragment C183/N-terminal NpuDnaE intein SEQ ID NO: 117 [1137] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDG SLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN [1138] C-terminal NpuDnaE intein/C-terminal GS fragment C183 SEQ ID NO: 118 [1139] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCLYAGVKIAGTNAEVMPAQW EFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNWNGAGCHTNFSTKAMREENG LKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQE KKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1140] N-terminal GS fragment C229/N-terminal NpuDnaE intein SEQ ID NO: 119 [1141] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCLSYETEILTVEYGLLPIG KIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDG QMLPIDEIFERELDLMRVDNLPN [1142] C-terminal NpuDnaE intein/C-terminal GS fragment C229 SEQ ID NO: 120 [1143] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCEDFGVIATFDPKPIPGNWNGA GCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAG VANRSASIRIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1144] N-terminal GS fragment C252/N-terminal NpuDnaE intein SEQ ID NO: 121 [1145] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNW NGAGCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFE YCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN [1146] C-terminal NpuDnaE intein/C-terminal GS fragment C252 SEQ ID NO: 122 [1147] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHTNFSTKAMREENGLKYIEE AIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNINDFSAGVANRSASIRIPRTVGQEKKGYF EDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1148] Full-length Thymidylate Synthase (TYMS) SEQ ID NO: 123 [1149] MPVAGSELPRRPLPPAAQERDAEPRPPHGELQYLGQIQHILRCGVRKDDRTGTGTLSVFG MQARYSLRDEFPLLTTKRVFWKGVLEELLWFIKGSTNAKELSSKGVKIWDANGSRDFLDSLGFS TREEGDLGPVYGFQWRHFGAEYRDMESDLPLMALPPCHALCQFYVVNSELSCQLYQRSGDMG LGVPFNIASYALLTYMIAHITGLKPGDFIHTLGDAHIYLNHIEPLKIQLQREPRPFPKLRILRKVEKI DDFKAEDFQIEGYNPHPTIKMEMAV [1150] N-terminal TYMS fragment C41/N-terminal NpuDnaE intein SEQ ID NO: 124 [1151] MPVAGSELPRRPLPPAAQERDAEPRPPHGELQYLGQIQHILRCLSYETEILTVEYGLLPIG KIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQML PIDEIFERELDLMRVDNLPN [1152] C-terminal NpuDnaE intein/C-terminal TYMS fragment C41 SEQ ID NO: 125 [1153] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCGVRKDDRTGTGTLSVFGMQ ARYSLRDEFPLLTTKRVFWKGVLEELLWFIKGSTNAKELSSKGVKIWDANGSRDFLDSLGFSTR EEGDLGPVYGFQWRHFGAEYRDMESDLPLMALPPCHALCQFYVVNSELSCQLYQRSGDMGLG VPFNIASYALLTYMIAHITGLKPGDFIHTLGDAHIYLNHIEPLKIQLQREPRPFPKLRILRKVEKIDD FKAEDFQIEGYNPHPTIKMEMAV [1154] N-terminal TYMS fragment C161/N-terminal NpuDnaE intein SEQ ID NO: 126 [1155] MPVAGSELPRRPLPPAAQERDAEPRPPHGELQYLGQIQHILRCGVRKDDRTGTGTLSVFG MQARYSLRDEFPLLTTKRVFWKGVLEELLWFIKGSTNAKELSSKGVKIWDANGSRDFLDSLGFS TREEGDLGPVYGFQWRHFGAEYRDMESDLPLMALPPCLSYETEILTVEYGLLPIGKIVEKRIE CTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEI FERELDLMRVDNLPN [1156] C-terminal NpuDnaE intein/C-terminal TYMS fragment C161 SEQ ID NO: 127 [1157] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCHALCQFYVVNSELSCQLYQR SGDMGLGVPFNIASYALLTYMIAHITGLKPGDFIHTLGDAHIYLNHIEPLKIQLQREPRPFPKLRIL RKVEKIDDFKAEDFQIEGYNPHPTIKMEMAV [1158] N-terminal TYMS fragment C165/N-terminal NpuDnaE intein SEQ ID NO: 128 [1159] MPVAGSELPRRPLPPAAQERDAEPRPPHGELQYLGQIQHILRCGVRKDDRTGTGTLSVFG MQARYSLRDEFPLLTTKRVFWKGVLEELLWFIKGSTNAKELSSKGVKIWDANGSRDFLDSLGFS TREEGDLGPVYGFQWRHFGAEYRDMESDLPLMALPPCHALCLSYETEILTVEYGLLPIGKIVE KRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLP IDEIFERELDLMRVDNLPN [1160] C-terminal NpuDnaE intein/C-terminal TYMS fragment C165 SEQ ID NO: 129 [1161] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCQFYVVNSELSCQLYQRSGDM GLGVPFNIASYALLTYMIAHITGLKPGDFIHTLGDAHIYLNHIEPLKIQLQREPRPFPKLRILRKVE KIDDFKAEDFQIEGYNPHPTIKMEMAV [1162] N-terminal TYMS fragment C176/N-terminal NpuDnaE intein SEQ ID NO: 130 [1163] MPVAGSELPRRPLPPAAQERDAEPRPPHGELQYLGQIQHILRCGVRKDDRTGTGTLSVFG MQARYSLRDEFPLLTTKRVFWKGVLEELLWFIKGSTNAKELSSKGVKIWDANGSRDFLDSLGFS TREEGDLGPVYGFQWRHFGAEYRDMESDLPLMALPPCHALCQFYVVNSELSCLSYETEILTVE YGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHK FMTVDGQMLPIDEIFERELDLMRVDNLPN [1164] C-terminal NpuDnaE intein/C-terminal TYMS fragment C176 SEQ ID NO: 131 [1165] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNCQLYQRSGDMGLGVPFNIASY ALLTYMIAHITGLKPGDFIHTLGDAHIYLNHIEPLKIQLQREPRPFPKLRILRKVEKIDDFKAEDFQI EGYNPHPTIKMEMAV [1166] Attenuated EF1alpha promoter SEQ ID NO: 132 [1167] AAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAG TCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCG GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGA ACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAA CACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGC CGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGC GTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTT GGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTAC GTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC [1168] EF1alpha promoter SEQ ID NO: 133 [1169] AAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCC ACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAA GGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGC AACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTC ACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCC GCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCT CAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCG GCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCT GTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTAC [1170] CMV promoter SEQ ID NO: 134 [1171] ACTAGTATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTAC ATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAAT GGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTC AATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAA CTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTTTATATAA GCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTG ACCTCCATAGAAGA [1172] IRES SEQ ID NO: 135 [1173] GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAA GGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATG TGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCC CTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTG GAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCC CCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTG CAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTC AAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACC CCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTC GAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAA AAACACGATGATAATATGGCCACAACC [1174] Rep2BFP-CODE/Cap5 construct SEQ ID NO: 136 [1175] GGAGGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTA GAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGGTATTTAAGC CCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACGCGCAGCCGCCATGC CGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATT TCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACA TGGATCTGAATCTGATTGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTT TCTGACGGAATGGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGA AGGGAGAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGT TTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCG AGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAA CAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTCC AGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGAGCGTAA ACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGAACAAAGAGAA TCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCAGCCAGGTACATGGAG CTGGTCGGGTGGCTCGTGGACAAGGTGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCT ATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAG ATGTCCCTTGTATCACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGA CAACCGTTGTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGC ATTTGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATC ACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACATA ACTTCGTATAAAGTATACTATACGAAGTTATCGGGCCCCTCTGCTAACCATGTTCATGCCTTC TTCTTTTTCCTACAGATGTCAGAACTCATTAAAGAGAATATGCACATGAAGCTGTATATGGA AGGTACTGTAGACAACCACCATTTCAAATGCACGTCCGAAGGTGAGGGGAAGCCATACGAG GGTACCCAAACTATGCGCATCAAAGTGGTTGAGGGTGGCCCCCTGCCATTCGCATTCGACAT CCTGGCAACTAGCTTTCTTTACGGTTCCAAGACATTCATAAATCATACCCAGGGTATTCCCG ATTTCTTCAAACAATCCTTCCCGGAAGGGTTTACTTGGGAGCGGGTCACGACATATGAAGAC GGGGGTGTTCTTACAGCCACACAGGATACGAGTTTGCAAGACGGTTGTCTTATCTATAACGT GAAGATTCGGGGTGTGAATTTCACATCCAATGGCCCGGTGATGCAGAAAAAAACACTGGGC TGGGAAGCATTTACGGAGACGTTGTATCCCGCCGATGGAGGTCTCGAGGGCCGAAACGATA TGGCCCTCAAGTTGGTAGGTGGTTCTCACCTTATAGCAAACATTAAGACCACGTATCGATCA AAAAAACCCGCTAAGAATCTGAAAATGCCAGGCGTGTATTATGTTGATTACAGACTGGAGC GAATAAAAGAGGCTAACAATGAGACCTACGTCGAACAGCATGAAGTCGCTGTAGCTAGATA TTGCGACCTCCCGTCAAAGTTGGGCCATAAATTGAATTAACCTCAGGTGCAGGCTGCCTATC AGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCC TCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAG GAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATA TGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATG CCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACA GCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTAT ATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCC AGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCA TAACTTCGTATAAAGTATACTATACGAAGTTATAATTGTTATAATTAAATGATAAGGTAGAA TATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACACC CTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAGGATA AAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTA CAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCTCATACATCTCCTTCA ATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTAT GAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCC AGCAATCGGATTTATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGT CTTTCTGGGATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCT GCAACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGT GCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGG TGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGA AGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGA TCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCGAACAC CAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTGGATCATGACTT TGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGCAAAGGATCACGTGGTT GAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGT GACGCAGATATAAGTGAGCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAG ACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGG CATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCT GCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTT TCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCC AGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAAT AAATGATTTGTAAATAAATTTAGTAGTCATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGA AGAAGTTGGTGAAGGTCTTCGCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAA CCCAATCAGCAGCATCAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGG ACCCGGAAACGGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGA GCACGACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAAC CACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCG GAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTTGAAGAGGG TGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAAAGAAAGAAGGCT CGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCTGGACCCAGCGGAT CCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGGAGCTGATACAATGTCTGC GGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTGCCGATGGAGTGGGCAATGCCTCG GGAGATTGGCATTGCGATTCCACGTGGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAA CCTGGGTGCTGCCCAGCTACAACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGA CGGAAGCAACGCCAACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACC GCTTCCACAGCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTT CAGACCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAG GACTCCACCACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGA CTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCTCCGC AGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACACAGAAAATCC CACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGATGCTGAGAACGGGC AACAACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCCACTCCAGCTTCGCTCCCAG TCAGAACCTGTTCAAGCTGGCCAACCCGCTGGTGGACCAGTACTTGTACCGCTTCGTGAGCA CAAATAACACTGGCGGAGTCCAGTTCAACAAGAACCTGGCCGGGAGATACGCCAACACCTA CAAAAACTGGTTCCCGGGGCCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTC AACCGCGCCAGTGTCAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTT ACCAGGTGCCCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGC CCTGGAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTAC CTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGT ACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCCACCACTGCCCCCGCGACCGG CACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGACGTGTACCTC CAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCACCCCTCTCCGGCCA TGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTCATCAAGAACACGCCTGTGCC CGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCAGCTTCATCACCCAGTACAGCACCG GGCAGGTCACCGTGGAGATGGAGTGGGAGCTCAAGAAGGAAAACTCCAAGAGGTGGAACC CAGAGATCCAGTACACAAACAACTACAACGACCCCCAGTTTGTGGACTTTGCCCCGGACAG CACCGGGGAATACAGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAA TTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCTGCGTATTTCTT TCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAGC CCGGGCGTTTAAACAGCGGGCGGAGGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTA GGGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGT [1176] N-terminal Blasticidin fragment/N-terminal NpuDnaE Intein SEQ ID NO: 137 [1177] GAAAACATTTAACATTTCTCAACAGGATCTAGAATTAGTAGAAGTAGCGACAGAGA AGATTACAATGCTTTATGAGGATAATAAACATCATGTGGGAGCGGCAATTCGTACGAAAAC AGGAGAAATCATTTCGGCAGTACATATTGAAGCGTATATAGGACGAGTAACTGTTTGTGCA GAAGCCATTGCGATTGGTAGTGCAGTTTCGAATGGACAAAAGGATTTTGACACGATTGTAG CTGTTAGACACCCTTATTCTGACGAAGTAGATAGAAGTATTCGAGTGGTAAGTCCTTGTGGT ATGTGCCTTTCATACGAGACCGAGATCCTGACTGTCGAGTACGGATTGCTTCCTATCGGCAA AATCGTGGAGAAGAGGATTGAATGTACCGTCTATTCAGTCGATAATAATGGGAACATCTAC ACACAGCCCGTGGCTCAATGGCACGACAGAGGAGAGCAGGAAGTTTTTGAATACTGTCTCG AGGACGGATCCCTCATCCGCGCTACTAAAGATCATAAGTTTATGACCGTGGACGGCCAGAT GCTGCCAATTGACGAAATTTTTGAACGAGAGCTGGATCTGATGAGAGTCGACAACCTTCCA AACTGA [1178] C-terminal NpuDnaE Intein/C-terminal Blasticidin fragment SEQ ID NO: 138 [1179] ATGATTAAGATCGCTACGCGGAAGTACCTGGGGAAACAGAACGTCTACGACATAGG TGTGGAGCGCGATCACAACTTTGCTCTGAAAAATGGATTTATCGCCAGCAACTGTAGGGAGT TGATTTCAGACTATGCACCAGATTGTTTTGTGTTAATAGAAATGAATGGCAAGTTAGTCAAA ACTACGATTGAAGAACTCATTCCACTCAAATATACCCGAAATT [1180] GFP AAV (ITR to ITR) SEQ ID NO: 139 [1181] GCTTTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAA GGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAG GGAGTGGCCAACTCCATCACTAGGGGTTCCTCTGCAGCCGCGACCGGCCAAGGTTTAATGAT AGGCTGCAACGGGATGTTGGGAATATGTTGCACTGGTCCGTGAGGGTACCAACTTGTTTATT GCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTT TTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGACCGGT TCACTTGAGCTCGAGATCTGAGTACTTGTACAGCTCGTCCATGCCGAGAGTGATCCCGGCGG CGGTCACGAACTCCAGCAGGACCATGTGATCGCGCTTCTCGTTGGGGTCTTTGCTCAGGGCG GACTGGGTGCTCAGGTAGTGGTTGTCGGGCAGCAGCACGGGGCCGTCGCCGATGGGGGTGT TCTGCTGGTAGTGGTCGGCGAGCTGCACGCTGCCGTCCTCGATGTTGTGGCGGATCTTGAAG TTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTG TACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATG CGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTC CTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCG TGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCA CGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTT GCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTGGCCGTTTACGTCGC CGTCCAGCTCGACCAGGATGGGCACCACCCCGGTGAACAGCTCCTCGCCCTTGCTCACCATG GTGGCGGCTTAAGGGTTCGATCCTCTAGAGTCCGGAGGCTGGATCGGTCCCGGTGTCTACTA TGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTC TGCTTATATAGACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTG TTACGACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAAT GGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCC AAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTGCCAAGTAGGAAA GTCCCATAAGGTCATGTACTGGGCATAATGCCAGGCGGGCCATTTACCGTCATTGACGTCAA TAGGGGGCGTACTTGGCATATGATACACTTGATGTACTGCCAAGTGGGCAGTTTACCGTAAA TACTCCACCCATTGACGTCAATGGAAAGTCCCTATTGGCGTTACTATGGGAACATACGTCAT TATTGACGTCAATGGGCGGGGGTCGTTGGGCGGTCAGCCAGGCGGGCCATTTACCGTAAGT TATGTAACGCGGAACTCCATATATGGGCTATGAACTAATGACCCCGTAATTGATTACTATTA ATAACTAGTCAATAATCAATGTCAACGCGTATGGTACCTGCGGAGGATGCCGAGGATAACC TTGTTACTAGCCTCCGCCTGGCCGTTGGACTGTGGATAATATGGCGTAGAGGATCCTCTGCG CGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGC CCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAA [1182] N-term NpuDnaE intein fragment SEQ ID NO: 140 [1183] CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCL EDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPN [1184] C-term NpuDnaE intein fragment SEQ ID NO: 141 [1185] MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN [1186] Mutant GS R324C SEQ ID NO: 142 [1187] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNW NGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNIND FSAGVANRSASICIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1188] Mutant GS R324S SEQ ID NO: 143 [1189] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNW NGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNIND FSAGVANRSASISIPRTVGQEKKGYFEDRRPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1190] Mutant GS R341C SEQ ID NO: 144 [1191] MTTSASSHLNKGIKQVYMSLPQGEKVQAMYIWIDGTGEGLRCKTRTLDSEPKCVEELPE WNFDGSSTLQSEGSNSDMYLVPAAMFRDPFRKDPNKLVLCEVFKYNRRPAETNLRHTCKRIMD MVSNQHPWFGMEQEYTLMGTDGHPFGWPSNGFPGPQGPYYCGVGADRAYGRDIVEAHYRAC LYAGVKIAGTNAEVMPAQWEFQIGPCEGISMGDHLWVARFILHRVCEDFGVIATFDPKPIPGNW NGAGCHTNFSTKAMREENGLKYIEEAIEKLSKRHQYHIRAYDPKGGLDNARRLTGFHETSNIND FSAGVANRSASIRIPRTVGQEKKGYFEDRCPSANCDPFSVTEALIRTCLLNETGDEPFQYKN [1192] P5 promoter sequence SEQ ID NO: 145 [1193] GGAGGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTA GAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTG [1194] Progranulin payload (ITR to ITR) SEQ ID NO: 146 [1195] CACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTC GCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGA GGGAGTGGCCAACTCCATCACTAGGGGTTCCTCTGCAGTGAGAGACACAAAAAATT CCAACACACTATTGCAATGAAAATAAATTTCCTTTATTTCACAGCAGCTGTCTCAAG GCTGGGTCCCTCAAAGGGGCGTCCCAGCGCGGGGCCTCCCTGCGCAAACACTTGGT ACCCCTGGCTGCGCAGCGGAAGCCAGCAGGACAGCAGTGGCGCCGATCAGCACAA CAGACGCCCTGGCGGTAGGGACAGCAGGCCCAGCCCTGTCGGTTGTCTCGGCAGCA GGTCTGGTTATCATGGCAGAAGTGTCCTTCCCCACACTCCACGTCCTTCACACCCAC GTGAGGGCTACGGGCCAGGAAGGTGGCAGGCTGGGCAGAGACCACTTCCTTCTCGC AGGATCGAGCCTTCACGTTGCAGGTGTAGCCAGCCGGGCAGCAGTGCTGGCGATCC TCGCAGCACACAGCATGGGGCAACTGGCAGCAGGCCCAGCTCCCACCCAGGCTCGG GCAGCAGGTCTGCCCCACCGGGCAGCTGGTGTGCTGGTCACAGCCGATGTCTCTGG GGTGGGATAAGGAAGCCCGGCGGGCAGGCATCTTCTCCAGTCCAGCCACGATCTCG CTTCCTCGCTGACACTGCCCCTCAGCTACACACGTGTAGCCCTGGGGGCAGCAGTGC TGGTGGTCCGAGCAGCAGACAGCCTCTGGGATTGGACAGCAGCCCCACTCCCCAGA CGTGAGTTGGCAGCAGGTATCGGAGGAGGGACAGCTGCTGACATTATCACAGGGGA CATCTCTCTTCAAGGCTTGTGGGTCTGGCAGGCTGAGGTGAGCTGGGGCCTTCTCCA TCCAGGGCACCTGGTGGGGCCCCTGTTCACAGGTACCCTTCTGCGTGTCACACGTAA ACCCCGCGGGACAGCAGTGTATGTGGTCCTCACAGCACACAGCCTGGGTAAAAGGG CAGCAGCCCCAGGCCCCCGACTGTAGACGGCAGCAGGTATAGCCATCTGGGCAGCT CACCTCCATGTCACATTTCACATCCCCCACTGTGTGCGCAGGCAGCTTAGTGAGGAG GTCCGTGGTAGCGTTCTCCTTGGAGAGGCACTTACTCTGGATCAGGTCACACACAGT GTCTTGGGGGCAGCAGTGCAGGTGATCGGAGCAGCAGGTGGCGTTGGGCATTGGGC AGCAGCCATACTTCCCACTGGGCAGCTCACAGCAGGTAGAACCATCAGGGCACCGG GACCGTGCGTCCGGACACATGACCGAGCTGGACAAGGCCACTGCCCTGTTAGTCCT CTGGGCAGGGAGCTTCTTTGCCAGGGGGTGGGTGCCCGTGGGTGTGATGCAGCGGG TGTGAACCAGGTCGCAGAAGGCACCGTGCGGACAGCAGTGCACCCTGTCTTCACAG CAGGAAGCCTGGGGCATGGGGCAGCACCCCCAGGAGCCATCGACCATAACACAGC ACGTGGAGAAGTCCGGGCATTCGAACTGACTATCAGGGCACTGGATGGCACCCACG GAGTTGTTACCTGATCTTTGGAAGCAGGATCGCCCGTCTGCACTGCAGTGGAAGCCC CGTGGGCAGCAGTGATGGCCATCCCCGCATGCCACGGCCTCTGGGAAGGGGCAGCA ACTGGAAGTCCCTGAGACGGTAAAGATGCAGGAGTGGCCGGCAGAGCAGTGGGCA TCAACCTGGCAGGGGCCACCCAGATGCCTGCTCAGTGTTGTGGGCCATTTGTCCAGA AGGGGACGGCAGCAGCTGTAGCTGGCTCCTCCGGGGTCCAGGCAGCAGGCCACAG GGCAGAACTGACCATCTGGGCACCGCGTTCCAGCCACCAGCCCTGCTGTTAAGGCC ACCCAGCTCACCAGGGTCCACATGGTGGCGGCGCTAGCGGTGCACACCAATGTGGT GAATGGTCAAATGGCGTTTATTGTATCGAGCTAGGCACTTAAATACAATATCTCTGC AATGCGGAATTCAGTGGTTCGTCCAATCCATGTCAGACCCGTCTGTTGCCTTCCTAA TAAGGCACGATCGTACCACCTTACTTCCACCAATCGGCATGCACGGTGCTTTTTCTC TCCTTGTAAGGCATGTTGCTAACTCATCGTTACCATGTTGCAAGACTACAAGAGTAT TGCATAAGACTACATTGGATCCTCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGG CAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCG CGCAGAGA [1196] Progranulin Payload Plasmid (STX_C0230) SEQ ID NO: 147 [1197] CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA CATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGC GTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAAT CCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGA ACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTC TATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCG AGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCG CCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACT GTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG GGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACG TTGTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGA GGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGA TGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATT GTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTG TATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTT ACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAA AAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATTTCTTTCCTGCG TTATCTTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA AAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCG CAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCTGCAGTGAGAGACACAA AAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTATTTCACAGCAGCTGT CTCAAGGCTGGGTCCCTCAAAGGGGCGTCCCAGCGCGGGGCCTCCCTGCGCAAACA CTTGGTACCCCTGGCTGCGCAGCGGAAGCCAGCAGGACAGCAGTGGCGCCGATCAG CACAACAGACGCCCTGGCGGTAGGGACAGCAGGCCCAGCCCTGTCGGTTGTCTCGG CAGCAGGTCTGGTTATCATGGCAGAAGTGTCCTTCCCCACACTCCACGTCCTTCACA CCCACGTGAGGGCTACGGGCCAGGAAGGTGGCAGGCTGGGCAGAGACCACTTCCTT CTCGCAGGATCGAGCCTTCACGTTGCAGGTGTAGCCAGCCGGGCAGCAGTGCTGGC GATCCTCGCAGCACACAGCATGGGGCAACTGGCAGCAGGCCCAGCTCCCACCCAGG CTCGGGCAGCAGGTCTGCCCCACCGGGCAGCTGGTGTGCTGGTCACAGCCGATGTC TCTGGGGTGGGATAAGGAAGCCCGGCGGGCAGGCATCTTCTCCAGTCCAGCCACGA TCTCGCTTCCTCGCTGACACTGCCCCTCAGCTACACACGTGTAGCCCTGGGGGCAGC AGTGCTGGTGGTCCGAGCAGCAGACAGCCTCTGGGATTGGACAGCAGCCCCACTCC CCAGACGTGAGTTGGCAGCAGGTATCGGAGGAGGGACAGCTGCTGACATTATCACA GGGGACATCTCTCTTCAAGGCTTGTGGGTCTGGCAGGCTGAGGTGAGCTGGGGCCT TCTCCATCCAGGGCACCTGGTGGGGCCCCTGTTCACAGGTACCCTTCTGCGTGTCAC ACGTAAACCCCGCGGGACAGCAGTGTATGTGGTCCTCACAGCACACAGCCTGGGTA AAAGGGCAGCAGCCCCAGGCCCCCGACTGTAGACGGCAGCAGGTATAGCCATCTGG GCAGCTCACCTCCATGTCACATTTCACATCCCCCACTGTGTGCGCAGGCAGCTTAGT GAGGAGGTCCGTGGTAGCGTTCTCCTTGGAGAGGCACTTACTCTGGATCAGGTCAC ACACAGTGTCTTGGGGGCAGCAGTGCAGGTGATCGGAGCAGCAGGTGGCGTTGGGC ATTGGGCAGCAGCCATACTTCCCACTGGGCAGCTCACAGCAGGTAGAACCATCAGG GCACCGGGACCGTGCGTCCGGACACATGACCGAGCTGGACAAGGCCACTGCCCTGT TAGTCCTCTGGGCAGGGAGCTTCTTTGCCAGGGGGTGGGTGCCCGTGGGTGTGATG CAGCGGGTGTGAACCAGGTCGCAGAAGGCACCGTGCGGACAGCAGTGCACCCTGTC TTCACAGCAGGAAGCCTGGGGCATGGGGCAGCACCCCCAGGAGCCATCGACCATAA CACAGCACGTGGAGAAGTCCGGGCATTCGAACTGACTATCAGGGCACTGGATGGCA CCCACGGAGTTGTTACCTGATCTTTGGAAGCAGGATCGCCCGTCTGCACTGCAGTGG AAGCCCCGTGGGCAGCAGTGATGGCCATCCCCGCATGCCACGGCCTCTGGGAAGGG GCAGCAACTGGAAGTCCCTGAGACGGTAAAGATGCAGGAGTGGCCGGCAGAGCAG TGGGCATCAACCTGGCAGGGGCCACCCAGATGCCTGCTCAGTGTTGTGGGCCATTT GTCCAGAAGGGGACGGCAGCAGCTGTAGCTGGCTCCTCCGGGGTCCAGGCAGCAGG CCACAGGGCAGAACTGACCATCTGGGCACCGCGTTCCAGCCACCAGCCCTGCTGTT AAGGCCACCCAGCTCACCAGGGTCCACATGGTGGCGGCGCTAGCGGTGCACACCAA TGTGGTGAATGGTCAAATGGCGTTTATTGTATCGAGCTAGGCACTTAAATACAATAT CTCTGCAATGCGGAATTCAGTGGTTCGTCCAATCCATGTCAGACCCGTCTGTTGCCT TCCTAATAAGGCACGATCGTACCACCTTACTTCCACCAATCGGCATGCACGGTGCTT TTTCTCTCCTTGTAAGGCATGTTGCTAACTCATCGTTACCATGTTGCAAGACTACAA GAGTATTGCATAAGACTACATTGGATCCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTA ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC ACTAGAGCTAGCGAATTCGAATTTAAATCGGATCCGCGGCCGCAAGGATCTGCGAT CGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTT GGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACC GTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG AACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTAC CTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGC CTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCT TTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCT GACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGA TCCAAGCTGTGACCGGCGCCTACGATATCGCCACCATGAAAACATTTAACATTTCTC AACAGGATCTAGAATTAGTAGAAGTAGCGACAGAGAAGATTACAATGCTTTATGAG GATAATAAACATCATGTGGGAGCGGCAATTCGTACGAAAACAGGAGAAATCATTTC GGCAGTACATATTGAAGCGTATATAGGACGAGTAACTGTTTGTGCAGAAGCCATTG CGATTGGTAGTGCAGTTTCGAATGGACAAAAGGATTTTGACACGATTGTAGCTGTTA GACACCCTTATTCTGACGAAGTAGATAGAAGTATTCGAGTGGTAAGTCCTTGTGGTA TGTGCCTTTCATACGAGACCGAGATCCTGACTGTCGAGTACGGATTGCTTCCTATCG GCAAAATCGTGGAGAAGAGGATTGAATGTACCGTCTATTCAGTCGATAATAATGGG AACATCTACACACAGCCCGTGGCTCAATGGCACGACAGAGGAGAGCAGGAAGTTTT TGAATACTGTCTCGAGGACGGATCCCTCATCCGCGCTACTAAAGATCATAAGTTTAT GACCGTGGACGGCCAGATGCTGCCAATTGACGAAATTTTTGAACGAGAGCTGGATC TGATGAGAGTCGACAACCTTCCAAACTGAGTGCATGACCCGCAAGCCCGGTGCCTG AAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCGTTAACT AAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAT GTATCTTATCATGTCTGGAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTC ATCTGGTCTCCCTTCCGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGT TCCCAAACTGGGTTCTTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGT CCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAAT TGACTGGATTCCTTTTTTAGGGCCCATTGGTATGGCTGATATCTATAACAAGAAAAT ATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGT ATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGC GGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGG GAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATT TAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCT TTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCA GTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTT TCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCAT TGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGC ACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGC TTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAAT ACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGC GCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGA CATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGC TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAA GCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC CTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGG TATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCAC [1198] Tet inducible Cap5 PolyA sequence SEQ ID NO: 148 [1199] GCCGCGAGTTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCC CTATCAGTGATAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGT ATAAGGAGTTTACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATC AGTGATAGAGAACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCC AGTTTACTCCCTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGC GCCTATAAAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCAC AACACTTTTGTCTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAAGTTGCGC AGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAAC AAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAG AGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGA GTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAA ACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGA TCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAG GTATGTCTTTTGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTC GCGAGTTTTTGGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCAT CAAGATCAAGCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAAC GGTCTCGATCGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACG ACATCTCGTACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAAC CACGCGGACGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGAAA CCTCGGAAAGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGG TTGAAGAGGGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCA AAAAGAAAGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACG CCGAAGCTGGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCA AGTTTGGGAGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAA CCAAGGTGCCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGT GGATGGGGGACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTAC AACAACCACCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACGCCA ACGCCTACTTTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACA GCCACTGGAGCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGA CCCCGGTCCCTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCA GGACTCCACCACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGA CGACGACTACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGG CCTTCCCTCCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCG ACAACACAGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCC AGCAAGATGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGAGGT GCCCTTCCACTCCAGCTTCGCTCCCAGTCAGAACCTGTTCAAGCTGGCCAACCCGCT GGTGGACCAGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGT TCAACAAGAACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGG CCCATGGGCCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGT CAGCGCCTTCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGC CCCCGCAGCCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTG GAGAACACTATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTA CCTCGAGGGCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGC GTGGCGTACAACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCCACCACTGC CCCCGCGACCGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGG AGAGGGACGTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGC GCACTTTCACCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCAT GATGCTCATCAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGC CCGTCAGCAGCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAG TGGGAGCTCAAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAA ACAACTACAACGACCCCCAGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATAC AGAACCACCAGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAACCGGTCAG ACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCT GCAATAAACAAG [1200] Tet inducible Cap5 SV40 PolyA plasmid (STX_C0150) SEQ ID NO: 149 [1201] TATAGGCATTTTGCTAGTGCCGCACTTCCGATCCCAGAAGTTTTGGATATTGG AGAGTTTTCTGAATCCCTGACATATTGTATTTCTCGACGAGCTCAAGGAGTTACGTT GCAAGATCTTCCAGAGACTGAGCTTCCCGCCGTTCTGCAGCCTGTCGCCGAGGCCAT GGACGCAATTGCTGCGGCAGACCTGAGCCAAACCTCCGGCTTTGGTCCATTTGGAC CGCAGGGAATCGGCCAGTATACAACATGGCGGGACTTCATCTGCGCAATAGCGGAC CCCCATGTATATCATTGGCAGACCGTAATGGACGACACTGTTTCTGCATCAGTTGCG CAAGCCTTGGATGAGCTGATGTTGTGGGCGGAAGATTGTCCCGAGGTTCGGCATCT CGTACACGCGGACTTTGGTAGCAATAATGTACTTACTGATAATGGTAGGATTACTGC CGTAATAGACTGGAGTGAGGCGATGTTCGGCGACAGTCAGTATGAGGTAGCGAATA TATTTTTTTGGAGACCATGGTTGGCTTGCATGGAACAACAAACCAGGTACTTCGAAC GCCGACACCCGGAGCTTGCAGGGAGCCCACGACTGAGAGCCTATATGCTTCGGATA GGTTTGGATCAGTTGTATCAGTCTCTCGTTGATGGTAATTTCGACGATGCAGCATGG GCACAGGGGCGATGTGATGCAATCGTTCGCAGCGGAGCTGGTACAGTTGGCAGAAC TCAAATTGCCAGGAGGAGCGCTGCGGTCTGGACGGACGGCTGCGTAGAAGTCCTGG CAGACTCTGGCAACCGGCGCCCCTCCACAAGACCCAGGGCCAAAGAATAATAGTGA AATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTT GCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCGTTAACTA AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTC ACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAT GTATCTTATCATGTCTGGAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTC ATCTGGTCTCCCTTCCGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGT TCCCAAACTGGGTTCTTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGT CCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAAT TGACTGGATTCCTTTTTTAGGGCCCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGG TGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCA CCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAG CGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGG GACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACA AGAAAATATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAA TTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGA CTCACGCGGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCC TCACGGGAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGC GCTATTTAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGA CTATCTTTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCG GCTCAGTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTG CCTTTTCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGC TGCATTGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGC CATGCACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTC GCGGCTTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGA ACAATACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGT GCGGCGCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAA ATGGACATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTC ATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGC CGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAA TTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATT AATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCT TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCT CACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGA ACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCT GGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAA GTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGA AGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCT TTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTT CGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCG ACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACT TATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGT ATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTC TTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCA GATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGT CTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCA AAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAA AGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCT ATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGA TAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA GACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGC CGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTG CCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCAT TGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGG TTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTA GCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCA TGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTC TGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGA GTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTA AAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACC GCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATC TTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAA AAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAA TATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGT ATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACC TAAATTGTAAGCGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCT CATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAG ACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAA CGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTAC GTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATC GGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGT GGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAG TGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTAC AGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTG CGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATT AAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTG AGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGAGGACGGGCAGACTCGCGGT GCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGATGCTCGACACGCTGCAGAA CACGCAGCTAGATTAACCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATA ATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATT AATTTGAATAGATATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTC AACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAACAAAAACTCAAAATTT CTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCCCCCCCCAAAGCCCCCAGG GATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCG CTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACAGCCCG GGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTG AGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCACGGCCAGCGGCCGC GAGTTTACTCCCTATCAGTGATAGAGAACGTATGAAGAGTTTACTCCCTATCAGTGA TAGAGAACGTATGCAGACTTTACTCCCTATCAGTGATAGAGAACGTATAAGGAGTT TACTCCCTATCAGTGATAGAGAACGTATGACCAGTTTACTCCCTATCAGTGATAGAG AACGTATCTACAGTTTACTCCCTATCAGTGATAGAGAACGTATATCCAGTTTACTCC CTATCAGTGATAGAGAACGTATAAGCTTTAGGCGTGTACGGTGGGCGCCTATAAAA GCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGCAATTCCACAACACTTTTGT CTTATACCAACTTTCCGTACCACTTCCTACCCTCGTAAAAGTTGCGCAGCCATCGAC GTCAGACGCGGAAGCTTCGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTC GTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAAT CAGAATTCAAATATCTGCTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCC GTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTAC ATTCATCATATCATGGGAAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAAT GTGGATTTGGATGACTGCATCTTTGAACAATAAATGATTTAAATCAGGTATGTCTTT TGTTGATCACCCTCCAGATTGGTTGGAAGAAGTTGGTGAAGGTCTTCGCGAGTTTTT GGGCCTTGAAGCGGGCCCACCGAAACCAAAACCCAATCAGCAGCATCAAGATCAA GCCCGTGGTCTTGTGCTGCCTGGTTATAACTATCTCGGACCCGGAAACGGTCTCGAT CGAGGAGAGCCTGTCAACAGGGCAGACGAGGTCGCGCGAGAGCACGACATCTCGT ACAACGAGCAGCTTGAGGCGGGAGACAACCCCTACCTCAAGTACAACCACGCGGA CGCCGAGTTTCAGGAGAAGCTCGCCGACGACACATCCTTCGGGGGAAACCTCGGAA AGGCAGTCTTTCAGGCCAAGAAAAGGGTTCTCGAACCTTTTGGCCTGGTTGAAGAG GGTGCTAAGACGGCCCCTACCGGAAAGCGGATAGACGACCACTTTCCAAAAAGAA AGAAGGCTCGGACCGAAGAGGACTCCAAGCCTTCCACCTCGTCAGACGCCGAAGCT GGACCCAGCGGATCCCAGCAGCTGCAAATCCCAGCCCAACCAGCCTCAAGTTTGGG AGCTGATACAATGTCTGCGGGAGGTGGCGGCCCATTGGGCGACAATAACCAAGGTG CCGATGGAGTGGGCAATGCCTCGGGAGATTGGCATTGCGATTCCACGTGGATGGGG GACAGAGTCGTCACCAAGTCCACCCGAACCTGGGTGCTGCCCAGCTACAACAACCA CCAGTACCGAGAGATCAAAAGCGGCTCCGTCGACGGAAGCAACGCCAACGCCTACT TTGGATACAGCACCCCCTGGGGGTACTTTGACTTTAACCGCTTCCACAGCCACTGGA GCCCCCGAGACTGGCAAAGACTCATCAACAACTACTGGGGCTTCAGACCCCGGTCC CTCAGAGTCAAAATCTTCAACATTCAAGTCAAAGAGGTCACGGTGCAGGACTCCAC CACCACCATCGCCAACAACCTCACCTCCACCGTCCAAGTGTTTACGGACGACGACT ACCAGCTGCCCTACGTCGTCGGCAACGGGACCGAGGGATGCCTGCCGGCCTTCCCT CCGCAGGTCTTTACGCTGCCGCAGTACGGTTACGCGACGCTGAACCGCGACAACAC AGAAAATCCCACCGAGAGGAGCAGCTTCTTCTGCCTAGAGTACTTTCCCAGCAAGA TGCTGAGAACGGGCAACAACTTTGAGTTTACCTACAACTTTGAGGAGGTGCCCTTCC ACTCCAGCTTCGCTCCCAGTCAGAACCTGTTCAAGCTGGCCAACCCGCTGGTGGACC AGTACTTGTACCGCTTCGTGAGCACAAATAACACTGGCGGAGTCCAGTTCAACAAG AACCTGGCCGGGAGATACGCCAACACCTACAAAAACTGGTTCCCGGGGCCCATGGG CCGAACCCAGGGCTGGAACCTGGGCTCCGGGGTCAACCGCGCCAGTGTCAGCGCCT TCGCCACGACCAATAGGATGGAGCTCGAGGGCGCGAGTTACCAGGTGCCCCCGCAG CCGAACGGCATGACCAACAACCTCCAGGGCAGCAACACCTATGCCCTGGAGAACAC TATGATCTTCAACAGCCAGCCGGCGAACCCGGGCACCACCGCCACGTACCTCGAGG GCAACATGCTCATCACCAGCGAGAGCGAGACGCAGCCGGTGAACCGCGTGGCGTAC AACGTCGGCGGGCAGATGGCCACCAACAACCAGAGCTCCACCACTGCCCCCGCGAC CGGCACGTACAACCTCCAGGAAATCGTGCCCGGCAGCGTGTGGATGGAGAGGGAC GTGTACCTCCAAGGACCCATCTGGGCCAAGATCCCAGAGACGGGGGCGCACTTTCA CCCCTCTCCGGCCATGGGCGGATTCGGACTCAAACACCCACCGCCCATGATGCTCAT CAAGAACACGCCTGTGCCCGGAAATATCACCAGCTTCTCGGACGTGCCCGTCAGCA GCTTCATCACCCAGTACAGCACCGGGCAGGTCACCGTGGAGATGGAGTGGGAGCTC AAGAAGGAAAACTCCAAGAGGTGGAACCCAGAGATCCAGTACACAAACAACTACA ACGACCCCCAGTTTGTGGACTTTGCCCCGGACAGCACCGGGGAATACAGAACCACC AGACCTATCGGAACCCGATACCTTACCCGACCCCTTTAACCGGTCAGACATGATAA GATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTT ATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAA CAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGGTGTGG GAGGTTTTTTAAACACGTGCGGACCGAGTGGCGTAATCATGGTCATAGAGCTAGCG AATTCGAATTTAAATCGGATCCGCGGCCGCAAGGATCTGCGATCGCTCCGGTGCCC GTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGT CGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGAT GTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCA GTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAG CTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCAT CCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCG TCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTC CCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCT CAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGAC CGGCGCCTACGATATCGCCACCATGGGGAAAAAACCAGAGCTCACTGCGACATCAG TGGAGAAATTCCTCATCGAGAAATTTGACTCAGTGAGCGACCTTATGCAGCTCTCCG AGGGAGAGGAGTCAAGGGCTTTCTCTTTTGATGTTGGCGGAAGGGGATATGTACTT CGAGTAAACAGTTGTGCGGATGGATTTTATAAAGATAGGTACGTT [1202] Tet inducible stuffer plasmid (STX_C0225) SEQ ID NO: 150 [1203] ttaaccctagaaagatagtctgcgtaaaattgacgcatgcattcttgaaatattgctctctctttctaaatagcgcgaatccgtcgctgtgcattta ggacatctcagtcgccgcttggagctcccgtgaggcgtgcttgtcaatgcggtaagtgtcactgattttgaactataacgaccgcgtgagtcaaaatgacg catgattatcttttacgtgacttttaagatttaactcatacgataattatattgttatttcatgttctacttacgtgataacttattatatatatattttcttgttatagatat catcaactttgtatagaaaagttgggtaccgagctcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatag ggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttca cttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgatagggagtggtaaactcgactttcacttttctctatcactgataggga gtggtaaactcgacctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccggg accgatccagcctccgcggccccgaattgcaagtttgtacaaaaaagcaggctgccaccgtcgttttacaacgtcgtgactgggaaaaccctggcgtta cccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctga acggcgagtggcgctttgcctggtttccggcaccagaagcggtgccggaaagctggctggagtgcgatcttcctgaggccgatactgtcgtcgtcccct caaactggcagacccagctttcttgtacaaagtggtgatcctcaggtgcaggctgcctatcagaaggtggtggctggtgtggccaatgccctggctcaca aataccactgagatctttttccctctgccaaaaattatggggacatcatgaagccccttgagcatctgacttctggctaataaaggaaatttattttcattgcaa tagtgtgttggaattttttgtgtctctcactcggaaggacatatgggagggcaaatcatttaaaacatcagaatgagtatttggtttagagtttggcaacatatg cccatatgctggctgccatgaacaaaggttggctataaagaggtcatcagtatatgaaacagccccctgctgtccattccttattccatagaaaagccttga cttgaggttagattttttttatattttgttttgtgttatttttttctttaacatccctaaaattttccttacatgttttactagccagatttttcctcctctcctgactactccca gtcatagctgtccctcttctcttatggagatccctcgacctgcagcccaagcttcgcgttgacattgattattgactagttattaatagtaatcaattacggggt cattagttcatagcccatatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgcccattgacgtcaata atgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggtggagtatttacggtaaactgcccacttggcagtacatcaagtgtatc atatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttggcagtaca tctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtggatagcggtttgactcacggggatttccaagtctccaccc cattgacgtcaatgggagtttgttttggcaccaaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgtg tacggtgggaggtctatataagcagagctctctggctaactagagaacccactgcgccaccatgaaaaagcctgaactcaccgcgacgtctgtcgaga agtttctgatcgaaaagttcgacagcgtctccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatat gtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattg gggaatttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcagc cggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactac atggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatg agctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataaca gcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcag acgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttg gttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgccc gcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaatagc tcgagtctagagggcccgtttaaacccgctgatcagcctcgactgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccct ggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcagg acagcaagggggaggattgggaagacaatagcaggcatgctggggatgcggtgggctctatggctcgagttaattaacgagagcataatattgatatgt gccaaagttgtttctgactgactaataagtataatttgtttctattatgtataggttaagctaattacttattttataatacaacatgactgtttttaaagtacaaaata agtttatttttgtaaaagagagaatgtttaaaagttttgttactttatagaagaaattttgagtttttgtttttttttaataaataaataaacataaataaattgtttgttga atttattattagtatgtaagtgtaaatataataaaacttaatatctattcaaattaataaataaacctcgatatacagaccgataaaacacatgcgtcaattttacg catgattatctttaacgtacgtcacaatatgattatctttctagggttaaataatagtttctaatttttttattattcagcctgctgtcgtgaataccgagctccaattc gccctatagtgagtcgtattacaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatcc ccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgccctgtagc ggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttct cgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgatta gggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaa caacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaat tttaacaaaatattaacgcttacaatttaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctc atgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgcc ttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggt aagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaag agcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaatta tgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgg gggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaaca acgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcg ctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccc tcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggt aactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaat cccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaat actgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcc cagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta tccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctct gacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctt ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcg cagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacag gtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtat gttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagctcgaaattaaccctcactaaagggaacaaaag ctggtacctcgcgcgacttggtttgccattctttagcgcgcgtcgcgtcacacagcttggccacaatgtggtttttgtcaaacgaagattctatgacgtgttta aagtttaggtcgagtaaagcgcaaatctttt [1204] bGH polyA SEQ ID NO: 151 [1205] CTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCAT CGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGC AAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTA TGG [1206] SV40 polyA SEQ ID NO: 152 [1207] CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGC AGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCA TTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGG TTCAGGGGGAGGTGTGGGAGGTTTTTTAAACACGTGCGGACCGAGTGGCGTAATCA T [1208] Helper construct - uninduced SEQ ID NO: 153 [1209] CAGAGGTGTAAAGTACTTGAGTAATTTTACTTGATTACTGTACTTAAGTATTATTTTTGGGGATT TTTACTTTACTTGAGTACAATTAAAAATCAATACTTTTACTTTTACTTAATTACATTTTTTTAGAAAAAAAA GTACTTTTTACTCCTTACAATTTTATTTACAGTCAAAAAGTACTTATTTTTTGGAGATCACTTCATTCTATTT TCCCTTGCTATTACCAAACCAATTGAATTGCGCTGATGCCCAGTTTAATTTATTTACACTTACATACTAAT AATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAACAAAAACTCAAAATTTCTTCTA TAAAGTAACAAAACTTTTATGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCC CCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGA GCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCT TCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCACGGCCACTAGTC CATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCA CCCCACCCCCTAGAATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAG GACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTG GCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGCGGTGGTGCT GCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAGGAATACAACATGGCAGTGGTC TCCTCAGCGATGATTCGCACCGCCCGCAGCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTG ATCTCACTTAAATCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGCAAG GCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAGGTA GATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGGCATGTTGTAATTCACC ACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCA AAACCTGCCCGCCGGCTATGCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGA CTCGTAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACACGTGCATACAC TTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGGGAACAACCCATTCCTGAATCAGCG TAAATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTGTGCATTGTCAAAGTGTTACATTCGG GCAGCAGCGGATGATCCTCCAGTATGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTA CTGTACGGAGTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGCCGGA CGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGG GGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACCGAGGCCCCAGATCAGATC CCATACAATGGGGTACCTTCTGGGCATCCTTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGAC TCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACA CGTGGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTAC AGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGCATT CCTTTGGCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCT CACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAA GCGGCTTCGGCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTCTGC CGCGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAACTCA GGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAACGCGTTT AGCAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATA CACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGG AGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTC CCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGGCATCAGAAGGTGACCGTGC CCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCCTTT GCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATG CACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTG GCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTC CTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCAC AACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAG GAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTC GTTTAGCCAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCTTT AGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACA CGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCACTTTCCGCTTCACTGGACTCTTCCTTTTCCTCT TGCGTCCGCATACCCCGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGC CGTGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTCG CTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTTTTTCTTTTTG GACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGCGCAT CTTGTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTT GTCGTCATCGTCTTTGTAGTCGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTG GTGGACGTCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCA TGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCAGACATGATAAGATACATT GATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCT ATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTT TCAGGTTCAGGGGGAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTG ATTATGATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCTCTGCCTCCCCCG TGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCAGTGGCCAAGTGGCTTTGGTCCGTCT CCTCCACGGATGCCCCTCCACGGCTAGTGGGCGCATGTAGGCGGTGGGCGTCCGCCGCCTCCAGCAGC AGGTCATAGAGGGGCACCACGTTCTTGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGT GTCGGATGTGGGAGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCAGGCC TGCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGTGGATATGGTCCTTCTCT TCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTCCAGAATTAAGCAAAATAATAGATTTGAG GCACACAAACTCCTCTCCCTGCAGATTCATCATGCGGAACCGAGATGATGTAGCCAGCAGCATGTCGAA GATCTCCACCATGCCCTCTACACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGC TTCACTGGGTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGGCACATTCTAGA AGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACCCTCTTCGCCCAGTTGATCATGTGA ACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAGCCCATCATCGAAGCTTCACTGAAGGGTCTGGTAGGA TCATACTCGGAATAGAGTATGGGGGGCTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAG GGACAAGGCCAGGCTGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAGCTCT CATGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTGCCCCTGTTTCACTATCC AGGTTACGGATATAGTTCATGACAATATTTACATTGGTCCAGCCACCAGCTTGCATGATCTCCGGTATTG AAACTCCAGCGCGGGCCATATCTCGCGCGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTAT CTCTGACCAGAGTCATCCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAA ATTTTATATTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCCAGGGCGCGAGT TGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCTGACCCGGCAAAACAGGTAGTTATT CGGATCATCAGCTACACCAGAGACGGAAATCCATCGCTCGACCAGTTTAGTGACTCCCAGGCTAAGTGC CTTCTCTACACCTGCGGTGCTAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAG TACGTGAGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAGCAATCCC CAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGAGTGAACGGACTTGGTCGAA ATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTTCACCGGCATCAACGTTTTCTTTTCGGATCCGC CGCATAACCAGTGAAACAGCATTGCTGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTT AGCTGGCCCAAATGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCG CGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCCGCCCACGACCGGC AAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTGGCGATCCCTGAACATGTCCATCAGGT TCTTGCGAACCTCATCACTCGTTGCATCGACCGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATT GGACATGGTGGCTACGTAATAACTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGG GTAGGAAGTGGTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGACGG TTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTATACGTTCTCTATCACTG ATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGGGAGTAAACTGTAGATACGTTCTCTATCA CTGATAGGGAGTAAACTGGTCATACGTTCTCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTAT CACTGATAGGGAGTAAAGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCT ATCACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCACTTGCACTGGGGACA GCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTTAGTACTATCGATTCACA CAAAAAACCAACACACAGATGTAATGAAAATAAAGATATTTTATTGGATCTGCGATCGCTCCGGTGCCC GTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAAC GGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTC CCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTT GCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGA GGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCC GCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCT AGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTT CTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGCGGATCCGCCGCCACCATGTCTAGAC TGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACG ACAAGGAAACTCGCTCAAAAGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCG GGCCCTGCTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGG CGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTCTCTCACATCGCGAC GGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAAACCCTGGAAAATCAGCTCGC GTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACA CTGGGCTGCGTATTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTTCCT TTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAAGCGGCGGGCCGACCG ACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCT GCCTGCTGACGCTCTTGACGATTTTGACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCG ACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGC CACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTC TGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGG GATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAA CCCTTAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAA TTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTATAATGTATGCTATA CGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAA AAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGG TACCTCAAGCGCCGGGTTTTCGCGTCATGCACCACGTCCGTGGGCCCTCGGGTACTTCAACGTCAGCAG TAACTGTAAATCCGAGCCGTTCATAGAAGGGCAAATTCCTTGGCGCTGACGTTTCAAGAAAGGCTGGCA CTCCGGCTCGTTCTGCGGCTTCTACTCCGGGCAATACCACCGCGGAACCAAGGCCCTTTCCCTGATGATC GGGGCTAACGCCCACAGTAGCGAGGAACCAAGCTGGTTCTTTAGGGCGGTGAGGGGCGAGGAGTCCT TCCATTTGTTGCTGAGCCGCGAGACGAGAGCCACTAAGCTCAGCCATTCGGGGACCAATTTCTGCAAAT ACAGCCCCGGCCTCAACGCTCTCCGGAGTCGTCCACACTGCCACTGCAGCCCCGTCGTCGGCGACCCAA ACTTTACCGATGTCCAATCCTACCCTGGTCAAAAAAAGTTCTTGCAATTCTGTAACCCGTTCAATATGTCT ATCAGGATCAACTGTGTGGCGTGTAGCGGGATAATCCGCGAAAGCGGCAGCCAATGTTCTCACGGCCC TAGGGACGTCGTCTCGAGTTGCCAGTCTGACAGTAGGTTTATATTCTGTCATGGTGGCGGCGAATTCTC TTCTATGGAGGTCAAAACAGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGC TTATATAAACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTACGACATTT TGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAATGGGGTGGAGACTTGGAAA TCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATGTACTGCCAAAACCGCATCACCATGGTAATAGCG ATGACTAATACGTAGATGTACTGCCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATACTAG TTCTTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATAATGTATGCTATACGAAGTTATTTTGCAGTTTT AAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTAT ATATCTTGTGGAAAGGACGAAACACCGGGCACTCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATC ATGGCGGACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGC GTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTGGGCCCATTGGTATGGCT TTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGGAAAGCGAT CCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGC CGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCC CTGGGGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATAAGTTATCAC GTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATAA TCATATCAAGCTTAAACAAGAATCTCTAGTTTTCTTTCTTGCTTTTACTTTTACTTCCTTAATACTCAAGTA CAATTTTAATGGAGTACTTTTTTACTTTTACTCAAGTAAGATTCTAGCCAGATACTTTTACTTTTAATTGAG TAAAATTTTCCCTAAGTACTTGTACTTTCACTTGAGTAAAATTTTTGAGTACTTTTTACACCTCTG [1210] Helper plasmid SEQ ID NO: 154 [1211] GGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGG GGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCAC AAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACC AGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTA CCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCAC GCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACG AACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCA ACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGG CTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGG AAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCT TTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATT TTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGA AGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGC TTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCT GACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGT GCTGCAATGATACCGCGAGATCCACGCTCACCGGCTCCAGATTTATCAGCAATAAA CCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCA TCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTT TGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTA TGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGT TGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGG CCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGC CATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAAT AGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCG CCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAA ACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACC CAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGG AAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTC ATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTT CCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTAT AAAAATAGGCGTATCACGAGGCCCTTTCGTCCAGAGGTGTAAAGTACTTGAGTAAT TTTACTTGATTACTGTACTTAAGTATTATTTTTGGGGATTTTTACTTTACTTGAGTAC AATTAAAAATCAATACTTTTACTTTTACTTAATTACATTTTTTTAGAAAAAAAAGTA CTTTTTACTCCTTACAATTTTATTTACAGTCAAAAAGTACTTATTTTTTGGAGATCAC TTCATTCTATTTTCCCTTGCTATTACCAAACCAATTGAATTGCGCTGATGCCCAGTTT AATTTATTTACACTTACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTAT TTATTTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTT ATGAGGGACAGCCCCCCCCCAAAGCCCCCAGGGATGTAATTACGTCCCTCCCCCGC TAGGGGGCAGCAGCGAGCCGCCCGGGGCTCCGCTCCGGTCCGGCGCTCCCCCCGCA TCCCCGAGCCGGCAGCGTGCGGGGACAGCCCGGGCACGGGGAAGGTGGCACGGGA TCGCTTTCCTCTGAACGCTTCTCGCTGCTCTTTGAGCCTGCAGACACCTGGGGGGAT ACGGGGAAAAGGCCTCCACGGCCACTAGTCCATAGAGCCCACCGCATCCCCAGCAT GCCTGCTATTGTCTTCCCAATCCTCCCCCTTGCTGTCCTGCCCCACCCCACCCCCTAG AATAGAATGACACCTACTCAGACAATGCGATGCAATTTCCTCATTTTATTAGGAAAG GACAGTGGGAGTGGCACCTTCCAGGGTCAAGGAAGGCACGGGGGAGGGGCAAACA ACAGATGGCTGGCAACTAGAAGGCACAGCTACATGGGGGTAGAGTCATAATCGTGC ATCAGGATAGGGCGGTGGTGCTGCAGCAGCGCGCGAATAAACTGCTGCCGCCGCCG CTCCGTCCTGCAGGAATACAACATGGCAGTGGTCTCCTCAGCGATGATTCGCACCGC CCGCAGCATGAGACGCCTTGTCCTCCGGGCACAGCAGCGCACCCTGATCTCACTTA AATCAGCACAGTAACTGCAGCACAGCACCACAATATTGTTCAAAATCCCACAGTGC AAGGCGCTGTATCCAAAGCTCATGGCGGGGACCACAGAACCCACGTGGCCATCATA CCACAAGCGCAGGTAGATTAAGTGGCGACCCCTCATAAACACGCTGGACATAAACA TTACCTCTTTTGGCATGTTGTAATTCACCACCTCCCGGTACCATATAAACCTCTGATT AAACATGGCGCCATCCACCACCATCCTAAACCAGCTGGCCAAAACCTGCCCGCCGG CTATGCACTGCAGGGAACCGGGACTGGAACAATGACAGTGGAGAGCCCAGGACTC GTAACCATGGATCATCATGCTCGTCATGATATCAATGTTGGCACAACACAGGCACA CGTGCATACACTTCCTCAGGATTACAAGCTCCTCCCGCGTCAGAACCATATCCCAGG GAACAACCCATTCCTGAATCAGCGTAAATCCCACACTGCAGGGAAGACCTCGCACG TAACTCACGTTGTGCATTGTCAAAGTGTTACATTCGGGCAGCAGCGGATGATCCTCC AGTATGGTAGCGCGGGTCTCTGTCTCAAAAGGAGGTAGGCGATCCCTACTGTACGG AGTGCGCCGAGACAACCGAGATCGTGTTGGTCGTAGTGTCATGCCAAATGGAACGC CGGACGTAGTCATGGTTGTGGCCATATTATCATCGTGTTTTTCAAAGGAAAACCACG TCCCCGTGGTTCGGGGGGCCTAGACGTTTTTTTAACCTCGACTAAACACATGTAAAG CATGTGCACCGAGGCCCCAGATCAGATCCCATACAATGGGGTACCTTCTGGGCATC CTTCAGCCCCTTGTTGAATACGCTTGAGGAGAGCCATTTGACTCTTTCCACAACTAT CCAACTCACAACGTGGCACTGGGGTTGTGCCGCCTTTGCAGGTGTATCTTATACACG TGGCTTTTGGCCGCAGAGGCACCTGTCGCCAGGTGGGGGGTTCCGCTGCCTGCAAA GGGTCGCTACAGACGTTGTTTGTCTTCAAGAAGCTTCCAGAGGAACTGCTTCCTTCA CGACATTCAACAGACCTTGCATTCCTTTGGCGAGAGGGGAAAGACCCCTAGGAATG CTCGTCAAGAAGACAGGGCCAGGTTTCCGGGCCCTCACATTGCCAAAAGACGGCAA TATGGTGGAAAATAACATATAGACAAACGCACACCGGCCTTATTCCAAGCGGCTTC GGCCAGTAACGTTAGGGGGGGGGGAGGGAGAGGGGCTTAAAAATCAAAGGGGTTC TGCCGCGCATCACTATGCGCCACTGGCAGGGACACGTTGCGATACTGGTGTTTAGTG CTCCACTTAAACTCAGGCACAACCATCCGCGGCAGCTCGGTGAAGTTTTCACTCCAC AGGCTGCGCACCATCACCAACGCGTTTAGCAGGTCGGGCGCCGATATCTTGAAGTC GCAGTTGGGGCCTCCGCCCTGCGCGCGCGAGTTGCGATACACAGGGTTGCAGCACT GGAACACTATCAGCGCCGGGTGGTGCACGCTGGCCAGCACGCTCTTGTCGGAGATC AGATCCGCGTCCAGGTCCTCCGCGTTGCTCAGGGCGAACGGAGTCAACTTTGGTAG CTGCCTTCCCAAAAAGGGTGCATGCCCAGGCTTTGAGTTGCACTCGCACCGTAGTGG CATCAGAAGGTGACCGTGCCCGGTCTGGGCGTTAGGATACAGCGCCTGCATGAAAG CCTTGATCTGCTTAAAAGCCACCTGAGCCTTTGCGCCTTCAGAGAAGAACATGCCGC AAGACTTGCCGGAAAACTGATTGGCCGGACAGGCCGCGTCATGCACGCAGCACCTT GCGTCGGTGTTGGAGATCTGCACCACATTTCGGCCCCACCGGTTCTTCACGATCTTG GCCTTGCTAGACTGCTCCTTCAGCGCGCGCTGCCCGTTTTCGCTCGTCACATCCATTT CAATCACGTGCTCCTTATTTATCATAATGCTCCCGTGTAGACACTTAAGCTCGCCTTC GATCTCAGCGCAGCGGTGCAGCCACAACGCGCAGCCCGTGGGCTCGTGGTGCTTGT AGGTTACCTCTGCAAACGACTGCAGGTACGCCTGCAGGAATCGCCCCATCATCGTC ACAAAGGTCTTGTTGCTGGTGAAGGTCAGCTGCAACCCGCGGTGCTCCTCGTTTAGC CAGGTCTTGCATACGGCCGCCAGAGCTTCCACTTGGTCAGGCAGTAGCTTGAAGTTT GCCTTTAGATCGTTATCCACGTGGTACTTGTCCATCAACGCGCGCGCAGCCTCCATG CCCTTCTCCCACGCAGACACGATCGGCAGGCTCAGCGGGTTTATCACCGTGCTTTCA CTTTCCGCTTCACTGGACTCTTCCTTTTCCTCTTGCGTCCGCATACCCCGCGCCACTG GGTCGTCTTCATTCAGCCGCCGCACCGTGCGCTTACCTCCCTTGCCGTGCTTGATTA GCACCGGTGGGTTGCTGAAACCCACCATTTGTAGCGCCACATCTTCTCTTTCTTCCTC GCTGTCCACGATCACCTCTGGGGATGGCGGGCGCTCGGGCTTGGGAGAGGGGCGCT TCTTTTTCTTTTTGGACGCAATGGCCAAATCCGCCGTCGAGGTCGATGGCCGCGGGC TGGGTGTGCGCGGCACCAGCGCATCTTGTGACGAGTCTTCTTCGTCCTCGGACTCGA GACGCCGCCTCAGCCGCTTTTTTGGGGGCGCGCGCTTGTCGTCATCGTCTTTGTAGT CGGGAGGCGGCGGCGACGGCGACGGGGACGACACGTCCTCCATGGTTGGTGGACG TCGCGCCGCACCGCGTCCGCGCTCGGGGGTGGTTTCGCGCTGCTCCTCTTCCCGACT GGCCATGGTGGCCGAGGATAACTTCGTATATGGTTTCTTATACGAAGTTATGATCCA GACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAA AAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGC TGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGG GAGGTGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTATGGCTGA TTATGATCCTCTAGAGTCGCAGATCTGCTACGTATCAAGCTGTGGCAGGGAAACCCT CTGCCTCCCCCGTGATGTAATACTTTTGCAAGGAATGCGATGAAGTAGAGCCCGCA GTGGCCAAGTGGCTTTGGTCCGTCTCCTCCACGGATGCCCCTCCACGGCTAGTGGGC GCATGTAGGCGGTGGGCGTCCGCCGCCTCCAGCAGCAGGTCATAGAGGGGCACCAC GTTCTTGCACTTCATGCTGTACAGATGCTCCATGCCTTTGTTACTCATGTGTCGGATG TGGGAGAGGATGAGGAGGAGCTGGGCCAGCCGCTGGTGCTGCTGCTGCAGGGTCA GGCCTGCCTTGGCCATCAGGTGGATCAAAGTGTCTGTGATCTTGTCCAGGACTCGGT GGATATGGTCCTTCTCTTCCAGAGACTTCAGGGTGCTGGACAGAAATGTGTACACTC CAGAATTAAGCAAAATAATAGATTTGAGGCACACAAACTCCTCTCCCTGCAGATTC ATCATGCGGAACCGAGATGATGTAGCCAGCAGCATGTCGAAGATCTCCACCATGCC CTCTACACATTTTCCCTGGTTCCTGTCCAAGAGCAAGTTAGGAGCAAACAGTAGCTT CACTGGGTGCTCCATGGAGCGCCAGACGAGACCAATCATCAGGATCTCTAGCCAGG CACATTCTAGAAGGTGGACCTGATCATGGAGGGTCAAATCCACAAAGCCTGGCACC CTCTTCGCCCAGTTGATCATGTGAACCAGCTCCCTGTCTGCCAGGTTGGTCAGTAAG CCCATCATCGAAGCTTCACTGAAGGGTCTGGTAGGATCATACTCGGAATAGAGTAT GGGGGGCTCAGCATCCAACAAGGCACTGACCATCTGGTCGGCCGTCAGGGACAAGG CCAGGCTGTTCTTCTTAGAGCGTTTGATCATGAGCGGGCTTGGCCAAAGGTTGGCAG CTCTCATGTCTCCAGCAGATGGCTCGAGATCGCCATCTTCCAGCAGGCGCACCATTG CCCCTGTTTCACTATCCAGGTTACGGATATAGTTCATGACAATATTTACATTGGTCC AGCCACCAGCTTGCATGATCTCCGGTATTGAAACTCCAGCGCGGGCCATATCTCGCG CGGCTCCGACACGGGCACTGTGTCCAGACCAGGCCAGGTATCTCTGACCAGAGTCA TCCTAAAATACACAAACAATTAGAATCAGTAGTTTAACACATTATACACTTAAAAA TTTTATATTTACCTTAGCGCCGTAAATCAATCGATGAGTTGCTTCAAAAATCCCTTCC AGGGCGCGAGTTGATAGCTGGCTGGTGGCAGATGGCGCGGCAACACCATTTTTTCT GACCCGGCAAAACAGGTAGTTATTCGGATCATCAGCTACACCAGAGACGGAAATCC ATCGCTCGACCAGTTTAGTGACTCCCAGGCTAAGTGCCTTCTCTACACCTGCGGTGC TAACCAGCGTTTTCGTTCTGCCAATATGGATTAACATTCTCCCACCGTCAGTACGTG AGATATCTTTAACCCTGATCCTGGCAATTTCGGCTATACGTAACAGGGTGTTATAAG CAATCCCCAGAAATGCCAGATTACGTATATCCTGGCAGCGATCGCTATTTTCCATGA GTGAACGGACTTGGTCGAAATCAGTGCGTTCGAACGCTAGAGCCTGTTTTGCACGTT CACCGGCATCAACGTTTTCTTTTCGGATCCGCCGCATAACCAGTGAAACAGCATTGC TGTCACTTGGTCGTGGCAGCCCGGACCGACGATGAAGCATGTTTAGCTGGCCCAAA TGTTGCTGGATAGTTTTTACTGCCAGACCGCGCGCTTGAAGATATAGAAGATAATCG CGAACATCTTCAGGTTCTGCGGGAAACCATTTCCGGTTATTCAACTTGCACCATGCC GCCCACGACCGGCAAACGGACAGAAGCATTTTCCAGGTATGCTCAGAAAACGCCTG GCGATCCCTGAACATGTCCATCAGGTTCTTGCGAACCTCATCACTCGTTGCATCGAC CGGTAATGCAGGCAAATTTTGGTGTACGGTCAGTAAATTGGACATGGTGGCTACGT AATAACTTCGTATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGG AAGTGGTACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGAT CTGACGGTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAG CTTATACGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTG ATAGGGAGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCAT ACGTTCTCTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGG GAGTAAAGTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTC TCTATCACTGATAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCA CTTGCACTGGGGACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGA GAGCGTATGTTAGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAA AATAAAGATATTTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGC GCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGT GCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCG CCTTTTTCCCGAGGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCA TCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTC GCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAA GTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTA GACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGT TTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGC GGATCCGCCGCCACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCT GGAATTACTCAATGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAA AGCTGGGAGTTGAGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTG CTCGATGCCCTGCCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTG GAAGGCGAGTCATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGC TCTTCTCTCACATCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGA AACAGTACGAAACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCC CTGGAGAACGCACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTA TTGGAGGAACAGGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCG ATTCTATGCCCCCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCC GAACCTGCCTTCCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTA AAGTGCGAAAGCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCT CCCAGCCGATGCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGA CGATTTTGACCTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGT GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTG GAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGT CTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGA GGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTG AGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCT ATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAAT TAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTAT AATGTATGCTATACGAAGTTATCAGACATGATAAGATACATTGATGAGTTTGGACA AACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTAT TGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAAGCGCCGGGTT TTCGCGTCATGCACCACGTCCGTGGGCCCTCGGGTACTTCAACGTCAGCAGTAACTG TAAATCCGAGCCGTTCATAGAAGGGCAAATTCCTTGGCGCTGACGTTTCAAGAAAG GCTGGCACTCCGGCTCGTTCTGCGGCTTCTACTCCGGGCAATACCACCGCGGAACCA AGGCCCTTTCCCTGATGATCGGGGCTAACGCCCACAGTAGCGAGGAACCAAGCTGG TTCTTTAGGGCGGTGAGGGGCGAGGAGTCCTTCCATTTGTTGCTGAGCCGCGAGAC GAGAGCCACTAAGCTCAGCCATTCGGGGACCAATTTCTGCAAATACAGCCCCGGCC TCAACGCTCTCCGGAGTCGTCCACACTGCCACTGCAGCCCCGTCGTCGGCGACCCAA ACTTTACCGATGTCCAATCCTACCCTGGTCAAAAAAAGTTCTTGCAATTCTGTAACC CGTTCAATATGTCTATCAGGATCAACTGTGTGGCGTGTAGCGGGATAATCCGCGAA AGCGGCAGCCAATGTTCTCACGGCCCTAGGGACGTCGTCTCGAGTTGCCAGTCTGA CAGTAGGTTTATATTCTGTCATGGTGGCGGCGAATTCTCTTCTATGGAGGTCAAAAC AGCGTGGATGGCGTCTCCAGGCGATCTGACGGTTCACTAAACGAGCTCTGCTTATAT AAACCTCCCACCGTACACGCCTACCGCCCATTTGCGTCAATGGGGCGGAGTTGTTAC GACATTTTGGAAAGTCCCGTTGATTTTGGTGCCAAAACAAACTCCCATTGACGTCAA TGGGGTGGAGACTTGGAAATCCCCGTGAGTCAAACCGCTATCCACGCCCATTGATG TACTGCCAAAACCGCATCACCATGGTAATAGCGATGACTAATACGTAGATGTACTG CCAAGTAGGAAAGTCCCATAAGGTCATGTACTGGGCATAATACTAGTTCTTGGGAA AAGCGCTCCCCTACCCATAACTTCGTATAATGTATGCTATACGAAGTTATTTTGCAG TTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTT CGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGGGCACTCTTCCGT GATCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGACCGGGATTCGAACCCC GGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGCGTGTCGAACCCAGGT GTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTGGGCCCATTGGTATGGCTTTTT CCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAGCAGCGAGAAGCGTTCAGAGG AAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCTGTCCCCGCACGCTGCCGGCTC GGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAGCCCCGGGCGGCTCGCTGCTGC CCCCTAGCGGGGGAGGGACGTAATTACATCCCTGGGGGCTTTGGGGGGGGGCTGTC CCTGATATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGTAGAACA TGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAAAAGATA ATCATATCAAGCTTAAACAAGAATCTCTAGTTTTCTTTCTTGCTTTTACTTTTACTTC CTTAATACTCAAGTACAATTTTAATGGAGTACTTTTTTACTTTTACTCAAGTAAGATT CTAGCCAGATACTTTTACTTTTAATTGAGTAAAATTTTCCCTAAGTACTTGTACTTTC ACTTGAGTAAAATTTTTGAGTACTTTTTACACCTCTG [1212] Helper construct - induced SEQ ID NO: 155 [1213] CAGAGGTGTAAAGTACTTGAGTAATTTTACTTGATTACTGTACTTAAGTATTA TTTTTGGGGATTTTTACTTTACTTGAGTACAATTAAAAATCAATACTTTTACTTTTAC TTAATTACATTTTTTTAGAAAAAAAAGTACTTTTTACTCCTTACAATTTTATTTACAG TCAAAAAGTACTTATTTTTTGGAGATCACTTCATTCTATTTTCCCTTGCTATTACCAA ACCAATTGAATTGCGCTGATGCCCAGTTTAATTTATTTACACTTACATACTAATAAT AAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAAAAACAAAAACTCA AAATTTCTTCTATAAAGTAACAAAACTTTTATGAGGGACAGCCCCCCCCCAAAGCCC CCAGGGATGTAATTACGTCCCTCCCCCGCTAGGGGGCAGCAGCGAGCCGCCCGGGG CTCCGCTCCGGTCCGGCGCTCCCCCCGCATCCCCGAGCCGGCAGCGTGCGGGGACA GCCCGGGCACGGGGAAGGTGGCACGGGATCGCTTTCCTCTGAACGCTTCTCGCTGC TCTTTGAGCCTGCAGACACCTGGGGGGATACGGGGAAAAGGCCTCCACGGCCACTA GTCCATAGAGCCCACCGCATCCCCAGCATGCCTGCTATTGTCTTCCCAATCCTCCCC CTTGCTGTCCTGCCCCACCCCACCCCCTAGAATAGAATGACACCTACTCAGACAATG CGATGCAATTTCCTCATTTTATTAGGAAAGGACAGTGGGAGTGGCACCTTCCAGGGT CAAGGAAGGCACGGGGGAGGGGCAAACAACAGATGGCTGGCAACTAGAAGGCACA GCTACATGGGGGTAGAGTCATAATCGTGCATCAGGATAGGGCGGTGGTGCTGCAGC AGCGCGCGAATAAACTGCTGCCGCCGCCGCTCCGTCCTGCAGGAATACAACATGGC AGTGGTCTCCTCAGCGATGATTCGCACCGCCCGCAGCATGAGACGCCTTGTCCTCCG GGCACAGCAGCGCACCCTGATCTCACTTAAATCAGCACAGTAACTGCAGCACAGCA CCACAATATTGTTCAAAATCCCACAGTGCAAGGCGCTGTATCCAAAGCTCATGGCG GGGACCACAGAACCCACGTGGCCATCATACCACAAGCGCAGGTAGATTAAGTGGCG ACCCCTCATAAACACGCTGGACATAAACATTACCTCTTTTGGCATGTTGTAATTCAC CACCTCCCGGTACCATATAAACCTCTGATTAAACATGGCGCCATCCACCACCATCCT AAACCAGCTGGCCAAAACCTGCCCGCCGGCTATGCACTGCAGGGAACCGGGACTGG AACAATGACAGTGGAGAGCCCAGGACTCGTAACCATGGATCATCATGCTCGTCATG ATATCAATGTTGGCACAACACAGGCACACGTGCATACACTTCCTCAGGATTACAAG CTCCTCCCGCGTCAGAACCATATCCCAGGGAACAACCCATTCCTGAATCAGCGTAA ATCCCACACTGCAGGGAAGACCTCGCACGTAACTCACGTTGTGCATTGTCAAAGTG TTACATTCGGGCAGCAGCGGATGATCCTCCAGTATGGTAGCGCGGGTCTCTGTCTCA AAAGGAGGTAGGCGATCCCTACTGTACGGAGTGCGCCGAGACAACCGAGATCGTGT TGGTCGTAGTGTCATGCCAAATGGAACGCCGGACGTAGTCATGGTTGTGGCCATATT ATCATCGTGTTTTTCAAAGGAAAACCACGTCCCCGTGGTTCGGGGGGCCTAGACGTT TTTTTAACCTCGACTAAACACATGTAAAGCATGTGCACCGAGGCCCCAGATCAGAT CCCATACAATGGGGTACCTTCTGGGCATCCTTCAGCCCCTTGTTGAATACGCTTGAG GAGAGCCATTTGACTCTTTCCACAACTATCCAACTCACAACGTGGCACTGGGGTTGT GCCGCCTTTGCAGGTGTATCTTATACACGTGGCTTTTGGCCGCAGAGGCACCTGTCG CCAGGTGGGGGGTTCCGCTGCCTGCAAAGGGTCGCTACAGACGTTGTTTGTCTTCAA GAAGCTTCCAGAGGAACTGCTTCCTTCACGACATTCAACAGACCTTGCATTCCTTTG GCGAGAGGGGAAAGACCCCTAGGAATGCTCGTCAAGAAGACAGGGCCAGGTTTCC GGGCCCTCACATTGCCAAAAGACGGCAATATGGTGGAAAATAACATATAGACAAAC GCACACCGGCCTTATTCCAAGCGGCTTCGGCCAGTAACGTTAGGGGGGGGGGAGGG AGAGGGGCTTAAAAATCAAAGGGGTTCTGCCGCGCATCACTATGCGCCACTGGCAG GGACACGTTGCGATACTGGTGTTTAGTGCTCCACTTAAACTCAGGCACAACCATCCG CGGCAGCTCGGTGAAGTTTTCACTCCACAGGCTGCGCACCATCACCAACGCGTTTAG CAGGTCGGGCGCCGATATCTTGAAGTCGCAGTTGGGGCCTCCGCCCTGCGCGCGCG AGTTGCGATACACAGGGTTGCAGCACTGGAACACTATCAGCGCCGGGTGGTGCACG CTGGCCAGCACGCTCTTGTCGGAGATCAGATCCGCGTCCAGGTCCTCCGCGTTGCTC AGGGCGAACGGAGTCAACTTTGGTAGCTGCCTTCCCAAAAAGGGTGCATGCCCAGG CTTTGAGTTGCACTCGCACCGTAGTGGCATCAGAAGGTGACCGTGCCCGGTCTGGG CGTTAGGATACAGCGCCTGCATGAAAGCCTTGATCTGCTTAAAAGCCACCTGAGCC TTTGCGCCTTCAGAGAAGAACATGCCGCAAGACTTGCCGGAAAACTGATTGGCCGG ACAGGCCGCGTCATGCACGCAGCACCTTGCGTCGGTGTTGGAGATCTGCACCACAT TTCGGCCCCACCGGTTCTTCACGATCTTGGCCTTGCTAGACTGCTCCTTCAGCGCGC GCTGCCCGTTTTCGCTCGTCACATCCATTTCAATCACGTGCTCCTTATTTATCATAAT GCTCCCGTGTAGACACTTAAGCTCGCCTTCGATCTCAGCGCAGCGGTGCAGCCACA ACGCGCAGCCCGTGGGCTCGTGGTGCTTGTAGGTTACCTCTGCAAACGACTGCAGG TACGCCTGCAGGAATCGCCCCATCATCGTCACAAAGGTCTTGTTGCTGGTGAAGGTC AGCTGCAACCCGCGGTGCTCCTCGTTTAGCCAGGTCTTGCATACGGCCGCCAGAGCT TCCACTTGGTCAGGCAGTAGCTTGAAGTTTGCCTTTAGATCGTTATCCACGTGGTAC TTGTCCATCAACGCGCGCGCAGCCTCCATGCCCTTCTCCCACGCAGACACGATCGGC AGGCTCAGCGGGTTTATCACCGTGCTTTCACTTTCCGCTTCACTGGACTCTTCCTTTT CCTCTTGCGTCCGCATACCCCGCGCCACTGGGTCGTCTTCATTCAGCCGCCGCACCG TGCGCTTACCTCCCTTGCCGTGCTTGATTAGCACCGGTGGGTTGCTGAAACCCACCA TTTGTAGCGCCACATCTTCTCTTTCTTCCTCGCTGTCCACGATCACCTCTGGGGATGG CGGGCGCTCGGGCTTGGGAGAGGGGCGCTTCTTTTTCTTTTTGGACGCAATGGCCAA ATCCGCCGTCGAGGTCGATGGCCGCGGGCTGGGTGTGCGCGGCACCAGCGCATCTT GTGACGAGTCTTCTTCGTCCTCGGACTCGAGACGCCGCCTCAGCCGCTTTTTTGGGG GCGCGCGCTTGTCGTCATCGTCTTTGTAGTCGGGAGGCGGCGGCGACGGCGACGGG GACGACACGTCCTCCATGGTTGGTGGACGTCGCGCCGCACCGCGTCCGCGCTCGGG GGTGGTTTCGCGCTGCTCCTCTTCCCGACTGGCCATGGTGGCCGAGGATAACTTCGT ATATGGTTTCTTATACGAAGTTATGCGGCCGCTTTACGAGGGTAGGAAGTGGTACG GAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGACGGTTC ACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTATACGTT CTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGGGAGTA AACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTCTCTAT CACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAAAGTCT GCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATCACTGA TAGGGAGTAAACTCGCGGCCGCAGAGAAATGTTCTGGCACCTGCACTTGCACTGGG GACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTT AGTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAAGATAT TTTATTGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCA CAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAG GTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGA GGGTGGGGGAGAACCGTATGTAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCA ACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCAC GCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGC CTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCA GGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGC TCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTT CTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGCTAGCGGATCCGCCGCC ACCATGTCTAGACTGGACAAGAGCAAAGTCATAAACTCTGCTCTGGAATTACTCAA TGGAGTCGGTATCGAAGGCCTGACGACAAGGAAACTCGCTCAAAAGCTGGGAGTTG AGCAGCCTACCCTGTACTGGCACGTGAAGAACAAGCGGGCCCTGCTCGATGCCCTG CCAATCGAGATGCTGGACAGGCATCATACCCACTCCTGCCCCCTGGAAGGCGAGTC ATGGCAAGACTTTCTGCGGAACAACGCCAAGTCATACCGCTGTGCTCTTCTCTCACA TCGCGACGGGGCTAAAGTGCATCTCGGCACCCGCCCAACAGAGAAACAGTACGAA ACCCTGGAAAATCAGCTCGCGTTCCTGTGTCAGCAAGGCTTCTCCCTGGAGAACGC ACTGTACGCTCTGTCCGCCGTGGGCCACTTTACACTGGGCTGCGTATTGGAGGAACA GGAGCATCAAGTAGCAAAAGAGGAAAGAGAGACACCTACCACCGATTCTATGCCC CCACTTCTGAAACAAGCAATTGAGCTGTTCGACCGGCAGGGAGCCGAACCTGCCTT CCTTTTCGGCCTGGAACTAATCATATGTGGCCTGGAGAAACAGCTAAAGTGCGAAA GCGGCGGGCCGACCGACGCCCTTGACGATTTTGACTTAGACATGCTCCCAGCCGAT GCCCTTGACGACTTTGACCTTGATATGCTGCCTGCTGACGCTCTTGACGATTTTGAC CTTGACATGCTCCCCGGGTGAACCGGTCGCTGATCAGCCTCGACTGTGCCTTCTAGT TGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGG TGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAA GAACCAGCTGGGGCTCGACTAGAGCTTGCGGAACCCTTAGAGGGCCTATTTCCCAT GATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAA TTTGACTGTAAACACAAAGATATTAGTACAAAATAATAACTTCGTATAATGTATGCT ATACGAAGTTATTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTT ACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGA AACACCGGGCACTCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATCATGGCGG ACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCG CCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTG GGCCCATTGGTATGGCTTTTTCCCCGTATCCCCCCAGGTGTCTGCAGGCTCAAAGAG CAGCGAGAAGCGTTCAGAGGAAAGCGATCCCGTGCCACCTTCCCCGTGCCCGGGCT GTCCCCGCACGCTGCCGGCTCGGGGATGCGGGGGGAGCGCCGGACCGGAGCGGAG CCCCGGGCGGCTCGCTGCTGCCCCCTAGCGGGGGAGGGACGTAATTACATCCCTGG GGGCTTTGGGGGGGGGCTGTCCCTGATATCTATAACAAGAAAATATATATATAATA AGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATC TTAAAAGTCACGTAAAAGATAATCATATCAAGCTTAAACAAGAATCTCTAGTTTTCT TTCTTGCTTTTACTTTTACTTCCTTAATACTCAAGTACAATTTTAATGGAGTACTTTTT TACTTTTACTCAAGTAAGATTCTAGCCAGATACTTTTACTTTTAATTGAGTAAAATTT TCCCTAAGTACTTGTACTTTCACTTGAGTAAAATTTTTGAGTACTTTTTACACCTCTG [1214] payload construct - sc SEQ ID NO: 156 [1215] CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAA ATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGA TATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTT ATTTATGTTTATTTATTTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGT AACAAAACTTTTATTTCTTTCCTGCGTTATCTTTGGCCACTCCCTCTCTGCGCGCTCG CTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGG CGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAG GGGTTCCTCTGCAGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAAT AAATTTCCTTTATTTCACAGCAGCTGTCTCAAGGCTGGGTCCCTCAAAGGGGCGTCC CAGCGCGGGGCCTCCCTGCGCAAACACTTGGTACCCCTGGCTGCGCAGCGGAAGCC AGCAGGACAGCAGTGGCGCCGATCAGCACAACAGACGCCCTGGCGGTAGGGACAG CAGGCCCAGCCCTGTCGGTTGTCTCGGCAGCAGGTCTGGTTATCATGGCAGAAGTGT CCTTCCCCACACTCCACGTCCTTCACACCCACGTGAGGGCTACGGGCCAGGAAGGT GGCAGGCTGGGCAGAGACCACTTCCTTCTCGCAGGATCGAGCCTTCACGTTGCAGG TGTAGCCAGCCGGGCAGCAGTGCTGGCGATCCTCGCAGCACACAGCATGGGGCAAC TGGCAGCAGGCCCAGCTCCCACCCAGGCTCGGGCAGCAGGTCTGCCCCACCGGGCA GCTGGTGTGCTGGTCACAGCCGATGTCTCTGGGGTGGGATAAGGAAGCCCGGCGGG CAGGCATCTTCTCCAGTCCAGCCACGATCTCGCTTCCTCGCTGACACTGCCCCTCAG CTACACACGTGTAGCCCTGGGGGCAGCAGTGCTGGTGGTCCGAGCAGCAGACAGCC TCTGGGATTGGACAGCAGCCCCACTCCCCAGACGTGAGTTGGCAGCAGGTATCGGA GGAGGGACAGCTGCTGACATTATCACAGGGGACATCTCTCTTCAAGGCTTGTGGGT CTGGCAGGCTGAGGTGAGCTGGGGCCTTCTCCATCCAGGGCACCTGGTGGGGCCCC TGTTCACAGGTACCCTTCTGCGTGTCACACGTAAACCCCGCGGGACAGCAGTGTATG TGGTCCTCACAGCACACAGCCTGGGTAAAAGGGCAGCAGCCCCAGGCCCCCGACTG TAGACGGCAGCAGGTATAGCCATCTGGGCAGCTCACCTCCATGTCACATTTCACATC CCCCACTGTGTGCGCAGGCAGCTTAGTGAGGAGGTCCGTGGTAGCGTTCTCCTTGGA GAGGCACTTACTCTGGATCAGGTCACACACAGTGTCTTGGGGGCAGCAGTGCAGGT GATCGGAGCAGCAGGTGGCGTTGGGCATTGGGCAGCAGCCATACTTCCCACTGGGC AGCTCACAGCAGGTAGAACCATCAGGGCACCGGGACCGTGCGTCCGGACACATGAC CGAGCTGGACAAGGCCACTGCCCTGTTAGTCCTCTGGGCAGGGAGCTTCTTTGCCAG GGGGTGGGTGCCCGTGGGTGTGATGCAGCGGGTGTGAACCAGGTCGCAGAAGGCA CCGTGCGGACAGCAGTGCACCCTGTCTTCACAGCAGGAAGCCTGGGGCATGGGGCA GCACCCCCAGGAGCCATCGACCATAACACAGCACGTGGAGAAGTCCGGGCATTCGA ACTGACTATCAGGGCACTGGATGGCACCCACGGAGTTGTTACCTGATCTTTGGAAG CAGGATCGCCCGTCTGCACTGCAGTGGAAGCCCCGTGGGCAGCAGTGATGGCCATC CCCGCATGCCACGGCCTCTGGGAAGGGGCAGCAACTGGAAGTCCCTGAGACGGTAA AGATGCAGGAGTGGCCGGCAGAGCAGTGGGCATCAACCTGGCAGGGGCCACCCAG ATGCCTGCTCAGTGTTGTGGGCCATTTGTCCAGAAGGGGACGGCAGCAGCTGTAGC TGGCTCCTCCGGGGTCCAGGCAGCAGGCCACAGGGCAGAACTGACCATCTGGGCAC CGCGTTCCAGCCACCAGCCCTGCTGTTAAGGCCACCCAGCTCACCAGGGTCCACAT GGTGGCGGCGCTAGCGGTGCACACCAATGTGGTGAATGGTCAAATGGCGTTTATTG TATCGAGCTAGGCACTTAAATACAATATCTCTGCAATGCGGAATTCAGTGGTTCGTC CAATCCATGTCAGACCCGTCTGTTGCCTTCCTAATAAGGCACGATCGTACCACCTTA CTTCCACCAATCGGCATGCACGGTGCTTTTTCTCTCCTTGTAAGGCATGTTGCTAACT CATCGTTACCATGTTGCAAGACTACAAGAGTATTGCATAAGACTACATTGGATCCTC TGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACC TTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAATTCACTGGCCGTCG TTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAG CACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTT CCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTA CGCATCTGTGCGGTATTTCACACCGCACTAGAGCTAGCGAATTCGAATTTAAATCGG ATCCGCGGCCGCAAGGATCTGCGATCGCTCCGGTGCCCGTCAGTGGGCAGAGCGCA CATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACGGGTGCC TAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCT TTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTC TTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCTTCGAGGGGCTCGCATCT CTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCG TTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTT TAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGAC TCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTC GTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCGGCGCCTACGATATCGCC ACCATGAAAACATTTAACATTTCTCAACAGGATCTAGAATTAGTAGAAGTAGCGAC AGAGAAGATTACAATGCTTTATGAGGATAATAAACATCATGTGGGAGCGGCAATTC GTACGAAAACAGGAGAAATCATTTCGGCAGTACATATTGAAGCGTATATAGGACGA GTAACTGTTTGTGCAGAAGCCATTGCGATTGGTAGTGCAGTTTCGAATGGACAAAA GGATTTTGACACGATTGTAGCTGTTAGACACCCTTATTCTGACGAAGTAGATAGAAG TATTCGAGTGGTAAGTCCTTGTGGTATGTGCCTTTCATACGAGACCGAGATCCTGAC TGTCGAGTACGGATTGCTTCCTATCGGCAAAATCGTGGAGAAGAGGATTGAATGTA CCGTCTATTCAGTCGATAATAATGGGAACATCTACACACAGCCCGTGGCTCAATGG CACGACAGAGGAGAGCAGGAAGTTTTTGAATACTGTCTCGAGGACGGATCCCTCAT CCGCGCTACTAAAGATCATAAGTTTATGACCGTGGACGGCCAGATGCTGCCAATTG ACGAAATTTTTGAACGAGAGCTGGATCTGATGAGAGTCGACAACCTTCCAAACTGA GTGCATGACCCGCAAGCCCGGTGCCTGAAATCAACCTCTGGATTACAAAATTTGTG AAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTG CTTTAATGCCTTTGTATCATGCGTTAACTAAACTTGTTTATTGCAGCTTATAATGGTT ACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATT CTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGAATTGACTCA AATGATGTCAATTAGTCTATCAGAAGCTCATCTGGTCTCCCTTCCGGGGGACAAGAC ATCCCTGTTTAATATTTAAACAGCAGTGTTCCCAAACTGGGTTCTTATATCCCTTGCT CTGGTCAACCAGGTTGCAGGGTTTCCTGTCCTCACAGGAACGAAGTCCCTAAAGAA ACAGTGGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCCTTTTTTAGGGCCCATT GGTATGGCTGATATCTATAACAAGAAAATATATATATAATAAGTTATCACGTAAGT AGAACATGAAATAACAATATAATTATCGTATGAGTTAAATCTTAAAAGTCACGTAA AAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGTTCAAAATCAGTGACA CTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCGGCGACTGAGATGTCC TAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGAGCAATATTTCAAGAA TGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGG [1216] payload plasmid - sc SEQ ID NO: 157 [1217] CCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAA TACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCAT GAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCA CATTTCCCCGAAAAGTGCCACCTAAATTGTAAGCGTTAATATTTTGTTAAAATTCGC GTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAAT CCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGA ACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTC TATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCG AGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTG ACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGC GGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCG CCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCATTCAGGCTGCGCAACT GTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGG GGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACG TTGTAAAACGACGGCCAGTGAGCGCGCCTCGTTCATTCACGTTTTTGAACCCGTGGA GGACGGGCAGACTCGCGGTGCAAATGTGTTTTACAGCGTGATGGAGCAGATGAAGA TGCTCGACACGCTGCAGAACACGCAGCTAGATTAACCCTAGAAAGATAATCATATT GTGACGTACGTTAAAGATAATCATGTGTAAAATTGACGCATGTGTTTTATCGGTCTG TATATCGAGGTTTATTTATTAATTTGAATAGATATTAAGTTTTATTATATTTACACTT ACATACTAATAATAAATTCAACAAACAATTTATTTATGTTTATTTATTTATTAAAAA AAACAAAAACTCAAAATTTCTTCTATAAAGTAACAAAACTTTTATTTCTTTCCTGCG TTATCTTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCA AAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCG CAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCTGCAGTGAGAGACACAA AAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTATTTCACAGCAGCTGT CTCAAGGCTGGGTCCCTCAAAGGGGCGTCCCAGCGCGGGGCCTCCCTGCGCAAACA CTTGGTACCCCTGGCTGCGCAGCGGAAGCCAGCAGGACAGCAGTGGCGCCGATCAG CACAACAGACGCCCTGGCGGTAGGGACAGCAGGCCCAGCCCTGTCGGTTGTCTCGG CAGCAGGTCTGGTTATCATGGCAGAAGTGTCCTTCCCCACACTCCACGTCCTTCACA CCCACGTGAGGGCTACGGGCCAGGAAGGTGGCAGGCTGGGCAGAGACCACTTCCTT CTCGCAGGATCGAGCCTTCACGTTGCAGGTGTAGCCAGCCGGGCAGCAGTGCTGGC GATCCTCGCAGCACACAGCATGGGGCAACTGGCAGCAGGCCCAGCTCCCACCCAGG CTCGGGCAGCAGGTCTGCCCCACCGGGCAGCTGGTGTGCTGGTCACAGCCGATGTC TCTGGGGTGGGATAAGGAAGCCCGGCGGGCAGGCATCTTCTCCAGTCCAGCCACGA TCTCGCTTCCTCGCTGACACTGCCCCTCAGCTACACACGTGTAGCCCTGGGGGCAGC AGTGCTGGTGGTCCGAGCAGCAGACAGCCTCTGGGATTGGACAGCAGCCCCACTCC CCAGACGTGAGTTGGCAGCAGGTATCGGAGGAGGGACAGCTGCTGACATTATCACA GGGGACATCTCTCTTCAAGGCTTGTGGGTCTGGCAGGCTGAGGTGAGCTGGGGCCT TCTCCATCCAGGGCACCTGGTGGGGCCCCTGTTCACAGGTACCCTTCTGCGTGTCAC ACGTAAACCCCGCGGGACAGCAGTGTATGTGGTCCTCACAGCACACAGCCTGGGTA AAAGGGCAGCAGCCCCAGGCCCCCGACTGTAGACGGCAGCAGGTATAGCCATCTGG GCAGCTCACCTCCATGTCACATTTCACATCCCCCACTGTGTGCGCAGGCAGCTTAGT GAGGAGGTCCGTGGTAGCGTTCTCCTTGGAGAGGCACTTACTCTGGATCAGGTCAC ACACAGTGTCTTGGGGGCAGCAGTGCAGGTGATCGGAGCAGCAGGTGGCGTTGGGC ATTGGGCAGCAGCCATACTTCCCACTGGGCAGCTCACAGCAGGTAGAACCATCAGG GCACCGGGACCGTGCGTCCGGACACATGACCGAGCTGGACAAGGCCACTGCCCTGT TAGTCCTCTGGGCAGGGAGCTTCTTTGCCAGGGGGTGGGTGCCCGTGGGTGTGATG CAGCGGGTGTGAACCAGGTCGCAGAAGGCACCGTGCGGACAGCAGTGCACCCTGTC TTCACAGCAGGAAGCCTGGGGCATGGGGCAGCACCCCCAGGAGCCATCGACCATAA CACAGCACGTGGAGAAGTCCGGGCATTCGAACTGACTATCAGGGCACTGGATGGCA CCCACGGAGTTGTTACCTGATCTTTGGAAGCAGGATCGCCCGTCTGCACTGCAGTGG AAGCCCCGTGGGCAGCAGTGATGGCCATCCCCGCATGCCACGGCCTCTGGGAAGGG GCAGCAACTGGAAGTCCCTGAGACGGTAAAGATGCAGGAGTGGCCGGCAGAGCAG TGGGCATCAACCTGGCAGGGGCCACCCAGATGCCTGCTCAGTGTTGTGGGCCATTT GTCCAGAAGGGGACGGCAGCAGCTGTAGCTGGCTCCTCCGGGGTCCAGGCAGCAGG CCACAGGGCAGAACTGACCATCTGGGCACCGCGTTCCAGCCACCAGCCCTGCTGTT AAGGCCACCCAGCTCACCAGGGTCCACATGGTGGCGGCGCTAGCGGTGCACACCAA TGTGGTGAATGGTCAAATGGCGTTTATTGTATCGAGCTAGGCACTTAAATACAATAT CTCTGCAATGCGGAATTCAGTGGTTCGTCCAATCCATGTCAGACCCGTCTGTTGCCT TCCTAATAAGGCACGATCGTACCACCTTACTTCCACCAATCGGCATGCACGGTGCTT TTTCTCTCCTTGTAAGGCATGTTGCTAACTCATCGTTACCATGTTGCAAGACTACAA GAGTATTGCATAAGACTACATTGGATCCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACC CTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTA ATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGC GAATGGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGC ACTAGAGCTAGCGAATTCGAATTTAAATCGGATCCGCGGCCGCAAGGATCTGCGAT CGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTT GGGGGGAGGGGTCGGCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACC GTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAG AACACAGCTGAAGCTTCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTAC CTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGC CTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCT TTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCT GACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGA TCCAAGCTGTGACCGGCGCCTACGATATCGCCACCATGAAAACATTTAACATTTCTC AACAGGATCTAGAATTAGTAGAAGTAGCGACAGAGAAGATTACAATGCTTTATGAG GATAATAAACATCATGTGGGAGCGGCAATTCGTACGAAAACAGGAGAAATCATTTC GGCAGTACATATTGAAGCGTATATAGGACGAGTAACTGTTTGTGCAGAAGCCATTG CGATTGGTAGTGCAGTTTCGAATGGACAAAAGGATTTTGACACGATTGTAGCTGTTA GACACCCTTATTCTGACGAAGTAGATAGAAGTATTCGAGTGGTAAGTCCTTGTGGTA TGTGCCTTTCATACGAGACCGAGATCCTGACTGTCGAGTACGGATTGCTTCCTATCG GCAAAATCGTGGAGAAGAGGATTGAATGTACCGTCTATTCAGTCGATAATAATGGG AACATCTACACACAGCCCGTGGCTCAATGGCACGACAGAGGAGAGCAGGAAGTTTT TGAATACTGTCTCGAGGACGGATCCCTCATCCGCGCTACTAAAGATCATAAGTTTAT GACCGTGGACGGCCAGATGCTGCCAATTGACGAAATTTTTGAACGAGAGCTGGATC TGATGAGAGTCGACAACCTTCCAAACTGAGTGCATGACCCGCAAGCCCGGTGCCTG AAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGT TGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCGTTAACT AAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTT CACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAAT GTATCTTATCATGTCTGGAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTC ATCTGGTCTCCCTTCCGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGT TCCCAAACTGGGTTCTTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGT CCTCACAGGAACGAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAAT TGACTGGATTCCTTTTTTAGGGCCCATTGGTATGGCTGATATCTATAACAAGAAAAT ATATATATAATAAGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGT ATGAGTTAAATCTTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGC GGTCGTTATAGTTCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGG GAGCTCCAAGCGGCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATT TAGAAAGAGAGAGCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCT TTCTAGGGTTAATCTAGCTGCATCAGGATCATATCGTCGGGTCTTTTTTCCGGCTCA GTCATCGCCCAAGCTGGCGCTATCTGGGCATCGGGGAGGAAGAAGCCCGTGCCTTT TCCCGCGAGGTTGAAGCGGCATGGAAAGAGTTTGCCGAGGATGACTGCTGCTGCAT TGACGTTGAGCGAAAACGCACGTTTACCATGATGATTCGGGAAGGTGTGGCCATGC ACGCCTTTAACGGTGAACTGTTCGTTCAGGCCACCTGGGATACCAGTTCGTCGCGGC TTTTCCGGACACAGTTCCGGATGGTCAGCCCGAAGCGCATCAGCAACCCGAACAAT ACCGGCGACAGCCGGAACTGCCGTGCCGGTGTGCAGATTAATGACAGCGGTGCGGC GCTGGGATATTACGTCAGCGAGGACGGGTATCCTGGCTGGATGCCGCAGAAATGGA CATGGATACCCCGTGAGTTACCCGGCGGGCGCGCTTGGCGTAATCATGGTCATAGC TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAA GCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCG TTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGA ATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCG CTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCA AAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGT GAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGA GGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCC CTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCC CTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGT AGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCT ACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGG TATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATC CGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTAC GCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACG CTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGG ATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCA GCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTA CGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCA CGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCG CAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGA AGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTAC AGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCA ACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCT TCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTA TGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGA CTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGC TCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGT GCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTT GAGATCCAGTTCGATGTAACCCACTCGTGCAC [1218] payload construct - ss SEQ ID NO: 158 [1219] CCCTAGAAAGATAATCATATTGTGACGTACGTTAAAGATAATCATGTGTAAA ATTGACGCATGTGTTTTATCGGTCTGTATATCGAGGTTTATTTATTAATTTGAATAGA TATTAAGTTTTATTATATTTACACTTACATACTAATAATAAATTCAACAAACAATTT ATTTATGTTTATTTATTTATTAAAAAAAACAAAAACTCAAAATTTCTTCTATAAAGT AACAAAACTTTTATTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTCTGCAGGAGA CCGAGTTGGATTGTCTCCTCTAAGAACTCTTGATTACAGTTAAGGAATCTGCCGATC AGTGGTGTGTCAAGGTCTACAGTGAGCGCCGCTTGACTCTTATCTGATCAACGGCTA TAACGTGGAATTGCCAGGCTTGCAGATAGCGTTAGGTATTCCAACCGAATAGACAA CACACTGTGTCTTGTTAACAGTTCTGCTACGACGGCAATAACACGAAGATAATACC GGTGAGAGACACAAAAAATTCCAACACACTATTGCAATGAAAATAAATTTCCTTTA TTTCACAGCAGCTGTCTCAAGGCTGGGTCCCTCAAAGGGGCGTCCCAGCGCGGGGC CTCCCTGCGCAAACACTTGGTACCCCTGGCTGCGCAGCGGAAGCCAGCAGGACAGC AGTGGCGCCGATCAGCACAACAGACGCCCTGGCGGTAGGGACAGCAGGCCCAGCC CTGTCGGTTGTCTCGGCAGCAGGTCTGGTTATCATGGCAGAAGTGTCCTTCCCCACA CTCCACGTCCTTCACACCCACGTGAGGGCTACGGGCCAGGAAGGTGGCAGGCTGGG CAGAGACCACTTCCTTCTCGCAGGATCGAGCCTTCACGTTGCAGGTGTAGCCAGCCG GGCAGCAGTGCTGGCGATCCTCGCAGCACACAGCATGGGGCAACTGGCAGCAGGCC CAGCTCCCACCCAGGCTCGGGCAGCAGGTCTGCCCCACCGGGCAGCTGGTGTGCTG GTCACAGCCGATGTCTCTGGGGTGGGATAAGGAAGCCCGGCGGGCAGGCATCTTCT CCAGTCCAGCCACGATCTCGCTTCCTCGCTGACACTGCCCCTCAGCTACACACGTGT AGCCCTGGGGGCAGCAGTGCTGGTGGTCCGAGCAGCAGACAGCCTCTGGGATTGGA CAGCAGCCCCACTCCCCAGACGTGAGTTGGCAGCAGGTATCGGAGGAGGGACAGCT GCTGACATTATCACAGGGGACATCTCTCTTCAAGGCTTGTGGGTCTGGCAGGCTGAG GTGAGCTGGGGCCTTCTCCATCCAGGGCACCTGGTGGGGCCCCTGTTCACAGGTACC CTTCTGCGTGTCACACGTAAACCCCGCGGGACAGCAGTGTATGTGGTCCTCACAGC ACACAGCCTGGGTAAAAGGGCAGCAGCCCCAGGCCCCCGACTGTAGACGGCAGCA GGTATAGCCATCTGGGCAGCTCACCTCCATGTCACATTTCACATCCCCCACTGTGTG CGCAGGCAGCTTAGTGAGGAGGTCCGTGGTAGCGTTCTCCTTGGAGAGGCACTTAC TCTGGATCAGGTCACACACAGTGTCTTGGGGGCAGCAGTGCAGGTGATCGGAGCAG CAGGTGGCGTTGGGCATTGGGCAGCAGCCATACTTCCCACTGGGCAGCTCACAGCA GGTAGAACCATCAGGGCACCGGGACCGTGCGTCCGGACACATGACCGAGCTGGAC AAGGCCACTGCCCTGTTAGTCCTCTGGGCAGGGAGCTTCTTTGCCAGGGGGTGGGT GCCCGTGGGTGTGATGCAGCGGGTGTGAACCAGGTCGCAGAAGGCACCGTGCGGAC AGCAGTGCACCCTGTCTTCACAGCAGGAAGCCTGGGGCATGGGGCAGCACCCCCAG GAGCCATCGACCATAACACAGCACGTGGAGAAGTCCGGGCATTCGAACTGACTATC AGGGCACTGGATGGCACCCACGGAGTTGTTACCTGATCTTTGGAAGCAGGATCGCC CGTCTGCACTGCAGTGGAAGCCCCGTGGGCAGCAGTGATGGCCATCCCCGCATGCC ACGGCCTCTGGGAAGGGGCAGCAACTGGAAGTCCCTGAGACGGTAAAGATGCAGG AGTGGCCGGCAGAGCAGTGGGCATCAACCTGGCAGGGGCCACCCAGATGCCTGCTC AGTGTTGTGGGCCATTTGTCCAGAAGGGGACGGCAGCAGCTGTAGCTGGCTCCTCC GGGGTCCAGGCAGCAGGCCACAGGGCAGAACTGACCATCTGGGCACCGCGTTCCAG CCACCAGCCCTGCTGTTAAGGCCACCCAGCTCACCAGGGTCCACATGGTGGCGGCG CTAGCGGTGCACACCAATGTGGTGAATGGTCAAATGGCGTTTATTGTATCGAGCTA GGCACTTAAATACAATATCTCTGCAATGCGGAATTCAGTGGTTCGTCCAATCCATGT CAGACCCGTCTGTTGCCTTCCTAATAAGGCACGATCGTACCACCTTACTTCCACCAA TCGGCATGCACGGTGCTTTTTCTCTCCTTGTAAGGCATGTTGCTAACTCATCGTTACC ATGTTGCAAGACTACAAGAGTATTGCATAAGACTACATTCCGGTTATAACGGTCCA ATCTAACAGTGATATTCACTGTGGTATCCTGTTCTAGGTTGGTTCTTGATAGGTCTCG GATCCAGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCT CACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCT CAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAAGAATTCACTGGCCGTCGT TTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGC ACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTC CCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCCTGATGCGGTATTTTCTCCTTAC GCATCTGTGCGGTATTTCACACCGCATATGAGGATCTGCGATCGCTCCGGTGCCCGT CAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCG GCAATTGAACGGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGT CGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGT AGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGCTGAAGCT TCGAGGGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCC ACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTC CGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCC TTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCA ACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCCAAGCTGTGACCG GCGCCTACGATATCGCCACCATGAAAACATTTAACATTTCTCAACAGGATCTAGAAT TAGTAGAAGTAGCGACAGAGAAGATTACAATGCTTTATGAGGATAATAAACATCAT GTGGGAGCGGCAATTCGTACGAAAACAGGAGAAATCATTTCGGCAGTACATATTGA AGCGTATATAGGACGAGTAACTGTTTGTGCAGAAGCCATTGCGATTGGTAGTGCAG TTTCGAATGGACAAAAGGATTTTGACACGATTGTAGCTGTTAGACACCCTTATTCTG ACGAAGTAGATAGAAGTATTCGAGTGGTAAGTCCTTGTGGTATGTGCCTTTCATACG AGACCGAGATCCTGACTGTCGAGTACGGATTGCTTCCTATCGGCAAAATCGTGGAG AAGAGGATTGAATGTACCGTCTATTCAGTCGATAATAATGGGAACATCTACACACA GCCCGTGGCTCAATGGCACGACAGAGGAGAGCAGGAAGTTTTTGAATACTGTCTCG AGGACGGATCCCTCATCCGCGCTACTAAAGATCATAAGTTTATGACCGTGGACGGC CAGATGCTGCCAATTGACGAAATTTTTGAACGAGAGCTGGATCTGATGAGAGTCGA CAACCTTCCAAACTGAGTGCATGACCCGCAAGCCCGGTGCCTGAAATCAACCTCTG GATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACG CTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCGTTAACTAAACTTGTTTATT GCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGC ATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAT GTCTGGAATTGACTCAAATGATGTCAATTAGTCTATCAGAAGCTATCTGGTCTCCCT TCCGGGGGACAAGACATCCCTGTTTAATATTTAAACAGCAGTGTTCCCAAACTGGGT TCTTATATCCCTTGCTCTGGTCAACCAGGTTGCAGGGTTTCCTGTCCTCACAGGAAC GAAGTCCCTAAAGAAACAGTGGCAGCCAGGTTTAGCCCCGGAATTGACTGGATTCC TTTTTTAGGGCCCATTGGTATGGCTGATATCTATAACAAGAAAATATATATATAATA AGTTATCACGTAAGTAGAACATGAAATAACAATATAATTATCGTATGAGTTAAATC TTAAAAGTCACGTAAAAGATAATCATGCGTCATTTTGACTCACGCGGTCGTTATAGT TCAAAATCAGTGACACTTACCGCATTGACAAGCACGCCTCACGGGAGCTCCAAGCG GCGACTGAGATGTCCTAAATGCACAGCGACGGATTCGCGCTATTTAGAAAGAGAGA GCAATATTTCAAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGG [1220] payload plasmid - ss SEQ ID NO: 159 [1221] TAAAAGTTTTGTTACTTTATAGAAGAAATTTTGAGTTTTTGTTTTTTTTAATAA ATAAATAAACATAAATAAATTGTTTGTTGAATTTATTATTAGTATGTAAGTGTAAAT ATAATAAAACTTAATATCTATTCAAATTAATAAATAAACCTCGATATACAGACCGAT AAAACACATGCGTCAATTTTACACATGATTATCTTTAACGTACGTCACAATATGATT ATCTTTCTAGGGTTAATCTAGCTGCGTGTTCTGCAGCGTGTCGAGCATCTTCATCTGC TCCATCACGCTGTAAAACACATTTGCACCGCGAGTCTGCCCGTCCTCCACGGGTTCA AAAACGTGAATGAACGAGGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTG GGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAG CTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCC TGAATGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCT TTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGG GGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTG ATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTT TGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACAC TCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTA TTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATT AACGCTTACAATTTAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGT TTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAGACAATAACCCTGATAA ATGCTTCAATAATATTGAAAAAGGAAGAGTATGAGTATTCAACATTTCCGTGTCGCC CTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGCTGG TGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGAACTG GATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGG CAAGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCA CCAGTCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGC TGCCATAACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATCGGAG GACCGAAGGAGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTT GATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCA CGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAACTACTT ACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGG ACCACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGC CGGTGAGCGTGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCT CCCGTATCGTAGTTATCTACACGACGGGGAGTCAGGCAACTATGGATGAACGAAAT AGACAGATCGCTGAGATAGGTGCCTCACTGATTAAGCATTGGTAACTGTCAGACCA AGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATC TAGGTGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCG TTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTT TTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTT TGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGA GCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAG AACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCT GCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGA TAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACG CTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAG GAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTC GGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGG AGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGG CCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTA CCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAG TCAGTGAGCGAGGAAGCGGAAGAGCGCCCAATACGCAAACCGCCTCTCCCCGCGCG TTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCA GTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTAC ACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACA CAGGAAACAGCTATGACCATGATTACGCCAAGCGCGCCCGCCGGGTAACTCACGGG GTATCCATGTCCATTTCTGCGGCATCCAGCCAGGATACCCGTCCTCGCTGACGTAAT ATCCCAGCGCCGCACCGCTGTCATTAATCTGCACACCGGCACGGCAGTTCCGGCTGT CGCCGGTATTGTTCGGGTTGCTGATGCGCTTCGGGCTGACCATCCGGAACTGTGTCC GGAAAAGCCGCGACGAACTGGTATCCCAGGTGGCCTGAACGAACAGTTCACCGTTA AAGGCGTGCATGGCCACACCTTCCCGAATCATCATGGTAAACGTGCGTTTTCGCTCA ACGTCAATGCAGCAGCAGTCATCCTCGGCAAACTCTTTCCATGCCGCTTCAACCTCG CGGGAAAAGGCACGGGCTTCTTCCTCCCCGATGCCCAGATAGCGCCAGCTTGGGCG ATGACTGAGCCGGAAAAAAGACCCGACGATATGATCCTGATGCAGCTAGATTAACC CTAGAAAGATAGTCTGCGTAAAATTGACGCATGCATTCTTGAAATATTGCTCTCTCT TTCTAAATAGCGCGAATCCGTCGCTGTGCATTTAGGACATCTCAGTCGCCGCTTGGA GCTCCCGTGAGGCGTGCTTGTCAATGCGGTAAGTGTCACTGATTTTGAACTATAACG ACCGCGTGAGTCAAAATGACGCATGATTATCTTTTACGTGACTTTTAAGATTTAACT CATACGATAATTATATTGTTATTTCATGTTCTACTTACGTGATAACTTATTATATATA TATTTTCTTGTTATAGATATCAGCCATACCAATGGGCCCTAAAAAAGGAATCCAGTC AATTCCGGGGCTAAACCTGGCTGCCACTGTTTCTTTAGGGACTTCGTTCCTGTGAGG ACAGGAAACCCTGCAACCTGGTTGACCAGAGCAAGGGATATAAGAACCCAGTTTGG GAACACTGCTGTTTAAATATTAAACAGGGATGTCTTGTCCCCCGGAAGGGAGACCA GATAGCTTCTGATAGACTAATTGACATCATTTGAGTCAATTCCAGACATGATAAGAT ACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATT TGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAA GTTTAGTTAACGCATGATACAAAGGCATTAAAGCAGCGTATCCACATAGCGTAAAA GGAGCAACATAGTTAAGAATACCAGTCAATCTTTCACAAATTTTGTAATCCAGAGG TTGATTTCAGGCACCGGGCTTGCGGGTCATGCACTCAGTTTGGAAGGTTGTCGACTC TCATCAGATCCAGCTCTCGTTCAAAAATTTCGTCAATTGGCAGCATCTGGCCGTCCA CGGTCATAAACTTATGATCTTTAGTAGCGCGGATGAGGGATCCGTCCTCGAGACAG TATTCAAAAACTTCCTGCTCTCCTCTGTCGTGCCATTGAGCCACGGGCTGTGTGTAG ATGTTCCCATTATTATCGACTGAATAGACGGTACATTCAATCCTCTTCTCCACGATTT TGCCGATAGGAAGCAATCCGTACTCGACAGTCAGGATCTCGGTCTCGTATGAAAGG CACATACCACAAGGACTTACCACTCGAATACTTCTATCTACTTCGTCAGAATAAGGG TGTCTAACAGCTACAATCGTGTCAAAATCCTTTTGTCCATTCGAAACTGCACTACCA ATCGCAATGGCTTCTGCACAAACAGTTACTCGTCCTATATACGCTTCAATATGTACT GCCGAAATGATTTCTCCTGTTTTCGTACGAATTGCCGCTCCCACATGATGTTTATTAT CCTCATAAAGCATTGTAATCTTCTCTGTCGCTACTTCTACTAATTCTAGATCCTGTTG AGAAATGTTAAATGTTTTCATGGTGGCGATATCGTAGGCGCCGGTCACAGCTTGGAT CTGTAACGGCGCAGAACAGAAAACGAAACAAAGACGTAGAGTTGAGCAAGCAGGG TCAGGCAAAGCGTGGAGAGCCGGCTGAGTCTAGGTAGGCTCCAAGGGAGCGCCGG ACAAAGGCCCGGTCTCGACCTGAGCTTTAAACTTACCTAGACGGCGGACGCAGTTC AGGAGGCACCACAGGCGGGAGGCGGCAGAACGCGACTCAACCGGCGTGGATGGCG GCCTCAGGTAGGGCGGCGGGCGCGTGAAGGAGAGATGCGAGCCCCTCGAAGCTTC AGCTGTGTTCTGGCGGCAAACCCGTTGCGAAAAAGAACGTTCACGGCGACTACTGC ACTTATATACGGTTCTCCCCCACCCTCGGGAAAAAGGCGGAGCCAGTACACGACAT CACTTTCCCAGTTTACCCCGCGCCACCTTCTCTAGGCACCCGTTCAATTGCCGACCC CTCCCCCCAACTTCTCGGGGACTGTGGGCGATGTGCGCTCTGCCCACTGACGGGCAC CGGAGCGATCGCAGATCCTCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGA GAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGG CGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGC AAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA CGGCCAGTGAATTCTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAG CGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCTGGATCCGAGA CCTATCAAGAACCAACCTAGAACAGGATACCACAGTGAATATCACTGTTAGATTGG ACCGTTATAACCGGAATGTAGTCTTATGCAATACTCTTGTAGTCTTGCAACATGGTA ACGATGAGTTAGCAACATGCCTTACAAGGAGAGAAAAAGCACCGTGCATGCCGATT GGTGGAAGTAAGGTGGTACGATCGTGCCTTATTAGGAAGGCAACAGACGGGTCTGA CATGGATTGGACGAACCACTGAATTCCGCATTGCAGAGATATTGTATTTAAGTGCCT AGCTCGATACAATAAACGCCATTTGACCATTCACCACATTGGTGTGCACCGCTAGCG CCGCCACCATGTGGACCCTGGTGAGCTGGGTGGCCTTAACAGCAGGGCTGGTGGCT GGAACGCGGTGCCCAGATGGTCAGTTCTGCCCTGTGGCCTGCTGCCTGGACCCCGG AGGAGCCAGCTACAGCTGCTGCCGTCCCCTTCTGGACAAATGGCCCACAACACTGA GCAGGCATCTGGGTGGCCCCTGCCAGGTTGATGCCCACTGCTCTGCCGGCCACTCCT GCATCTTTACCGTCTCAGGGACTTCCAGTTGCTGCCCCTTCCCAGAGGCCGTGGCAT GCGGGGATGGCCATCACTGCTGCCCACGGGGCTTCCACTGCAGTGCAGACGGGCGA TCCTGCTTCCAAAGATCAGGTAACAACTCCGTGGGTGCCATCCAGTGCCCTGATAGT CAGTTCGAATGCCCGGACTTCTCCACGTGCTGTGTTATGGTCGATGGCTCCTGGGGG TGCTGCCCCATGCCCCAGGCTTCCTGCTGTGAAGACAGGGTGCACTGCTGTCCGCAC GGTGCCTTCTGCGACCTGGTTCACACCCGCTGCATCACACCCACGGGCACCCACCCC CTGGCAAAGAAGCTCCCTGCCCAGAGGACTAACAGGGCAGTGGCCTTGTCCAGCTC GGTCATGTGTCCGGACGCACGGTCCCGGTGCCCTGATGGTTCTACCTGCTGTGAGCT GCCCAGTGGGAAGTATGGCTGCTGCCCAATGCCCAACGCCACCTGCTGCTCCGATC ACCTGCACTGCTGCCCCCAAGACACTGTGTGTGACCTGATCCAGAGTAAGTGCCTCT CCAAGGAGAACGCTACCACGGACCTCCTCACTAAGCTGCCTGCGCACACAGTGGGG GATGTGAAATGTGACATGGAGGTGAGCTGCCCAGATGGCTATACCTGCTGCCGTCT ACAGTCGGGGGCCTGGGGCTGCTGCCCTTTTACCCAGGCTGTGTGCTGTGAGGACC ACATACACTGCTGTCCCGCGGGGTTTACGTGTGACACGCAGAAGGGTACCTGTGAA CAGGGGCCCCACCAGGTGCCCTGGATGGAGAAGGCCCCAGCTCACCTCAGCCTGCC AGACCCACAAGCCTTGAAGAGAGATGTCCCCTGTGATAATGTCAGCAGCTGTCCCT CCTCCGATACCTGCTGCCAACTCACGTCTGGGGAGTGGGGCTGCTGTCCAATCCCAG AGGCTGTCTGCTGCTCGGACCACCAGCACTGCTGCCCCCAGGGCTACACGTGTGTA GCTGAGGGGCAGTGTCAGCGAGGAAGCGAGATCGTGGCTGGACTGGAGAAGATGC CTGCCCGCCGGGCTTCCTTATCCCACCCCAGAGACATCGGCTGTGACCAGCACACCA GCTGCCCGGTGGGGCAGACCTGCTGCCCGAGCCTGGGTGGGAGCTGGGCCTGCTGC CAGTTGCCCCATGCTGTGTGCTGCGAGGATCGCCAGCACTGCTGCCCGGCTGGCTAC ACCTGCAACGTGAAGGCTCGATCCTGCGAGAAGGAAGTGGTCTCTGCCCAGCCTGC CACCTTCCTGGCCCGTAGCCCTCACGTGGGTGTGAAGGACGTGGAGTGTGGGGAAG GACACTTCTGCCATGATAACCAGACCTGCTGCCGAGACAACCGACAGGGCTGGGCC TGCTGTCCCTACCGCCAGGGCGTCTGTTGTGCTGATCGGCGCCACTGCTGTCCTGCT GGCTTCCGCTGCGCAGCCAGGGGTACCAAGTGTTTGCGCAGGGAGGCCCCGCGCTG GGACGCCCCTTTGAGGGACCCAGCCTTGAGACAGCTGCTGTGAAATAAAGGAAATT TATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACCGGTATTATCTTCG TGTTATTGCCGTCGTAGCAGAACTGTTAACAAGACACAGTGTGTTGTCTATTCGGTT GGAATACCTAACGCTATCTGCAAGCCTGGCAATTCCACGTTATAGCCGTTGATCAGA TAAGAGTCAAGCGGCGCTCACTGTAGACCTTGACACACCACTGATCGGCAGATTCC TTAACTGTAATCAAGAGTTCTTAGAGGAGACAATCCAACTCGGTCTCCTGCAGAGG AACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGC GAGCGAGCGCGCAGAGAGGGAGTGGCCAAA [1222] Rep2BFP-CODE without Cap5 sequence - uninduced SEQ ID NO: 160 [1223] GGAGGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCTGT ATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTGGG TATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAACG CGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTTG ACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGAA TGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCCT GACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGTA AGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCAC ATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCTG AGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTTT GCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAAG GTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCTC CAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGGA GCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAGA ACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTCA GCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGTGAGTTTGGGGACCCT TGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGTT TTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATGA TAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCGTTGTCTCCTCTTATTTTCTT TTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAAA TTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAGG CAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACATAACTTCGTAT AAAGTATACTATACGAAGTTATCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTTC TTTTTCCTACAGATGTCAGAACTCATTAAAGAGAATATGCACATGAAGCTGTATATG GAAGGTACTGTAGACAACCACCATTTCAAATGCACGTCCGAAGGTGAGGGGAAGCC ATACGAGGGTACCCAAACTATGCGCATCAAAGTGGTTGAGGGTGGCCCCCTGCCAT TCGCATTCGACATCCTGGCAACTAGCTTTCTTTACGGTTCCAAGACATTCATAAATC ATACCCAGGGTATTCCCGATTTCTTCAAACAATCCTTCCCGGAAGGGTTTACTTGGG AGCGGGTCACGACATATGAAGACGGGGGTGTTCTTACAGCCACACAGGATACGAGT TTGCAAGACGGTTGTCTTATCTATAACGTGAAGATTCGGGGTGTGAATTTCACATCC AATGGCCCGGTGATGCAGAAAAAAACACTGGGCTGGGAAGCATTTACGGAGACGTT GTATCCCGCCGATGGAGGTCTCGAGGGCCGAAACGATATGGCCCTCAAGTTGGTAG GTGGTTCTCACCTTATAGCAAACATTAAGACCACGTATCGATCAAAAAAACCCGCT AAGAATCTGAAAATGCCAGGCGTGTATTATGTTGATTACAGACTGGAGCGAATAAA AGAGGCTAACAATGAGACCTACGTCGAACAGCATGAAGTCGCTGTAGCTAGATATT GCGACCTCCCGTCAAAGTTGGGCCATAAATTGAATTAACCTCAGGTGCAGGCTGCC TATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGA TCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGAC TTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGT CTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTA TTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTG GCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCA TAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTT CTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTC CTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCATAACTTCGTATA AAGTATACTATACGAAGTTATAATTGTTATAATTAAATGATAAGGTAGAATATTTCT GCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACACCCT GGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGAG GATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCTT CTTTTTCCTACAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCCT CATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTGG ACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGGC CAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAAA CGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAGTT CGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAACA TCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCAAT GAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAGGG GAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCAAG GTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTGAT CGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTTCG AACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTCTG GATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGGGC AAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGCCA AGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGCGA GTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGACA GGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTGCA GACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGAAA GACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAAAG GCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCTTG CACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA [1224] Rep2BFP-CODE without Cap5 sequence - induced SEQ ID NO: 161 [1225] ATGGAGGGGTGGAGTCGTGACGTGAATTACGTCATAGGGTTAGGGAGGTCCT GTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACACCATGTGGTCACGCTG GGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAAGCGGGAGGTTTGAA CGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAGGTCCCCAGCGACCTT GACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGGGTGGCCGAGAAGGA ATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGATTGAGCAGGCACCCC TGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAATGGCGCCGTGTGAGT AAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGAGAGAGCTACTTCCA CATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGTTTTGGGACGTTTCCT GAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGGGATCGAGCCGACTT TGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCGGAGGCGGGAACAA GGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAAACCCAGCCTGAGCT CCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTGTTTGAATCTCACGG AGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGCAGACGCAGGAGCAG AACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGATCAGATCAAAAACTTC AGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGGTGAGTTTGGGGACCC TTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATATGGAGGGGGCAAAGT TTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATCACCATGGACCCTCATG ATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCGTTGTCTCCTCTTATTTTCT TTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATTTGTAACGAATTTTTAA ATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTAATCACTTTTTTTTCAAG GCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAGAGAACATAACTTCGTA TAAAGTATACTATACGAAGTTATAATTGTTATAATTAAATGATAAGGTAGAATATTT CTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGGTAGAAACAACTACACC CTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATATACACTGTTTGAGATGA GGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTAACCATGTTCATGCCTTCT TCTTTTTCCTACAGGGGATTACCTCGGAGAAGCAGTGGATCCAGGAGGACCAGGCC TCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCCAAATCAAGGCTGCCTTG GACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCCCCCGACTACCTGGTGGG CCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTATAAAATTTTGGAACTAA ACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGGATGGGCCACGAAAAAG TTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAACTACCGGGAAGACCAA CATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGGTGCGTAAACTGGACCA ATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGTGATCTGGTGGGAGGAG GGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCCATTCTCGGAGGAAGCA AGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGATAGACCCGACTCCCGTG ATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACGGGAACTCAACGACCTT CGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTTGAACTCACCCGCCGTC TGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAAAGACTTTTTCCGGTGG GCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACGTCAAAAAGGGTGGAGC CAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGAGCCCAAACGGGTGCGC GAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTTCGATCAACTACGCAGA CAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAATCTGATGCTGTTTCCCTG CAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTGCTTCACTCACGGACAGA AAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACCCGTTTCTGTCGTCAAAA AGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGGAAAGGTGCCAGACGCT TGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACTGCATCTTTGAACAATAA [1226] AAV native polyA SEQ ID NO: 162 [1227] TTGCTTGTTAATCAATAAACCGTTTAATTCGTTTCAGTTGAACTTTGGTCTCT GCGTATTTCTTTCTTATCTAGTTTCCATGGCTACGTAGATAAGTAGCATGGCGGGTT AATCATTAACTACAGCCCGGGCGTTTAAACAGCGGGCGGAGGGGTGGAGTCGTGAC GTGAATTACGTCATAGGGTTAGGGAGGTCCTGTATTAGAGGTCACGTGAGTGTTTTG CGACATTTTGCGACACCATGTG [1228] Full SV40 polyA SEQ ID NO: 163 [1229] CAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGC AGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCA TTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGG TTCAGGGGGAGGTGTGGGAGGTTTTTTAAACACGTGCGGACCGAGTGGCGTAATCA T [1230] Inducible Cap + Rep construct - uninduced SEQ ID NO: 164 [1231] TTAAAGGGGTCGGGTAAGGTATCGGGTTCCGATAGGTCTGGTGGTTCTGTAT TCCCCGGTGCTGTCCGGGGCAAAGTCCACAAACTGGGGGTCGTTGTAGTTGTTTGTG TACTGGATCTCTGGGTTCCACCTCTTGGAGTTTTCCTTCTTGAGCTCCCACTCCATCT CCACGGTGACCTGCCCGGTGCTGTACTGGGTGATGAAGCTGCTGACGGGCACGTCC GAGAAGCTGGTGATATTTCCGGGCACAGGCGTGTTCTTGATGAGCATCATGGGCGG TGGGTGTTTGAGTCCGAATCCGCCCATGGCCGGAGAGGGGTGAAAGTGCGCCCCCG TCTCTGGGATCTTGGCCCAGATGGGTCCTTGGAGGTACACGTCCCTCTCCATCCACA CGCTGCCGGGCACGATTTCCTGGAGGTTGTACGTGCCGGTCGCGGGGGCAGTGGTG GAGCTCTGGTTGTTGGTGGCCATCTGCCCGCCGACGTTGTACGCCACGCGGTTCACC GGCTGCGTCTCGCTCTCGCTGGTGATGAGCATGTTGCCCTCGAGGTACGTGGCGGTG GTGCCCGGGTTCGCCGGCTGGCTGTTGAAGATCATAGTGTTCTCCAGGGCATAGGTG TTGCTGCCCTGGAGGTTGTTGGTCATGCCGTTCGGCTGCGGGGGCACCTGGTAACTC GCGCCCTCGAGCTCCATCCTATTGGTCGTGGCGAAGGCGCTGACACTGGCGCGGTT GACCCCGGAGCCCAGGTTCCAGCCCTGGGTTCGGCCCATGGGCCCCGGGAACCAGT TTTTGTAGGTGTTGGCGTATCTCCCGGCCAGGTTCTTGTTGAACTGGACTCCGCCAG TGTTATTTGTGCTCACGAAGCGGTACAAGTACTGGTCCACCAGCGGGTTGGCCAGCT TGAACAGGTTCTGACTGGGAGCGAAGCTGGAGTGGAAGGGCACCTCCTCAAAGTTG TAGGTAAACTCAAAGTTGTTGCCCGTTCTCAGCATCTTGCTGGGAAAGTACTCTAGG CAGAAGAAGCTGCTCCTCTCGGTGGGATTTTCTGTGTTGTCGCGGTTCAGCGTCGCG TAACCGTACTGCGGCAGCGTAAAGACCTGCGGAGGGAAGGCCGGCAGGCATCCCTC GGTCCCGTTGCCGACGACGTAGGGCAGCTGGTAGTCGTCGTCCGTAAACACTTGGA CGGTGGAGGTGAGGTTGTTGGCGATGGTGGTGGTGGAGTCCTGCACCGTGACCTCT TTGACTTGAATGTTGAAGATTTTGACTCTGAGGGACCGGGGTCTGAAGCCCCAGTA GTTGTTGATGAGTCTTTGCCAGTCTCGGGGGCTCCAGTGGCTGTGGAAGCGGTTAAA GTCAAAGTACCCCCAGGGGGTGCTGTATCCAAAGTAGGCGTTGGCGTTGCTTCCGTC GACGGAGCCGCTTTTGATCTCTCGGTACTGGTGGTTGTTGTAGCTGGGCAGCACCCA GGTTCGGGTGGACTTGGTGACGACTCTGTCCCCCATCCACGTGGAATCGCAATGCCA ATCTCCCGAGGCATTGCCCACTCCATCGGCACCTTGGTTATTGTCGCCCAATGGGCC GCCACCTCCCGCAGACATTGTATCAGCTCCCAAACTTGAGGCTGGTTGGGCTGGGAT TTGCAGCTGCTGGGATCCGCTGGGTCCAGCTTCGGCGTCTGACGAGGTGGAAGGCT TGGAGTCCTCTTCGGTCCGAGCCTTCTTTCTTTTTGGAAAGTGGTCGTCTATCCGCTT TCCGGTAGGGGCCGTCTTAGCACCCTCTTCAACCAGGCCAAAAGGTTCGAGAACCC TTTTCTTGGCCTGAAAGACTGCCTTTCCGAGGTTTCCCCCGAAGGATGTGTCGTCGG CGAGCTTCTCCTGAAACTCGGCGTCCGCGTGGTTGTACTTGAGGTAGGGGTTGTCTC CCGCCTCAAGCTGCTCGTTGTACGAGATGTCGTGCTCTCGCGCGACCTCGTCTGCCC TGTTGACAGGCTCTCCTCGATCGAGACCGTTTCCGGGTCCGAGATAGTTATAACCAG GCAGCACAAGACCACGGGCTTGATCTTGATGCTGCTGATTGGGTTTTGGTTTCGGTG GGCCCGCTTCAAGGCCCAAAAACTCGCGAAGACCTTCACCAACTTCTTCCAACCAA TCTGGAGGGTGATCAACAAAAGACATACCTGATTTAAATCATTTATTGTTCAAAGAT GCAGTCATCCAAATCCACATTGACCAGATCGCAGGCAGTGCAAGCGTCTGGCACCT TTCCCATGATATGATGAATGTAGCACAGTTTCTGATACGCCTTTTTGACGACAGAAA CGGGTTGAGATTCTGACACGGGAAAGCACTCTAAACAGTCTTTCTGTCCGTGAGTG AAGCAGATATTTGAATTCTGATTCATTCTCTCGCATTGTCTGCAGGGAAACAGCATC AGATTCATGCCCACGTGACGAGAACATTTGTTTTGGTACCTGTCTGCGTAGTTGATC GAAGCTTCCGCGTCTGACGTCGATGGCTGCGCAACTTTTACGAGGGTAGGAAGTGG TACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGACG GTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTATA CGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGGG AGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTCT CTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAAA GTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATCA CTGATAGGGAGTAAACTCGCGAGAGAAATGTTCTGGCACCTGCACTTGCACTGGGG ACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTTA GTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAAGATATT TTATTTAGCGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACAC CATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAA GCGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAG GTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGG GTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGAT TGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAAT GGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGA GAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGT TTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGG GATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCG GAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAA ACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTG TTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGC AGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGAT CAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGG TGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATA TGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATC ACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATT GTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATT TGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTA ATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAG AGAACATAACTTCGTATAAAGTATACTATACGAAGTTATCGGGCCCCTCTGCTAACC ATGTTCATGCCTTCTTCTTTTTCCTACAGATGTCAGAACTCATTAAAGAGAATATGC ACATGAAGCTGTATATGGAAGGTACTGTAGACAACCACCATTTCAAATGCACGTCC GAAGGTGAGGGGAAGCCATACGAGGGTACCCAAACTATGCGCATCAAAGTGGTTG AGGGTGGCCCCCTGCCATTCGCATTCGACATCCTGGCAACTAGCTTTCTTTACGGTT CCAAGACATTCATAAATCATACCCAGGGTATTCCCGATTTCTTCAAACAATCCTTCC CGGAAGGGTTTACTTGGGAGCGGGTCACGACATATGAAGACGGGGGTGTTCTTACA GCCACACAGGATACGAGTTTGCAAGACGGTTGTCTTATCTATAACGTGAAGATTCG GGGTGTGAATTTCACATCCAATGGCCCGGTGATGCAGAAAAAAACACTGGGCTGGG AAGCATTTACGGAGACGTTGTATCCCGCCGATGGAGGTCTCGAGGGCCGAAACGAT ATGGCCCTCAAGTTGGTAGGTGGTTCTCACCTTATAGCAAACATTAAGACCACGTAT CGATCAAAAAAACCCGCTAAGAATCTGAAAATGCCAGGCGTGTATTATGTTGATTA CAGACTGGAGCGAATAAAAGAGGCTAACAATGAGACCTACGTCGAACAGCATGAA GTCGCTGTAGCTAGATATTGCGACCTCCCGTCAAAGTTGGGCCATAAATTGAATTAA CCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGC TCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAA GCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGT GTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTT AAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGC TGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTG CTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATT TTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAG CCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATG GAGATCATAACTTCGTATAAAGTATACTATACGAAGTTATAATTGTTATAATTAAAT GATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTG GTAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGAT ATACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCT AACCATGTTCATGCCTTCTTCTTTTTCCTACAGGGGATTACCTCGGAGAAGCAGTGG ATCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCC CAAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGC CCCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTT ATAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGG GATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCA ACTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGG GTGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGG TGATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGC CATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGA TAGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACG GGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTT GAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAA AGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACG TCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTT CGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAAT CTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTG CTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGG AAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACT GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCA GATTGGCTCGAGGACACTCTCTCTGAATAACTAGCTA [1232] Inducible Cap + Rep construct - induced SEQ ID NO: 165 [1233] TTAAAGGGGTCGGGTAAGGTATCGGGTTCCGATAGGTCTGGTGGTTCTGTAT TCCCCGGTGCTGTCCGGGGCAAAGTCCACAAACTGGGGGTCGTTGTAGTTGTTTGTG TACTGGATCTCTGGGTTCCACCTCTTGGAGTTTTCCTTCTTGAGCTCCCACTCCATCT CCACGGTGACCTGCCCGGTGCTGTACTGGGTGATGAAGCTGCTGACGGGCACGTCC GAGAAGCTGGTGATATTTCCGGGCACAGGCGTGTTCTTGATGAGCATCATGGGCGG TGGGTGTTTGAGTCCGAATCCGCCCATGGCCGGAGAGGGGTGAAAGTGCGCCCCCG TCTCTGGGATCTTGGCCCAGATGGGTCCTTGGAGGTACACGTCCCTCTCCATCCACA CGCTGCCGGGCACGATTTCCTGGAGGTTGTACGTGCCGGTCGCGGGGGCAGTGGTG GAGCTCTGGTTGTTGGTGGCCATCTGCCCGCCGACGTTGTACGCCACGCGGTTCACC GGCTGCGTCTCGCTCTCGCTGGTGATGAGCATGTTGCCCTCGAGGTACGTGGCGGTG GTGCCCGGGTTCGCCGGCTGGCTGTTGAAGATCATAGTGTTCTCCAGGGCATAGGTG TTGCTGCCCTGGAGGTTGTTGGTCATGCCGTTCGGCTGCGGGGGCACCTGGTAACTC GCGCCCTCGAGCTCCATCCTATTGGTCGTGGCGAAGGCGCTGACACTGGCGCGGTT GACCCCGGAGCCCAGGTTCCAGCCCTGGGTTCGGCCCATGGGCCCCGGGAACCAGT TTTTGTAGGTGTTGGCGTATCTCCCGGCCAGGTTCTTGTTGAACTGGACTCCGCCAG TGTTATTTGTGCTCACGAAGCGGTACAAGTACTGGTCCACCAGCGGGTTGGCCAGCT TGAACAGGTTCTGACTGGGAGCGAAGCTGGAGTGGAAGGGCACCTCCTCAAAGTTG TAGGTAAACTCAAAGTTGTTGCCCGTTCTCAGCATCTTGCTGGGAAAGTACTCTAGG CAGAAGAAGCTGCTCCTCTCGGTGGGATTTTCTGTGTTGTCGCGGTTCAGCGTCGCG TAACCGTACTGCGGCAGCGTAAAGACCTGCGGAGGGAAGGCCGGCAGGCATCCCTC GGTCCCGTTGCCGACGACGTAGGGCAGCTGGTAGTCGTCGTCCGTAAACACTTGGA CGGTGGAGGTGAGGTTGTTGGCGATGGTGGTGGTGGAGTCCTGCACCGTGACCTCT TTGACTTGAATGTTGAAGATTTTGACTCTGAGGGACCGGGGTCTGAAGCCCCAGTA GTTGTTGATGAGTCTTTGCCAGTCTCGGGGGCTCCAGTGGCTGTGGAAGCGGTTAAA GTCAAAGTACCCCCAGGGGGTGCTGTATCCAAAGTAGGCGTTGGCGTTGCTTCCGTC GACGGAGCCGCTTTTGATCTCTCGGTACTGGTGGTTGTTGTAGCTGGGCAGCACCCA GGTTCGGGTGGACTTGGTGACGACTCTGTCCCCCATCCACGTGGAATCGCAATGCCA ATCTCCCGAGGCATTGCCCACTCCATCGGCACCTTGGTTATTGTCGCCCAATGGGCC GCCACCTCCCGCAGACATTGTATCAGCTCCCAAACTTGAGGCTGGTTGGGCTGGGAT TTGCAGCTGCTGGGATCCGCTGGGTCCAGCTTCGGCGTCTGACGAGGTGGAAGGCT TGGAGTCCTCTTCGGTCCGAGCCTTCTTTCTTTTTGGAAAGTGGTCGTCTATCCGCTT TCCGGTAGGGGCCGTCTTAGCACCCTCTTCAACCAGGCCAAAAGGTTCGAGAACCC TTTTCTTGGCCTGAAAGACTGCCTTTCCGAGGTTTCCCCCGAAGGATGTGTCGTCGG CGAGCTTCTCCTGAAACTCGGCGTCCGCGTGGTTGTACTTGAGGTAGGGGTTGTCTC CCGCCTCAAGCTGCTCGTTGTACGAGATGTCGTGCTCTCGCGCGACCTCGTCTGCCC TGTTGACAGGCTCTCCTCGATCGAGACCGTTTCCGGGTCCGAGATAGTTATAACCAG GCAGCACAAGACCACGGGCTTGATCTTGATGCTGCTGATTGGGTTTTGGTTTCGGTG GGCCCGCTTCAAGGCCCAAAAACTCGCGAAGACCTTCACCAACTTCTTCCAACCAA TCTGGAGGGTGATCAACAAAAGACATACCTGATTTAAATCATTTATTGTTCAAAGAT GCAGTCATCCAAATCCACATTGACCAGATCGCAGGCAGTGCAAGCGTCTGGCACCT TTCCCATGATATGATGAATGTAGCACAGTTTCTGATACGCCTTTTTGACGACAGAAA CGGGTTGAGATTCTGACACGGGAAAGCACTCTAAACAGTCTTTCTGTCCGTGAGTG AAGCAGATATTTGAATTCTGATTCATTCTCTCGCATTGTCTGCAGGGAAACAGCATC AGATTCATGCCCACGTGACGAGAACATTTGTTTTGGTACCTGTCTGCGTAGTTGATC GAAGCTTCCGCGTCTGACGTCGATGGCTGCGCAACTTTTACGAGGGTAGGAAGTGG TACGGAAAGTTGGTATAAGACAAAAGTGTTGTGGAATTGCTCCAGGCGATCTGACG GTTCACTAAACGAGCTCTGCTTTTATAGGCGCCCACCGTACACGCCTAAAGCTTATA CGTTCTCTATCACTGATAGGGAGTAAACTGGATATACGTTCTCTATCACTGATAGGG AGTAAACTGTAGATACGTTCTCTATCACTGATAGGGAGTAAACTGGTCATACGTTCT CTATCACTGATAGGGAGTAAACTCCTTATACGTTCTCTATCACTGATAGGGAGTAAA GTCTGCATACGTTCTCTATCACTGATAGGGAGTAAACTCTTCATACGTTCTCTATCA CTGATAGGGAGTAAACTCGCGAGAGAAATGTTCTGGCACCTGCACTTGCACTGGGG ACAGCCTATTTTGCTAGTTTGTTTTGTTTCGTTTTGTTTTGATGGAGAGCGTATGTTA GTACTATCGATTCACACAAAAAACCAACACACAGATGTAATGAAAATAAAGATATT TTATTTAGCGGTCCTGTATTAGAGGTCACGTGAGTGTTTTGCGACATTTTGCGACAC CATGTGGTCACGCTGGGTATTTAAGCCCGAGTGAGCACGCAGGGTCTCCATTTTGAA GCGGGAGGTTTGAACGCGCAGCCGCCATGCCGGGGTTTTACGAGATTGTGATTAAG GTCCCCAGCGACCTTGACGAGCATCTGCCCGGCATTTCTGACAGCTTTGTGAACTGG GTGGCCGAGAAGGAATGGGAGTTGCCGCCAGATTCTGACATGGATCTGAATCTGAT TGAGCAGGCACCCCTGACCGTGGCCGAGAAGCTGCAGCGCGACTTTCTGACGGAAT GGCGCCGTGTGAGTAAGGCCCCGGAGGCCCTTTTCTTTGTGCAATTTGAGAAGGGA GAGAGCTACTTCCACATGCACGTGCTCGTGGAAACCACCGGGGTGAAATCCATGGT TTTGGGACGTTTCCTGAGTCAGATTCGCGAAAAACTGATTCAGAGAATTTACCGCGG GATCGAGCCGACTTTGCCAAACTGGTTCGCGGTCACAAAGACCAGAAATGGCGCCG GAGGCGGGAACAAGGTGGTGGATGAGTGCTACATCCCCAATTACTTGCTCCCCAAA ACCCAGCCTGAGCTCCAGTGGGCGTGGACTAATATGGAACAGTATTTAAGCGCCTG TTTGAATCTCACGGAGCGTAAACGGTTGGTGGCGCAGCATCTGACGCACGTGTCGC AGACGCAGGAGCAGAACAAAGAGAATCAGAATCCCAATTCTGATGCGCCGGTGAT CAGATCAAAAACTTCAGCCAGGTACATGGAGCTGGTCGGGTGGCTCGTGGACAAGG TGAGTTTGGGGACCCTTGATTGTTCTTTCTTTTTCGCTATTGTAAAATTCATGTTATA TGGAGGGGGCAAAGTTTTCAGGGTGTTGTTTAGAATGGGAAGATGTCCCTTGTATC ACCATGGACCCTCATGATAATTTTGTTTCTTTCACTTTCTACTCTGTTGACAACCATT GTCTCCTCTTATTTTCTTTTCATTTTCTGTAACTTTTTCGTTAAACTTTAGCTTGCATT TGTAACGAATTTTTAAATTCACTTTTGTTTATTTGTCAGATTGTAAGTACTTTCTCTA ATCACTTTTTTTTCAAGGCAATCAGGGTATATTATATTGTACTTCAGCACAGTTTTAG AGAACATAACTTCGTATAAAGTATACTATACGAAGTTATCATTGTTATAATTAAATG ATAAGGTAGAATATTTCTGCATATAAATTCTGGCTGGCGTGGAAATATTCTTATTGG TAGAAACAACTACACCCTGGTCATCATCCTGCCTTTCTCTTTATGGTTACAATGATA TACACTGTTTGAGATGAGGATAAAATACTCTGAGTCCAAACCGGGCCCCTCTGCTA ACCATGTTCATGCCTTCTTCTTTTTCCTACAGGGGATTACCTCGGAGAAGCAGTGGA TCCAGGAGGACCAGGCCTCATACATCTCCTTCAATGCGGCCTCCAACTCGCGGTCCC AAATCAAGGCTGCCTTGGACAATGCGGGAAAGATTATGAGCCTGACTAAAACCGCC CCCGACTACCTGGTGGGCCAGCAGCCCGTGGAGGACATTTCCAGCAATCGGATTTA TAAAATTTTGGAACTAAACGGGTACGATCCCCAATATGCGGCTTCCGTCTTTCTGGG ATGGGCCACGAAAAAGTTCGGCAAGAGGAACACCATCTGGCTGTTTGGGCCTGCAA CTACCGGGAAGACCAACATCGCGGAGGCCATAGCCCACACTGTGCCCTTCTACGGG TGCGTAAACTGGACCAATGAGAACTTTCCCTTCAACGACTGTGTCGACAAGATGGT GATCTGGTGGGAGGAGGGGAAGATGACCGCCAAGGTCGTGGAGTCGGCCAAAGCC ATTCTCGGAGGAAGCAAGGTGCGCGTGGACCAGAAATGCAAGTCCTCGGCCCAGAT AGACCCGACTCCCGTGATCGTCACCTCCAACACCAACATGTGCGCCGTGATTGACG GGAACTCAACGACCTTCGAACACCAGCAGCCGTTGCAAGACCGGATGTTCAAATTT GAACTCACCCGCCGTCTGGATCATGACTTTGGGAAGGTCACCAAGCAGGAAGTCAA AGACTTTTTCCGGTGGGCAAAGGATCACGTGGTTGAGGTGGAGCATGAATTCTACG TCAAAAAGGGTGGAGCCAAGAAAAGACCCGCCCCCAGTGACGCAGATATAAGTGA GCCCAAACGGGTGCGCGAGTCAGTTGCGCAGCCATCGACGTCAGACGCGGAAGCTT CGATCAACTACGCAGACAGGTACCAAAACAAATGTTCTCGTCACGTGGGCATGAAT CTGATGCTGTTTCCCTGCAGACAATGCGAGAGAATGAATCAGAATTCAAATATCTG CTTCACTCACGGACAGAAAGACTGTTTAGAGTGCTTTCCCGTGTCAGAATCTCAACC CGTTTCTGTCGTCAAAAAGGCGTATCAGAAACTGTGCTACATTCATCATATCATGGG AAAGGTGCCAGACGCTTGCACTGCCTGCGATCTGGTCAATGTGGATTTGGATGACT GCATCTTTGAACAATAAATGATTTAAATCAGGTATGGCTGCCGATGGTTATCTTCCA GATTGGCTCGAGGACACTCTCTCTGAATAACTAGCTA [1234] Progranulin Sequence (with stop codon) SEQ ID NO: 166 [1235] ATGTGGACCCTGGTGAGCTGGGTGGCCTTAACAGCAGGGCTGGTGGCTGGAA CGCGGTGCCCAGATGGTCAGTTCTGCCCTGTGGCCTGCTGCCTGGACCCCGGAGGA GCCAGCTACAGCTGCTGCCGTCCCCTTCTGGACAAATGGCCCACAACACTGAGCAG GCATCTGGGTGGCCCCTGCCAGGTTGATGCCCACTGCTCTGCCGGCCACTCCTGCAT CTTTACCGTCTCAGGGACTTCCAGTTGCTGCCCCTTCCCAGAGGCCGTGGCATGCGG GGATGGCCATCACTGCTGCCCACGGGGCTTCCACTGCAGTGCAGACGGGCGATCCT GCTTCCAAAGATCAGGTAACAACTCCGTGGGTGCCATCCAGTGCCCTGATAGTCAG TTCGAATGCCCGGACTTCTCCACGTGCTGTGTTATGGTCGATGGCTCCTGGGGGTGC TGCCCCATGCCCCAGGCTTCCTGCTGTGAAGACAGGGTGCACTGCTGTCCGCACGGT GCCTTCTGCGACCTGGTTCACACCCGCTGCATCACACCCACGGGCACCCACCCCCTG GCAAAGAAGCTCCCTGCCCAGAGGACTAACAGGGCAGTGGCCTTGTCCAGCTCGGT CATGTGTCCGGACGCACGGTCCCGGTGCCCTGATGGTTCTACCTGCTGTGAGCTGCC CAGTGGGAAGTATGGCTGCTGCCCAATGCCCAACGCCACCTGCTGCTCCGATCACCT GCACTGCTGCCCCCAAGACACTGTGTGTGACCTGATCCAGAGTAAGTGCCTCTCCAA GGAGAACGCTACCACGGACCTCCTCACTAAGCTGCCTGCGCACACAGTGGGGGATG TGAAATGTGACATGGAGGTGAGCTGCCCAGATGGCTATACCTGCTGCCGTCTACAG TCGGGGGCCTGGGGCTGCTGCCCTTTTACCCAGGCTGTGTGCTGTGAGGACCACATA CACTGCTGTCCCGCGGGGTTTACGTGTGACACGCAGAAGGGTACCTGTGAACAGGG GCCCCACCAGGTGCCCTGGATGGAGAAGGCCCCAGCTCACCTCAGCCTGCCAGACC CACAAGCCTTGAAGAGAGATGTCCCCTGTGATAATGTCAGCAGCTGTCCCTCCTCCG ATACCTGCTGCCAACTCACGTCTGGGGAGTGGGGCTGCTGTCCAATCCCAGAGGCT GTCTGCTGCTCGGACCACCAGCACTGCTGCCCCCAGGGCTACACGTGTGTAGCTGA GGGGCAGTGTCAGCGAGGAAGCGAGATCGTGGCTGGACTGGAGAAGATGCCTGCC CGCCGGGCTTCCTTATCCCACCCCAGAGACATCGGCTGTGACCAGCACACCAGCTG CCCGGTGGGGCAGACCTGCTGCCCGAGCCTGGGTGGGAGCTGGGCCTGCTGCCAGT TGCCCCATGCTGTGTGCTGCGAGGATCGCCAGCACTGCTGCCCGGCTGGCTACACCT GCAACGTGAAGGCTCGATCCTGCGAGAAGGAAGTGGTCTCTGCCCAGCCTGCCACC TTCCTGGCCCGTAGCCCTCACGTGGGTGTGAAGGACGTGGAGTGTGGGGAAGGACA CTTCTGCCATGATAACCAGACCTGCTGCCGAGACAACCGACAGGGCTGGGCCTGCT GTCCCTACCGCCAGGGCGTCTGTTGTGCTGATCGGCGCCACTGCTGTCCTGCTGGCT TCCGCTGCGCAGCCAGGGGTACCAAGTGTTTGCGCAGGGAGGCCCCGCGCTGGGAC GCCCCTTTGAGGGACCCAGCCTTGAGACAGCTGCTGTGA [1236] VA RNA only construct - uninduced SEQ ID NO: 167 [1237] GGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTA GAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATAAT AACTTCGTATAATGTATGCTATACGAAGTTATCAGACATGATAAGATACATTGATGA GTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTT GTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGGTACCTCAA GCGCCGGGTTTTCGCGTCATGCACCACGTCCGTGGTAGAACTAGTATTATGCCCAGT ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTA TTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACT CACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATG GGCGGTAGGCGTGTACGGTGGGAGGTTTATATAAGCAGAGCTCGTTTAGTGAACCG TCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGAGAATTCGC CGCCACCATGACAGAATATAAACCTACTGTCAGACTGGCAACTCGAGACGACGTCC CTAGGGCCGTGAGAACATTGGCTGCCGCTTTCGCGGATTATCCCGCTACACGCCACA CAGTTGATCCTGATAGACATATTGAACGGGTTACAGAATTGCAAGAACTTTTTTTGA CCAGGGTAGGATTGGACATCGGTAAAGTTTGGGTCGCCGACGACGGGGCTGCAGTG GCAGTGTGGACGACTCCGGAGAGCGTTGAGGCCGGGGCTGTATTTGCAGAAATTGG TCCCCGAATGGCTGAGCTTAGTGGCTCTCGTCTCGCGGCTCAGCAACAAATGGAAG GACTCCTCGCCCCTCACCGCCCTAAAGAACCAGCTTGGTTCCTCGCTACTGTGGGCG TTAGCCCCGATCATCAGGGAAAGGGCCTTGGTTCCGCGGTGGTATTGCCCGGAGTA GAAGCCGCAGAACGAGCCGGAGTGCCAGCCTTTCTTGAAACGTCAGCGCCAAGGAA TTTGCCCTTCTATGAACGGCTCGGATTTACAGTTACTGCTGACGTTGAAGTACCCGA GGGCCCACGGACGTGGTGCATGACGCGAAAACCCGGCGCTTGAACCGGTCGCTGAT CAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCC TTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAAT TGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGG ACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGG CTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCGACTAGAGCTTGCGGAA CCCTTAGGTTGGGAAAAGCGCTCCCCTACCCATAACTTCGTATAATGTATGCTATAC GAAGTTATTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGT AACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACAC CGGGCACTCTTCCGTGATCTGGTGGATAAATTCGCAAGGGTATCATGGCGGACGAC CGGGATTCGAACCCCGGATCCGGCCGTCCGCCGTGATCCATGCGGTTACCGCCCGC GTGTCGAACCCAGGTGTGCGACGTCAGACAACGGGGGAGCGCTCCTTTTTGGGCCC [1238] VA RNA only construct - induced SEQ ID NO: 168 [1239] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGT TAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATA ATAACTTCGTATAATGTATGCTATACGAAGTTATTGCAGTTTTAAAATTATGTTTTA AAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTAT ATATCTTGTGGAAAGGACGAAACACCGGGCACTCTTCCGTGATCTGGTGGATAAAT TCGCAAGGGTATCATGGCGGACGACCGGGATTCGAACCCCGGATCCGGCCGTCCGC CGTGATCCATGCGGTTACCGCCCGCGTGTCGAACCCAGGTGTGCGACGTCAGACAA CGGGGGAGCGCTCCTTTTTGGGCCCAT [1240] TBE sequence SEQ ID NO: 169 [1241] AATAAAATATCTTTATTTTCATTACATCTGTGTGTTGGTTTTTTGTGTGAATCGATAGTACTAACA TACGCTCTCCATCAAAACAAAACGAAACAAAACAAACTAGCAAAATAGGCTGTCCCCAGTGCAAGTGCA GGTGCCAGAACATTTCTCT [1242] Rabbit Beta Globin PolyA SEQ ID NO: 170 [1243] AATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTC TCA [1244] Exemplary Construct 1/Rep/Cap Features
Figure imgf000496_0001
EQUIVALENTS AND INCORPORATION BY REFERENCE [1245] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, was specifically and individually indicated to be incorporated by reference in its entirety, for all purposes. This statement of incorporation by reference is intended by Applicants, pursuant to 37 C.F.R. §1.57(b)(1), to relate to each and every individual publication, database entry (e.g., Genbank sequences or GeneID entries), patent application, or patent, each of which is clearly identified in compliance with 37 C.F.R. §1.57(b)(2), even if such citation is not immediately adjacent to a dedicated statement of incorporation by reference. The inclusion of dedicated statements of incorporation by reference, if any, within the specification does not in any way weaken this general statement of incorporation by reference. Citation of the references herein is not intended as an admission that the reference is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. [1246] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.

Claims

WHAT IS CLAIMED IS: 1. A polynucleotide comprising: (i) a sequence encoding AAV Cap proteins operably linked to an inducible promoter; and (ii) a polyadenylation signal sequence, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence and is a 3’ of the sequence encoding AAV Cap proteins.
2. The polynucleotide of claim 1, wherein the polyadenylation signal sequence is a SV40 polyadenylation signal sequence, a bovine growth hormone polyadenylation signal sequence, or a Rabbit Beta Globin polyadenylation signal sequence.
3. The polynucleotide of claim 1 or 2, wherein the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152, has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151, or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 170.
4. The polynucleotide construct of claim 1, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162.
5. The polynucleotide of any one of claims 1-4, wherein the polynucleotide is not flanked by inverted terminal repeat sequences.
6. The polynucleotide of claim 1, wherein the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof.
7. The polynucleotide of claim 1, wherein the inducible promoter is a tetracycline- inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter.
8. The polynucleotide of claim 1, wherein the inducible promoter comprises a tetracycline- responsive promoter element (TRE).
9. The polynucleotide of claim 8, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
10. The polynucleotide of claim 9, wherein the minimal promoter is a human cytomegalovirus promoter.
11. The polynucleotide of claim 1, wherein the inducible promoter is a Tet-On promoter.
12. The polynucleotide of claim 1, wherein the inducible promoter comprises a first inducible promoter.
13. The polynucleotide of claim 1, wherein the polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163.
14. The polynucleotide of claim 1, wherein the AAV Cap proteins comprise VP1, VP2, and VP3.
15. The polynucleotide of claim 1, wherein the AAV Cap proteins encode for AAV5 Cap proteins, AAV9 Cap proteins, PhP.EB Cap proteins, AAV8 Cap proteins, AAV2 proteins, or AAV6 Cap proteins.
16. A polynucleotide comprising: a) a first sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second sequence encoding the polynucleotide of any one of claims 1-15.
17. The polynucleotide of claim 16, wherein transcription of the first sequence is driven by native AAV promoters; optionally, wherein transcription of the first sequence is driven by the P5 and P19 native AAV promoters.
18. The polynucleotide of claim 16, wherein the one or more promoters comprise P5 and P19 native promoters.
19. The polynucleotide of claim 16, wherein the first sequence has: a) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161; b) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or c) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.
20. The polynucleotide of any one of claims 16-19, wherein the first sequence is separated from the second sequence by an intervening sequence.
21. The polynucleotide of claim 20, wherein the intervening sequence comprises a transcriptional blocking element (TBE); optionally, wherein a sequence of the TBE has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 169.
22. The polynucleotide of any one of claims 16-21, wherein the first sequence encoding AAV Rep proteins comprises: a. a first part of an AAV Rep proteins coding sequence, b. an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and c. a second part of the AAV Rep proteins coding sequence.
23. The polynucleotide of any one of claims 16-22, the first sequence encoding AAV Rep proteins comprises from 5’ to 3’: one or more promoters operably linked to a sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.
24. The system of polynucleotides of claim 23, wherein the coding sequence encoding the stop signaling sequence of the first sequence encodes for, from 5’ to 3’: an exon and the stop signaling sequence.
25. The polynucleotide of claim 23, wherein the coding sequence encoding the stop signaling sequence of the first sequence further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.
26. The polynucleotide of any one of claims 16-25, further comprising a first constitutive promoter operably linked to a sequence encoding a first selectable marker or a first portion or a second portion of a split selectable marker.
27. The polynucleotide of any one of claims 16-26, wherein the polynucleotide is a polynucleotide construct.
28. The polynucleotide of any one of claims 16-27, wherein the polynucleotide further comprises a selectable marker operably linked to a promoter; optionally wherein the promoter is a constitutive promoter.
29. A system of polynucleotides comprising: a) a first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters; and b) a second polynucleotide comprising the sequence of the polynucleotide of any one of claims 1-15; and one or more of: c) a third polynucleotide comprising a sequence encoding one or more adenoviral helper proteins; and d) a fourth polynucleotide comprising a sequence encoding a payload.
30. The system of polynucleotides of claim 29, wherein transcription of the first polynucleotide is driven by native AAV promoters; optionally, wherein transcription of the first polynucleotide is driven by the P5 and P19 native AAV promoters.
31. The system of polynucleotides of claim 29, wherein the one or more promoters comprise a P5 native AAV promoter and a P19 native AAV promoter.
32. The system of polynucleotides of any one of claims 29-31, wherein the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises: a) a first part of an AAV Rep proteins coding sequence, b) an excisable element comprising a first recombination site, a coding sequence encoding a stop signaling sequence, a second recombination site, wherein the first and second recombination sites flank the coding sequence encoding a stop signaling sequence and wherein the first recombination site and the second recombination site are oriented in the same direction, and c) a second part of the AAV Rep proteins coding sequence.
33. The system of polynucleotides of any one of claims 29-32, wherein the first polynucleotide comprising a sequence encoding AAV Rep proteins operably linked to one or more promoters comprises from 5’ to 3’: one or more promoters operably linked to a first sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a first recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a second recombination site, a second part of the intron, a second 3’ splice site, and a second sequence comprising a second part of the AAV Rep coding sequence, wherein the first recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the second recombination site form an excisable element, wherein the first recombination site and the second recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the second sequence comprising the second part of the AAV Rep coding sequence, wherein the first and second recombination sites are recombined by the inducible recombinase in the presence of a first triggering agent and a second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins.
34. The system of polynucleotides of claim 32 or 33, wherein the coding sequence encoding the stop signaling sequence of the first polynucleotide encodes for, from 5’ to 3’: an exon and the stop signaling sequence.
35. The system of polynucleotides of any one of claims 32-34, wherein the coding sequence encoding the stop signaling sequence of the first polynucleotide further comprises a sequence encoding a protein marker, wherein the sequence encoding the protein marker is in-frame with the stop signaling sequence.
36. The system of polynucleotides of any one of claims 29-35, wherein the first polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160 or 161.
37. The system of polynucleotides of any one of claims 29-36, the first polynucleotide further comprises a sequence encoding AAV Cap proteins.
38. The system of polynucleotides of claim 37, wherein the sequence encoding the AAV Cap proteins in the second polynucleotide is substantially identical to the sequence encoding the AAV Cap proteins in the first polynucleotide.
39. The system of polynucleotides of claim 37, wherein transcription of the sequence encoding the AAV Cap proteins on the first polynucleotide is driven by a native AAV Cap proteins promoter; optionally, wherein the native AAV Cap proteins promoter is a P40 native AAV promoter.
40. The system of polynucleotides of any one of claims 29-39, wherein the first polynucleotide has: a) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136; or b) at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 136 but lacks the sequence of SEQ ID NO: 145 downstream of the sequence corresponding to the Cap sequence of SEQ ID NO: 136.
41. The system of polynucleotides of any one of claims 29-40, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a second inducible promoter operably linked to a self-excising element; the self-excising element comprising a third recombination site and a fourth recombination site flanking a sequence encoding an inducible recombinase, wherein the third recombination site and the fourth recombination site are oriented in the same direction, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a first constitutive promoter operably linked to a sequence encoding an activator, wherein the third polynucleotide constitutively expresses the activator and the activator is unable to activate the first inducible promoter or the second inducible promoter in absence of a first triggering agent; wherein in absence of activation of the first inducible promoter and the second inducible promoter, detectable levels of the Rep proteins from the first polynucleotide or if present the Cap proteins from the first polynucleotide, the Cap proteins from the second polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins are not expressed, and wherein the inducible recombinase is activated in the presence of a second triggering agent.
42. The system of polynucleotides of any one of claims 29-41, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises: a first sequence comprising from 5’ to 3’: a second inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a third recombination site, the sequence encoding the inducible recombinase, and a fourth recombination site, wherein the third recombination site and the fourth recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the second inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the second inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the third recombination site and the fourth recombination site resulting in excision of the self-excising element, thereby operably linking the second inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins
43. The system of polynucleotides of claim 41 or 42, wherein the one or more adenoviral helper proteins comprise one or more of adenovirus E1A protein, E1B protein, E2A protein, and E4 protein, and optionally comprises E2A protein and E4 protein.
44. The system of polynucleotides of any one of claims 29-43, wherein the third polynucleotide comprising the sequence encoding for one or more AAV helper proteins comprises a bicistronic open reading frame encoding two AAV helper proteins; optionally, wherein the SEQ ID NO: 30 comprises the third polynucleotide.
45. The system of polynucleotides of any one of claims 41-44, wherein the one or more adenoviral helper proteins are separated by a bicistronic open reading frame; optionally, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
46. The system of polynucleotides of any one of claims 41-45, wherein the second inducible promoter operably linked to the self-excising element in the third polynucleotide is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same; further optionally, wherein the first inducible promoter and the second inducible promoter are a tetracycline-inducible promoter.
47. The system of polynucleotides of claim 46, wherein the tetracycline-inducible promoter comprises a tetracycline-responsive promoter element (TRE).
48. The system of polynucleotides of claim 47, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
49. The system of polynucleotides of claim 48, wherein the minimal promoter is a human cytomegalovirus promoter.
50. The system of polynucleotides of any one of claims 41-49, wherein the first constitutive promoter is EF1alpha promoter or human cytomegalovirus promoter.
51. The system of polynucleotides of any one of claims 41-50, wherein the activator is reverse tetracycline-controlled transactivator (rTA) comprising a Tet Repressor binding protein (TetR) fused to a VP16 transactivation domain.
52. The system of polynucleotides of claim 47, wherein a triggering agent for inducing the tetracycline-inducible promoter is tetracycline or doxycycline.
53. The system of polynucleotides of any one of claims 41-53, wherein the inducible recombinase is fused to an estrogen response element (ER) and translocates to the nucleus in the presence of tamoxifen.
54. The system of polynucleotides of any one of claims 41-53, wherein the recombination sites in the first polynucleotide and the third polynucleotide are lox sites and the inducible recombinase is a cre recombinase or wherein the recombination sites in the first polynucleotide and the third polynucleotide are flippase recognition target (FRT) sites and the inducible recombinase is a flippase (Flp) recombinase.
55. The system of polynucleotides of any one of claims 41-54, wherein presence of the triggering agent activates the activator for activation of the first inducible promoter to express AAV Cap proteins from the second polynucleotide encoding the AAV Cap proteins.
56. The system of polynucleotides of any one of claims 41-55, wherein presence of the triggering agent activates the activator for activation of the second inducible promoter to express the AAV Rep proteins of the first polynucleotide, if present the AAV Cap proteins of the first polynucleotide, the inducible recombinase, and the one or more adenoviral helper proteins.
57. The system of polynucleotides of any one of claims 41-56, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide and if present the AAV Cap Proteins of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins.
58. The system of polynucleotides of any one of claims 29-57, wherein the third polynucleotide further comprises a third selectable marker operably linked to a third promoter.
59. The system of polynucleotides of any one of claims 29-58, wherein the third polynucleotide further comprises a sequence encoding a viral associated RNA (VA- RNA); optionally, wherein the VA-RNA is a mutated VA-RNA.
60. The system of polynucleotides of claim 59, wherein the VA-RNA is wild-type VA- RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.
61. The system of polynucleotides of claim 59 or 60, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a second constitutive promoter and a second part of the second constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete second constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.
62. The system of polynucleotides of claim 61, wherein the first part of the second constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the second constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the second constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.
63. The system of polynucleotides of any one of claims 59-62, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.
64. The system of polynucleotides of any one of claims 59-63, wherein the third polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 153.
65. The system of polynucleotides of any one of claims 29-64, wherein the payload of the fourth polynucleotide comprises a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the payload of the fourth polynucleotide is progranulin.
66. The system of polynucleotides of any one of claims 29-65, wherein the sequence encoding the payload of the fourth polynucleotide comprises a sequence encoding a reporter gene, a therapeutic gene, or a transgene encoding a protein of interest; optionally, wherein the sequence encoding the payload of the fourth polynucleotide is a sequence encoding progranulin; further optionally, wherein the sequence encoding progranulin has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 166.
67. The system of polynucleotides of any one of claims 29-66, wherein the sequence encoding the payload comprises a sequence encoding a suppressor tRNA, a guide RNA, or a homology region for homology-directed repair.
68. The system of polynucleotides of any one of claims 29-67, wherein the fourth polynucleotide comprising the sequence encoding the payload comprises the sequence encoding the payload flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR).
69. The system of polynucleotides of any one of claims 29-68, wherein the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR) has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 146; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 158; optionally, wherein a sequence that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the SEQ ID NO: 147, SEQ ID NO: 157, or SEQ ID NO: 159 comprises the sequence encoding the payload is flanked by a 5’ AAV inverted terminal repeat (5’ ITR) and a 3’ AAV inverted terminal repeat (3’ ITR).
70. The system of polynucleotides of any one of claims 29-69 further comprising a fifth polynucleotide comprising a sequence encoding a viral associated RNA (VA-RNA); optionally, wherein the VA-RNA is a mutated VA-RNA.
71. The system of polynucleotides of claim 70, wherein the VA-RNA is wild-type VA- RNA or VA-RNA comprising one or more mutations in the VA-RNA internal promoter.
72. The system of polynucleotides of claim 70 or 71, wherein the sequence encoding the VA-RNA is operably linked to an inactive promoter comprising a first part of a constitutive promoter and a second part of the constitutive promoter separated by a second excisable element comprising a fifth recombination site and a sixth recombination site flanking a stuffer sequence, the fifth and sixth recombination sites are oriented in the same direction, and excision of the second excisable element by the inducible recombinase generates a functional complete constitutive promoter operably linked to the VA-RNA coding sequence to allow expression of the VA-RNA.
73. The system of polynucleotides of claim 72, wherein the first part of the constitutive promoter comprises a distal sequence element (DSE) of an RNA polymerase III promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of an RNA polymerase III promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U6 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U6 promoter, or the first part of the constitutive promoter comprises a distal sequence element (DSE) of a U7 promoter, and the second part of the constitutive promoter comprises a proximal sequence element (PSE) of a U7 promoter.
74. The system of polynucleotides of any one of claims 70-73, wherein the VA-RNA comprises a G16A mutation or a G60A mutation, or a combination thereof.
75. The system of polynucleotides of any one of claims 70-74, wherein: a) the first polynucleotide further comprises a sequence encoding a first selectable marker operably linked to a first promoter, b) the second polynucleotide further comprises a sequence encoding a second selectable marker operably linked to a second promoter, c) the third polynucleotide further comprises a sequence encoding a third selectable marker operably linked to a third promoter, d) the fourth polynucleotide further comprises a sequence encoding a fourth selectable marker operably linked to a fourth promoter, e) the fifth polynucleotide further comprises a sequence encoding a fifth selectable marker operably linked to a fifth promoter, or g) any combination of thereof; optionally, wherein any combinations of the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker are different selectable markers; the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, the fifth selectable marker, or any combinations thereof are the same selectable marker but as a different split portion of the selectable marker; or any combinations thereof; further optionally, wherein any combinations of the first promoter, the second promoter, the third promoter, the fourth promoter, and the fifth promoter are the same constitutive promoter or different constitutive promoters.
76. The system of polynucleotides of claim 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is an antibiotic resistance gene; optionally, wherein the antibiotic resistance gene is a blasticidin resistance gene, a hygromycin resistance gene, or a puromycin resistance gene.
77. The system of polynucleotides of claim 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a first split portion of an antibiotic resistance gene; optionally, wherein the first split portion of the antibiotic resistance gene is a first split portion of the blasticidin resistance gene.
78. The system of polynucleotides of claim 75, wherein the first selectable marker, the second selectable marker, the third selectable marker, the fourth selectable marker, and the fifth selectable marker, or any combination thereof is a second split portion of an antibiotic resistance gene; optionally, wherein the second split portion of the antibiotic resistance gene is a second split portion of the blasticidin resistance gene.
79. The system of polynucleotides of claim 75, wherein the first promoter, the second promoter, the third promoter, the fourth promoter, the fifth promoter, or any combination thereof, is an EF1alpha promoter or an attenuated version thereof, wherein the attenuated version comprises a mutation in the TATA box, optionally wherein the attenuated EF1alpha promoter has weaker promoter activity than an EF1alpha promoter.
80. The system of polynucleotides of any one of claims 29-79, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5’ ITR and the sequence encoding the fourth selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof.
81. The system of polynucleotides of any one of claims 29-80, wherein: (i) the first polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the split selectable marker is a split antibiotic resistance protein; (ii) the second polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally wherein the constitutive promoter is an EF1 alpha promoter and/or the selectable marker is a first antibiotic resistance protein; (iii) the third polynucleotide further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the fourth polynucleotide further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter.
82. The system of polynucleotides of any one of claims 29-81, wherein the fourth polynucleotide comprising the sequence encoding the payload further comprises a spacer between the 5’ ITR and the sequence encoding the selectable marker or a spacer between the sequence encoding the fourth selectable marker and the 3’ ITR, or a combination thereof.
83. The system of polynucleotides of claim 82, wherein the spacer ranges in length from 500 base pairs to 5000 base pairs.
84. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; and c) the third polynucleotide of any one of claims 41-64 and 75-83.
85. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; and c) the fourth polynucleotide of any one of claims 65-69 and 75-83.
86. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 6-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the third polynucleotide of any one of claims 41-64 and 75-83; d) the fourth polynucleotide of any one of claims 65-69 and 75-83.
87. The system of polynucleotides of any one of claims 29-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the third polynucleotide of any one of claims 41-64 and 75-83; and d) the fifth polynucleotide of any one of claims 70-83.
88. The system of polynucleotides of any one of claims, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the fourth polynucleotide of any one of claims 65-69 and 75-83; and d) the fifth polynucleotide of any one of claims 70-83.
89. The system of polynucleotides of any one of claims 16-40 and 75-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; and c) the fifth polynucleotide of any one of claims 70-83.
90. The system of polynucleotides of any one of claims 16-40 and 75-83, comprising: a) the first polynucleotide of any one of claims 16-40 and 75-83; b) the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) the third polynucleotide of any one of claims 41-64 and 75-83; d) the fourth polynucleotide of any one of claims 65-69 and 75-83; and e) the fifth polynucleotide of any one of claims 70-83.
91. A system of polynucleotide constructs comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and one or more of: b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.
92. The system of polynucleotide constructs of claim 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83.
93. The system of polynucleotide constructs of claim 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and b) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.
94. The system of polynucleotide constructs of claim 91, comprising: a) a first polynucleotide construct comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; and b) a second polynucleotide construct comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83; c) a third polynucleotide construct comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.
95. The system of polynucleotide constructs of any one of claims 91-94, further comprising d) a fourth polynucleotide construct comprising the sequence of the fifth polynucleotide of any one of claims 70-83.
96. The system of polynucleotide constructs of any one of claims 91-95, wherein the sequence of the first polynucleotide is separated from the sequence of the second polynucleotide by an intervening sequence.
97. The system of polynucleotide constructs of claim 96, wherein the intervening sequence comprises a transcriptional blocking element (TBE).
98. The system of polynucleotide constructs of any one of claims 91-97, wherein the first polynucleotide construct comprises a sequence encoding a single selectable marker.
99. The system of polynucleotide constructs of any one of claims 91-98, wherein: (i) the first polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a first portion of a split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or first portion of the split selectable marker is a first portion of a split of a first antibiotic resistance protein; (ii) the second polynucleotide construct further comprises a sequence encoding a selectable marker operably linked to a constitutive promoter, optionally, wherein the constitutive promoter is an CMV promoter and/or the selectable marker is a second antibiotic resistance protein; and/or (iv) the third polynucleotide construct further comprises a constitutive promoter operably linked to a sequence encoding a selectable marker or a second part of the split selectable marker, optionally, wherein the constitutive promoter is an EF1 alpha promoter and/or the second part of the split selectable maker is a second portion of the first antibiotic resistance protein.
100. The system of polynucleotide constructs of claim 99, wherein the first antibiotic resistance protein is a blasticidin resistance protein and the second antibiotic resistance protein is a puromycin resistance protein.
101. A vector comprising the polynucleotide of any one of claims 1-100.
102. A vector system comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83; b) a second vector comprising the sequence of the second polynucleotide of any one of claims 1-15, 29 and 75-83; c) a third vector comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83; and d) a fourth vector comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83.
103. The vector system of claim 102, further comprising d) a fifth vector comprising the sequence of the fifth polynucleotide of any one of claims 70-83.
104. The vector system of any one of claims 102 or 103, wherein the first vector is a first plasmid, the second vector is a second plasmid, the third vector is a third plasmid, and the fourth vector is a fourth plasmid.
105. The vector system of claim 103 or 104, wherein the fifth vector is a fifth plasmid.
106. The vector system of any one of claims 102-105, wherein the first plasmid has least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 32 or SEQ ID NO: 160.
107. The vector system of any one of claims 102-105, wherein the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149.
108. The vector system of any one of claims 102-105, wherein the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154.
109. The vector system of any one of claims 102-105, wherein the fourth plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159.
110. A vector system comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of claims 91-100; and one or more of: b) a second vector comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83 or the second polynucleotide construct of any one of claims 1- 15, 29 and 75-83; and c) a third vector comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83 or the third polynucleotide construct of any one of claims 41-64 and 75-83.
111. The vector system of claim 110, comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of claims 91-100; and b) a second vector comprising the sequence of the third polynucleotide of any one of claims 41-64 and 75-83 or the second polynucleotide construct of any one of claims 91- 100.
112. The vector system of claim 110 or 111, comprising: a) a first vector comprising the sequence of the first polynucleotide of any one of claims 16-40 and 75-83 and the sequence of the second polynucleotide of any one of claims 1- 15, 29 and 75-83 or the first polynucleotide construct of any one of claims 91-100; and b) a third vector comprising the sequence of the fourth polynucleotide of any one of claims 65-69 and 75-83 or the third polynucleotide construct of any one of claims 91- 100.
113. The vector system of any one of claims 110-112, further comprising a fourth vector comprising the sequence of the fifth polynucleotide of any one of claims 70-83 or the fourth polynucleotide construct of any one of claims 91-100.
114. The vector system of any one of claims 110-113, wherein the first vector is a first plasmid, the second vector is a second plasmid, and the third vector is a third plasmid.
115. The vector system of claim 113 or 114, wherein the fourth vector is a fourth plasmid.
116. The vector system of claim 114, wherein the first plasmid comprises a nucleic acid sequence having least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 160.
117. The vector system of claim 114, wherein the second plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 30 or SEQ ID NO: 154.
118. The vector system of claim 114, wherein the third plasmid has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 157 or SEQ ID NO: 159.
119. A plasmid comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
120. A plasmid comprising at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 149.
121. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148.
122. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
123. A plasmid comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 and a sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164 or 165.
124. A cell comprising the polynucleotide of any one of claims 1-28.
125. A cell comprising the polynucleotide system of any one of claims 29-100.
126. The cell of claim 125, wherein one or more of the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are integrated into the nuclear genome of the cell.
127. The cell of claim 126, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
128. The cell of claim 126, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, and the fourth polynucleotide are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
129. The cell of any one of claims 125-128, wherein the first polynucleotide, the second polynucleotide, the third polynucleotide, the fourth polynucleotide, and the fifth polynucleotide are, in any combination, on one or more polynucleotide constructs.
130. A cell comprising the polynucleotide construct system of any one of claims 91- 100.
131. The cell of claim 130, wherein one or more of the first polynucleotide construct, the second polynucleotide construct, the third polynucleotide construct, and the fourth polynucleotide are integrated into the nuclear genome of the cell.
132. The cell of claim 131, wherein the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
133. The cell of claim 131, wherein the first polynucleotide construct, the second polynucleotide construct, and the third polynucleotide construct are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
134. A cell comprising the vector system of any of claims 102-118.
135. The cell of claim 134, wherein one or more of the first vector, the second vector, the third vector, and the fourth vector are integrated into the nuclear genome of the cell.
136. The cell of claim 135, wherein the first vector, the second vector, and the third vector are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
137. The cell of claim 135, wherein the first vector, the second vector, and the third vector, are integrated into the nuclear genome of the cell, and wherein the AAV Rep proteins, the AAV Cap proteins, the VA-RNA, and the one or more adenoviral helper proteins are conditionally expressible and when the cell expresses the AAV Rep, the Cap proteins, the VA-RNA, and the helper proteins, the cell conditionally produces recombinant AAV (rAAV) virions.
138. The cell of any one of claims 124-137, wherein the cell comprises a polynucleotide comprising a sequence encoding an adenovirus E1A protein, a polynucleotide comprising a sequence encoding an E1B protein, a polynucleotide comprising a sequence encoding an E2A protein, a polynucleotide comprising a sequence encoding an E4 protein, or any combination thereof.
139. The cell of any one of claims 124-138, wherein the third polynucleotide comprising the sequence encoding one or more adenoviral helper proteins comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide comprising the sequence encoding the E4 protein, or any combination thereof; optionally, wherein the second polynucleotide construct comprises the polynucleotide comprising the sequence encoding the adenovirus E1A protein, the polynucleotide comprising the sequence encoding the E1B protein, the polynucleotide comprising the sequence encoding the E2A protein, the polynucleotide sequence comprising the encoding the E4 protein, or any combination thereof.
140. The cell of any one of claims 124-139, wherein the cell comprises an adenovirus E1A protein and E1B protein, and the one or more adenoviral helper proteins expressed by the third polynucleotide are an adenovirus E2A protein and E4 protein or wherein the cell comprises an adenovirus E2A protein and E4 protein, and the one or more AAV helper proteins expressed by the second polynucleotide construct are an adenovirus E1A protein and E1B protein.
141. The cell of any one of claims 124-140, wherein the cell constitutively expresses any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein, from a nucleic acid integrated in the nuclear genome; optionally, wherein the two proteins are the adenovirus E1A protein and the adenovirus E1B protein or the adenovirus E2A protein and the adenovirus E4 protein.
142. The cell of any one of claims 124-141, wherein the third polynucleotide comprising the sequence encoding for one or more adenoviral helper proteins comprises a bicistronic open reading frame encoding two adenoviral helper proteins; optionally, wherein the third polynucleotide comprises SEQ ID NO: 30.
143. The cell of any one of claims 126-142, wherein the two adenoviral helper proteins are any two proteins from a group consisting of an adenovirus E1A protein, an adenovirus E1B protein, an adenovirus E2A protein, and adenovirus E4 protein; optionally, wherein the any two proteins are E2A and E4 or E1A and E1B.
144. The cell of claim 142, wherein the bicistronic open reading frame comprises an internal ribosome entry site (IRES) or a peptide 2A (P2A) sequence.
145. The cell of any one of claims 126-144, wherein the cell is a mammalian cell.
146. The cell of claim 145, wherein the mammalian cell is a HEK293 cell.
147. The cell of claim 146, wherein the HEK293 cell is DHFR-deficient or GS- deficient.
148. The cell of any one of claims 126-147, wherein the cell expresses adenoviral helper proteins E1A and E1B.
149. The cell of any one of claims 126-148, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.
150. The cell of any one of claims 126-149, wherein upon expression of the inducible recombinase, recombination between the first recombination site and the second recombination site in the first polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein of the first polynucleotide construct; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins.
151. The cell of any one of claims 126-150, wherein after induction, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 155.
152. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 161.
153. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 165.
154. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 164.
155. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or SEQ ID NO: 163.
156. The cell of any one of claims 126-151, wherein in the presence of the first triggering agent and the second triggering agent, the cell comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 147; has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156; or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 156 or SEQ ID NO: 158.
157. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the third polynucleotide of any one of claims 41-64 and 75-83; selecting for a cell expressing a selectable marker of the third polynucleotide; introducing into the cell expressing the selectable marker of the third polynucleotide, the first polynucleotide of any one of claims 16-40 and 75-83, the second polynucleotide of any one of claims 1-15, 29 and 75-83, and the fourth polynucleotide of any one of claims 65-69 and 75-83; selecting for a cell expressing the selectable marker of the third polynucleotide, a selectable marker of the first polynucleotide, a selectable marker of the second polynucleotide, and a selectable marker of the fourth polynucleotide; expanding the cell expressing the selectable marker of the third polynucleotide, the selectable marker of the first polynucleotide, the selectable marker of the second polynucleotide, and the selectable marker of the fourth polynucleotide into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.
158. The method of claim 157, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the third polynucleotide results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide.
159. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the second polynucleotide construct of any one of claims 91-100; selecting for a cell expressing a selectable marker of the second polynucleotide construct; introducing into the cell expressing the selectable marker of the second polynucleotide construct, the first polynucleotide construct of any one of claims 91-100, and the third polynucleotide construct of any one of claims 91-100; selecting for a cell expressing the selectable marker of the second polynucleotide construct, a selectable marker of the first polynucleotide construct, and a selectable marker of the third polynucleotide construct; expanding the cell expressing the selectable marker of the second polynucleotide construct, the selectable marker of the first polynucleotide construct, and the selectable marker of the third polynucleotide construct into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.
160. The method of claim 159, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide construct results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second polynucleotide construct.
161. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the third vector of any one of claims 102-118; selecting for a cell expressing a selectable marker of the third vector; introducing into the cell expressing the selectable marker of the third vector, the first vector of any one of claims 102-118, the second vector of any of any one of claims 102- 118, and the fourth vector of any one of claims 102-118; selecting for a cell expressing the selectable marker of the third vector, a selectable marker of the first vector, a selectable marker of the vector polynucleotide, and a selectable marker of the fourth vector; expanding the cell expressing the selectable marker of the third vector, the selectable marker of the first vector, the selectable marker of the second vector, and the selectable marker of the fourth vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.
162. The method of claim 161, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the first polynucleotide, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the third vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector.
163. A method of generating a cell line for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell the second vector of any one of claims 102-118; selecting for a cell expressing a selectable marker of the second vector; introducing into the cell expressing the selectable marker of the second vector, the first vector of any one of claims 102-118, and the third vector of any one of claims 102-118; selecting for a cell expressing the selectable marker of the second vector, a selectable marker of the first vector, and a selectable marker of the third vector; expanding the cell expressing the selectable marker of the second vector, the selectable marker of the first vector, and the selectable marker of the third vector into a plurality of cells, thereby generating the plurality of cells as the cell line for inducibly producing the rAAV virions.
164. The method of claim 163, further comprising contacting a cell of the cell line to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter resulting in expression of the recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector results in excision of the excisable element, and the first part of the AAV Rep proteins coding sequence and the second part of the AAV Rep proteins coding sequence are joined to form a complete AAV Rep proteins coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, wherein the one or more promoters are operably linked to the complete AAV Rep proteins coding sequence to allow expression of an AAV Rep protein and, if present in the second polynucleotide construct, expression of an AAV Cap protein; and recombination between the third recombination site and the fourth recombination site in the second vector results in excision of the self-excising element comprising the sequence encoding the second inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; and wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of an AAV Cap protein of the second vector.
165. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second polynucleotide by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more AAV helper proteins to allow expression of the one or more AAV helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second polynucleotide; wherein the expression of the one or more AAV helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.
166. The method of claim 165, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide.
167. The method of claim 165, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide.
168. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first polynucleotide construct by the recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first polynucleotide construct; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.
169. The method of claim 168, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first polynucleotide construct.
170. The method of claim 168, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first polynucleotide construct.
171. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the second vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the second vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.
172. The method of claim 171, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second vector.
173. The method of claim 171, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second vector.
174. A method for generating a recombinant adenovirus associated virus (rAAV) virion comprising a sequence encoding a payload, the method comprising contacting the cell according to any one of claims 124-156 to a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent and the second triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein recombination between the first recombination site and the second recombination site in the first vector by the inducible recombinase results in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein and, if present, an AAV Cap protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the AAV Cap proteins of the first vector; wherein the expression of the one or more adenoviral helper proteins results in expression of the one or more Rep proteins and the AAV Cap proteins, thereby generating an rAAV virion comprising the sequence encoding the payload.
175. The method of claim 174, wherein the generated rAAV is at least 35-fold higher than rAAV generated from a cell lacking the second polynucleotide of the first vector.
176. The method of claim 174, wherein the generated rAAV titer is at least 35-fold higher than rAAV generated titer from a cell lacking the second polynucleotide of the first vector.
177. A method for producing recombinant AAV, the method comprising performing transfection of a cell with the polynucleotides of the system of any one of claims 29- 100, the polynucleotide constructs of the system of any one of claims 91-100, or the vectors of the system of any one of claims 102-118 and contacting the cell to a first triggering agent and a second triggering agent.
178. The method of claim 177, wherein the polynucleotides of the system of any one of claims 29-100, the polynucleotide constructs of the system of any one of claims 91- 100, or the vectors of the system of any one of claims 102-118 are integrated into a nuclear genome of the cell.
179. The method of claim 177, wherein the polynucleotides of the system of any one of claims 29-100, the polynucleotide constructs of the system of any one of claims 91- 100, or the vectors of the system of any one of claims 102-118 are not integrated into a nuclear genome of the cell.
180. The method of any one of claims 177-179, wherein one or more of the polynucleotides of the system of any one of claims 29-100, of the polynucleotide constructs of the system of any one of claims 91-100, or of the vectors of the system of any one of claims 102-118 are integrated into a nuclear genome of the cell and one or more of the polynucleotides of the system of any one of claims 29-100, of the polynucleotide constructs of the system of any one of claims 91-100, or of the vectors of the system of any one of claims 102-118 are not integrated into the nuclear genome of the cell.
181. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotides of the system of any one of claims 29- 100 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third polynucleotide resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third polynucleotide by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.
182. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the polynucleotide constructs of the system of any one of claims 91-100 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second polynucleotide construct resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first polynucleotide construct resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second polynucleotide construct by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.
183. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the vectors of the system of any one of claims 102-118 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the third vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the third vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.
184. A method for inducibly producing recombinant AAV, the method comprising: culturing a cell comprising the vectors of the system of any one of claims 102-118 integrated into a nuclear genome of the cell in the presence of a first triggering agent and a second triggering agent, wherein in the presence of the first triggering agent, the activator activates the second inducible promoter of the second vector resulting in expression of the inducible recombinase, wherein in the presence of the second triggering agent the inducible recombinase translocates to the nucleus and catalyzes recombination between the first recombination site and the second recombination site in the first vector resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the second part of the AAV Rep coding sequence are joined to form a complete AAV Rep coding sequence, wherein the one or more promoters are operably linked to the complete AAV Rep coding sequence to allow expression of an AAV Rep protein, and recombination between the third recombination site and the fourth recombination site in the second vector by the inducible recombinase results in excision of the self-excising element comprising the sequence encoding the inducible recombinase, wherein the second inducible promoter becomes operably linked to the sequence encoding the one or more adenoviral helper proteins to allow expression of the one or more adenoviral helper proteins; wherein the first triggering agent and the activator activate the first inducible promoter operably linked to the AAV Cap proteins coding sequence; wherein the expression of the one or more adenoviral helper proteins, one or more Rep proteins, and the AAV Cap proteins results in generation of rAAV virions comprising the sequence encoding the payload.
185. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third polynucleotide of any one of claims 41-64 and 75-83; (ii) selecting for cells expressing a selectable marker encoded by third polynucleotide; (iii) introducing into the cells selected in (ii) the first polynucleotide and the fourth polynucleotide of any one of claims 65-69 and 75-83, wherein the first polynucleotide encodes a first part of a split selectable marker and the fourth polynucleotide encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third polynucleotide and the selectable marker encoded by the first polynucleotide and the fourth polynucleotide, thereby generating the cells for inducibly producing the rAAV virions.
186. The method of claim 185, further comprising: (iii) introducing into the cells selected in (iv) or the cells expanded in (v), the second polynucleotide of any one of claims 1-15 and 75-83; and (iv) selecting for cells expressing a selectable marker encoded by the second polynucleotide.
187. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second polynucleotide construct of any one of claims 91- 100; (ii) selecting for cells expressing a selectable marker encoded by second polynucleotide construct; (iii) introducing into the cells selected in (ii) the first polynucleotide construct and the third polynucleotide construct of any one of claims 91-100, wherein the first polynucleotide construct encodes a first part of a split selectable marker and the third polynucleotide construct encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second polynucleotide construct and the selectable marker encoded by the first polynucleotide construct and the third polynucleotide construct, thereby generating the cells for inducibly producing the rAAV virions.
188. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the third vector of any one of claims 102-118; (ii) selecting for cells expressing a selectable marker encoded by third vector; (iii) introducing into the cells selected in (ii) the first vector and the fourth vector of any one of claims 102-118, wherein the first vector encodes a first part of a split selectable marker and the fourth vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the third vector and the selectable marker encoded by the first vector and the fourth vector, thereby generating the cells for inducibly producing the rAAV virions.
189. The method of claim 188, further comprising: (vi) introducing into the cells selected in (iv) or the cells expanded in (v), the second vector of any one of claims 102-118; and (vii) selecting for cells expressing a selectable marker encoded by the second vector.
190. A method of generating a cell for inducibly producing recombinant AAV (rAAV) comprising a payload, the method comprising: (i) introducing into cells the second vector of any one of claims 102-118; (ii) selecting for cells expressing a selectable marker encoded by second vector; (iii) introducing into the cells selected in (ii) the first vector and the third vector of any one of claims 102-118, wherein the first vector encodes a first part of a split selectable marker and the vector encodes a second part of a split selectable marker; (iv) selecting for cells expressing an active selectable marker formed from the first part and the second part of the split selectable marker; and (v) expanding the cells expressing the selectable marker encoded by the second vector and the selectable marker encoded by the first vector and the third vector, thereby generating the cells for inducibly producing the rAAV virions.
191. A method of generating a cell for inducibly producing recombinant AAV (rAAV) virions comprising a payload, the method comprising: introducing into a cell a first polynucleotide comprising: a first sequence comprising from 5’ to 3’: a first inducible promoter operably linked to a sequence encoding an inducible recombinase; a self-excising element comprising a first recombination site, the sequence encoding the inducible recombinase, and a second recombination site, wherein the first recombination site and the second recombination site are oriented in the same direction; and a sequence encoding one or more adenoviral helper proteins, wherein the first inducible promoter is not operably linked to the sequence encoding the one or more adenoviral helper proteins; a second sequence comprising a first constitutive promoter operably linked to a sequence encoding an activator, wherein the cell constitutively expresses the activator and the activator is unable to activate the first inducible promoter in absence of a first triggering agent, wherein in the presence of the first triggering agent, the activator activates the first inducible promoter resulting in expression of the inducible recombinase, and the inducible recombinase is expressed and wherein in the presence of a second triggering agent, the inducible recombinase translocates to a nucleus of the cell and causes recombination between the first recombination site and the second recombination site resulting in excision of the self-excising element, thereby operably linking the first inducible promoter to the sequence encoding the one or more adenoviral helper proteins and allowing expression of the one or more adenoviral helper proteins; and a third sequence comprising a second constitutive promoter operably linked to a sequence encoding a first selectable marker, wherein the cell constitutively expresses the first selectable marker, selecting for a cell expressing the first selectable marker; introducing a second polynucleotide and a third polynucleotide into the cell expressing the first selectable marker, the second polynucleotide comprising: a first sequence encoding AAV Cap proteins operably linked to a second inducible promoter; and from 5’ to 3’: one or more promoters operably linked to a second sequence comprising a first part of an AAV Rep coding sequence, a 5’ splice site, a first part of an intron, a third recombination site, a first 3’ splice site, a coding sequence comprising a stop signaling sequence, a fourth recombination site, a second part of the intron, a second 3’ splice site, and a third sequence comprising a second part of the AAV Rep coding sequence, wherein the third recombination site, the first 3’ splice site, the coding sequence comprising the stop signaling sequence, and the fourth recombination site form an excisable element, wherein the third recombination site and the fourth recombination site are oriented in the same direction, and wherein the one or more promoters are not operably linked to the third sequence comprising the second part of the AAV Rep coding sequence, wherein the third and fourth recombination sites are recombined by the inducible recombinase in the presence of the first triggering agent and the second triggering agent resulting in excision of the excisable element, and the first part of the AAV Rep coding sequence and the first part of the intron are joined to the second part of the intron and the second part of the AAV Rep coding sequence to form a complete AAV Rep coding sequence, allowing expression of AAV Rep proteins; and a third constitutive promoter operably linked to a sequence encoding a first portion of a second selectable marker, the third polynucleotide comprising a sequence encoding the payload and a fourth constitutive promoter operably linked to a second portion of the second selectable marker, wherein the sequence encoding the payload is flanked by AAV inverted terminal repeats (ITRs); selecting for a cell expressing the first selectable marker and the second selectable marker, thereby generating the cell for inducibly producing recombinant AAV (rAAV) virions comprising the payload.
192. The method of claim 191, further comprising contacting the cell with the first triggering agent and the second triggering agent for inducibly producing recombinant AAV (rAAV) virions comprising the payload.
193. The method of claim 191 or 192, wherein the coding sequence encoding the stop signaling sequence of the second polynucleotide encodes for from 5’ to 3’: an exon and the stop signaling sequence.
194. The method of any one of claims 191-193, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide further comprises a polyadenylation signal.
195. The method of claim 194, wherein the polyadenylation signal sequence encodes a stronger polyadenylation signal than a native AAV Cap polyadenylation signal sequence.
196. The polynucleotide of claim 194 or 195, wherein the polyadenylation signal sequence is a 3’ of the sequence encoding AAV Cap proteins.
197. The method of any one of claims 194-196, wherein the polyadenylation signal sequence is a SV40 polyadenylation signal sequence or a bovine growth hormone polyadenylation signal sequence.
198. The method of any one of claims 194-197, wherein the polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 152 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 151.
199. The method of claim 195, wherein the native AAV Cap polyadenylation signal sequence has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 162.
200. The method of any one of claims 191-199, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide is not flanked by inverted terminal repeat sequences.
201. The method of claim 195, wherein the stronger polyadenylation signal enhances RNA processing, RNA stability, RNA translation efficiency, or any combination thereof.
202. The method of any one of claims 191-201, wherein the first inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate- inducible promoter and second inducible promoter is a tetracycline-inducible promoter, an ecdysone-inducible promoter, or a cumate-inducible promoter; optionally, wherein the first inducible promoter and the second inducible promoter are the same.
203. The method of any one of claims 191-202, wherein the first inducible promoter comprises a tetracycline-responsive promoter element (TRE) and the second inducible promoter comprises a tetracycline-responsive promoter element (TRE).
204. The method of claim 203, wherein the TRE comprises Tet operator (tetO) sequence concatemers fused to a minimal promoter.
205. The method of claim 204, wherein the minimal promoter is a human cytomegalovirus promoter.
206. The method of any one of claims 191-203, wherein the first inducible promoter is a Tet-On promoter and the second inducible promoter is a Tet-On promoter.
207. The method of any one of claims 191-206, wherein the first sequence encoding AAV Cap proteins operably linked to the second inducible promoter of the second polynucleotide has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 148 or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 163.
208. The method of any one of claims 191-207, wherein the AAV Cap proteins comprise VP1, VP2, and VP3.
209. The method of any one of claims 157-208, wherein the first triggering agent is tetracycline and the second triggering agent is tamoxifen.
210. The method of any one of claims 157-208, wherein the first triggering agent is doxycycline and the second triggering agent is tamoxifen.
211. The rAAV virion produced by the method of any one of claims 157-210.
PCT/US2023/080783 2022-11-21 2023-11-21 Systems for amplification of aav cap protein Ceased WO2024112813A2 (en)

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WO2025054007A1 (en) * 2023-09-06 2025-03-13 Shape Therapeutics Inc. Genetic elements to increase recombinant aav production
WO2025122676A1 (en) 2023-12-05 2025-06-12 Shape Therapeutics Inc. Systems and methods for raav production using a biphasic culture
EP4502166A4 (en) * 2022-03-25 2025-09-24 Univ Jichi Medical EFFICIENT SYSTEM FOR THE PRODUCTION OF AN ADENOASOCIATED VIRUS (AAV) VECTOR

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JP7406783B2 (en) * 2015-12-14 2023-12-28 ザ ユニバーシティ オブ ノース カロライナ アット チャペル ヒル Modified capsid proteins for enhanced delivery of parvovirus vectors
JP2023536148A (en) * 2020-07-30 2023-08-23 シェイプ セラピューティクス インク． Stable cell lines for inducible production of RAAV virus particles
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EP4502166A4 (en) * 2022-03-25 2025-09-24 Univ Jichi Medical EFFICIENT SYSTEM FOR THE PRODUCTION OF AN ADENOASOCIATED VIRUS (AAV) VECTOR
WO2025054007A1 (en) * 2023-09-06 2025-03-13 Shape Therapeutics Inc. Genetic elements to increase recombinant aav production
WO2025122676A1 (en) 2023-12-05 2025-06-12 Shape Therapeutics Inc. Systems and methods for raav production using a biphasic culture

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