AU2018241075A1 - Novel Methods of Constructing Libraries Comprising Displayed and/or Expressed Members of a Diverse Family of Peptides, Polypeptides or Proteins and the Novel Libraries - Google Patents

Abstract

Methods useful in constructing libraries that collectively display and/or express members of diverse families of peptides, polypeptides or proteins and the libraries produced using those methods. Methods of screening those libraries and the peptides, polypeptides or proteins identified by such screens.

Description

TECHNICAL FIELD

This application is a divisional of Australian Patent Application No. 2016225923, which is a divisional of Australian Patent Application No. 2013205033, which is a divisional of Australian

Patent Application No. 2011253898. Australian Patent Application No. 2011253898 is a divisional application of Australian Patent Application No. 200900092, which is a divisional of Australian Patent Application No. 2002307422, which claims the benefit of US Patent Application No. 10/045,674, filed October 25, 2001, US Patent Application No. 10/000,516, filed October 24, 2001 and US Patent Application No. 09/837,306, filed April 17, 2001. All of the above referenced applications are herein incorporated by reference in their entireties.

The present invention relates to libraries of genetic packages that display and/or express a member of a diverse family of peptides, polypeptides or proteins and collectively display and/or express at least a portion of the diversity of the family. In an alternative embodiment, the invention relates to libraries that include a member of a diverse family of peptides, polypeptides or proteins and collectively comprise at least a portion of the diversity of the family. In a preferred embodiment, the displayed and/or expressed polypeptides are human Fabs .

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More specifically, the invention is directed to the methods of cleaving single-stranded nucleic acids at chosen locations, the cleaved nucleic acids encoding, at least in part, the peptides, polypeptides or proteins displayed on the genetic packages of, and/or expressed in, the libraries of the invention.

In a preferred embodiment, the genetic packages are filamentous phage or phagemids or yeast.

The present invention further relates to 10 vectors for displaying and/or expressing a diverse family of peptides, polypeptides or proteins.

The present invention further relates to methods of screening the libraries of the invention and to the peptides, polypeptides and proteins identified by such screening.

BACKGROUND OF THE INVENTION

It is now common practice in the art to prepare libraries of genetic packages that display, express or comprise a member of a diverse family of peptides, polypeptides or proteins and collectively display, express or comprise at least a portion of the diversity of the family. In many common libraries, the peptides, polypeptides or proteins are related to antibodies. Often, they are Fabs or single chain antibodies.

In general, the DNAs that encode members of the families to be displayed and/or expressed must be amplified before they are cloned and used to display and/or express the desired member. Such amplification typically makes use of forward and backward primers.

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- 3 Such primers can be complementary to sequences native to the DNA to be amplified or complementary to oligonucleotides attached at the 5' or 3' ends of that DNA. Primers that are complementary to sequences native to the DNA to be amplified are disadvantaged in that they bias the members of the families to be displayed. Only those members that contain a sequence in the native DNA that is substantially complementary to the primer will be amplified. Those that do not will be absent from the family. For those members that are amplified, any diversity within the primer region will be suppressed.

For example, in European patent 368,684 BI, the primer that is used is at the 5' end of the V_H region of an antibody gene. It anneals to a sequence region in the native DNA that is said to be sufficiently well conserved within a single species. Such primer will bias the members amplified to those having this conserved region. Any diversity within this region is extinguished.

It is generally accepted that human antibody genes arise through a process that involves a combinatorial selection of V and J or V, D, and J followed by somatic mutations. Although most diversity occurs in the Complementary Determining Regions (CDRs) , diversity also occurs in the more conserved Framework Regions (FRs) and at least some of this diversity confers or enhances specific binding to antigens (Ag). As a consequence, libraries should contain as much of the CDR and FR diversity as possible.

To clone the amplified DNAs of the peptides, polypeptides or proteins that they encode for display on a genetic package and/or for expression, the DNAs

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- 4 must be cleaved to produce appropriate ends for ligation to a vector. Such cleavage is generally effected using restriction endonuclease recognition sites carried on the primers. When the primers are at the 5' end of DNA produced from reverse transcription of RNA, such restriction leaves deleterious 5' untranslated regions in the amplified DNA. These regions interfere with expression of the cloned genes and thus the display of the peptides, polypeptides and proteins coded for by them.

SUMMARY OF THE INVENTION

It is an object of this invention to provide novel methods for constructing libraries that display, express or comprise a member of a diverse family of 15 peptides, polypeptides or proteins and collectively display, express or comprise at least a portion of the diversity of the family. These methods are not biased toward DNAs that contain native sequences that are complementary to the primers used for amplification.

They also enable any sequences that may be deleterious to expression to be removed from the amplified DNA before cloning and displaying and/or expressing.

It is another object of this invention to provide a method for cleaving single-stranded nucleic 25 acid sequences at a desired location, the method comprising the steps of:

(i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement

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- 5 in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

the contacting and the cleaving steps being performed 10 at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

It is a further object of this invention to provide an alternative method for cleaving single20 stranded nucleic acid sequences at a desired location, the method comprising the steps of;

(i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid solely at the cleavage site formed by the

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- 6 complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

the contacting and the cleaving steps being performed 5 at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

In an alternative embodiment of this objeci of the invention, the restriction endonuclease recognition site is not initially located in the double-stranded part of the oligonucleotide. Instead, it is part of an amplification primer, which primer is complementary to the double-stranded region of the oligonucleotide. On amplification of the DNA-partially double-stranded combination, the restriction endonuclease recognition site carried on the primer becomes part of the DNA. It can then be used to cleave the DNA.

Preferably, the restriction endonuclease recognition site is that of a Type II-S restriction endonuclease whose cleavage site is located at a known distance from its recognition site.

It is another object of the present invention to provide a method of capturing DNA molecules that comprise a member of a diverse family of DNAs and collectively comprise at least a portion of the diversity of the family. These DNA molecules in

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- 7 single-stranded form have been cleaved by one of the methods of this invention. This method involves ligating the individual single-stranded DNA members of the family to a partially duplex DNA complex. The method comprises the steps of:

(i) contacting a single-stranded nucleic acid sequence that has been cleaved with a restriction endonuclease with a partially double-stranded oligonucleotide, the single10 stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region that remains after cleavage, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper reading frame for expression and containing a restriction endonuclease recognition site 5' of those sequences; and (ii) cleaving the partially doublestranded oligonucleotide sequence solely at the restriction endonuclease cleavage site contained within the double-stranded region of the partially double-stranded oligonucleotide.

As before, in this object of the invention, the restriction endonuclease recognition site need not be located in the double-stranded portion of the oligonucleotide. Instead, it can be introduced on amplification with an amplification primer that is used to amplify the DNA-partially double-stranded oligonucleotide combination.

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It is another object of this invention to prepare libraries, that display, express or comprise a diverse family of peptides, polypeptides or proteins and collectively display, express or comprise at least part of the diversity of the family, using the methods and

DNAs described above.

It is an object of this invention to screen those libraries to identify useful peptides, polypeptides and proteins and to use those substances in human therapy.

Additional objects of the invention are reflected in the claims. 10 Each of these claims is specifically incorporated by reference in this specification.

A definition of the specific embodiment of the invention claimed herein follows.

In a broad format, the invention provides a method for cleaving 15 a nucleic acid at a desired location, the method comprising the steps of :

(i) contacting a single-stranded nucleic acid with a singlestranded oligonucleotide, the single-stranded oligonucleotide being complementary to the single-stranded nucleic acid in the region in which cleavage is desired; wherein the single - stranded nucleic acid and the single-stranded oligonucleotide associate to form a locally double-stranded region of the single-stranded nucleic acid, wherein the locally double-stranded region comprises a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid at the restriction endonuclease recognition, wherein the cleaving comprises contacting a restriction endonuclease to the locally double-stranded region, wherein the restriction endonuclease is specific for the restriction endonuclease recognition site;

the contacting and the cleaving steps being performed at a temperature wherein the single-stranded nucleic acid and the single-stranded oligonucleotide associate to form a locally double-stranded region of the single-stranded nucleic acid, wherein the remainder of the single-stranded nucleic acid is single-stranded, and wherein the restriction endonuclease is active at the temperature.

8a

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BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic of various methods that may be employed to amplify VH genes without using primers specific for VH sequences.

FIG. 2 is a schematic of various methods that may be employed to amplify VL genes without using primers specific for VL sequences.

FIG. 3 is a schematic of RACE amplification of antibody heavy and light chains.

FIG. 4 depicts gel analysis of amplification products obtained after the primary PCR reaction from 4 different patient samples.

FIG. 5 depicts gel analysis of cleaved kappa DNA from Example 2.

FIG. 6 depicts gel analysis of extender-cleaved kappa DNA from

Example 2.

[Text continues on page 9.]

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FIG. 7 depicts gel analysis of the PCR product from the extender-kappa amplification from Example 2.

FIG. 8 depicts gel analysis of purified PCR product from the extender-kappa amplification from Example 2.

FIG. 9 depicts gel analysis of cleaved and ligated kappa light chains from Example 2.

FIG. 10 is a schematic of the design for CDR1 and CDR2 synthetic diversity.

FIG. 11 is a schemaitc of the cloning schedule for construction of the heavy chain repertoire .

FIG. 12 is a schematic of the cleavage and ligation of the antibody light chain.

FIG. 13 depicts gel analysis of cleaved and ligated lambda light chains from Example 4.

FIG. 14 is a schematic of the cleavage and ligation of the antibody heavy chain.

FIG. 15 depicts gel analysis of cleaved and ligated lambda light chains from Example 5.

FIG. 16 is a schematic of a phage display vector.

FIG. 17 is a schematic of a Fab cassette.

FIG. 18 is a schematic of a process for incorporating fixed FR1 residues in an antibody lambda sequence.

FIG. 19 is a schematic of a process for incorporating fixed FR1 residues in an antibody kappa sequence.

FIG. 20 is a schematic of a process for incorporating fixed FR1 residues in an antibody heavy chain sequence.

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- 10 TERMS

In this application, the following terms and abbreviations are used:

Sense strand 5

Antisense strand

Forward primer

Backward primer

The upper strand of ds DNA as usually written. In the sense strand, 5'-ATG-3’ codes for Met.

The lower strand of ds DNA as usually written. In the antisense strand, 3'-TAC-5* would correspond to a Met codon in the sense strand.

A forward primer is complementary to a part of the sense strand and primes for synthesis of a new antisensestrand molecule. Forward primer and lower-strand primer are equivalent.

A backward primer is complementary to a part of the antisense strand and primes for synthesis of a new sensestrand molecule. Backward primer and top-strand primer are equivalent.

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Bases

Sv

Ap ap^R

RERS

RE

URE

- 11 Bases are specified either by their position in a vector or gene as their position within a gene by codon and base. For example, 89.1 is the first base of codon 89, 89.2 is the second base of codon 89.

Streptavidin

Ampicillin

A gene conferring ampicillin resistance.

Restriction endonuclease recognition site

Restriction endonuclease cleaves preferentially at RERS

Universal restriction endonuclease

Functionally complementary

Two sequences are sufficiently complementary so as to anneal under the chosen conditions.

AA

Amino acid

PCR

Polymerization chain reaction

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GLGS

Ab

Fab scFv

w. t.

HC

LC

VK

VH

- 12 Germline genes

Antibody: an immunoglobin.

The term also covers any protein having a binding domain which is homologous to an immunoglobin binding domain. A few examples of antibodies within this definition are, inter alia, immunoglobin isotypes and the Fab, Flab¹) 2, scfv, Fv, dAb and Fd fragments.

Two chain molecule comprising an Ab light chain and part of a heavy-chain.

A single-chain Ab comprising either VH: .-linker: :VL or VL:: linker::VH

Wild type

Heavy chain

Light chain

A variable domain of a Kappa light chain.

A variable domain of a heavy chain.

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- 13 VL A variable domain of a lambda light chain.

In this application when it is said that nucleic acids are cleaved solely at the cleavage site of a restriction endonuclease, it should be understood that minor cleavage may occur at random, e.g., at nonspecific sites other than the specific cleavage site that is characteristic of the restriction endonuclease. The skilled worker will recognize that such non10 specific, random cleavage is the usual occurrence. Accordingly, solely at the cleavage site of a restriction endonuclease means that cleavage occurs preferentially at the site characteristic of that endonuclease.

As used in this application and claims, the term cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide includes cleavage sites formed by the single-stranded portion of the partially double20 stranded ologonucleotide duplexing with the singlestranded DNA, cleavage sites in the double-stranded portion of the partially double-stranded oligonucleotide, and cleavage sites introduced by the amplification primer used to amplify the single25 stranded DNA-partially double-stranded oligonucleotide combination.

In the two methods of this invention for preparing single-stranded nucleic acid sequences, the first of those cleavage sites is preferred. In the methods of this invention for capturing diversity and cloning a family of diverse nucleic acid sequences, the latter two cleavage sites are preferred.

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- 14 In this application, all references referred to are specifically incorporated by reference.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The nucleic acid sequences that are useful in 5 the methods of this invention, i.e., those that encode at least in part the individual peptides, polypeptides and proteins displayed, or expressed in or comprising the libraries of this invention, may be native, synthetic or a combination thereof. They may be mRNA,

DNA or cDNA. In the preferred embodiment, the nucleic acids encode antibodies. Most preferably, they encode Fabs .

The nucleic acids useful in this invention may be naturally diverse, synthetic diversity may be introduced into those naturally diverse members, or the diversity may be entirely synthetic. For example, synthetic diversity can be introduced into one or more CDRs of antibody genes. Preferably, it is introduced into CDR1 and CDR2 of immunoglobulins. Preferably, natural diversity is captured in the CDR3 regions of the immunoglogin genes of this invention from B cells. Most preferably, the nucleic acids of this invention comprise a population of immunoglobin genes that comprise synthetic diversity in at least one, and more preferably both of the CDR1 and CDR2 and diversity in CDR3 captured from B cells.

Synthetic diversity may be created, for example, through the use of TRIM technology (U.S. 5,869,644). TRIM technology allows control over exactly which amino-acid types are allowed at variegated positions and in what proportions. In TRIM technology, codons to be diversified are synthesized

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- 15 using mixtures of trinucleotides. This allows any set of amino acid types to be included in any proportion.

Another alternative that may be used to generate diversified DNA is mixed oligonucleotide synthesis. With TRIM technology, one could allow Ala and Trp. With mixed oligonucleotide synthesis, a mixture that included Ala and Trp would also necessarily include Ser and Gly. The amino-acid types allowed at the variegated positions are picked with reference to the structure of antibodies, or other peptides, polypeptides or proteins of the family, the observed diversity in germline genes, the observed somatic mutations frequently observed, and the desired areas and types of variegation.

In a preferred embodiment of this invention, the nucleic acid sequences for at least one CDR or other region of the peptides, polypeptides or proteins of the family are cDNAs produced by reverse transcription from mRNA. More preferably, the mRNAs are obtained from peripheral blood cells, bone marrow cells, spleen cells or lymph node cells (such as B-lymphocytes or plasma cells) that express members of naturally diverse sets of related genes. More preferable, the mRNAs encode a diverse family of antibodies. Most preferably, the mRNAs are obtained from patients suffering from at least one autoimmune disorder or cancer. Preferably, mRNAs containing a high diversity of autoimmune diseases, such as systemic lupus erythematosus, systemic sclerosis, rheumatoid arthritis, antiphospholipid syndrome and vasculitis are used.

In a preferred embodiment of this invention, the cDNAs are produced from the mRNAs using reverse

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- 16 transcription. In this preferred embodiment, the mRNAs are separated from the cell and degraded using standard methods, such that only the full length (i.e., capped) mRNAs remain. The cap is then removed and reverse transcription used to produce the cDNAs.

The reverse transcription of the first (antisense) strand can be done in any manner with any suitable primer. See, e.g·., HJ de Haard et al.,

Journal of Biological Chemistry, 274(26):18218-30 (1999). In the preferred embodiment of this invention where the mRNAs encode antibodies, primers that are complementary to the constant regions of antibody genes may be used. Those primers are useful because they do not generate bias toward subclasses of antibodies. In another embodiment, poly-dT primers may be used (and may be preferred for the heavy-chain genes) . Alternatively, sequences complementary to the primer may be attached to the termini of the antisense strand.

In one preferred embodiment of this invention, the reverse transcriptase primer may be biotinylated, thus allowing the cDNA product to be immobilized on streptavidin (Sv) beads. Immobilization can also be effected using a primer labeled at the 5' end with one of a) free amine group, b) thiol, c) carboxylic acid, or d) another group not found in DNA that can react to form a strong bond to a known partner on an insoluble medium. If, for example, a free amine (preferably primary amine) is provided at the 5' end of a DNA primer, this amine can be reacted with carboxylic acid groups on a polymer bead using standard amideforming chemistry. If such preferred immobilization is used during reverse transcription, the top strand RNA is degraded using well-known enzymes, such as a

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- 17 combination of RNAseH and RNAseA, either before or after immobilization.

The nucleic acid sequences useful in the methods of this invention are generally amplified before being used to display and/or express the peptides, polypeptides or proteins that they encode. Prior to amplification, the single-stranded DNAs may be cleaved using either of the methods described before. Alternatively, the single-stranded DNAs may be amplified and then cleaved using one of those methods.

Any of the well known methods for amplifying nucleic acid sequences may be used for such amplification. Methods that maximize, and do not bias, diversity are preferred. In a preferred embodiment of this invention where the nucleic acid sequences are derived from antibody genes, the present invention preferably utilizes primers in the constant regions of the heavy and light chain genes and primers to a synthetic sequence that are attached at the 5' end of the sense strand. Priming at such synthetic sequence avoids the use of sequences within the variable regions of the antibody genes. Those variable region priming sites generate bias against V genes that are either of rare subclasses or that have been mutated at the priming sites'. This bias is partly due to suppression of diversity within the primer region and partly due to lack of priming when many mutations are present in the region complementary to the primer. The methods disclosed in this invention have the advantage of not biasing the population of amplified antibody genes for particular V gene types.

The synthetic sequences may be attached to the 5' end of the DNA strand by various methods well

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- 18 known for ligating DNA seguences together. RT CapExtention is one preferred method.

In RT CapExtention (derived from Smart PCR'™’), a short overlap (5'-...GGG-3' in the upper5 strand primer (USP-GGG) complements 3'-CCC....5' in the lower strand) and reverse transcriptases are used so that the reverse complement of the upper-strand primer is attached to the lower strand.

FIGs. 1 and 2 show schematics to amplify VH 10 and VL genes using RT CapExtention. FIG. 1 shows a schematic of the amplification of VH genes. FIG. 1, Panel A shows a primer specific to the poly-dT region of the 3' UTR priming synthesis of the first, lower strand. Primers that bind in the constant region are also suitable. Panel B shows the lower strand extended at its 3' end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3' terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5' biotinylated synthetic top-strand primer that replicates the 5' end of the synthetic primer of panel

C and a bottom-strand primer complementary to part of the constant domain. Panel E shows immobilized doublestranded (ds) cDNA obtained by using a 5’-biotinylated top-strand primer.

FIG. 2 shows a similar schematic for amplification of VL genes. FIG. 2, Panel A shows a primer specific to the constant region at or near the 3' end priming synthesis of the first, lower strand. Primers that bind in the poly-dT region are also

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- 19 suitable. Panel B shows the lower strand extended at its 3' end by three Cs that are not complementary to the mRNA. Panel C shows the result of annealing a synthetic top-strand primer ending in three GGGs that hybridize to the 3' terminal CCCs and extending the reverse transcription extending the lower strand by the reverse complement of the synthetic primer sequence. Panel D shows the result of PCR amplification using a 5' biotinylated synthetic top-strand primer that replicates the 5' end of the synthetic primer of panel C and a bottom-strand primer complementary to part of the constant domain. The bottom-strand primer also contains a useful restriction endonuclease site, such as Ascl. Panel E shows immobilized ds cDNA obtained by using a 5'-biotinylated top-strand primer.

In FIGs. 1 and 2, each V gene consists of a

5' untranslated region (UTR) and a secretion signal, followed by the variable region, followed by a constant region, followed by a 3' untranslated region (which typically ends in poly-A). An initial primer for reverse transcription may be complementary to the constant region or to the poly A segment of the 3'-UTR. For human heavy-chain genes, a primer of 15 T is preferred. Reverse transcriptases attach several C residues to the 3' end of the newly synthesized DNA.

RT CapExtention exploits this feature. The reverse transcription reaction is first run with only a lowerstrand primer. After about 1 hour, a primer ending in GGG (USP-GGG) and more RTase are added. This causes the lower-strand cDNA to be extended by the reverse complement of the USP-GGG up to the final GGG. Using one primer identical to part of the attached synthetic sequence and a second primer complementary to a region

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- 20 of known sequence at the 3' end of the sense strand, all the V genes are amplified irrespective of their V gene subclass.

In another preferred embodiment, synthetic 5 sequences may be added by Rapid Amplification of cDNA

Ends (RACE) (see Frohman, M.A., Dush, M.K., & Martin, G.R. (1988) Proc. Natl. Acad. Sci. USA (85):

8998-9002).

FIG. 1 shows a schematic of RACE amplification of antibody heavy and light chains.

First, mRNA is selected by treating total or poly(A+) RNA with calf intestinal phosphatase (CIP) to remove the 5'-phosphate from all molecules that have them such as ribosomal RNA, fragmented mRNA, tRNA and genomic

DNA. Full length mRNA (containing a protective 7methyl cap structure) is uneffected. The RNA is then treated with tobacco acid pyrophosphatase (TAP) to remove the cap structure from full length mRNAs leaving a 5'-monophosphate group. Next, a synthetic RNA adaptor is ligated to the RNA population, only molecules which have a 5-phosphate (uncapped, full length mRNAs) will accept the adaptor. Reverse trascriptase reactions using an oligodT primer, and nested PCR (using one adaptor primer (located in the 5' synthetic adaptor) and one primer for the gene) are then used to amplify the desired transcript.

In a preferred embodiment of this invention, the upper strand or lower strand primer may be also biotinylated or labeled at the 5' end with one of a) free amino group, b) thiol, c) carboxylic acid and d) another group not found in DNA that can react to form a strong bond to a known partner as an insoluble medium. These can then be used to immobilize the labeled strand

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- 21 after amplification. The immobilized DNA can be either single or double-stranded.

After amplification (using e.g., RT CapExtension or RACE), the DNAs of this invention are 5 rendered single-stranded. For example, the strands can be separated by using a biotinylated primer, capturing the biotinylated product on streptavidin beads, denaturing the DNA, and washing away the complementary strand. Depending on which end of the captured DNA is 10 wanted, one will choose to immobilize either the upper (sense) strand or the lower (antisense) strand.

To prepare the single-stranded amplified DNAs for cloning into genetic packages so as to effect display of, or for expression of, the peptides, polypeptides or proteins encoded, at least in part, by those DNAs, they must be manipulated to provide ends suitable for cloning and display and/or expression. In particular, any 5' untranslated regions and mammalian signal sequences must be removed and replaced, in 20 frame, by a suitable signal sequence that functions in the display or expression host. Additionally, parts of the variable domains (in antibody genes) may be removed and replaced by synthetic segments containing synthetic diversity. The diversity of other gene families may 25 likewise be expanded with synthetic diversity.

According to the methods of this invention, there are two ways to manipulate the single-stranded DNAs for display and/or expression. The first method comprises the steps of:

(i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the

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- 22 region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

In this first method, short oligonucleotides are annealed to the single-stranded DNA so that restriction endonuclease recognition sites formed within the now locally double-stranded regions of the DNA can be cleaved. In particular, a recognition site that occurs at the same position in a substantial fraction of the single-stranded DNAs is identical.

For antibody genes, this can be done using a catalog of germline sequences. See, e.g., http://www.mrc-cpe. cam.ac.uk/imt-doc/restricted/ok.htm

1. Updates can be obtained from this site under the heading Amino acid and nucleotide sequence alignments. For other families, similar comparisons

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- 23 exist and may be used to select appropriate regions for cleavage and to maintain diversity.

For example, Table 1 depicts the DNA sequences of the FR3 regions of the 51 known human VH germline genes. In this region, the genes contain restriction endonuclease recognition sites shown in Table 2. Restriction endonucleases that cleave a large fraction of germline genes at the same site are preferred over endonucleases that cut at a variety of sites. Furthermore, it is preferred that there be only one site for the restriction endonucleases within the region to which the short oligonucleotide binds on the single-stranded DNA, e.g., about 10 bases on either side of the restriction endonuclease recognition site.

An enzyme that cleaves downstream in FR3 is also more preferable because it captures fewer mutations in the framework. This may be advantageous is some cases. However, it is well known that framework mutations exist and confer and enhance antibody binding. The present invention, by choice of appropriate restriction site, allows all or part of FR3 diversity to be captured. Hence, the method also allows extensive diversity to be captured.

Finally, in the methods of this invention restriction endonucleases that are active between about 37°C and about 75°C are used. Preferably, restriction endonucleases that are active between about 45°C and about 75°C may be used. More preferably, enzymes that are active above 50°C, and most preferably active about

55°C, are used. Such temperatures maintain the nucleic acid sequence to be cleaved in substantially singlestranded form.

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- 24 Enzymes shown in Table 2 that cut many of the heavy chain FR3 germline genes at a single position include: Maelll(2404), Tsp45I(2104), Bphl(4405),

BsaJI(23065), Alul(23047), BlpI(21048), Ddel(29058),

Bglll(10061), Msll(44072), BsiEI (23074), Bael(23074), Eagl(23074), Haelll(25@75), Bst4CI(51086) ,

BpyCH4III(51086), Hint I (3802), Mlyl(1802), Biel(1802), Mnll(31067), BpyCH4V (21044 ) , BsmAI(16Θ11) , Bpml(19012) , Xmnl(12030), and Sacl(11051). (The notation used means, for example, that BsmAI cuts 16 of the FR3 germline genes with a restriction endonuclease recognition site beginning at base 11 of FR3.)

For cleavage of human heavy chains in FR3, the preferred restriction endonucleases are: Bst4CI (or

Taal or HpyCH4III) , BlpI, BpyCH4V, and Msll. Because ACNGT (the restriction endonuclease recognition site for Bs£4CI, Taal, and BpyCH4III) is found at a consistent site in all the human FR3 germline genes, one of those enzymes is the most preferred for capture of heavy chain CDR3 diversity. BlpI and BpyCH4V are complementary. BlpI cuts most members of the VH1 and VH4 families while BpyCH4V cuts most members of the VH3, VH5, VH6, and VH7 families. Neither enzyme cuts VH2s, but this is a very small family, containing only three members. Thus, these enzymes may also be used in preferred embodiments of the methods of this invention.

The restriction endonucleases HpyCH4III, Bst4CI, and Taal all recognize 5'-ACnGT-3' and cut upper strand DNA after n and lower strand DNA before the base complementary to n. This is the most preferred restriction endonuclease recognition site for this method on human heavy chains because it is found in all germline genes. Furthermore, the restriction

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- 25 endonuclease recognition region (ACnGT) matches the second and third bases of a tyrosine codon (tay) and the following cysteine codon (tqy) as shown in Table 3. These codons are highly conserved, especially the cysteine in mature antibody genes.

Table 4 E shows the distinct oligonucleotides of length 22 (except the last one which is of length 20) bases. Table 5 C shows the analysis of 1617 actual heavy chain antibody genes. Of these, 1511 have the site and match one of the candidate oligonucleotides to within 4 mismatches. Eight oligonucleotides account for most of the matches and are given in Table 4 F.l. The 8 oligonucleotides are very similar so that it is likely that satisfactory cleavage will be achieved with only one oligonucleotide (such as H43.77.97.l-02#l) by adjusting temperature, pH, salinity, and the like. One or two oligonucleotides may likewise suffice whenever the germline gene sequences differ very little and especially if they differ very little close to the restriction endonuclease recognition region to be cleaved. Table 5 D shows a repeat analysis of 1617 actual heavy chain antibody genes using only the 8 chosen oligonucleotides. This shows that 1463 of the sequences match at least one of the oligonucleotides to within 4 mismatches and have the site as expected.

Only 7 sequences have a second HpyCH4III restriction endonuclease recognition region in this region.

Another illustration of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of human heavy chains.

Cleavage in FR1 allows capture of the entire CDR diversity of the heavy chain.

2018241075 03 Oct 2018

- 26 The germline genes for human heavy chain FR1 are shown in Table 6. Table 7 shows the restriction endonuclease recognition sites found in human germline genes FRls. The preferred sites are Bsgl (GTGCAG;3904),

BsoFI(GCngc;4306,1109,203,1012),

Tsel(Gcwgc;4306, 1109, 203, 1012) ,

MspAlI(CMGckg;4607,201), PvuII(CAGctg;4607,201),

Alul(AGct;4808202), Ddel(Ctnag;22052,9048),

Hphl(tcacc;22080), BssKI(Nccngg;35039, 2040),

BsaJI(Ccnngg;32040,2041), BstNI(CCwgg;33040),

ScrFI(CCngg;35040,2041), £co0109I(RGgnccy;22046,

11043), Sau96I(Ggncc;23047,11044),

Avail(Ggwcc;23047,4044), PpuMI(RGgwccy;22046,4043), BsmFI(gtccc;20048), HinfI(Gantc;34016, 21056,21077),

Tfil(21077), Mlyl(GAGTC;34016) , Mlyl(gactc;21056), and AlwNI(CAGnnnctg;22068). The more preferred sites are MspAI and PvuII. MspAI and PvuII have 4 6 sites at 7-12 and 2 at 1-6. To avoid cleavage at both sites, oligonucleotides are used that do not fully cover the site at 1-6. Thus, the DNA will not be cleaved at that site. We have shown that DNA that extends 3, 4, or 5 bases beyond a PvuII-site can be cleaved efficiently.

Another illustration of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of human kappa light chains. Table 8 shows the human kappa FR1 germline genes and Table 9 shows restriction endonuclease recognition sites that are found in a substantial number of human kappa FR1 germline genes at consistent locations. Of the restriction endonuclease recognition sites listed, BsmAI and PflFI are the most preferred enzymes. BsmAI sites are found at base 18 in 35 of 40 germl’ine genes.

2018241075 03 Oct 2018

- 27 PflFI sites are found in 35 of 40 germline genes at base 12.

Another example of choosing an appropriate restriction endonuclease recognition site involves cleavage in FR1 of the human lambda light chain. Table 10 shows the 31 known human lambda FR1 germline gene sequences. Table 11 shows restriction endonuclease recognition sites found in human lambda FR1 germline genes. Hinfl and Ddel are the most preferred restriction endonucleases for cutting human lambda chains in FR1.

After the appropriate site or sites for cleavage are chosen, one or more short oligonucleotides are prepared so as to functionally complement, alone or in combination, the chosen recognition site. The oligonucleotides also include sequences that flank the recognition site in the majority of the amplified genes. This flanking region allows the sequence to anneal to the single-stranded DNA sufficiently to allow cleavage by the restriction endonuclease specific for the site chosen.

The actual length and sequence of the oligonucleotide depends on the recognition site and the conditions to be used for contacting and cleavage. The 25 length must be sufficient so that the oligonucleotide is functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location.

Typically, the oligonucleotides of this preferred method of the invention are about 17 to about 30 nucleotides in length. Below about 17 bases, annealing is too weak and above 30 bases there can be a

2018241075 03 Oct 2018

- 28 loss of specificity. A preferred length is 18 to 24 bases .

Oligonucleotides of this length need not be identical complements of the germline genes. Rather, a few mismatches taken may be tolerated. Preferably, however, no more than 1-3 mismatches are allowed. Such mismatches do not adversely affect annealing of the oligonucleotide to the single-stranded DNA. Hence, the two DNAs are said to be functionally complementary.

The second method to manipulate the singlestranded DNAs of this invention for display and/or expression comprises the steps of:

(i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid solely at the cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur

2018241075 03 Oct 2018

- 29 at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

As explained above, the cleavage site may be 5 formed by the single-stranded portion of the partially double-stranded oligonucleotide duplexing with the single-stranded DNA, the cleavage site may be carried in the double-stranded portion of the partially doublestranded oligonucleotide, or the cleavage site may be introduced by the amplification primer used to amplify the single-stranded DNA-partially double-stranded oligonucleotide combination. In this embodiment, the first is preferred. And, the restriction endonuclease recognition site may be located in either the double15 stranded portion of the oligonucleotide or introduced by the amplification primer, which is complementary to that double-stranded region, as used to amplify the combination.

Preferably, the restriction endonuclease site is that of a Type II-S restriction endonuclease, whose cleavage site is located at a known distance from its recognition site.

This second method, preferably, employs Universal Restriction Endonucleases (URE). UREs are partially double-stranded oligonucleotides. The single-stranded portion or overlap of the URE consists of a DNA adapter that is functionally complementary to the sequence to be cleaved in the single-stranded DNA. The double-stranded portion consists of a restriction endonuclease recognition site, preferably type II-S.

The URE method of this invention is specific and precise and can tolerate some (e.g., 1-3) mismatches in the complementary regions, i.e., it is

- 30 2018241075 03 Oct 2018 functionally complementary to that region. Further, conditions under which the URE is used can be adjusted so that most of the genes that are amplified can be cut, reducing bias in the library produced from those genes.

The sequence of the single-stranded DNA adapter or overlap portion of the URE typically consists of about 14-22 bases. However, longer or shorter adapters may be used. The size depends on the ability of the adapter to associate with its functional complement in the single-stranded DNA and the temperature used for contacting the URE and the singlestranded DNA at the temperature used for cleaving the DNA with the restriction enzyme. The adapter must be functionally complementary to the single-stranded DNA over a large enough region to allow the two strands to associate such that the cleavage may occur at the chosen temperature and at the desired location. We prefer singe-stranded or overlap portions of 14-17 bases in length, and more preferably 18-20 bases in length.

The site chosen for cleavage using the URE is preferably one that is substantially conserved in the family of amplified DNAs. As compared to the first cleavage method of this invention, these sites do not need to be endonuclease recognition sites. However, like the first method, the sites chosen can be synthetic rather than existing in the native DNA. Such sites may be chosen by references to the -sequences of known antibodies or other families of genes. For example, the sequences of many germline genes are reported at http://www.mrc-cpe. cam.ac.uk/imtdoc/restricted/ok.html. For example, one preferred

2018241075 03 Oct 2018

- 31 site occurs near the end of FR3 -- codon 89 through the second base of codon 93. CDR3 begins at codon 95.

The sequences of 79 human heavy-chain genes are also available at http://www.ncbi.nlm.nih.qov/entre2/nucleotide.html.

This site can be used to identify appropriate sequences for URE cleavage according to the methods of this invention. See, e.g., Table 12B.

Most preferably, one or more sequences are 10 identified using these sites or other available sequence information. These sequences together are present in a substantial fraction of the amplified DNAs. For example, multiple sequences could be used to allow for known diversity in germline genes or for frequent somatic mutations. Synthetic degenerate sequences could also be used. Preferably, a sequence(s) that occurs in at least 65% of genes examined with no more than 2-3 mismatches is chosen

URE single-stranded adapters or overlaps are then made to be complementary to the chosen regions. Conditions for using the UREs are determined empirically. These conditions should allow cleavage of DNA that contains the functionally complementary sequences with no more than 2 or 3 mismatches but that do not allow cleavage of DNA lacking such sequences.

As described above, the double-stranded portion of the URE includes an endonuclease recognition site, preferably a Type II-S recognition site. Any enzyme that is active at a temperature necessary to maintain the single-stranded DNA substantially in that form and to allow the single-stranded DNA adapter portion of the URE to anneal long enough to the single- 32 2018241075 03 Oct 2018 stranded DNA to permit cleavage at the desired site may be used.

The preferred Type II-S enzymes for use in the URE methods of this invention provide asymmetrical cleavage of the single-stranded DNA. Among these are the enzymes listed in Table 13. The most preferred Type II-S enzyme is Fokl.

When the preferred Fokl containing URE is used, several conditions are preferably used to effect cleavage:

1) Excess of the URE over target DNA should be present to activate the enzyme. URE present only in equimolar amounts to the target DNA would yield poor cleavage of ssDNA because the amount of active enzyme available would be limiting.

2) An activator may be used to activate part of the Fokl enzyme to dimerize without causing cleavage. Examples of appropriate activators are shown in Table 14.

3) The cleavage reaction is performed at a temperature between 45°-75°C, preferably above 50°C and most preferably above 55°C.

The UREs used in the prior art contained a

14-base single-stranded segment, a 10-base stem (containing a Fokl site), followed by the palindrome of the 10-base stem. While such UREs may be used in the methods of this invention, the preferred UREs of this invention also include a segment of three to eight bases (a loop) between the Fokl restriction endonuclease recognition site containing segments. In the preferred embodiment, the stem (containing the Fokl

2018241075 03 Oct 2018

- 33 site) and its palindrome are also longer than 10 bases. Preferably, they are 10-14 bases in length. Examples of these lollipop URE adapters are shown in Table 15.

One example of using a URE to cleave an 5 single-stranded DNA involves the FR3 region of human heavy chain. Table 16 shows an analysis of 840 fulllength mature human heavy chains with the URE recognition sequences shown. The vast majority (718/840=0.85) will be recoqnized with 2 or fewer mismatches usinq five UREs (VHS881-1.1, VHS881-1.2, VHS881-2.1, VHS881-4.1, and VHS881-9.1). Each has a 20-base adaptor sequence to complement the germline gene, a ten-base stem segment containing a FokI site, a five base loop, and the reverse complement of the first stem segment. Annealing those adapters, alone or in combination, to single-stranded antisense heavy chain DNA and treating with FokI in the presence of, e.g., the activator FOKIact, will lead to cleavage of the antisense strand at the position indicated.

Another example of using a URE(s) to cleave a single-stranded DNA involves the FR1 region of the human Kappa light chains. Table 17 shows an analysis of 182 full-length human kappa chains for matching by the four 19-base probe sequences shown. Ninety-six percent of the sequences match one of the probes with 2 or fewer mismatches. The URE adapters shown in Table 17 are for cleavage of the sense strand of kappa chains. Thus, the adaptor sequences are the reverse complement of the germline gene sequences. The URE consists of a ten-base stem, a five base loop, the reverse complement of the stem and the complementation sequence. The loop shown here is TTGTT, but other sequences could be used. Its function is to interrupt

2018241075 03 Oct 2018

- 34 the palindrome of the stems so that formation of a lollypop monomer is favored over dimerization. Table 17 also shows where the sense strand is cleaved.

Another example of using a URE to cleave a 5 single-stranded DNA involves the human lambda light chain. Table 18 shows analysis of 128 human lambda light chains for matching the four 19-base probes shown. With three or fewer mismatches, 88 of 128 (69%) of the chains match one of the probes. Table 18 also shows URE adapters corresponding to these probes. Annealing these adapters to upper-strand ssDNA of lambda chains and treatment with FokI in the presence of FOKIact at a temperature at or above 45°C will lead to specific and precise cleavage of the chains.

The conditions under which the short oligonucleotide sequences of the first method and the UREs of the second method are contacted with the single-stranded DNAs may be empirically determined.

The conditions must be such that the single-stranded

DNA remains in substantially single-stranded form.

More particularly, the conditions must be such that the single-stranded DNA does not form loops that may int .rfere with its association with the oligonucleotide sequence or the URE or that may themselves provide sites for cleavage by the chosen restriction endonuclease.

The effectiveness and specificity of short oligonucleotides (first method) and UREs (second method) can be adjusted by controlling the concentrations of the URE adapters/oligonucleotides and substrate DNA, the temperature, the pH, the concentration of metal ions, the ionic strength, the concentration of chaotropes (such as urea and

2018241075 03 Oct 2018

- 35 formamide), the concentration of the restriction endonuclease(e. g., FokI), and the time of the digestion. These conditions can be optimized with synthetic oligonucleotides having: 1) target germline gene sequences, 2) mutated target gene sequences, or 3) somewhat related non-target sequences. The goal is to cleave most of the target sequences and minimal amounts of non-targets.

In accordance with this invention, the 10 single-stranded DNA is maintained in substantially that form using a temperature between about 37°C and about 75°C. Preferably, a temperature between about 4 5°C and about 75°C is used. More preferably, a temperature between 50°C and 60°C, most preferably between 55°C and

60°C, is used. These temperatures are employed both when contacting the DNA with the oligonucleotide or URE and when cleaving the DNA using the methods of this invention.

The two cleavage methods of this invention have several advantages. The first method allows the individual members of the family of single-stranded DNAs to be cleaved preferentially at one substantially conserved endonuclease recognition site. The method also does not require an endonuclease recognition site to be built into the reverse transcription or amplification primers. Any native or synthetic site in the family can be used.

The second method has both of these advantages. In addition, the preferred URE method allows the single-stranded DNAs to be cleaved at positions where no endonuclease recognition site naturally occurs or has been synthetically constructed.

2018241075 03 Oct 2018

- 36 Most importantly, both cleavage methods permit the use of 5' and 3' primers so as to maximize diversity and then cleavage to remove unwanted or deleterious sequences before cloning, display and/or expression.

After cleavage of the amplified DNAs using one of the methods of this invention, the DNA is prepared for cloning, display and/or expression. This is done by using a partially duplexed synthetic DNA adapter, whose terminal sequence is based on the specific cleavage site at which the amplified DNA has been cleaved.

The synthetic DNA is designed such that when it is ligated to the cleaved single-stranded DNA in proper reading frame so that the desired peptide, polypeptide or protein can be displayed on the surface of the genetic package and/or expressed. Preferably, the double-stranded portion of the adapter comprises the sequence of several codons that encode the amino acid sequence characteristic of the family of peptides, polypeptides or proteins up to the cleavage site. For human heavy chains, the amino acids of the 3-23 framework are preferably used to provide the sequences required for expression of the cleaved DNA.

Preferably, the double-stranded portion of the adapter is about 12 to 100 bases in length. More preferably, about 20 to 100 bases are used. The double-standard region of the adapter also preferably contains at least one endonuclease recognition site useful for cloning the DNA into a suitable display and/or expression vector {or a recipient vector used to archive the diversity). This endonuclease restriction site may be native to the germline gene sequences used

- 37 2018241075 03 Oct 2018 to extend the DNA sequence. It may be also constructed using degenerate sequences to the native germline gene sequences. Or, it may be wholly synthetic.

The single-stranded portion of the adapter is 5 complementary to the region of the cleavage in the single-stranded DNA. The overlap can be from about 2 bases up to about 15 bases. The longer the overlap, the more efficient the ligation is likely to be. A preferred length for the overlap is 7 to 10. This allows some mismatches in the region so that diversity in this region may be captured.

The single-stranded region or overlap of the partially duplexed adapter is advantageous because it allows DNA cleaved at the chosen site, but not other fragments to be captured. Such fragments would contaminate the library with genes encoding sequences that will not fold into proper antibodies and are likely to be non-specifically sticky.

One illustration of the use of a partially duplexed adaptor in the methods of this invention involves ligating such adaptor to a human FR3 region that has been cleaved, as described above, at 5'-ACnGT3' using HpyCH4III, Bst4CI or Taal.

Table 4 F.2 shows the bottom strand of the double-stranded portion of the adaptor for ligation to the cleaved bottom-strand DNA. Since the HpyCH4IIISite is so far to the right (as shown in Table 3), a sequence that includes the Aflll-site as well as the Xbal site can be added. This bottom strand portion of the partially-duplexed adaptor, H43.XAExt, incorporates both Xbal and Aflll-sites. The top strand of the double-stranded portion of the adaptor has neither site (due to planned mismatches in the segments

2018241075 03 Oct 2018

- 38 opposite the Xbal and Aflll-Sites of H43.XAExt), but will anneal very tightly to H43.XAExt. H43AExt contains only the AfUI-site and is to be used with the top strands H43.ABrl and H43.ABr2 (which have intentional alterations to destroy the AfUI-site).

After ligation, the desired, captured DNA can be PCR amplified again, if desired, using in the preferred embodiment a primer to the downstream constant region of the antibody gene and a primer to part of the double-standard region of the adapter. The primers may also carry restriction endonuclease sites for use in cloning the amplified DNA.

After ligation, and perhaps amplification, of the partially double-stranded adapter to the single15 stranded amplified DNA, the composite DNA is cleaved at chosen 5' and 3' endonuclease recognition sites.

The cleavage sites useful for cloning depend on the phage or phagemid or other vectors into which the cassette will be inserted and the available sites in the antibody genes. Table 19 provides restriction endonuclease data for 75 human light chains. Table 20 shows corresponding data for 79 human heavy chains. In each Table, the endonucleases are ordered by increasing frequency of cutting. In these Tables, Nch is the number of chains cut by the enzyme and Ns is the number of sites (some chains have more than one site).

From this analysis, Sfil, Notl, Aflll, ApaLI, and Ascl are very suitable. Sfil and Notl are preferably used in pCESl to insert the heavy-chain display segment. ApaLI and Ascl are preferably used in pCESl to insert the light-chain display segment.

BstEII-sites occur in 97% of germ-line JH genes. In rearranged V genes, only 54/79 (68%) of

2018241075 03 Oct 2018

- 39 heavy-chain genes contain a BstEII-Site and 7/61 of these contain two sites. Thus, 47/79 (59%) contain a single BstEII-Site. An alternative to using BstEII is to cleave via UREs at the end of JH and ligate to a synthetic oligonucleotide that encodes part of CHI.

One example of preparing a family of DNA sequences using the methods of this invention involves capturing human CDR 3 diversity. As described above, mRNAs from various autoimmune patients are reverse transcribed into lower strand cDNA. After the top strand RNA is degraded, the lower strand is immobilized and a short oligonucleotide used to cleave the cDNA upstream of CDR3. A partially duplexed synthetic DNA adapter is then annealed to the DNA and the DNA is amplified using a primer to the adapter and a primer to the constant region (after FR4). The DNA is then cleaved using BstEII (in FR4) and a restriction endonuclease appropriate to the partially doublestranded adapter (e.g., Xbal and Aflll (in FR3)). The

DNA is then ligated into a synthetic VH skeleton such as 3-23.

One example of preparing a single-stranded DNA that was cleaved using the URE method involves the human Kappa chain. The cleavage site in the sense strand of this chain is depicted in Table 17. The oligonucleotide kapextURE is annealed to the oligonucleotides (kaBROlUR, kaBR02UR, kaBR03UR, and kaBR04UR) to form a partially duplex DNA. This DNA is then ligated to the cleaved soluble kappa chains. The ligation product is then amplified using primers kapextUREPCR and CKForeAsc (which inserts a Ascl site after the end of C kappa). This product is then cleaved with ApaLI and Ascl and ligated to similarly

- 40 2018241075 03 Oct 2018 cut recipient vector.

Another example involves the cleavage of lambda light chains, illustrated in Table 18. After cleavage, an extender (ON_LamExi33) and four bridge oligonucleotides (ON_LamBl-133, ON_LamB2-133, ON_LainB3-133, and ON_LamB4-i33) are annealed to form a partially duplex DNA. That DNA is ligated to the cleaved lambda-chain sense strands. After ligation, the DNA is amplified with ON_Laml33PCR and a forward primer specific to the lambda constant domain, such as CL2ForeAsc or CL7ForeAsc (Table 130) .

In human heavy chains, one can cleave almost all genes in FR4 (downstream, i.e., toward the 3' end of the sense strand, of CDR3) at a BstEII-Site that occurs at a constant position in a very large fraction of human heavy-chain V genes. One then needs a site in FR3, if only CDR3 diversity is to be captured, in FR2,_e if CDR2 and CDR3 diversity is wanted, or in FR1, if all the CDR diversity is wanted. These sites are preferably inserted as part of the partially doublestranded adaptor.

The preferred process of this invention is to provide recipient vectors (e.g., for display and/or expression) having sites that allow cloning of either light or heavy chains. Such vectors are well known and widely used in the art. A preferred phage display vector in accordance with this invention is phage MALIA3. This displays in gene III. The sequence of the phage MALIA3 is shown in Table 21A (annotated) and

Table 21B (condensed).

The DNA encoding the selected regions of the light or heavy chains can be transferred to the vectors using endonucleases that cut either light or heavy

2018241075 03 Oct 2018

- 41 chains only very rarely. For example, light chains may be captured with ApaLI and Ascl. Heavy-chain genes are preferably cloned into a recipient vector having Sfil, Ncol, Xbal, Aflll, BstEIl, Apal, and Notl sites. The light chains are preferably moved into the library as ApaLI-AscI fragments. The heavy chains are preferably moved into the library as Sfil-Notl fragments.

Most preferably, the display is had on the surface of a derivative of M13 phage. The most preferred vector contains all the genes of M13, an antibiotic resistance gene, and the display cassette. The preferred vector is provided with restriction sites that allow introduction and excision of members of the diverse family of genes, as cassettes. The preferred vector is stable against rearrangement under the growth conditions used to amplify phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a phagemid vector (e.g., pCESl) that displays and/or expresses the peptide, polypeptide or protein. Such vectors may also be used to store the diversity for subsequent display and/or expression using other vectors or phage.

In another embodiment of this invention, the diversity captured by the methods of the present invention may be displayed and/or expressed in a yeast vector.

In another embodiment, the mode of display may be through a short linker to anchor domains — one possible anchor comprising the final portion of M13 III (Illstump) and a second possible anchor being the full length III mature protein.

2018241075 03 Oct 2018

- 42 The Illstump fragment contains enough of M13 III to assemble into phage but not the domains involved in mediating infectivity. Because the w.t. Ill proteins are present the phage is unlikely to delete the antibody genes and phage that do delete these segments receive only a very small growth advantage.

For each of the anchor domains, the DNA encodes the w.t. AA sequence, but differs from the w.t. DNA sequence to a very high extent. This will greatly reduce the potential for homologous recombination between the anchor and the w.t. gene that is also present (see Example 6) .

Most preferably, the present invention uses a complete phage carrying an antibiotic-resistance gene (such as an ampicillin-resistance gene) and the display cassette. Because the w.t. iii and possibly viii genes are present, the w.t. proteins are also present. The display cassette is transcribed from a regulatable promoter (e.g., P_LacZ) . Use of a regulatable promoter allows control of the ratio of the fusion display gene to the corresponding w.t. coat protein. This ratio determines the average number of copies of the display fusion per phage (or phagemid) particle.

Another aspect of the invention is a method of displaying peptides, polypeptides or proteins (and particularly Fabs) on filamentous phage. In the most preferred embodiment this method displays FABs and comprises :

a) obtaining a cassette capturing a diversity of segments of DNA encoding the elements:

P_reg: :RBS1: :SS1: :VL: :CL: :stop: :RBS2 : :SS2: :VH: :CH1: : linker:: anchor: : stop::,

- 43 2018241075 03 Oct 2018 where P_reg is a regulatable promoter, RBS1 is a first ribosome binding site, SSI is a signal sequence operable in the host strain, VL is a member of a diverse set of light-chain variable regions, CL is a light-chain constant region, stop is one or more stop codons, RBS2 is a second ribosome binding site, SS2 is a second signal sequence operable in the host strain,

VH is a member of a diverse set of heavy-chain variable regions, CHI is an antibody heavy-chain first constant domain, linker is a sequence of amino acids of one to about 50 residues, anchor is a protein that will assemble into the filamentous phage particle and stop is a second example of one or more stop codons; and

b) positioning that cassette within the phage genome to maximize the viability of the phage and to minimize the potential for deletion of the cassette or parts thereof.

The DNA encoding the anchor protein in the above preferred cassette should be designed to encode the same (or a closely related) amino acid sequence as is found in one of the coat proteins of the phage, but with a distinct DNA sequence. This is to prevent unwanted homologous recombination with the w.t. gene.

In addition, the cassette should be placed in the intergenic region. The positioning and orientation of the display cassette can influence the behavior of the phage.

In one embodiment of the invention, a transcription terminator may be placed after the second stop of the display cassette above (e.g., Trp). This will reduce interaction between the display cassette

2018241075 03 Oct 2018

- 44 and other genes in the phage antibody display vector.

In another embodiment of the methods of this invention, the phage or phagemid can display and/or express proteins other than Fab, by replacing the Fab portions indicated above, with other protein genes.

Various hosts can be used the display and/or expression aspect of this invention. Such hosts are well known in the art. In the preferred embodiment, where Fabs are being displayed and/or expressed, the preferred host should grow at 30°C and be RecA' (to reduce unwanted genetic recombination) and EndA' (to make recovery of RF DNA easier). It is also preferred that the host strain be easily transformed by electroporation 15 XLl-Blue MRF' satisfies most of these preferences, but does not grow well at 30°C. XLl-Blue MRF^! does grow slowly at 38°C and thus is an acceptable host. TG-1 is also an acceptable host although it is RecA⁺ and EndA⁺. XLl-Blue MRF' is more preferred for the intermediate host used to accumulate diversity prior to final construction of the library.

After display and/or expression, the libraries of this invention may be screened using well known and conventionally used techniques. The selected peptides, polypeptides or proteins may then be used to treat disease. Generally, the peptides, polypeptides or proteins for use in therapy or in pharmaceutical compositions are produced by isolating the DNA encoding the desired peptide, polypeptide or protein from the member of the library selected. That DNA is then used in conventional methods to produce the peptide, polypeptides or protein it encodes in appropriate host cells, preferably mammalian host cells, e.g., CHO

2018241075 03 Oct 2018

- 45 cells. After isolation, the peptide, polypeptide or protein is used alone or with pharmaceutically acceptable compositions in therapy to treat disease.

EXAMPLES

Example 1: RACE amplification of heavy and light chain antibody repertoires from autoimmune patients.

Total RNA was isolated from individual blood samples (50 ml) of 11 patients using a RNAzolTM kit (CINNA/Biotecx), as described by the manufacturer. The patients were diagnosed as follows:

1. SLE and phospholipid syndrome

2. limited systemic sclerosis

3. SLE and Sjogren syndrome

4. Limited Systemic sclerosis

5. Reumatoid Arthritis with active vasculitis

6. Limited systemic sclerosis and Sjogren Syndrome

7. Reumatoid Artritis and (not active) vasculitis

8. SLE and Sjogren syndrome

9. SLE

10. SLE and (active) glomerulonephritis

11. Polyarthritis/ Raynauds Phenomen

From these 11 samples of total RNA, Poly-A+ RNA was isolated using Promega PolyATtract® mRNA Isolation kit (Promega).

250 ng of each poly-A+ RNA sample was used to amplify antibody heavy and light chains with the GeneRAacerTM kit (Invitrogen cat no. L1500-01). A schematic overview of the RACE procedure is shown in

- 46 2018241075 03 Oct 2018

FIG. 3.

Using the general protocol of the GeneRAacer”’ kit, an RNA adaptor was ligated to the 5'end of all mRNAs. Next, a reverse transcriptase reaction was performed in the presence of oligo(dT15) specific primer under conditions described by the manufacturer

TM m the GeneRAacer kit.

1/5 of the cDNA from the reverse transcriptase reaction was used in a 20 ul PCR reaction. For amplification of the heavy chain IgM repertoire, a forward primer based on the CHI chain of IgM [HuCmFOR] and a backward primer based on the ligated synthetic adaptor sequence [5Ά] were used.

(See Table 22)

For amplification of the kappa and lambda light chains, a forward primer that contains the 3’ coding-end of the cDNA [HuCkFor and HuCLFor2+HuCLf or7 ] and a backward primer based on the ligated synthetic adapter sequence [5Ά] was used (See Table 22).

Specific amplification products after 30 cycles of primary PCR were obtained.

FIG. 4 shows the amplification products obtained after the primary PCR reaction from 4 different patient samples. 8 ul primary PCR product from 4 different patients was analyzed on a agarose gel [labeled 1,2, 3 and 4]. For the heavy chain, a product of approximately 950 nt is obtained while for the kappa and lambda light chains the product is approximately 850 nt. Ml-2 are molecular weight markers.

PCR products were also analyzed by DNA sequencing [10 clones from the lambda, kappa or heavy chain repertoires]. All sequenced antibody genes recovered contained the full coding sequence as well as

- 47 2018241075 03 Oct 2018 the 5' leader sequence and the V gene diversity was the expected diversity (compared to literature data).

ng of all samples from all 11 individual amplified samples were mixed for heavy, lambda light or kappa light chains and used in secondary PCR reactions.

In all secondary PCRs approximately 1 ng template DNA from the primary PCR mixture was used in multiple 50 ul PCR reactions [25 cycles].

For the heavy chain, a nested biotinylated 10 forward primer [HuCm-Nested] was used, and a nested

5'end backward primer located in the synthetic adapter-sequence [5'NA] was used. The 5'end lower-strand of the heavy chain was biotinylated.

For the light chains, a 5'end biotinylated 15 nested primer in the synthetic adapter was used [5'NA] in combination with a 3'end primer in the constant region of Ckappa and Clambda, extended with a sequence coding for the Ascl restriction site [ kappa:

HuCkForAscI, Lambda: HuCL2-FOR-ASC + HuCL7-FOR-ASC] .

[5'end Top strand DNA was biotinylated]. After gel-analysis the secondary PCR products were pooled and purified with Promega Wizzard PCR cleanup.

Approximately 25 ug biotinylated heavy chain, lambda and kappa light chain DNA was isolated from the 11 patients.

Example 2: Capturing kappa chains with BsmAI.

A repertoire of human-kappa chain mRNAs was prepared using the RACE method of Example 1 from a collection of patients having various autoimmune diseases.

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- 48 This Example followed the protocol of Example 1. Approximately 2 micrograms (ug) of human kappachain (Igkappa) gene RACE material with biotin attached to 5'-end of upper strand was immobilized as in Example

1 on 200 microliters (pL) of Seradyn magnetic beads.

The lower strand was removed by washing the DNA with 2 aliquots 200 pL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by 30 seconds for the second aliquot. The beads were neutralized with 200 pL of 10 mM Tris (pH 7.5) 100 mM NaCl. The short oligonucleotides shown in Table 23 were added in 40 fold molar excess in 100 pL of NEB buffer 2 (50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol pH 7.9) to the dry beads. The mixture was incubated at

95°C for 5 minutes then cooled down to 55°C over 30 minutes. Excess oligonucleotide was washed away with 2 washes of NEB buffer 3 (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl₂, 1 mM dithiothreitol pH 7.9). Ten units of BsmAI (NEB) were added in NEB buffer 3 and incubated for 1 h at 55°C. The cleaved downstream DNA was collected and purified over a Qiagen PCR purification column (FIGs. 5 and 6).

FIG. 5 shows an analysis of digested kappa single-stranded DNA. Approximately 151.5 pmol of adapter was annealed to 3.79 pmol of immobilized kappa single-stranded DNA followed by digestion with 15 U of BsmAI. The supernatant containing the desired DNA was removed and analyzed by 5% polyacrylamide gel along with the remaining beads which contained uncleaved full length kappa DNA. 189 pmol of cleaved single-stranded DNA was purified for further analysis. Five percent of the original full length ssDNA remained on the beads.

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- 49 FIG. 6 shows an analysis of the extender cleaved kappa ligation. 180 pmol of pre-annealed bridge/extender was ligated to 1.8 pmol of BsmAI digested single-stranded DNA. The ligated DNA was purified by Qiagen PCR purification column and analyzed on a 5% polyacrylamide gel. Results indicated that the ligation of extender to single-stranded DNA was 95% efficient.

A partially double-stranded adaptor was 10 prepared using the oligonucleotide shown in Table 23.

The adaptor was added to the single-stranded DNA in 100 fold molar excess along with 1000 units of T4 DNA ligase and incubated overnight at 16°C. The excess oligonucleotide was removed with a Qiagen PCR purification column. The ligated material was amplified by PCR using the primers kapPCRtl and kapfor shown in Table 23 for 10 cycles with the program shown in Table 24.

The soluble PCR product was run on a gel and showed a band of approximately 700 n, as expected (FIGs. 7 and 8). The DNA was cleaved with enzymes ApaLI and Ascl, gel purified, and ligated to similarly cleaved vector pCESl.

FIG. 7 shows an analysis of the PCR product from the extender-kappa amplification. Ligated extender-kappa single-stranded DNA was amplified with primers specific to the extender and to the constant region of the light chain. Two different template concentrations, 10 ng versus 50 ng, were used as template and 13 cycles were used to generate approximately 1.5 ug of dsDNA as shown by 0.8% agarose gel analysis.

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- 50 FIG. 8 shows an analysis of the purified PCR product from the extender-kappa amplification. Approximately 5 ug of PCR amplified extender-kappa double-stranded DNA was run out on a 0.8% agarose gel, cut out, and extracted with a GFX gel purification column. By gel analysis, 3.5 ug of double-stranded DNA was prepared.

The assay for capturing kappa chains with BsmAl was repeated and produced similar results.

FIG 9A shows the DNA after it was cleaved and collected and purified over a Qiagen PCR purification column.

FIG. 9B shows the partially double-stranded adaptor ligated to the single-stranded DNA. This ligated material was then amplified (FIG. 9C). The gel showed a band of approximately 700 n.

Table 25 shows the DNA sequence of a kappa light chain captured by this procedure. Table 26 shows a second sequence captured by this procedure. The closest bridge sequence was complementary to the sequence 5'-agccacc-3’, but the sequence captured reads 5'-Tgccacc-3', showing that some mismatch in the overlapped region is tolerated.

Example 3: Construction of Synthetic CDR1 and CDR2 Diversity in V-3-23 VH Framework.

Synthetic diversity in Complementary

Determinant Region (CDR) 1 and 2 was created in the 323 VH framework in a two step process: first, a vector containing the 3-23 VH framework was constructed; and then, a synthetic CDR 1 and 2 was assembled and cloned into this vector.

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- 51 For construction of the 3-23 VH framework, 8 oligonucleotides and two PCR primers (long oligonucleotides - TOPFR1A, BOTFR1B, B0TFR2, BOTFR3, F06, BOTFR4, ON-vgCl, and ON-vgC2 and primers - SFPRMET and

BOTPCRPRIM, shown in Table 27) that overlap were designed based on the Genebank sequence of 3-23 VH framework region. The design incorporated at least one useful restriction site in each framework region, as shown in Table 27. In Table 27, the segments that were synthesized are shown as bold, the overlapping regions are underscored, and the PCR priming regions at each end are underscored.

A mixture of these 8 oligos was combined at a final concentration of 2.5uM in a 20ul PCR reaction.

The PCR mixture contained 200uM dNTPs, 2.5mM MgCl₂,

0.020 Pfu Turbo™ DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase, and IX Qiagen PCR buffer. The PCR program consisted of 10 cycles of 94 °C for 30s, 55°C for 30s, and 72°C for 30s.

The assembled 3-23 VH DNA sequence was then amplified, using 2.5ul of a 10-fold dilution from the initial PCR in lOOul PCR reaction. The PCR reaction contained 200uM dNTPs, 2.5mM MgCl₂, 0.02U Pfu Turbo™

DNA Polymerase, 1U Qiagen HotStart Taq DNA Polymerase,

IX Qiagen PCR Buffer and 2 outside primers (SFPRMET and BOTPCRPRIM) at a concentration of luM. The PCR program consisted of 23 cycles at 94°C for 30s, 55°C for 30s, and 72°C for 60s. The 3-23 VH DNA sequence was digested and cloned into pCESl (phagemid vector) using the Sfil and BstEII restriction endonuclease sites.

All restriction enzymes mentioned herein were supplied by New England BioLabs, Beverly, MA and used as per the manufacturer's instructions.

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- 52 Stuffer sequences (shown in Table 28 and Table 29) were introduced into pCESl to replace CDR1/CDR2 sequences (900 bases between BspEI and Xbal RE sites) and CDR3 sequences (358 bases between Aflll and BstEII) prior to cloning the CDR1/CDR2 diversity. This new vector was termed pCES5 and its sequence is given in Table 29.

Having stuffers in place of the CDRs avoids the risk that a parental sequence would be over10 represented in the library. The stuffer sequences are fragments from the penicillase gene of E. coli. The CDR1-2 stuffer contains restriction sites for Bglll, Bsu36I, Bell, Xcml, Mini, BvuII, Hpal, and Hindi, the underscored sites being unique within the vector pCES5.

The stuffer that replaces CDR3 contains the unique restriction endonuclease site RsrII.

A schematic representation of the design for CDR1 and CDR2 synthetic diversity is shown FIG. 10.

The design was based on the presence of mutations in

DP47/3-23 and related germline genes. Diversity was designed to be introduced at the positions within CDR1 and CDR2 indicated by the numbers in FIG. 10. The diversity at each position was chosen to be one of the three following schemes: 1 = ADEFGHIKLMNPQRSTVWY; 2 =

YRWVGS; 3 = PS, in which letters encode equimolar mixes of the indicated amino acids.

For the construction of the CDR1 and CDR2 diversity, 4 overlapping oligonucleotides (ON-vgCl, ON_Brl2, ON_CD2Xba, and 0N-vgC2, shown in Table 27 and

Table 30) encoding CDR1/2, plus flanking regions, were designed. A mixture of these 4 oligos was combined at a final concentration of 2.5uM in a 40ul PCR reaction. Two of the 4 oligos contained variegated sequences

2018241075 03 Oct 2018

- 53 positioned at the CDR1 and the CDR2. The PCR mixture contained 200uM dNTPs, 2.5U Pwo DNA Polymerase (Roche), and IX Pwo PCR buffer with 2mM MgSO₄. The PCR program consisted of 10 cycles at 94°C for 30s, 60°C for 30s, and 72°C for 60s. This assembled CDR1/2 DNA sequence was amplified, using 2.5ul of the mixture in lOOul PCR reaction. The PCR reaction contained 200uM dNTPs, 2.5U Pwo DNA Polymerase, IX Pwo PCR Buffer with 2mM MgSO„ and 2 outside primers at a concentration of luM. The PCR program consisted of 10 cycles at 94°C for 30s, 60°C for 30s, and 72°C for 60s. These variegated sequences were digested and cloned into the 3-23 VH framework in place of the CDR1/2 stuffer.

We obtained approximately 7 X 10⁷ independent transformants. CDR3 diversity either from donor populations or from synthetic DNA can be cloned into the vector containing synthetic CDR1 and CDR 2 diversity.

A schematic representation of this procedure is shown in FIG. 11. A sequence encoding the FRregions of the human V3-23 gene segment and CDR regions with synthetic diversity was made by oligonucleotide assembly and cloning via BspEl and Xbal sites into a vector that complements the FR1 and FR3 regions. Into this library of synthetic VH segments, the complementary VH-CDR3 sequence (top right) was cloned via Xbal an BstEll sites. The resulting cloned CH genes contain a combination of designed synthetic diversity and natural diversity (see FIG. 11).

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- 54 Example 4: Cleavage and ligation of the lambda light chains with HinfI.

A schematic of the cleavage and ligation of antibody light chains is shown in FIGs. 12A and 12B.

Approximately 2 ug of biotinylated human Lambda DNA prepared as described in Example 1 was immobilized on 200 ul Seradyn magnetic beads. The lower strand was removed by incubation of the DNA with 200 ul of 0.1 M NaOH (pH=13) for 3 minutes, the supernatant was removed and an additional washing of 30 seconds with 200 ul of 0.1 M NaOH was performed. Supernatant was removed and the beads were neutralized with 200 ul of 10 mM Tris (pH=7.5), 100 mM NaCl. 2 additional washes with 200 ul NEB2 buffer 2, containing 10 mM Tris (pH=7.9), 50 mM

NaCl, 10 mM MgCl2 and 1 mM dithiothreitol, were performed. After immobilization, the amount of ssDNA was estimated on a 5% PAGE-UREA gel.

About 0.8 ug ssDNA was recovered and incubated in 100 ul NEB2 buffer 2 containing 80 molar fold excess of an equimolar mix of ON_LamlaB7,

ON_Lam2aB7, ON_Lam3lB7 and ON_Lam3rB7 [each oligo in 20 fold molar excess] (see Table 31).

The mixture was incubated at 95° C for 5 minutes and then slowly cooled down to 50° C over a period of 30 minutes. Excess of oligonucleotide was washed away with 2 washes of 200 ul of NEB buffer 2.

U/ug of Hinf I was added and incubated for 1 hour at 50° C. Beads were mixed every 10 minutes.

After incubation the sample was purified over a Qiagen PCR purification column and was subsequently analysed on a 5% PAGE-urea gel (see FIG. 13A, cleavage was more than 70% efficient).

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- 55 A schematic of the ligation of the cleaved light chains is shown in FIG. 12B. A mix of bridge/extender pairs was prepared from the Brg/Ext oligo's listed in Table 31 {total molar excess 100 fold) in 1000 U of T4 DNA Ligase (NEB) and incubated overnight at 16 ^c C. After ligation of the DNA, the excess oligonucleotide was removed with a Qiagen PCR purification column and ligation was checked on a Urea-PAGE gel (see FIG. 13B; ligation was more than 95% efficient).

Multiple PCRs were performed containing 10 ng of the ligated material in an 50 ul PCR reaction using 25 pMol ON lamPlePCR and 25 pmol of an equimolar mix of Hu-CL2AscI/HuCL7AscI primer (see Example 1).

PCR was performed at 60° C for 15 cycles using Pfu polymerase. About 1 ug of dsDNA was recovered per PCR (see FIG. 13C) and cleaved with ApaLl and Ascl for cloning the lambda light chains in pCES2.

Example 5: Capture of human heavy-chain CDR3

0 population.

A schematic of the cleavage and ligation of antibody light chains is shown in FIGs. 14A and 14B.

Approximately 3 ug of human heavy-chain (IgM) gene RACE material with biotin attached to 5'-end of lower strand was immobilized on 300 uL of Seradyn magnetic beads. The upper strand was removed by washing the DNA with 2 aliquots 300 uL of 0.1 M NaOH (pH 13) for 3 minutes for the first aliquot followed by

30 seconds for the second aliquot. The beads were neutralized with 300 uL of 10 mM Tris (pH 7.5) 100 mM

NaCl. The REdaptors (oligonucleotides used to make

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- 56 single-stranded DNA locally double-stranded) shown in Table 32 were added in 30 fold molar excess in 200 uL of NEB buffer 4 (50 mM Potasium Acetate, 20 mM Tris-Acetate, 10 mM Magnesuim Acetate, 1 mM dithiothreitol pH 7.9) to the dry beads. The

REadaptors were incubated with the single-stranded DNA at 80 °C for 5 minutes then cooled down to 55 °C over 30 minutes. Excess REdaptors were washed away with 2 washes of NEB buffer 4. Fifteen units of HpyCH4III (NEB) were added in NEB buffer 4 and incubated for 1 hour at 55 °C. The cleaved downstream DNA remaining on the beads was removed from the beads using a Qiagen Nucleotide removal column (see FIG. 15).

The Bridge/Extender pairs shown in Table 33 were added in 25 molar excess along with 1200 units of T4 DNA ligase and incubated overnight at 16 °C. Excess Bridge/Extender was removed with a Qiagen PCR purification column. The ligated material was amplified by PCR using primers H43.XAExtPCR2 and

Hucumnest shown in Table 34 for 10 cycles with the program shown in Table 35.

The soluble PCR product was run on a gel and showed a band of approximately 500 n, as expected (see FIG. 15B) . The DNA was cleaved with enzymes Sfil and

Notl, gel purified, and ligated to similarly cleaved vector PCES1.

Example 6: Description of Phage Display Vector CJRA05, a member of the library built in vector DY3F7.

Table 36 contains an annotated DNA sequence 30 of a member of the library, CJRA05, see FIG. 16. Table is to be read as follows: on each line everything

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- 57 that follows an exclamation mark ! is a comment. All occurrences of A, C, G, and T before !” are the DNA sequence. Case is used only to show that certain bases constitute special features, such as restriction sites, ribosome binding sites, and the like, which are labeled below the DNA. CJRA05 is a derivative of phage DY3F7, obtained by cloning an ApaLI to Notl fragment into these sites in DY3F31. DY3F31 is like DY3F7 except that the light chain and heavy chain genes have been replaced by stuffer DNA that does not code for any antibody. DY3F7 contains an antibody that binds streptavidin, but did not come from the present library.

The phage genes start with gene ii and continue with genes x, v, vii, ix, viii, iii, vi, i, and iv. Gene iii has been slightly modified in that eight codons have been inserted between the signal sequence and the mature protein and the final amino acids of the signal sequence have been altered. This allows restriction enzyme recognition sites Eagl and Xbal to be present. Following gene iv is the phage origin of replication (ori). After ori is bla which confers resistance to ampiciliin (ApR) . The phage genes and bla are transcribed in the same sense.

After bla, is the Fab cassette (illustrated in FIG. 17) comprising:

a)	PlacZ promoter,
b)	A first Ribosome Binding Site (RBS1),
c)	The signal sequence form M13 iii,
d)	An ApaLI RERS,
e)	A light chain (a kappa L20::JKl shortened by one
	codon at the V-J boundary in this case),
f)	An Ascl RERS,

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- 58 g) A second Ribosome Binding Site (RBS2),

h) A signal sequence, preferably PelB, which contains,

i) An Sfil RERS,

j) A synthetic 3-23 V region with diversity in CDR1 and CDR2,

k) A captured CDR3,

l) A partially synthetic J region (FR4 after BstEII),

m) CHI,

10	n)	A	Notl	RERS,
	o)	A	His6	tag,
	P)	A	cMyc	tag,

q) An amber codon,

r) An anchor DNA that encodes the same amino-acid 15 sequence as codons 273 to 424 of M13 iii (as shown in

Table 37).

	s)	Two stop codons,
	t)	An Avril RERS, and
	u)	A trp terminator.
20		The anchor (item r) encodes the same

amino-acid sequence as do codons 273 to 424 of M13 iii but the DNA is approximately as different as possible from the wild-type DNA sequence. In Table 36, the III' stump runs from base 8997 to base 9455. Below the

DNA, as comments, are the differences with wild-type iii for the comparable codons with !W.T at the ends of these lines. Note that Met and Trp have only a single codon and must be left as is. These AA types are rare. Ser codons can be changed at all three base, while Leu and Arg codons can be changed at two.

In most cases, one base change can be introduced per codon. This has three advantages: 1) recombination with the wild-type gene carried elsewhere

2018241075 03 Oct 2018

- 59 on the phage is less likely, 2) new restriction sites can be introduced, facilitating construction; and 3) sequencing primers that bind in only one of the two regions can be designed.

The fragment of M13 III shown in CJRA05 is the preferred length for the anchor segment.

Alternative longer or shorter anchor segments defined by reference to whole mature III protein may also be utilized.

The sequence of M13 III consists of the following elements: Signal Sequence:: Domain 1 (Dl)::Linker 1 (Ll)::Domain 2 (D2)::Linker 2 (L2):: Domain 3 (D3) : : Transmembrane Segment (TM) :: Intracellular anchor (IC) (see Table 38).

The pill anchor (also known as trpIII) preferably consists of D2::L2::D3::TM::IC. Another embodiment for the pill anchor consists of

D2'::L2::D3::TM::IC (where D2' comprises the last 21 residues of D2 with the first 109 residues deleted). A further embodiment of the pill anchor consists of

D2 ’ (C>S) ::L2::D3::TM::IC (where D2'(C>S) is D2’ with the single C converted to S), and d) D3::TM::IC.

Table 38 shows a gene fragment comprising the Notl site, His6 tag, cMyc tag, an amber codon, a recombinant enterokinase cleavage site, and the whole of mature M13 III protein. The DNA used to encode this sequence is intentionally very different from the DNA of wild-type gene iii as shown by the lines denoted W.T. containing the w.t. bases where these differ from this gene. Ill is divided into domains denoted domain 1, linker 1, domain 2, linker 2, domain

3, transmembrane segment, and intracellular anchor.

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- 60 Alternative preferred anchor segments (defined by reference to the sequence of Table 38) include :

codons 1-29 joined to codons 104-435, deleting 5 domain 1 and retaining linker 1 to the end;

codons 1-38 joined to codons 104-435, deleting domain land retaining the rEK cleavage site plus linker 1 to the end from III;

codons 1-29 joined to codons 236-435, deleting 10 domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end;

codons 1-38 joined to codons 236-435, deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end and the rEK cleavage site;

codons 1-29 joined to codons 236-435 and changing codon 240 to Ser (e.g., age), deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end; and codons 1-38 joined to codons 236-435 and changing codon 240 to Ser (e.g., age), deleting domain 1, linker 1, and most of domain 2 and retaining linker 2 to the end and the rEK cleavage site.

The constructs would most readily be made by methods similar to those of Wang and Wilkinson (Biotechniques 2001: 31(4)722-724) in which PCR is used to copy the vector except the part to be deleted and matching restriction sites are introduced or retained at either end of the part to be kept. Table 39 shows the oligonucleotides to be used in deleting parts of the III anchor segment. The DNA shown in Table 38 has an Nhel site before the DINDDRMA recombinant enterokinase cleavage site (rEKCS). If Nhel is used in the deletion process with this DNA, the rEKCS site

2018241075 03 Oct 2018

- 61 would be lost. This site could be quite useful in cleaving Fabs from the phage and might facilitate capture of very high-afffinity antibodies. One could mutagenize this sequence so that the Nhel site would follow the rEKCS site, an Ala Ser amino-acid sequence is already present. Alternatively, one could use SphI for the deletions. This would involve a slight change in amino acid sequence but would be of no consequence.

Example 7 : Selection of antigen binders from an 10 enriched library of human antibodies using phage vector

DY3F31.

In this example the human antibody library used is described in de Haard et al., (Journal of Biological Chemistry. 274 (26): 18218-30 (1999). This library, consisting of a large non-immune human Fab phagemid library, was first enriched on antigen, either on streptavidin or on phenyl-oxazolone (phOx). The methods for this are well known in the art. Two preselected Fab libraries, the first one selected once on immobilized phOx-BSA (Rl-ox) and the second one selected twice on streptavidin (R2-strep), were chosen for recloning.

These enriched repertoires of phage antibodies, in which only a very low percentage have binding activity to the antigen used in selection, were confirmed by screening clones in an ELISA for antigen binding. The' selected Fab genes were transferred from the phagemid vector of this library to the DY3F31 vector via ApaLl-Notl restriction sites.

DNA from the DY3F31 phage vector was pretreated with ATP dependent DNAse to remove

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- 62 chromosomal DNA and then digested with ApaLl and Notl. An extra digestion with Ascl was performed in between to prevent self-ligation of the vector. The ApaLl/Notl Fab fragment from the preselected libraries was subsequently ligated to the vector DNA and transformed into competent XLl-blue MRF' cells.

Libraries were made using vector: insert ratios of 1:2 for phOx-library and 1:3 for STREP library, and using 100 ng ligated DNA per 50 μΐ of electroporation-competent cells (electroporation conditions : one shock of 1700 V, 1 hour recovery of cells in rich SOC medium, plating on amplicillincontaining agar plates).

This transformation resulted in a library size of 1.6 x 10⁶ for Rl-ox in DY3F31 and 2.1 x 10⁶ for R2-strep in DY3F31. Sixteen colonies from each library were screened for insert, and all showed the correct size insert (±1400 bp) (for both libraries).

Phage was prepared from these Fab libraries as follows. A representative sample of the library was inoculated in medium with ampicillin and glucose, and at OD 0.5, the medium exchanged for ampicillin and 1 mM IPTG. After overnight growth at 37 °C, phage was harvested from the supernatant by PEG-NaCl precipitation. Phage was used for selection on antigen. Rl-ox was selected on phOx-BSA coated by passive adsorption onto immunotubes and R2-strep on streptavidin coated paramagnetic beads (Dynal, Norway), in procedures described in de Haard et. al. and Marks et. al., Journal of Molecular Biology. 222(3): 581-97 (1991). Phage titers and enrichments are given in

Table 40.

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- 63 Clones from these selected libraries, dubbed R2-ox and R3-strep respectively, were screened for binding to their antigens in ELISA. 44 clones from each selection were picked randomly and screened as phage or soluble Fab for binding in ELISA. For the libraries in DY3F31, clones were first grown in 2TY-2% glucose-50 pg/ml AMP to an OD600 of approximately 0.5, and then grown overnight in 2TY-50 pg/ml AMP +/- ImM IPTG. Induction with IPTG may result in the production of both phage-Fab and soluble Fab. Therefore the (same) clones were also grown without IPTG. Table 41 shows the results of an ELISA screening of the resulting supernatant, either for the detection of phage particles with antigen binding (Anti-M13 HRP = anti-phage antibody), or for the detection of human Fabs, be it on phage or as soluble fragments, either with using the anti-myc antibody 9E10 which detects the myc-tag that every Fab carries at the C-terminal end of the heavy chain followed by a HRP-labeled rabbit-anti-Mouse serum (column 9E10/RAM-HRP), or with anti-light chain reagent followed by a HRP-labeled goat-anti-rabbit antiserum(anti-CK/CL Gar-HRP) .

The results shows that in both cases antigen-binders are identified in the library, with as

Fabs on phage or with the anti-Fab reagents (Table 41). IPTG induction yields an increase in the number of positives. Also it can be seen that for the phOx-clones, the phage ELISA yields more positives than the soluble Fab ELISA, most likely due to the avid binding of phage. Twenty four of the ELISA-positive clones were screened using PCR of the Eab-insert from the vector, followed by digestion with BstNI. This yielded 17 different patterns for the phOx-binding

- 64 2018241075 03 Oct 2018

Fab's in 23 samples that were correctly analyzed, and 6 out of 24 for the streptavidin binding clones. Thus, the data from the selection and screening from this pre-enriched non-immune Fab library show that the

DY3F31 vector is suitable for display and selection of Fab fragments, and provides both soluble Fab and Fab on phage for screening experiments after selection.

Example 8: Selection of Phage-antibody libraries on streptavidin magnetic beads.

The following example describes a selection in which one first depletes a sample of the library of binders to streptavidin and optionally of binders to a non-target (i.e., a molecule other than the target that one does not want the selected Fab to bind). It is hypothesized that one has a molecule, termed a competitive ligand, which binds the target and that an antibody which binds at the same site would be especially useful.

For this procedure Streptavidin Magnetic

Beads (Dynal) were blocked once with blocking solution (2% Marvel Milk, PBS (pH 7.4), 0.01% Tween-20 (2%MPBST)) for 60 minutes at room temperature and then washed five times with 2%MPBST. 450 pL of beads were blocked for each depletion and subsequent selection set.

Per selection, 6.25 pL of biotinylated depletion target (1 mg/mL stock in PBST) was added to 0.250 mL of washed, blocked beads (from step 1) . The target was allowed to bind overnight, with tumbling, at

4°C. The next day, the beads are washed 5 times with

PBST.

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- 65 Per selection, 0.010 mL of biotinylated target antigen (1 mg/mL stock in PBST) was added to 0.100 mL of blocked and washed beads (from step 1).

The antigen was allowed to bind overnight, with tumbling, at 4 °C. The next day, the beads were washed 5 times with PBST.

In round 1, 2 X 10¹² up to 10¹³ plaque forming units (pfu) per selection were blocked against non-specific binding by adding to 0.500 mL of 2%MPBS (=2%MPBST without Tween) for 1 hr at RT (tumble). In later rounds, 1011 pfu per selection were blocked as done in round 1.

Each phage pool was incubated with 50 pL of depletion target beads (final wash supernatant removed just before use) on a Labquake rotator for 10 min at room temperature. After incubation, the phage supernatant was removed and incubated with another 50 pL of depletion target beads. This was repeated 3 more times using depletion target beads and twice using blocked streptavidin beads for a total of 7 rounds of depletion, so each phage pool required 350 pL of depletion beads.

A small sample of each depleted library pool was taken for titering. Each library pool was added to

0.100 mL of target beads (final wash supernatant was removed just before use) and allowed to incubate for 2 hours at room temperature (tumble).

Beads were then washed as rapidly as possible (e.g.,3 minutes total) with 5 X 0.500 mL PBST and then

2X with PBS. Phage still bound to beads after the washing were eluted once with 0.250 mL of competitive ligand (~1 ppM) in PBST for 1 hour at room temperature on a Labquake rotator. The eluate was removed, mixed

- 66 2018241075 03 Oct 2018 with 0.500 mL Minimal A salts solution and saved. For a second selection, 0.500 mL 100 mM TEA was used for elution for 10 min at RT, then neutralized in a mix of 0.250 mL of 1 M Tris, pH 7.4 + 0.500 mL Min A salts.

After the first selection elution, the beads can be eluted again with 0.300 mL of non-biotinylated target (1 mg/mL) for 1 hr at RT on a Labquake rotator. Eluted phage are added to 0.450 mL Minimal A salts.

Three eluates (competitor from 1st selection, 10 target from 1st selection and neutralized TEA elution from 2nd selection) were kept separate and a small aliquot taken from each for titering. 0.500 mL Minimal A salts were added to the remaining bead aliquots after competitor and target elution and after TEA elution.

Take a small aliquot from each was taken for tittering.

Each elution and each set of eluted beads was mixed with 2X YT and an aliquot (e.g., 1 mL with 1. E 10/mL) of XLl-Blue MRF’ E. coli cells (or other F' cell line) which had been chilled on ice after having been grown to mid-logarithmic phase, starved and concentrated (see procedure below - Mid-Log prep of XL-1 blue MRF' cells for infection).

After approximately 30 minutes at room temperature, the phage/cell mixtures were spread onto

Bio-Assay Dishes (243 X 243 X 18 mm, Nalge Nunc) containing 2XYT, ImM IPTG agar. The plates were incubated overnight at 30°C. The next day, each amplified phage culture was harvested from its respective plate. The plate was flooded with 35 mL TBS or LB, and cells were scraped from the plate. The resuspended cells were transferred to a centrifuge bottle. An additional 20 mL TBS or LB was used to remove any cells from the plate and pooled with the

2018241075 03 Oct 2018

- 67 cells in the centrifuge bottle. The cells were centrifuged out, and phage in the supernatant was recovered by PEG precipitation. Over the next day, the amplified phage preps were titered.

In the first round, two selections yielded five amplified eluates. These amplified eluates were panned for 2-3 more additional rounds of selection using ~1. E 12 input phage/round. For each additional round, the depletion and target beads were prepared the night before the round was initiated.

For the elution steps in subsequent rounds, all elutions up to the elution step from which the amplified elution came from were done, and the previous elutions were treated as washes. For the bead infection amplified·phage, for example, the competitive ligand and target elutions were done and then tossed as washes (see below). Then the beads were used to infect E. coli. Two pools, therefore, yielded a total of 5 final elutions at the end of the selection.

1st selection set

A. Ligand amplified elution: elute w/ ligand for 1 hr, keep as elution

B. Target amplified elution: elute w/ ligand for 1 hr, toss as wash elute w/ target for 1 hr, keep as elution

C. Bead infect, amp. elution: elute w/ ligand for 1 hr, toss as wash elute w/ target for 1 hr, toss as wash elute w/ cell infection, keep as elution

- 68 2018241075 03 Oct 2018

2nd selection set

A. TEA amplified elution; elute w/ TEA lOmin, keep as elution

B. Bead infect, amp. elution; elute w/

TEA lOmin, toss as wash elute w/ cell infection, keep as elution

Mid-log prep of XLl blue MRF' cells for infection (based on Barbas et al. Phage Display manual procedure)

Culture XLl blue MRF' in NZCYM (12.5 mg/mL tet) at 37°C and 250 rpm overnight. Started a 500 mL culture in 2 liter flask by diluting cells 1/50 in NZCYM/tet (10 mL overnight culture added) and incubated at 37°C at 250 rpm until OD600 of 0.45 (1.5-2 hrs) was reached. Shaking was reduced to 100 rpm for 10 min.

When OD600 reached between 0.55-0.65, cells were transferred to 2 x 250 mL centrifuge bottles, centrifuged at 600 g for 15 min at 4°C. Supernatant was poured off. Residual liquid was removed with a pipette.

The pellets were gently resuspended (not pipetting up and down) in the original volume of 1 X Minimal A salts at room temp. The resuspended cells were transferred back into 2-liter flask, shaken at 100 rpm for 45 min at 37°C. This process was performed in order to starve the cells and restore pili. The cells were transferred to 2 x 250 mL centrifuge bottles, and centrifuged as earlier.

The cells were gently resuspended in ice cold Minimal A salts (5 mL per 500 mL original culture).

2018241075 03 Oct 2018

- 69 The cells were put on ice for use in infections as soon as possible.

The phage eluates were brought up to 7.5 mL with 2XYT medium and 2.5 mL of cells were added. Beads were brought up to 3 mL with 2XYT and 1 mL of cells were added. Incubated at 37oC for 30 min. The cells were plated on 2XYT, 1 mM IPTG agar large NUNC plates and incubated for 18 hr at 30°C.

Example 9: Incorporation of synthetic region in FR1/3 10 region.

Described below are examples for incorporating of fixed residues in antibody sequences for light chain kappa and lambda genes, and for heavy chains. The experimental conditions and oligonucleotides used for the examples below have been described in previous examples (e.g., Examples 3 & 4).

The process for incorporating fixed FR1 residues in an antibody lambda sequence consists of 3 steps (see FIG. 18): (1) annealing of single-stranded

DNA material encoding VL genes to a partially complementary oligonucleotide mix (indicated with Ext and Bridge) , to anneal in this example to the region encoding residues 5-7 of the FR1 of the lambda genes (indicated with X..X; within the lambda genes the overlap may sometimes not be perfect); (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VL gene. In this process the first few residues of all lambda genes will be encoded by the sequences present in the oligonucleotides (Ext., Bridge

2018241075 03 Oct 2018

- 70 or PCRpr). After the PCR, the lambda genes can be cloned using the indicated restriction site for ApaLI.

The process for incorporating fixed FRl residues in an antibody kappa sequence (FIG. 19) consists of 3 steps : (1) annealing of single-stranded

DNA material encoding VK genes to a partially complementary oligonucleotide mix (indicated with Ext and Bri), to anneal in this example to the region encoding residues 8-10 of the FRl of the kappa genes (indicated with X..X; within the kappa genes the overlap may sometimes not be perfect) ; (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VK gene. In this process the first few (8) residues of all kappa genes will be encode by the sequences present in the oligonucleotides (Ext., Bridge or PCRpr.). After the PCR, the kappa genes can be cloned using the indicated restriction site for ApaLI.

The process of incorporating fixed FR3 residues in a antibody heavy chain sequence (FIG. 20) consists of 3 steps : (1) annealing of single-stranded DNA material encoding part of the VH genes (for example encoding FR3, CDR3 and FR4 regions) to a partially complementary oligonucleotide mix (indicated with Ext and Bridge), to anneal in this example to the region encoding residues 92-94 (within the FR3 region) of VH genes (indicated with X..X; within the VH genes the overlap may sometimes not be perfect); (2) ligation of this complex; (3) PCR of the ligated material with the indicated primer ('PCRpr') and for example one primer based within the VH gene (such as in the FR4 region).

In this process certain residues of all VH genes will be encoded by the sequences present in the

2018241075 03 Oct 2018

- 71 oligonucleotides used here, in particular from PCRpr (for residues 70-73), or from Ext/Bridge oligonucleotides (residues 74-91). After the PCR, the partial VH genes can be cloned using the indicated restriction site for Xbal.

It will be understood that the foregoing is only illustrative of the principles of this invention and that various modifications can be made by those skilled in the art without departing from the scope of and sprit of the invention.

The term comprise and variants of the term such as comprises or comprising are used herein to denote the inclusion of a stated integer or stated integers but not to exclude any other integer or any other integers, ¹⁵ unless in the context or usage an exclusive interpretation of the term is required.

Any reference to publications cited in this specification is not an admission that the disclosures constitute common general knowledge in Australia.

- 72 2018241075 03 Oct 2018

Table 1

.: Human

GLG

FR3 sequences

! VH1

! 66

67

68

69

70 71

72

73

74

75

76

77

78

79

80

agg

gtc

acc

atg

acc agg

gac

aeg

tee

ate

age

aca

gee

tac

atg

! 81

82

82a

82b

82c 83

84

85

86

87

88

89

90

91

92

gag

ctg

age

agg

ctg aga

tet

gac

gac

aeg

gee

gtg

tat

tac

tgt

! 93

94

95

gcg

aga

ga '

! 1-

02# 1

aga

gtc

acc

att

acc agg

gac

aca

tee

gcg

age

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gaa

gac

aeg

get

gtg

tat

tac

tgt

gcg

aga

ga !

! 1-

03# 2

aga

gtc

acc

atg

acc agg

aac

acc

tee

ata

age

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

gg !

! 1-

08# 3

aga

gtc

acc

atg

acc aca

gac

aca

tee

aeg

age

aca

gee

tac

atg

gag

ctg

agg

age

ctg aga

tet

gac

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

ga )

! 1-

18# 4

aga

gtc

acc

atg

acc gag

gac

aca

tet

aca

gac

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aeg

gee

gtg

tat

tac

tgt

gca

aca

ga :

! 1-

24# 5

aga

gtc

acc

att

acc agg

gac

agg

tet

atg

age

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aca

gee

atg

tat

tac

tgt

gca

aga

ta :

! 1-

45# 6

aga

gtc

acc

atg

acc agg

gac

aeg

tee

aeg

age

aca

gtc

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

ga :

! 1-

46# 7

aga

gtc

acc

att

acc agg

gac

atg

tee

aca

age

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tee

gag

gac

aeg

gee

gtg

tat

tac

tgt

gcg

gca

ga '

! 1-

58# 8

aga

gtc

aeg

att

acc gcg

gac

gaa

tee

aeg

age

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

ga

! 1-

69# 9

aga

gtc

aeg

att

acc gcg

gac

aaa

tee

aeg

age

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

ga

! 1-

e# 10

aga

gtc

acc

ata

acc gcg

gac

aeg

tet

aca

gac

aca

gee

tac

atg

gag

ctg

age

age

ctg aga

tet

gag

gac

aeg

gee

gtg

tat

tac

tgt

gca

aca

ga !

! 1-

f# 11

- 73 2018241075 03 Oct 2018 ! VH2

agg

etc

acc

ate acc aag

gac

acc

tee

aaa

aac

cag

gtg

gtc

ett

aca

atg

acc

aac atg gac

cct

gtg

gac

aca

gee

aca

tat

tac

tgt

gca

cac

aga

c! 2-05# 12

5

agg

etc

acc

ate tee aag

gac

acc

tee

aaa

age

cag

gtg

gtc

ett

acc

atg

acc

aac atg gac

cct

gtg

gac

aca

gee

aca

tat

tac

tgt

gca

egg

ata

c! 2-26# 13

agg

etc

acc

ate tee aag

gac

acc

tee

aaa

aac

cag

gtg

gtc

ett

aca

atg

acc

aac atg gac

cct

gtg

gac

aca

gee

aeg

tat

tac

tgt

10

gca

egg

ata

c! 2-70# 14

! VH3

cga

ttc

acc

ate tee aga

gac

aac

gee

aag

aac

tea

ctg

tat

ctg

caa

atg

aac

age ctg aga

gee

gag

gac

aeg

get

gtg

tat

tac

tgt

gcg

aga

ga !

i 3-07# 15

15

cga

ttc

acc

ate tee aga

gac

aac

gee

aag

aac

tee

ctg

tat

ctg

caa

atg

aac

agt ctg aga

get

gag

gac

aeg

gee.

ttg

tat

tac

tgt

gca

aaa

gat

a! 3-09#16

cga

ttc

acc

ate tee agg

gac

aac

gee

aag

aac

tea

ctg

tat

ctg

caa

atg

aac

age ctg aga

gee

gag

gac

aeg

gee

gtg

tat

tac

tgt

20

gcg

aga

ga !

! 3-11# 17

cga

ttc

acc

ate tee aga

gaa

aat

gee

aag

aac

tee

ttg

tat

ett

caa

atg

aac

age ctg aga

gee

ggg

gac

aeg

get

gtg

tat

tac

tgt

gca

aga

ga !

! 3-13# 18

aga

ttc

acc

ate tea aga

gat

gat

tea

aaa

aac

aeg

ctg

tat

ctg

25

caa

atg

aac

age ctg aaa

acc

gag

gac

aca

gee

gtg

tat

tac

tgt

acc

aca

ga !

1 3-15# 19

cga

ttc

acc

ate tee aga

gac

aac

gee

aag

aac

tee

ctg

tat

ctg

caa

atg

aac

agt ctg aga

gee

gag

gac

aeg

gee

ttg

tat

cac

tgt

gcg

aga

ga !

! 3-20# 20

30

cga

ttc

acc

ate tee aga

gac

aac

gee

aag

aac

tea

ctg

tat

ctg

caa

atg

aac

age ctg aga

gee

gag

gac

aeg

get

gtg

tat

tac

tgt

gcg

aga

ga )

! 3-21# 21

egg

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

tat

ctg

caa

atg

aac

age ctg aga

gee

gag

gac

aeg

gee

gta

tat

tac

tgt

35

gcg

aaa

ga :

! 3-23# 22

cga

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

tat

ctg

caa

atg

aac

age ctg aga

get

gag

gac

aeg

get

gtg

tat

tac

tgt

gcg

aaa

ga i

! 3-30# 23

cga

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

tat

ctg

40

caa

atg

aac

age ctg aga

get

gag

gac

aeg

get

gtg

tat

tac

tgt

gcg

aga

ga

! 3303# 24

- 74 2018241075 03 Oct 2018

cga caa gcg cga

ttc acc ate tee aga atg aac age ctg aga

gac aat

tee gac tee

aag aeg aag

aac get aac

aeg gtg aeg

ctg tat ctg

tat tac tat

ctg tgt ctg

get gac

gag aat

aaa ttc

ga ! acc

3305# 25

ate tee aga

5

caa gcg

atg aga

aac ga !

age ctg aga 3-33# 26

gee

gag

gac

aeg

get

gtg

tat

tac

tgt

cga

ttc

acc

ate tee aga

gac

aac

age

aaa

aac

tee

ctg

tat

ctg

caa gca

atg aaa

aac gat

agt ctg aga a! 3-43#27

act

gag

gac

acc

gee

ttg

tat

tac

tgt

10

cga

ttc

acc

ate tee aga

gac

aat

gee

aag

aac

tea

ctg

tat

ctg

caa gcg

atg aga

aac ga !

age ctg aga 3-48# 28

gac

gag

gac

aeg

get

gtg

tat

tac

tgt

aga

ttc

acc

ate tea aga

gat

ggt

tee

aaa

age

ate

gee

tat

ctg

15

caa act

atg aga

aac ga !

age ctg aaa 3-49# 29

acc

gag

gac

aca

gee

gtg

tat

tac

tgt

cga

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

tat

ett

caa gcg

atg aga

aac ga !

age ctg aga . 3-53# 30

gee

gag

gac

aeg

gee

gtg

tat

tac

tgt

aga

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

tat

ett

20

caa gcg

atg aga

ggc ga !

age ctg aga i 3-64# 31

get

gag

gac

atg

get

gtg

tat

tac

tgt

aga

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

tat

ett

caa gcg

atg aga

aac ga !

age ctg aga ! 3-66# 32

get

gag

gac

aeg

get

gtg

tat

tac

tgt

25

aga

ttc

acc

ate tea aga

gat

gat

tea

aag

aac

tea

ctg

tat

ctg

caa get

atg aga

aac ga !

age ctg aaa ! 3-72# 33

acc

gag

gac

aeg

gee

gtg

tat

tac

tgt

agg

ttc

acc

ate tee aga

gat

gat

tea

aag

aac

aeg

gcg

tat

ctg

30

caa act

atg aga

aac ca !

age ctg aaa ! 3-73# 34

acc

gag

gac

aeg

gee

gtg

tat

tac

tgt

cga

ttc

acc

ate tee aga

gac

aac

gee

aag

aac

aeg

ctg

tat

ctg

caa gca

atg aga

aac ga :

agt ctg aga ! 3-74# 35

gee

gag

gac

aeg

get

gtg

tat

tac

tgt

aga

ttc

acc

ate tee aga

gac

aat

tee

aag

aac

aeg

ctg

cat

ett

35

caa aag ! VH4

atg aaa

aac ga

age ctg aga ! 3-d# 36

get

gag

gac

aeg

get

gtg

tat

tac

tgt

cga

gtc

acc

ata tea gta

gac

aag

tee

aag

aac

cag

ttc

tee

ctg

40

aag gcg

ctg aga

age ga

tet gtg acc ! 4-04# 37

gee

gcg

gac

aeg

gee

gtg

tat

tac

tgt

cga

gtc

acc

atg tea gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

- 75 2018241075 03 Oct 2018

aag

ctg

age

tet gtg acc

gee

gtg

gac

aeg

gcc

gtg

tat

tac

tgt

gcg

aga

aa

! 4-28# 38

cga

gtt

acc

ata tea gta

gac

aeg

tet

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg act

gcc

gcg

gac

aeg

gee

gtg

tat

tac

tgt

5

gcg

aga

ga

! 4301# 39

cga

gtc

acc

ata tea gta

gac

agg

tee

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg acc

gcc

gcg

gac

aeg

gcc

gtg

tat

tac

tgt

gcc

aga

ga

ί 4302# 40

cga

gtt

acc

ata tea gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

10

aag

ctg

age

tet gtg act

gcc

gca

gac

aeg

gcc

gtg

tat

tac

tgt

gcc

aga

ga

! 4304# 41

cga

gtt

acc

ata tea gta

gac

aeg

tet

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg act

gcc

gcg

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

ga

! 4-31# 42

15

cga

gtc

acc

ata tea gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg acc

gcc

gcg

gac

aeg

get

gtg

tat

tac

tgt

gcg

aga

ga

! 4-34# 43

cga

gtc

acc

ata tee gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg acc

gcc

gca

gac

aeg

get

gtg

tat

tac

tgt

20

gcg

aga

ca

! 4-39# 44

cga

gtc

acc

ata tea gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg acc

get

gcg

gac

aeg

gcc

gtg

tat

tac

tgt

gcg

aga

ga

! 4-59# 45

cga

gtc

acc

ata tea gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

25

aag

ctg

age

tet gtg acc

get

gcg

gac

aeg

gcc

gtg

tat

tac

tgt

gcg

aga

ga

! 4-61# 46

cga

gtc

acc

ata tea gta

gac

aeg

tee

aag

aac

cag

ttc

tee

ctg

aag

ctg

age

tet gtg acc

gcc

gca

gac

aeg

gee

gtg

tat

tac

tgt

gcg

aga

ga

! 4-b# 47

30

! VH5

cag

gtc

acc

ate tea gcc

gac

aag

tee

ate

age

acc

gcc

tac

ctg

cag

tgg

age

age ctg aag

gcc

teg

gac

acc

gcc

atg

tat

tac

tgt

gcg

aga

ca

! 5-51# 48

cac

gtc

acc

ate tea get

gac

aag

tee

ate

age

act

gcc

tac

ctg

35

cag

tgg

age

age ctg aag

gcc

teg

gac

acc

gcc

atg

tat

tac

tgt

gcg

aga

1 i

5-a# 49

! VH6

cga

ata

acc

ate aac cca

gac

aca

tee

aag

aac

cag

ttc

tee

ctg

cag

ctg

aac

tet gtg act

ccc

gag

gac

aeg

get

gtg

tat

tac

tgt

40

gca

aga

ga

! 6-1# 50

! VH7

2018241075 03 Oct 2018

egg

ttt

gtc

ttc

tee

ttg

gac

ace

tet

gtc

age

aeg

gca

tat

ctg

cag

ate

tgc

age

eta

aag

get

gag

gac

act

gee

gtg

tat

tac

tgt

geg

aga

ga !

! 74

.1#

51

- 77 2018241075 03 Oct 2018 _

BstEII Ggtnacc 2

Table 2: Enzymes that either cut 15 or more human GLGs or have 5+-base recognition in FR3 Typical entry:

REname Recognition #sites

GLGid#:base# GLGid#:base# GLGid#:base#.....

	1: The:	3 re	48: are 2	3	3
hits	at	base#
10	Maelll	gtnac					36
	1:	4	2:	4	3:	4	4 :	4	5:	4	6:	4
	7:	4	8:	4	9:	4	10:	4	11:	4	37:	4
	37:	58	38:	4	38:	58	39:	4	39:	58	40:	4
	40:	58	41:	4	41:	58	42:	4	42:	58	43:	4
15	43:	58	44:	4	44 :	58	45:	4	45:	58	46:	4
	46:	58	47:	4	47:	58	48:	4	49:	4	50:	58
	There	are 24	hits	at	base#	4
	Tsp45I	gtsac					33
20	1:	4	2:	4	3:	4	4 :	4	5:	4	6:	4
	7:	4	8:	4	9:	4	10:	4	11:	4	37:	4
	37:	58	38:	4	38:	58	39:	58	40:	4	40:	58
	41:	58	42:	58	43:	4	43:	58	44 :	4	44 :	58
	45:	4	45:	58	46:	4	46:	58	47:	4	47:	58
25	48:	4	49:	4	50:	58

There are 21 hits at base# 4

HphI tcacc 45

	1:	5	2:	5	3:	5	4 :	5	5:	5	6:	5
30	7 :	5	8:	5	11:	5	12:	5	12:	11	13:	5
	14 :	5	15:	5	16:	5	17:	5	18:	5	19:	5
	20:	5	21:	5	22:	5	23:	5	24 :	5	25:	5
	26:	5	27:	5	28:	5	29:	5	30:	5	31:	5
	32:	5	33:	5	34:	5	35:	5	36:	5	37:	5
35	38:	5	40:	5	43:	5	44:	5	45:	5	46:	5
	47:	5	48:	5	49:	5
	There	are 44	hits	at	base#	5

- 78 2018241075 03 Oct 2018

Nlal	II	CATG					26
1:	9	1	: 42	2:	42	3:	9	3:	42	4 :	9
4 :	42	5	: 9	5:	42	6:	42	6:	78	7 :	9
7:	42	8	: 21	8:	42	9:	42	10:	42	11:	42
12:	57	13	: 48	13:	57	14:	57	31:	72	38:	9
48:	78	49	: 78
The	re	are	11 hits	at	base#	42

There are 1 hits at base# 48 Could cause raggedness

BsaJI Ccnngg 37

1:	14	2:	14	5:	14	6:	14	7:	14	8:	14
8:	65	9:	14	10:	14	11:	14	12:	14	13:	14
14 :	14	15:	65	17 :	14	17:	65	18:	65	19:	65
20:	65	21:	65	22:	65	26:	65	29:	65	30:	65
33:	65	34 :	65	35:	65	37:	65	38:	65	39:	65
40:	65	42:	65	43:	65	48:	65	49:	65	50:	65

51: 14

There are 23 hits at base# 65

There are 14 hits at base# 14

Alul AGct 42

1:	47	2:	47	3:	47	4 :	47	5:	47	6:	47
7:	47	8:	47	9:	47	10:	47	11:	47	16:	63
23:	63	24 :	63	25:	63	31:	63	32:	63	36:	63
37:	47	37:	52	38:	47	38:	52	39:	47	39:	52
40:	47	40:	52	41:	47	41:	52	42:	47	42 :	52
43:	47	43:	52	44 :	47	44:	52	45:	47	45:	52
46:	47	46:	52	47:	47	47:	52	49:	15	50:	47

There are 23 hits at base# 47

There are 11 hits at base# 52 Only 5 bases from 47

BlpI GCtnagc 21

	1:	48	2:	48	3:	48	5:	48	6:	48	7:	48
	8:	48	9:	48	10:	48	11:	48	37:	48	38:	48
35	39:	48	40:	48	41:	48	42:	48	43:	48	44:	48
	45:	48	46:	48	47:	48

There are 21 hits at base# 48

2018241075 03 Oct 2018

- 79 Mwol GCNNNNNnngc 19

	1:	48	2:	28	19:	36	22:	36	23:	36	24 :	36
	25:	36	26:	36	35:	36	37:	67	39:	67	40:	67
	41:	67	42:	67	43:	67	44:	67	45:	67	46:	67
5	47:	67
	There are	10	hits	at	base#	67
	There are	7	hits	at	base#	36
	Ddel	Ctnag					71
10	1:	49	1:	58	2:	49	2:	58	3:	49	3:	58
	3:	65	4 :	49	4 :	58	5:	49	5:	58	5:	65
	6:	49	6:	58	6:	65	7:	49	7:	58	7:	65
	8:	49	8 :	58	9:	49	9:	58	9:	65	10:	49
	10:	58	10:	65	11:	49	11:	58	11:	65	15:	58
15	16:	58	16:	65	17:	58	18:	58	20:	58	21:	58
	22:	58	23:	58	23:	65	24:	58	24 :	65	25:	58
	25:	65	26:	58	27:	58	27:	65	28:	58	30:	58
	31:	58	31:	65	32:	58	32:	65	35:	58	36:	58
	36:	65	37:	49	38:	49	39:	26	39:	49	40:	49
20	41:	49	42:	26	42:	49	43:	49	44 :	49	45:	49
	46:	49	47 :	49	48:	12	49:	12	51:	65
	There are	29	hits	at	basei	58
	There are	22	hits	at	basei	49	Only	nine base from 58

There are 16 hits at base# 65 Only seven bases from 58

BglXI Agatct 11

1: 61 7: 61	2: 9:	61 61	3: 10:	61 61	4: 11:	61 61	5: 51:	61 47	6:	61
There are	10	hits	at	base#	61
BstYI Rgatcy					12
1: 61	2:	61	3:	61	4:	61	5:	61	6:	61
7: 61	8:	61	9:	61	10:	61	11:	61	51:	47
There are	11	hits	at	base#	61

- 80 2018241075 03 Oct 2018

	Hpyl88I	TCNga 2: 8: 27:	17
1: 7: 20:	64 64 57	64 64 57	3: 9: 35:	64 64 57	4: 64 10: 64 48: 67	5 : 11: 49:	64 64 67	6: 16:	64 57
5	There are 11	hits	at	base#	64
	There are 4	hits	at	base#	57
	There are 2	hits	at	base#	67 Could be	ragged.
	MslI	CAYNNnnRTG			44
10	1:	72	2:	72	3:	72	4: 72	5:	72	6:	72
	7:	72	8:	72	9:	72	10: 72	11:	72	15:	72
	17:	72	18:	72	19:	72	21: 72	23:	72	24:	72
	25:	72	26:	72	28:	72	29: 72	30:	72	31:	72
	32:	72	33:	72	34:	72	35: 72	36:	72	37:	72
15	38:	72	39:	72	40:	72	41: 72	42:	72	43:	72
	44:	72	45:	72	46:	72	47: 72	48:	72	49:	72
	50:	72	51:	72
	There are 44	hits	at	base#	72
20	BsiEI CGRYcg				23
	1:	74	3:	74	4:	74	5: 74	7 :	74	8:	74
	9:	74	10:	74	11:	74	17: 74	22:	74	30:	74
	33:	74	34:	74	37:	74	38: 74	39:	74	40:	74
	41:	74	42:	74	45:	74	46: 74	47:	74
25	There are 23	t hits	ι at	base#	< 74
	Eael	Yggccr				23
	1:	74	3:	74	4:	74	5: 74	7:	74	8:	74
	9:	74	10:	74	11:	74	17: 74	22:	74	30:	74
30	33:	74	34:	74	37:	74	38: 74	39:	74	40:	74
	41:	74	42:	74	45:	74	46: 74	47:	74
	There i	are 23 hits	ι at	base#	74
	Eagl	Cggccg				23
35	1:	74	3:	74	4:	74	5: 74	7:	74	8:	74
	9:	74	10:	74	11:	74	17: 74	22:	74	30:	74
	33:	74	34:	74	37:	74	38: 74	39:	74	40:	74
	41:	74	42:	74	45:	74	46: 74	47:	74

There are 23 hits at base# 74

- 81 2018241075 03 Oct 2018

Haelll GGcc

	1:	75	3:	75	4 :	75	5:	75	7 :	75	8:	75
	9:	75	10:	75	11:	75	16:	75	17:	75	20:	75
5	22:	75	30:	75	33:	75	34 :	75	37:	75	38:	75
	39:	75	40:	75	41:	75	42 :	75	45:	75	46:	75
	47 :	75	48 :	63	49:	63
	There are 25 hits	i at	base# 75
10	Bst4CI ACNgt	65°C		63	Sites There	is a	third is
	1:	86	2:	86	3:	86	4:	86	5:	86	6:	86
	7:	34	7:	86	8:	86	9:	86	10:	86	11:	86
	12:	86	13:	86	14:	86	15:	36	15:	86	16:	53
	16:	86	17:	36	17:	86	18:	86	19:	86	20:	53
15	20:	86	21:	36	21:	86	22:	0	22:	86	23:	86
	24:	86	25:	86	26:	86	27:	53	27:	86	28:	36
	28:	86	29:	86	30:	86	31:	86	32:	: 86	33:	36
	33:	86	34:	86	35:	53	35:	86	36:	: 86	37:	86
	38:	86	39:	86	40:	86	41:	86	42:	: 86	43:	86
20	44:	86	45:	86	46:	86	47:	86	48:	: 86	49:	86
	50:	86	51:	0	51:	86
	There are 51 hits at	base# 86	All	the	other	sites ;
	HpyCH4III	ACNgt				63
25	1:	86	2:	86	3:	86	4:	86	5:	: 86	6:	86
	7:	34	7:	86	8:	86	9:	86	10:	: 86	11:	86
	12:	86	13:	86	14:	86	15:	36	15:	: 86	16:	53
	16:	86	17:	36	17:	86	18:	86	19:	: 86	20:	53
	20:	86	21:	36	21:	86	22:	0	22:	: 86	23:	86
30	24:	86	25:	86	26:	86	27:	53	27:	: 86	28:	36
	28:	86	29:	86	30:	86	31:	86	32:	: 86	33:	36
	33:	86	34:	86	35:	53	35:	86	36:	: 86	37:	86
	38:	86	39:	86	40:	86	41:	86	42:	: 86	43:	86
	44:	86	45:	86	46:	86	47:	86	48:	: 86	49:	86
35	50:	86	51:	0	51:	86

There are 51 hits at base# 86

- 82 2018241075 03 Oct 2018

Hinfl Gantc 43

2:	2	3:	2	4:	2	5:	2	6:	2	7:	2
8:	2	9:	2	9:	22	10:	2	11:	2	15:	2
16:	2	17:	2	18:	2	19:	2	19:	22	20:	2
21:	2	23:	2	24:	2	25:	2	26:	2	27:	2
28:	2	29:	2	30:	2	31:	2	32:	2	33:	2
33:	22	34:	22	35:	2	36:	2	37:	2	38:	2
40:	2	43:	2	44:	2	45:	2	46:	2	47:	2

50: 60

There are 38 hits at base# 2

	Mlyl GAGTCNNNNNn				18
	2: 2		3	:	2	4 :	2	5:	2	6:	2	7 :	2
	8: 2		9	:	2	10:	2	11:	2	37:	2	38 :	2
15	40: 2		43	:	2	44:	2	45:	2	4 6:	2	47 :	2
	There	are	18	hits	at	base#	2
	Plel gagtc						18
	2: 2		3	:	2	4:	2	5:	2	6:	2	7:	2
20	8: 2		9	:	2	10:	2	11:	2	37:	2	38:	2
	40: 2		43	:	2	44:	2	45:	2	46:	2	47:	2
	There	are	18	hits	at	base#	2
	Acil Ccgc							24
	2: 26		9	:	14	10:	14	11:	14	27:	74	37:	62
25	37: 65		38		62	39:	65	40:	62	40:	65	41:	65
	42: 65		43	.	62	43:	65	44 :	62	44:	65	45:	62
	46: 62		47	.	62	47:	65	48:	35	48:	74	49:	74
	There	are		8	hits	at	base#	62
	There	are		8	hits	at	base#	65
30	There	are		3	hits	at	base#	14
	There	are		3	hits	at	base#	74
	There	are		1	hits	at	base#	26
	There	are		1	hits	at	base#	35
	Gcgg							11
35	8: 91		9	:	16	10:	16	11:	16	37:	67	39:	67
	40: 67		42	:	67	43:	67	45:	67	46:	67
	There	are		7	hits	at	base#	67
	There	are		3	hits	at	base#	16
	There	are		1	hits	at	base#	91

- 83 2018241075 03 Oct 2018

BsiHKAI GWGCWc	6: 14 : 42: at	20	10: 39: 45:	30 51 51
2: 12: 5 40: 4 6: Ther	30 89 51 51 e	4 13 41 47 are	: 30 : 89 : 51 : 51 11 hits	30 89 51 base#	7 : 37: 43: 51	30 51 51	9: 38: 44 :	30 51 51
Bspl286I GDGCHc				20
0 2:	30	4	: 30	6:	30	7:	30	9:	30	10:	30
12:	89	13	: 89	14 :	89	37:	51	38:	51	39:	51
40:	51	41	: 51	42:	51	43:	51	44 :	51	45:	51
46:	51	47	: 51
There . ζ	are	11 hits	at	base#	51
HgiAI	: GWGCWc					20
2:	30	4	: 30	6:	30	7 :	30	9:	30	10:	30
12:	89	13	: 89	14 :	89	37 :	51	38 :	51	39:	51
40:	51	41	: 51	42 :	51	43:	51	44 :	51	45:	51
0 46:	51	47	: 51
There .	are	11 hits	at	base#	51
BsoFI	: GCngc					26
2:	53	3	: 53	5:	53	6:	53	7:	53	8:	53
5 8:	91	9	: 53	10:	53	11:	53	31:	53	36:	36
37:	64	39	: 64	40:	64	41:	64	42:	64	43:	64
44:	64	45	: 64	46:	64	47:	64	48:	53	49:	53
50:	45	51	: 53
There	are	13 hits	at	base#	53
0 There	are	10 hits	at	base#	64
Tsel	Gcwgc					17
2:	53	3	: 53	5:	53	6:	53	7:	53	8:	53
9:	53	10	: 53	11:	53	31:	53	36:	36	45:	64
46:	64	48	: 53	49:	53	50:	45	51:	53
;5 There	are	13 hits	; at	base#	53

2018241075 03 Oct 2018

Mnll gagg

	3:	67 3:	: 95	4:	51	5:	16	5:	67	6:	67
	7:	67 8:	: 67	9:	67	10:	67	11:	67	15:	67
	16:	67 17:	: 67	19:	67	20:	67	21:	67	22:	67
5	23:	67 24:	: 67	25:	67	26:	67	27:	67	28:	67
	29:	67 -30:	: 67	31:	67	32:	67	33:	67	34:	67
	35:	67 36	: 67	50:	67	51:	67
	There are 31 hits	at	base#	67
10	HpyCH4V TGca				34
	5:	90 6	: 90	11:	90	12:	90	13:	90	14:	90
	15:	44 16	: 44	16:	90	17:	44	18:	90	19:	44
	20:	44 21	: 44	22:	44	23:	44	24:	44	25 :	44
	26:	44 27	: 44	27:	90	28:	44	29:	44	33:	44
15	34:	44 35	: 44	35:	90	36:	38	48:	44	49:	44
	50:	44 50	: 90	51:	44	51:	52
	There are 21 hits	. at	base#	44
	There are	1 hits	at	base#	: 52
20	AccI	GTmkac					13	5-base recognition
	7:	37 11	: 24	37:	16	38:	16	39:	16	40:	16
	41:	16 42	: 16	43:	16	44:	16	45:	16	46:	16
	47:	16
	There are	11 hits	: at	base#	ί 16
25
	SaclI CCGCgg					8	6-base :	recognitior
	9:	14 10	: 14	11:	14	37:	65	39:	65	40:	65
	42:	65 43	: 65
	There are	5 hits	: at	base# 65
30	There are	3 hits	: at	base# 14
	Tfil	Gawtc					24
	9:	22 15	: 2	16:	2	17:	2	18:	2	19:	2
	19:	22 20	: 2	21:	2	23:	2	24:	2	25:	2
35	26:	2 27	: 2	28:	2	29:	2	30:	2	31:	2
	32:	2 33	: 2	33:	22	34:	22	35:	2	36:	2
	There are	20 hits at	base# 2

- 85 2018241075 03 Oct 2018

BsmAI Nnnnnngagac 19

	15: 11 16 24: 11 25 30: 11 31	: 11 : 11 : 11	20: 26: 32:	11 11 11	21: 11 27: 11 35: 11	22: 28: 36:	11 11 11	23 : 28 : 44 :	11 56 87
5	48: 87
	There are	16 hits	at	base#	11
	Bpml ctccag				19
	15: 12 16	: 12	17:	12	18 : 12	20:	12	21:	12
10	22: 12 23	: 12	24:	12	25: 12	26:	12	27:	12
	28: 12 30	: 12	31:	12	32: 12	34 :	12	35:	12
	36: 12
	There are	19 hits	at	base#	12
15	XmnI GAANNnnttc			12
	37: 30 38	: 30	39:	30	40: 30	41:	30	42 :	30
	43: 30 44	: 30	45:	30	46: 30	47:	30	50:	30
	There are	12 hits	at	base#	30
20	BsrI NCcagt				12
	37: 32 38	: 32	39:	32	40: 32	41:	32	42:	32
	43: 32 44	: 32	45:	32	46: 32	47:	32	50:	32
	There are	12 hits	at	base#	32
25	Banll GRGCYc				11
	37: 51 38	: 51	39:	51	40: 51	41:	51	42:	51
	43: 51 44	: 51	45:	51	46: 51	47:	51
	There are	11 hits	at	base#	51
30	EC1136I GAGctc			11
	37: 51 38	: 51	39:	51	40: 51	41:	51	42:	51
	43: 51 44	: 51	45:	51	46: 51	47:	51
	There are	11 hits	: at	base#	51
35	Sacl GAGCTc				11
	37: 51 38	: 51	39:	51	40: 51	41:	51	42:	51
	43: 51 44	: 51	45:	51	46: 51	47:	51

There are 11 hits at base# 51

2018241075 03 Oct 2018

Table 3: Synthetic 3-23 FR3 of human heavy chains showning positions of possible cleavage sites ! Sites engineered into the synthetic gene are shown in upper case DNA

	! with the RE name	between vertical bars	(as	in	I Xbal	1 ) -
5	! RERSs frequently	found in GLGs are shown below	the synthetic
	sequence
	! with the name to	the right (as in gtn	ac=MaeIII(24),	indicating
	that
	! 24 of the 51 GLGs	contain the site).
10	1
	1				1 —	FR3-	—
	1				89	90	(codon
	in
	1				R	F
15	synthetic 3-23)
					1 ege	1 ttc	1 6
	! Allowed DNA				1 cgn	Itty	1
	1				1 agr	1
	1				ga	ntc	=
20	Hinfl(38)
	J				ga	gtc	=
	Plel(18)
	1				ga	wtc	=
	Tfil(20)
25	}					gtn	ac =
	MaeIXI(24)
	1					gts	ac -
	Tsp45l(21)
	1					tc	acc =
30	Hphl(44)
	! 91 92 93	94 95 96 97 98 99	100	101	102 103	104	105
	! Τ I S	R D N S Κ N	T	L	Y L	Q	M
35	lactI ate|TCT1 AGA|gac|aac1tet1aaglaat	lactl	etc |	tacIttg1	cag |	atg| 5
	!allowed|acn1ath|ten	cgn|gay|aay1 ten 1aar1aay	1 acn	ttr |	tayIttr1	car|atg|
	! |agy	agri lagyl		ctn |	1 ctn 1
	! 1	galgac = BsmAX(16)				ag	ct =

g ctn age

Alul{23) !

BlpI(21)

- 87 2018241075 03 Oct 2018

I Xbal I g aan nrm ttc = Xmnl(12) tg ca = HpyCH4V(21)

J

---FR3—

------->|

5

1

106

107

108

109

110

111 112

113

114

115

116

117 118

119 120

1

N

S

L

R

A

E D

T

A

V

Y

Y C

A K

1 aac

agC

TTAIAGg

get I gag 1gac

aCT

GCA

Gtc1tac

tat Itgc

get|aaaI 96

1

allowed 1aay|ten

ttr

cgn

gen

gar 1 gay

acn|gen

gtn

tay

tayltgy

gen|aar1

1

agy

ctn

agr

1

1

10

I

1

1

CC

nng g =

BsaJI(23)

ac ngt

= Bst4CI(51)

1

1

aga

tet

= Bglll(lO)

1

ac ngt

= HpyCH4III (51,

1

1

Rga

tcY

= BstYI(ll)

1

ac ngt

= Taal<51)

1

1

1

c

ayn

nnn

rtc

= Msll<44)

1

1

1

eg

rye

g =

BsiEI(23)

15

f

1

1

yg

gee

r =

Eael (23)

1

1

1

eg

gee

g =

Eagl (23)

f

ί

1

ig

gee

= Haelll(25)

gag g = Mnll(31)I I Pstl I

Aflll

- 88 2018241075 03 Oct 2018

Table 4: REdaptors, Extenders, and Bridges used for Cleavage and Capture of Human Heavy Chains in FR3.

A: HpyCH4V Probes of actual human HC genes !HpyCH4V in FR3 of human HC, bases 35-56; only those with TGca site TGca;10,

RE recognition:tgca of length 4 is expected at

9

6-1

3-11,3-07,3-21,3-72,3-48 3-09,3-43,3-20

5-51

3-15,3-30, 3-30.5,3-30.3,3-74,3-23, 3-33 7-4 .1

3-73

5-a

3-49

B: HpyCH4V REdaptors, Extenders, and Bridges

B.1 REdaptors ! Cutting HC lower strand:

! TmKeller for 100 mM NaCl, zero formamide agttctccctgcagctgaactc cactgtatctgcaaatgaacag ccctgtatctgcaaatgaacag ccgcctacctgcagtggagcag cgc tg tate tgcaaa tgaacag cggcatatctgcagatctgcag cggcgtatctgcaaatgaacag ctgcctacctgcagtggagcag t cgcct a tctgcaaa tgaacag

! Edapters for cleavage	rp W -*· tn	T,
(ON_HCFR36-1)	5'-agttctcccTGCAgctgaactc-3'	68.0	64
<ON_HCFR36-1A)	5'-ttctcccTGCAgctgaactc-3’	62.0	62
<ON_HCFR36-1B)	5'-ttctcccTGCAgctgaac-3'	56.0	59
<ON_HCFR33-15)	5'-cgctgtatcTGCAaatgaacag-3'	64.0	60
(0N_HCFR33-15A)	5'-ctgtatcTGCAaatgaacag-3'	56.0	56
(ON_HCFR33-15B)	5'-ctgtatcTGCAaatgaac-3'	50.0	53
(ON_HCFR33-11)	5'-cactgtatcTGCAaatgaacag-3’	62.0	58
(ON_HCFR35-51) 1	5'-ccgcctaccTGCAgtggagcag-3’	74.0	70
B.2 Segment of	synthetic 3-23 gene into which	captured CDR3	is to
be cloned
1	Xbal...
!D323* coCttcacTaaq tcT aqa qae aaC tcT aaq aaT	acT etc taC
! scab....

- 89 2018241075 03 Oct 2018

HpyCH4V

.. .. Aflll...

Ttg caG atg aac age TtA aqG . . .

B.3 Extender and Bridges ! Extender (bottom strand):

I (ON_HCHpyEx01) 5 ' -cAAgTAgAgAgTATTcTTAgAgTTgTcTcTAgAcTTAgTgAAgcg-3 ' ! ON_HCHpyEx01 is the reverse complement of ! 5'-cgCttcacTaag tcT aqa gac aaC tcT aag aaT acT etc taC Ttg -3'

I ! Bridges (top strand, 9-base overlap):

I (ON_HCHpyBr016-l) 5'-cgCttcacTaag tcT aqa gac aaC tcT aagaaT acT ctC taC Ttg CAgctgaac-3' {3'-term C is blocked)

I ! 3-15 et al. + 3-11 (ON_HCHpyBr023-15) 5’-cgCttcacTaag tcT aqa gac aaC tcT aagaaT acT ctC taC Ttg CAaatgaac-3’ {3’-term C is blocked) ! 5-51 (ON_HCHpyBr045-51) 5’-cgCttcacTaag tcT aqa gac aaC tcT aagaaT acT ctC taC Ttg CAgtggagc-3' {3'-term C is blocked) ! PCR primer (top strand) (ON_HCHpyPCR) 5'-cgCttcacTaag tcT aqa gac-3'

C: BlpI Probes from human HC GLGs

1-58,1-03,1-08,1-69,1-24,1-45,1-46, 1-f, 1-e 35 acatggaGCTGAGCagcctgag

1-02 acatggaGCTGAGCaggctgag

2018241075 03 Oct 2018

- 90 3 1-18 acatggagctgaggagcctgag

5-51,5-a acctgcagtggagcagcctgaa

5 3-15,3-73,3-49,3-72 atctgcaaatgaacagcctgaa

3303,3-33,3-07,3-11,3-30, 3-21,3-23,3305,3-4 8 atctgcaaatgaacagcctgag

3-20,3-74,3-09,3-43 10 atctgcaaatgaacagtctgag

74.1 atctgcagatctgcagcctaaa

3-66,3-13,3-53,3-d atcttcaaatgaacagcctgag

10 3-64 atcttcaaatgggcagcctgag

4301,4-28,4302,4-04,4304,4-31, 4-34,4-39,4-59, 4-61,4-b ccctgaaGCTGAGCtctgtgac

6-1 20 ccctgcagctgaactctgtgac

2-70,2-05 tccttacaatgaccaacatgga

2-26 tccttaccatgaccaacatgga

D: BlpT REdaptors , Extenders, and Bridges D.1 REdaptors

T_m ^w 1 (BlpF3HCl-58) 5'-ac atg gaG CTG AGC age ctg ag-3' 70 66 (BlpF3HC6-l) 5'-cc ctg aag ctg age tet gtg ac-3' 70 66 ! BlpF3HC6-l matches 4-30.1, not 6-1.

D.2 Segment of synthetic 3-23 gene into which captured CDR3 is to be cloned !

BlpI ί Xbal...

- 91 2018241075 03 Oct 2018 !D323* cgCttcacTaag TCT AGA gac aaC tcT aag aaT acT etc taC Ttg caG atg aac

Aflll...

aqC TTA AGG

D.3 Extender and Bridges ! Bridges (BlpF3Brl) 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtGtaC Ttg caG Ctg a IGC age ctg-3' (BlpF3Br2) 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtGtaC Ttg caG Ctg a|gc tet gtg-3' ! I lower strand is cut here ! Extender (BlpF3Ext) 5 ' -TcAgcTgcAAgTAcAAAgTATTTTTAcTgTTATcTcTAgA_'cTgAgTgAAgcg15 3' ! BlpF3Ext is the reverse complement of:

! 5'-cgCttcacTcag tcT aga gaT aaC AGT aaA aaT acT TtG taC Ttg caG Ctg a-3' ι (BlpF3PCR) 5'-cgCttcacTcag tcT aga gaT aaC-3'

E: HpyCH4III Distinct GLG sequences surrounding site, bases 77-98

10201,11804,14607,16909,leOlO,311017,353030,404037,4301 ccgtgtattactgtgcgagaga

10302,307015,321021,3303024,333026,348028,364031,366032

ctgtgtattactgtgcgagaga
3	10803
ccgtgtattactgtgcgagagg 4	12405,lfOll
ccgtgtattactgtgcaacaga 5	14506
ccatgtattactgtgcaagata 6	15808
ccgtgtattactgtgcggcaga 7	205012
ccacatattactgtgcacacag 8	226013

ccacatattactgtgcacggat

- 92 2018241075 03 Oct 2018 ccacgtattactgtgcacggat ccttgtattactgtgcaaaaga ctgtgtattactgtgcaagaga ccgtgtattactgtaccacaga ccttgtatcactgtgcgagaga ccgtatattactgtgcgaaaga ctgtgtattactgtgcgaaaga ccgtgtattactgtactagaga ccgtgtattactgtgctagaga ccgtgtattactgtactagaca ctgtgtattactgtaagaaaga ccgtgtattactgtgcgagaaa ccgtgtattactgtgccagaga ctgtgtattactgtgcgagaca ccatgtattactgtgcgagaca ccatgtattactgtgcgaga

270414

309416,343427

313418,374435,61450

315419

320420

323422

330423,3305425

349429

372433

373434

3d436

428438

4302440,4304441

439444

551448

5a#49

F: HpyCH411I REdaptors, Extenders, and Bridges F.1 REdaptors ! ONs for cleavage of HC(lower) in FR3(bases 77-97) ! For cleavage with HpyCH4III, Bst4CI, or Taal ! cleavage is in lower chain before base 88.

	77 78	788 901	888 234	888 567	889 999	999 456	9
890	123	7	<p W
Tm (H43.77.97.l-02#l)	5' -cc	gtg	tat	tAC	TGT	gcg	aga	g-3'	6462.6
(H43.77.97.l-03#2)	5' -cS	gtg	tat	tAC	TGT	gcg	aga	g-3'	6260.6
(H43.77.97.108#3)	5' -cc	gtg	tat	tAC	TGT	gcg	aga	g-3’	6462.6
(H43.77.97.323422)	5' -cc	gttjr	tat	tac	tgt	gcg	a3a	g-3'	6058.7
(H43.77.97.330423)	5' -cf?	gtg	tat	tac	tgt	gcg	aga	g-3'	6058.7

- 93 2018241075 03 Oct 2018

(H43.77.97.439#44)	5’-cl	gtg	tat	tac	tgt	geg	aga	1-3'	6260
(H4 3.77.97.551#48 )	5' -cc	atg	tat	tac	tgt	geg	aga	1-3'	6260
(H43.77.97.5a#49)	5'-cc	'a’tg	tat	tAC	TGT	geg	aga	1-3'	5858

F.2 Extender and Bridges 5 ! Xbal and Aflll sites in bridges are bunged (H43.XABrl) 5'-ggtgtagtga| TCT | AGt | gac | aac | tct | aag I aat | act | etc | tac | ttg | cag | atg | | aac| agC£TTA_:l^^₌Lgcti3aglgacXaCT_LGCAJ_gtcJ_tacJXgX tgt geg aga-3’ (H43.XABr2) 5'-ggtgtagtga10 | TCT | AGt | gac | aac | tct | aag | aat | act | etc | tac | ttg | cag | atg | I aac I agC I TTt|AGg I get I gag I gac I aCT I GCA I Gt c I tac I tat tgt geg aaa-3' (H43.XAExt) 5'-ATAgTAgAcT gcAgTgTccT cAgcccTTAA gcTgTTcATc TgcAAgTAgAgAgTATTeTT AgAgTTgTcT cTAgATcAcT AcAcc-3' !H43.XAExt is the reverse complement of ! 5'-ggtgtagtga! I TCT | AGA | gac | aac | tct | aag | aat | act | etc | tac| ttg | cag | atg| ’ | aac | aqC I TTA I AGg I get I gag I gac I aCT I GCA I Gtc I tac I tat -3 ' (H43.XAPCR) 5'-ggtgtagtga |TCT|AGA|gac|aac-3' ! Xbal and Aflll sites in bridges are bunged (H43.ABrl) 5'-ggtgtagtgaIaac|agglTOtJ^^gLggtlgaslaacLaCTjqgALSXcJ^apJiat. tgt geg aga-3' (H43.ABr2) 5'-ggtgtagtgaI aac | agC I TTt 1 AGg I get I gag I gac I aCT i GCA (Gt c I tac i tat tgt geg aaa-3' 25 (H4 3 . AExt) 5 '-ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTTcAcTAcAcc-3 ' ! (H43.AExt) is the reverse complement of 5'-ggtgtagtga! | aac | agCpT^X^^LgctlgggJ^acJ^CTiGgAl^cJ^aclXa^ -3 ' (H43.APCR) 5 '-ggtgtagtga I aac | agqj^TAiA^lgc^Lg^L

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- 101 2018241075 03 Oct 2018

Table 5D:

Analysis repeated using only 8 best REdaptors

	Id Ntot 0123	4	5	6	7	8 +
5	1 301 78 101 54 32 ccgtgtattactgtgcgagaga	16	9	10	1	0	281	102#l
	2 493 69 155 125 73 ctgtgtattactgtgcgagaga	37	14	11	3	6	459	103#2
10	3 189 52 45 38 23 ccgtgtattactgtgcgagagg	18	5	4	1	3	176	108#3
	4 127 29 23 28 24 ccgtatattactgtgcgaaaga	10	6	5	2	0.	114	323#22
	5 78 21 25 14 11	1	4	2	0	0	72	330#23
15	ctgtgtattactgtgcgaaaga 6 79 439#44 ctgtgtattactgtgcgagaca	15	17	25	8	11 1
	7 43 14 15 5 5 ccatgtattactgtgcgagaca	3	0	1	0	0	42	551#48
	8 307 26 63 72 51	38	24	14	13	6	250	5a#49

76 ccatgtattactgtgcgaga

20	1	102#l	ccgtgtattactgtgcgagaga	ccgtgtattactgtgcgagaga
	2	103#2	ctgtgtattactgtgcgagaga	. t..........
	3	108#3	ccgtgtattactgtgcgagagg		..........g
	4	323#22	ccgtatattactgtgcgaaaga		.......a. . .
	5	330#23	ctgtgtattactgtgcgaaaga	.t..........	.......a. . .
25	6	439#44	ctgtgtattactgtgcgagaca	.t.........	.........c.
	7	551#48	ccatgtattactgtgcgagaca	. . a.........	.........c.
	8	5a#49	ccatgtattactgtgcgagaAA	. .a........	.........AA

Seqs with the expected RE site only.......1463 / 1617

Seqs with only an unexpected site......... 0

Seqs with both expected and unexpected.... 7 Seqs with no sites........................ 0

- 102 2018241075 03 Oct 2018

Table 6: Human HC GLG FR1 Sequences

VH Exon - Nucleotide sequence alignment

VH1

1-02

CAG

GTG

CAG

CTG

GTG

CAG

TCT

GGG

GCT

GAG

GTG

AAG

AAG

CCT

GGG

GCC

TCA

GTG

AAG

GTC

5

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TCT

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ACC

TTC

ACC

1-03

cag

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cag

ctT

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cag

tet

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get

gag

gtg

aag

aag

cct

ggg

gee

tea

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get

tet

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tac

acc

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1-08

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cag

ctg

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cag

tet

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get

gag

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aag

aag

cct

ggg

gee

tea

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aag

gtc

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get

tet

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tac

acc

ttc

acc

10

1-18

cag

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cag

ctg

gtg

cag

tet

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gag

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aag

aag

cct

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gee

tea

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aag

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aag

get

tet

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tac

acc

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acc

1-24

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cag

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tet

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get

gag

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aag

aag

cct

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gee

tea

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acc

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1-45

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cag

tet

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get

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aag

aag

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ggg

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tea

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aag

gtT

15

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acc

ttc

acc

1-46

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gee

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tet

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acc

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acc

1-58

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ggg

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tea

gtg

aag

gtc

tcc

tgc

aag

get

tet

gga

tTc

acc

ttT

acT

20

1-69

cag

gtg

cag

ctg

gtg

cag

tet

ggg

get

gag

gtg

aag

aag

cct

ggg

Tcc

teG

gtg

aag

gtc

tcc

tgc

aag

get

tet

gga

GGc

acc

ttc

aGc

1-e

cag

gtg

cag

ctg

gtg

cag

tet

ggg

get

gag

gtg

aag

aag

cct

ggg

Tcc

teG

gtg

aag

gtc

tcc

tgc

aag

get

tet

gga

GGc

acc

ttc

aGc

.

1-f

Gag

gtc

cag

ctg

gtA

cag

tet

ggg

get

gag

gtg

aag

aag

cct

ggg

geT

Aca

gtg

aaA

Ate

25

VH2

tcc

tgc

aag

gTt

tet

gga

tac

acc

ttc

acc

2-05

CAG

ATC

ACC

TTG

AAG

GAG

TCT

GGT

CCT

ACG

CTG

GTG

AAA

CCC

ACA

CAG

ACC

CTC

ACG

CTG

ACC

TGC

ACC

TTC

TCT

GGG

TTC

TCA

CTC

AGC

2-26

cag

Gtc

acc

ttg

aag

gag

tet

ggt

cct

GTg

ctg

gtg

aaa

ccc

aca

Gag

acc

etc

aeg

ctg

30

acc

tgc

acc

Gtc

tet

ggg

ttc

tea

etc

age

2-70

cag

Gtc

acc

ttg

aag

gag

tet

ggt

cct

Gcg

ctg

gtg

aaa

CCC

aca

cag

acc

etc

acA

ctg

acc

tgc

acc

ttc

tet

ggg

ttc

tea

etc

age

VH3

3-07

GAG

GTG

CAG

CTG

GTG

GAG

TCT

GGG

GGA

GGC

TTG

GTC

CAG

CCT

GGG

GGG

TCC

CTG

AGA

CTC

TCC TGT GCA GCC TCT GGA TTC ACC TTT AGT

3-09

gaA gtg cag ctg gtg gag

tet ttc

ggg gga

ggc GAt

ttg gtA

cag cct

ggC Agg

tee

ctg

aga

etc

tcc tgt gca

gee

tet

gga

acc

ttt

3-11

Cag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtc

Aag

cct

ggA

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-13

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtA

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-15

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtA

Aag

cct

ggg

ggg

tcc

ctT

aga

etc

tee

tgt

gca

gee

tet

gga

ttc

acT

ttc

agt

- 103 2018241075 03 Oct 2018

3-20 3-21

gag tcc gag tcc

gtg tgt gtg tgt

cag gca cag gca

ctg gtg gag

tet ggg ttc acc tet ggg

gga ttt gga ttc

ggT Gtg

gtA eGg

cct cct

ggg ggg

tcc tcc

ctg ctg

aga aga

etc etc

gee tet ctg gtg

gga gag gga

GAt ggc agt

Ctg

ggg

ggg

gtc

Aag

gee

tet

ttc

acc

5

3-23

gag

gtg

cag

ctg

Ttg

gag

tet

ggg

gga

ggc

ttg

gtA

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttt

agC

3-30

Cag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

Gtg

gtc

cag

cct

ggg

Agg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-30.3

Cag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

Gtg

gtc

cag

cct

ggg

Agg

tcc

ctg

aga

etc

10

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-30.5

Cag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

Gtg

gtc

cag

cct

ggg

Agg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-33

Cag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

Gtg

gtc

cag

cct

ggg

Agg

tcc

ctg

aga

etc

tcc

tgt

gca

geG

tet

gga

ttc

acc

ttc

agt

15

3-43

gaA

gtg

cag

ctg

gtg

gag

tet

ggg

gga

gTc

Gtg

gtA

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttt

GAt

3-48

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtA

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-49

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtA

cag

ccA

ggg

egg

tcc

ctg

aga

etc

20

tcc

tgt

Aca

geT

tet

gga

ttc

acc

ttt

Ggt

3-53

gag

gtg

cag

ctg

gtg

gag

Act

ggA

gga

ggc

ttg

Ate

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

ggG

ttc

acc

Gtc

agt

3-64

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtc

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

25

3-66

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtc

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

GtC

agt

3-72

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtc

cag

cct

ggA

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-73

gag

gtg

cag

ctg

gtg

gag

tet

ggg

gga

ggc

ttg

gtc

cag

cct

ggg

ggg

tcc

ctg

aAa

etc

30

tcc

tgt

gca

gee

tet

ggG

ttc

acc

ttc

agt

3-74

gag

gtg

cag

ctg

gtg

gag

teC

ggg

gga

ggc

ttA

gtT

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

ttc

agt

3-d

gag

gtg

cag

ctg

gtg

gag

tet

Cgg

gga

gTc

ttg

gtA

cag

cct

ggg

ggg

tcc

ctg

aga

etc

tcc

tgt

gca

gee

tet

gga

ttc

acc

GtC

agt

35

VH4

4-04

CAG

GTG

CAG

CTG

CAG

GAG

TCG

GGC

CCA

GGA

CTG

GTG

AAG

CCT

TCG

GGG

ACC

CTG

TCC

CTC

ACC

TGC

GCT

GTC

TCT

GGT

GGC

TCC

ATC

AGC

4-28

cag

gtg

cag

ctg

cag

gag

teg

ggc

cca

gga

ctg

gtg

aag

cct

teg

gAC

acc

ctg

tcc

etc

acc

tgc

get

gtc

tet

ggt

TAc

tcc

ate

age

40

4-30.1

cag

gtg

cag

ctg

cag

gag

teg

ggc

cca

gga

ctg

gtg

aag

cct

tcA

CAg

acc

ctg

tcc

etc

acc

tgc

Act

gtc

tet

ggt

ggc

tcc

ate

age

4-30.2

cag

Ctg

cag

ctg

cag

gag

teC

ggc

Tea

gga

ctg

gtg

aag

cct

tcA

CAg

acc

ctg

tcc

etc

acc

tgc

get

gtc

tet

ggt

ggc

tcc

ate

age

- 104 2018241075 03 Oct 2018

4-30.4 4-31

cag gtg cag

ctg cag gtc tet

gag ggt gag ggt

teg ggc teg ggc

ggc cca gga ctg

gtg aag cct

tcA tcA

CAg CAg

acc acc

ctg ctg

tcc tcc

et eti

acc cag acc

tgc Act gtg cag tgc Act

tcc ate age

ctg

ctg gtc

cag tet

ggc tcc

cca gga ate age

gtg

aag cct

5

4-34

cag

gtg

cag

ctA

cag

Cag

tGg

ggc

Gca

gga

ctg

Ttg

aag

cct

teg

gAg

acc

ctg

tcc

Ct(

acc

tgc

get

gtc

tAt

ggt

ggG

tee

Ttc

agT

4-39

cag

Ctg

cag

ctg

cag

gag

teg

ggc

cca

gga

ctg

gtg

aag

cct

teg

gAg

acc

ctg

tcc

Ctl

acc

tgc

Act

gtc

tet

ggt

ggc

tee

ate

age

4-59

cag

gtg

cag

ctg

cag

gag

teg

ggc

cca

gga

ctg

gtg

aag

cct

teg

gAg

acc

ctg

tcc

ctl

10

acc

tgc

Act

gtc

tet

ggt

ggc

tee

ate

agT

4-61

cag

gtg

cag

ctg

cag

gag

teg

ggc

cca

gga

ctg

gtg

aag

cct

teg

gAg

acc

ctg

tcc

eti

acc

tgc

Act

gtc

tet

ggt

ggc

tee

Gtc

age

4-b

cag

gtg

cag

ctg

cag

gag

teg

ggc

cca

gga

ctg

gtg

aag

cct

teg

gAg

acc

ctg

tcc

Ctl

acc

tgc

get

gtc

tet

ggt

TAc

tee

ate

age

15

VH5

5-51

GAG

GTG

CAG

CTG

GTG

CAG

TCT

GGA

GCA

GAG

GTG

AAA

AAG

ccc

GGG

GAG

TCT

CTG

AAG

ATi

TCC

TGT

AAG

GGT

TCT

GGA

TAC

AGC

TTT

ACC

5-a

gaA

gtg

cag

ctg

gtg

cag

tet

gga

gca

gag

gtg

aaa

aag

ccc

ggg

gag

tet

ctg

aGg

at'

tcc

tgt

aag

ggt

tet

gga

tac

age

ttt

acc

20

VH6

6-1

CAG

GTA

CAG

CTG

CAG

CAG

TCA

GGT

CCA

GGA

CTG

GTG

AAG

CCC

TCG

CAG

ACC

CTC

TCA

CT'

ACC

TGT

GCC

ATC

TCC

GGG

GAC

AGT

GTC

TCT

VH7

7-4.1

CAG

GTG

CAG

CTG

GTG

CAA

TCT

GGG

TCT

GAG

TTG

AAG

AAG

CCT

GGG

GCC

TCA

GTG

AAG

GT'

25

TCC

TGC

AAG

GCT

TCT

GGA

TAC

ACC

TTC

ACT

- 105 2018241075 03 Oct 2018

Table 7: RERS sites in Human HC GLG FRls where there are at least 20 GLGs cut Bsgl GTGCAG 71 (cuts 16/14 bases to right)

	1:	4	1:	13	2:	13	3:	4	3:	13	4 :	13
	6:	13	7:	4	7:	13	8 :	13	9:	4	9:	13
5	10:	4	10:	13	15:	4	15:	65	16:	4	16:	65
	17:	4	17:	65	18:	4	18:	65	19:	4	19:	65
	20:	4	20:	65	21:	4	21:	65	22:	4	22:	65
	23:	4	23:	65	24 :	4	24 :	65	25:	4	25:	65
	26:	4	26:	65	27:	4	27:	65	28:	4	28:	65
10	29:	4	30:	4	30:	65	31:	4	31:	65	32:	4
	32:	65	33:	4	33:	65	34 :	4	34:	65	35:	4
	35:	65	36:	4	36:	65	37:	4	38:	4	39:	4
	41:	4	42:	4	43:	4	45:	4	46:	4	47:	4
	48:	4	48:	13	49:	4	49:	13	51:	4
15	There are 39	1 hits	: at	base#	4
	There are	! 21	. hits	i at	base#	: 65
	_ If _	ctgcac					9
	12:	63	13:	63	14 :	63	39:	63	41:	63	42:	63
20	44 :	63	45:	63	46:	63
	Bbvl	GCAGC					65
	1:	6	3:	6	6:	6	7:	6	8:	6	9:	6
	10:	6	15:	6	15:	67	16:	6	16:	67	17 :	6
	17:	67	18:	6	18:	67	19:	6	19:	67	20:	6
25	20:	67	21:	6	21:	67	22:	6	22:	67	23:	6
	23:	67	24:	6	24:	67	25:	6	25:	67	26:	6
	26:	67	27:	6	27:	67	28:	6	28:	67	29:	6
	30:	6	30:	67	31:	6	31:	67	32:	6	32:	67
	33:	6	33:	67	34:	6	34 :	67	35:	6	35:	67
30	36:	6	36:	67	37:	6	38:	6	39:	6	40:	6
	41:	6	42:	6	43:	6	44:	6	45:	6	46:	6
	47:	6	48:	6	49:	6	50:	12	51:	6

There are 43 hits at base# 6 Bolded sites very near sites listed below

There are 21	hits	at	base#	67
gctgc				13
37: 9 38:	9	39:	9	40: 3	40:	9	41:	9
42: 9 44:	3	44:	9	45: 9	46:	9	47:	9

- 106 2018241075 03 Oct 2018

50: 9

	There are	11 hits at	base#
	BsoFI	GCngc
5	1:	6	3:	6	6:	6
	10:	6	15:	6	15:	67
	17:	67	18:	6	18:	67
	20:	67	21:	6	21:	67
	23:	67	24:	6	24:	67
10	26:	67	27:	6	27:	67
	30:	6	30:	67	31:	6
	33:	6	33:	67	34:	6
	36:	6	36:	67	37:	6
	39:	6	39:	9	40:	3
15	41:	9	42:	6	42:	9
	44 :	9	45:	6	45:	9
	47:	9	48:	6	49:	6
	There are	43	hits	at	base#
	There are	11	hits	at	base#
20	There are	2	hits	at	base#
	There are	21	hits	at	base#
	Tsel	Gcwgc
	1:	6	3:	6	6:	6
25	10:	6	15:	6	15:	67
	17:	67	18:	6	18:	67
	20:	67	21:	6	21:	67
	23:	67	24 :	6	24:	67
	26:	67	27:	6	27:	67
30	30:	6	30:	67	31:	6
	33:	6	33:	67	34:	6
	36:	6	36:	67	37:	6
	39:	6	39:	9	40:	3
	41:	9	42:	6	42:	9
35	44:	9	45:	6	45:	9
	47:	9	48:	6	49:	6
	There are	43	hits	at	base#
	There are 11	hits	at	base#

7:	6	8:	6	9:	6
16:	6	16:	67	17 :	6
19:	6	19:	67	20:	6
22:	6	22:	67	23:	6
25:	6	25:	67	26:	6
28:	6	28:	67	29:	6
31:	67	32:	6	32:	67
34:	67	35:	6	35:	67
37:	9	38:	6	38:	9
40:	6	40:	9	41:	6
43:	6	4 4 :	3	44 :	6
46:	6	46:	9	47 :	6
50:	9	50:	12	51:	6

These often occur together 9

7:	6	8:	6	9:	6
16:	6	16:	67	17:	6
19:	6	19:	67	20:	6
22:	6	22:	67	23:	6
25:	6	25:	67	26:	6
28:	6	28:	67	29:	6
31:	67	32:	6	32:	67
34:	67	35:	6	35:	67
37:	9	38:	6	38:	9
40:	6	40:	9	41:	6
43:	6	44:	3	44 :	6
46:	6	46:	9	47:	6
50:	9	50:	12	51:	6

Often together. 9

- 107 2018241075 03 Oct 2018

There	are	2	hits	at	base#	3
There	are	1	hits	at	base#	12
There	are	21	hits	at	base#	67

5	MspAlI 1:	1 7	CMGckg 3:	7	4 :	7	5:	48 7	6:	7	7:	7
	8:	7	9:	7	10:	7	11:	7	15:	7	16:	7
	17:	7	18:	7	19:	7	20:	7	21:	7	22:	7
	23:	7	24 :	7	25:	7	26:	7	27:	7	28 :	7
10	29:	7	30:	7	31:	7	32:	7	33:	7	34 :	7
	35:	7	36:	7	37:	7	38:	7	39:	7	40:	1
	40:	7	41:	7	42:	7	44 :	1	44 :	7	45:	7
	46:	7	47:	7	48:	7	49:	7	50:	7	51:	7

There are 46 hits at base# 7

	PvuII CAGctg	48	7: 16: 22:	7 7 7
1: 8: 17:	7 7 7	3: 9: 18:	7 7 7	4: 10: 19:	7 7 7	5: 11: 20:	7 7 7	6: 15: 21:	7 7 7
20	23:	7	24:	7	25:	7	26:	7	27:	7	28:	7
	29:	7	30:	7	31:	7	32:	7	33:	7	34:	7
	35:	7	36:	7	37:	7	38:	7	39:	7	40:	1
	40:	7	41:	7	42:	7	44:	1	44:	7	45:	7
	46:	7	47:	7	48:	7	49:	7	50:	7	51:	7
25	There are 4 6	hits	i at	base#	I 7
	There are 2	hits	i at	base# 1
	Alul	AGct						54
	1:	8	2:	8	3:	8	4 :	8	4 :	24	5:	8
30	6:	8	7 :	8	8:	8	9:	8	10:	8	11:	8
	15:	8	16:	8	17:	8	18:	8	19:	8	20:	8
	21:	8	22:	8	23:	8	24 :	8	25:	8	26:	8
	27:	8	28:	8	29:	8	29:	69	30:	8	31:	8
	32:	8	33:	8	34:	8	35:	8	36:	8	37:	8
35	38:	8	39:	8	40:	2	40:	8	41:	8	42:	8
	43:	8	44 :	2	44 :	8	45:	8	46:	8	47:	8
	48:	8	48:	82	49:	8	49:	82	50:	8	51:	8

- 108 2018241075 03 Oct 2018

	There are There are	48 hits at base# 2 hits at base#	8 2
	Ddel Ctnag					48
5	1:-26	1:	48	2:	26	2:	48	3:	26	3:	48
	4 : 26	4 :	48	5:	26	5:	48	6:	26	6:	48
	7: 26	7 :	48	8:	26	8:	48	9:	26	10:	26
	11: 26	12:	85	13:	85	14:	85	15:	52	16:	52
	17: 52	18:	52	19:	52	20:	52	21:	52	22:	52
10	23: 52	24 :	52	25:	52	26:	52	27:	52	28:	52
	29: 52	30:	52	31:	52	32:	52	33:	52	35:	30
	35: 52	36:	52	40:	24	49:	52	51:	26	51:	48
	There are	22	hits	at	base#	52	52	and 48	never together
	There are	9	hits	at	base#	48
15	There are	12	hits	at	base#	26	26	and 24	never together
	HphI tcacc					42
	1: 86	3:	86	6:	86	7:	86	8:	80	11:	86
	12: 5	13:	5	14 :	5	15:	80	16:	80	17:	80
20	18: 80	20:	80	21:	80	22:	80	23:	80	24 :	80
	25: 80	26:	80	27:	80	28:	80	29:	80	30:	80
	31: 80	32:	80	33:	80	34:	80	35:	80	36:	80
	37: 59	38:	59	39:	59	40:	59	41:	59	42:	59
	43: 59	44 :	59	45:	59	46:	59	47:	59	50:	59
25	There are	22	hits	at	base#	80	80	and 86 never together
	There are	, 5	’ hits	ι at	base#	86
	There are	12	hits	at	base#	59
	BssKI Nccngg					50
30	1: 39	2:	39	3:	39	4 :	39	5:	39	7:	39
	8: 39	9:	39	10:	39	11:	39	15:	39	16:	39
	17: 39	18:	39	19:	39	20:	39	21:	29	21:	39
	22: 39	23:	39	24 :	39	25:	39	26:	39	27:	39
	28: 39	29:	39	30:	39	31:	39	32:	39	33:	39
35	34: 39	35:	19	35:	39	36:	39	37:	24	38:	24
	39: 24	41:	24	42:	24	44:	24	45:	24	46:	24
	47: 24	48:	39	48:	40	49:	39	49:	40	50:	24
	50: 73	51:	39

There are 35 hits at base# 39 39 and 40 together twice

- 109 2018241075 03 Oct 2018

	There are	2 hits at base# 40
	BsaJI	Ccnngg					47
	1:	40	2:	40	3:	40	4 :	40	5:	40	7:	40
5	8:	40	9:	40	9:	47	10:	40	10:	47	11:	40
	15:	40	18:	40	19:	40	20:	40	21:	40	22:	40
	23:	40	24 :	40	25:	40	26:	40	27:	40	28:	40
	29:	40	30:	40	31:	40	32:	40	34:	40	35:	20
	35:	40	36:	40	37:	24	38:	24	39:	24	41:	24
10	42:	24	44 :	24	45:	24	46:	24	47:	24	48:	40
	48:	41	49:	40	49:	41	50:	74	51:	40
	There are	32 hits	at	base#	40	40	and 41	together twice
	There are	2 hits	at	base#	41
	There are		9 hits	at	base#	24
15	There are	2 hits	at	base#	47
	BstNI	CCwgg					44
	PspGI	ccwgg
	ScrFI	($M.HpaII) CCwgg
20	1:	40	2:	40	3:	40	4 :	40	5:	40	7:	40
	8:	40	9:	40	10:	40	11:	40	15:	40	16:	40
	17:	40	18:	40	19:	40	20:	40	21:	30	21:	40
	22:	40	23:	40	24:	40	25:	40	26:	40	27:	40
	28:	40	29:	40	30:	40	31:	40	32:	40	33:	40
25	34:	40	35:	40	36:	40	37:	25	38:	25	39:	25
	41:	25	42:	25	44 :	25	45:	25	46:	25	47:	25
	50:	25	51:	40
	There are	33 hits	at	base#	40
30	ScrFI	CCngg					50
	1:	40	2:	40	3:	40	4 :	40	5:	40	7:	40
	8:	40	9:	40	10:	40	11:	40	15:	40	16:	40
	17:	40	18:	40	19:	40	20:	40	21:	30	21:	40
	22:	40	23:	40	24 :	40	25:	40	26:	40	27:	40
35	28:	40	29:	40	30:	40	31:	40	32:	40	33:	40
	34:	40	35:	20	35:	40	36:	40	37:	25	38:	25
	39:	25	41:	25	42:	25	44 :	25	45:	25	46:	25

- 110 2018241075 03 Oct 2018

47:	25	48	: 40	48:	41	49:	40	49	: 41	50: 25
50:	74	51	: 40
There are	35 hits	at	base#	40
There are	2 hits	at	base#	41
EcoO109I	RGgnccy				34
1:	43	2	: 43	3:	43	4 :	43	5	: 43	6: 43
7:	43	8	: 43	9:	43	10:	43	15	: 4 6	16: 46
17:	46	18	: 46	19:	46	20:	46	21	: 46	22: 46
23:	46	24	: 46	25:	46	26:	46	27	: 46	28: 46
30:	46	31	: 46	32:	46	33:	46	34	: 46	35: 46
36:	46	37	: 46	43:	79	51:	43
There are	22 hits	at	base#	46	46	and	43 never together

There are 11 hits at base# 43 15 NlalV GGNncc 71

1:	43	2:	43	3:	43	4 :	43	5:	43	6:	43
7:	43	8:	43	9:	43	9:	79	10:	43	10:	79
15:	46	15:	47	16:	47	17 :	46	17:	47	18 :	46
18:	47	19:	46	19:	47	20:	46	20:	47	21:	46
21:	47	22:	46	22:	47	23:	47	24:	47	25:	47
26:	47	27:	46	27:	47	28:	46	28:	47	29:	47
30:	46	30:	47	31:	46	31:	47	32:	46	32:	47
33:	46	33:	47	34 :	46	34 :	47	35:	46	35:	47

	36:	46	36:	47	37: 21	37:	46	37:	47	37: 79
25	38:	21	39:	21	39: 79	40:	79	41:	21	41: 79
	42:	21	42:	79	43: 79	44 :	21	44 :	79	45: 21
	45:	79	46:	21	46: 79	47:	21	51:	43

There are	23	hits	at base#	47	46	& 47 often together
There are	17	hits	at	base#	46		There are	11	hits	at	base#	43
Sau96I Ggncc					70
1:	44	2:	3	2:	44	3:	44	4:	44	5:	3	5:	44	6:	44
7:	44	8:	22	8:	44	9:	44	10:	44	11:	3	12:	22	13:	22
14:	22	15:	33	15:	47	16:	47	17:	47	18:	47	19:	47	20:	47
21:	47	22:	47	23:	33	23:	47	24 :	33	24 :	47	25:	33	25:	47
26:	33	26:	47	27:	47	28:	47	29:	47	30:	47	31:	33	31:	47
32:	33	32:	47	33:	33	33:	47	34:	33	34 :	47	35:	47	36:	47
37:	21	37:	22	37:	47	38:	21	38:	22	39:	21	39:	22	41:	21
41:	22	42:	21	42:	22	43:	80	44 :	21	44 :	22	45:	21	45:	22
46:	21	46:	22	47 :	21	47:	22	50:	22	51:	44

2018241075 03 Oct 2018

- Ill -

There	are	23	hits	at	base#	47	These	do
There	are	11	hits	at	base#	44
There	are	14	hits	at	base#	22	These	do
There	are	9	hits	at	base#	21

not occur together occur together.

BsmAI GTCTCNnnnn 22

1:	58	3	: 58	4 :	58	5:	58	8:	58	9:	58
10:	58	13	: 70	36:	18	37:	70	38:	70	39:	70
40:	70	41	: 70	42:	70	44 :	70	45:	70	46:	70
47 :	70	48	: 48	49:	48	50:	85
There are		11 hits	at	base#	70

	_ It _ 13:	Nnnnnngagac	27	20:	48
40	15: 48	16: 48	17:	48	18 :	48
15	21:	48	22: 48	23: 48	24 :	48	25:	48	26:	48
	27:	48	28 : 48	29: 48	30:	10	30:	48	31:	48
	32:	48	33: 48	35: 48	36:	48	43:	40	44 :	40
	45:	40	46: 40	47: 40
	There are 20 hits at base#	48
20
	Avail	Ggwcc			44
	Sau96I($M.	Haelll)	Ggwcc		44
	2:	3	5: 3	6: 44	8:	44	9:	44	10:	44
	11:	3	12: 22	13: 22	14:	22	15:	33	15:	47
25	16:	47	17: 47	18: 47	19:	47	20:	47	21:	47
	22:	47	23: 33	23: 47	24:	33	24 :	47	25:	33
	25:	47	26: 33	26: 47	27:	47	28:	47	29:	47
	30:	47	31: 33	31: 47	32:	33	32:	47	33:	33
	33:	47	34: 33	34: 47	35:	47	36:	47	37:	47

43: 80 50: 22

There are 23 hits at base# 47 44 & 47 never together There are 4 hits at base# 44

PpuMI RGgwccy

6:	43	8:	43
17:	46	18:	46
23:	46	24 :	46

9:	43	10:	43
19:	46	20:	46
25:	46	26:	46

15:	46	16:	46
21:	46	22:	46
27:	46	28:	46

- 112 2018241075 03 Oct 2018

30: 4 6	31: 4 6	32:	46	33:	46	34	: 46	35: 46
36: 46	37: 46	43:	79
There are	22 hits at	base#	46	43	and	46 never occur together
There are	4 hits at	base#	43

	BsmFI 8: _ H _ 15:	GGGAC	77 48	3 33
43 gtcc 48	37: 46 :c 16: 48	50: 17:
1:	0	1:	0	20 :	48
10	21:	48	22: 48	23:	48	24 :	48	25:	48	26:	48
	27:	48	28: 48	29:	48	30:	48	31:	48	32:	48
	33:	48	34: 48	35:	48	36:	48	37 :	54	38 :	54
	39:	54	40: 54	41:	54	42 :	54	43:	54	44 :	54
	45:	54	46: 54	47:	54
15	There are 20 hits	; at	base#	48
	There are 11 hits at	base#	54
	Hinfl	; Gantc				80
	8:	77	12: 16	13:	16	14:	16	15:	16	15:	56
20	15:	77	16: 16	16:	56	16:	77	17:	16	17:	56
	17:	77	18: 16	18:	56	18:	77	19:	16	19:	56
	19:	77	20: 16	20:	56	20:	77	21:	16	21:	56
	21:	77	22: 16	22:	56	22:	77	23:	16	23:	56
	23:	77	24: 16	24:	56	24 :	77	25:	16	25:	56
25	25:	77	26: 16	26:	56	26:	77	27:	16	27:	26
	27:	56	27: 77	28:	16	28:	56	28:	77	29:	16
	29:	56	29: 77	30:	56	31:	16	31:	56	31:	77
	32:	16	32: 56	32:	77	33:	16	33:	56	33:	77
	34:	16	35: 16	35:	56	35:	77	36:	16	36:	26
30	36:	56	36: 77	37:	16	38:	16	39:	16	40:	16
	41:	16	42: 16	44:	16	45:	16	46:	16	47:	16
	48:	46	49: 46
	There are 34 hits at	base# 16
35	Tfil	Gawtc				21
	8:	77	15: 77	16:	77	17:	77	18:	77	19:	77
	20:	77	21: 77	22:	77	23:	77	24:	77	25:	77
	26:	77	27: 77	28:	77	29:	77	31:	77	32:	77

2018241075 03 Oct 2018

- 113

33: 77 There are	35: 21	77 hits	36: at	77 base#	77
Mlyl	GAGTC					38
12:	16	13:	16	14 :	16	15:	16	16:	16	17:	16
18:	16	19:	16	20:	16	21:	16	22:	16	23:	16
24 :	16	25 :	16	26:	16	27:	16	27:	26	28:	16
29:	16	31:	16	32:	16	33:	16	34 :	16	35:	16
36:	16	36:	26	37:	16	38:	16	39:	16	40:	16
41:	16	42:	16	44 :	16	45:	16	46:	16	47:	16
48:	46	49:	46
There are	34	hits	at	base#	16

GACTC

15:	56	16:	56	17 :	56	18:	56	19:	56	20:	56
21:	56	22:	56	23:	56	24 :	56	25:	56	26:	56
27:	56	28:	56	29:	56	30:	56	31:	56	32:	56
33:	56	35:	56	36:	56
There are	21	hits	ί at	base#	56
Plel	gagtc					38
12:	16	13:	16	14 :	16	15:	16	16:	16	17:	16
18:	16	19:	16	20:	16	21:	16	22:	16	23:	16
24:	16	25:	16	26:	16	27:	16	27:	26	28:	16
29:	16	31:	16	32:	16	33:	16	34 :	16	35:	16
36:	16	36:	26	37:	16	38:	16	39:	16	40:	16
41:	16	42 :	16	44 :	16	45:	16	46:	16	47:	16
48:	46	49:	46
There are	34	hits	; at	base#	’ 16
_ M __	gactc					21
15:	56	16:	56	.17 :	56	18:	56	19:	56	20:	56
21:	56	22:	56	23:	56	24 :	56	25:	56	26:	56
27:	56	28:	56	29:	56	30:	56	31:	56	32:	56

35: 56

36:

33: 56 There are 21 hits at AlwNI CAGNNNctg

16: 68 17:

15: 68 base# 56

18: 68

19: 68 20: 68

- 114 18241075 03 Oct 2018

21:	68	22:	68	23:	68	24:	68	25:	68	26:	68
27:	68	28:	68	29:	68	30:	68	31:	68	32:	68
33:	68	34:	68	35:	68	36:	68	39:	46	40:	46
41:	46	42:	46
There ,	are 22	hits	at	base#	68

Ο

CM

- 115 3 Oct 2018

Table 8: Kappa FR1 GLGs

o

! 1

2

3

4

5

6

7

8

9

10

11

12

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

! 13

14

15

16

17

18

19

20

21

22

23

O

5

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

i

012

τ—H T-f-

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

CM

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

t

02

OO

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

o

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

1

018

KN

10

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

1

08

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

1

A20

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

15

GCA

TCT

GTA

GGA

GAC'

AGA

GTC

ACC

ATC

ACT

TGC

1

A30

AAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCT

GCC

ATG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGT

1

L14

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCA

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGT

1

LI

20

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCC

TCA

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGT

t

L15

GCC

ATC

CAG

TTG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

1

L4

GCC

ATC

CAG

TTG

ACC

CAG

TCT

CCA

TCC

TCC

CTG

TCT

25

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

1

L18

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCT

TCC

GTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGT

I

L5

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCA

TCT

TCT

GTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGT

1

L19

30

GAC

ATC

CAG

TTG

ACC

CAG

TCT

CCA

TCC

TTC

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

I

L8

GCC

ATC

CGG

ATG

ACC

CAG

TCT

CCA

TTC

TCC

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

L23

GCC

ATC

CGG

ATG

ACC

CAG

TCT

CCA

TCC

TCA

TTC

TCT

35

GCA

TCT

ACA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGT

I

L9

- 116 2018241075 03 Oct 2018

GTC ATC GCA TCT GCC ATC GCA TCT

TGG ATG ACC ACA GGA GAC CAG ATG ACC

CAG TCT AGA GTC CAG TCT

CCA ACC CCA ACC

TCC ATC TCC ATC

TTA CTC AGT TGT TCC CTG

TCT

1 TCT 1

L24 Lll

GTA

GGA

GAC

AGA

GTC

ACT

TGC

5

GAC

ATC

CAG

ATG

ACC

CAG

TCT

CCT

TCC

ACC

CTG

TCT

GCA

TCT

GTA

GGA

GAC

AGA

GTC

ACC

ATC

ACT

TGC

1

L12

GAT

ATT

GTG

ATG

ACC

CAG

ACT

CCA

CTC

TCC

CTG

ccc

GTC

ACC

CCT

GGA

GAG

CCG

GCC

TCC

ATC

TCC

TGC

1

Oil

GAT

ATT

GTG

ATG

ACC

CAG

ACT

CCA

CTC

TCC

CTG

CCC

10

GTC

ACC

CCT

GGA

GAG

CCG

GCC

TCC

ATC

TCC

TGC

ι

01

GAT

GTT

GTG

ATG

ACT

CAG

TCT

CCA

CTC

TCC

CTG

CCC

GTC

ACC

CTT

GGA

CAG

CCG

GCC

TCC

ATC

TCC

TGC

I

A17

GAT

GTT

GTG

ATG

ACT

CAG

TCT

CCA

CTC

TCC

CTG

CCC

GTC

ACC

CTT

GGA

CAG

CCG

GCC

TCC

ATC

TCC

TGC

1

Al

15

GAT

ATT

GTG

ATG

ACC

CAG

ACT

CCA

CTC

TCT

CTG

TCC

GTC

ACC

CCT

GGA

CAG

CCG

GCC

TCC

ATC

TCC

TGC

1

A18

GAT

ATT

GTG

ATG

ACC

CAG

ACT

CCA

CTC

TCT

CTG

TCC

GTC

ACC

CCT

GGA

CAG

CCG

GCC

TCC

ATC

TCC

TGC

1

A2

GAT

ATT

GTG

ATG

ACT

CAG

TCT

CCA

CTC

TCC

CTG

CCC

20

GTC

ACC

CCT

GGA

GAG

CCG

GCC

TCC

ATC

TCC

TGC

1

A19

GAT

ATT

GTG

ATG

ACT

CAG

TCT

CCA

CTC

TCC

CTG

CCC

GTC

ACC

CCT

GGA

GAG

CCG

GCC

TCC

ATC

TCC

TGC

A3

GAT

ATT

GTG

ATG

ACC

CAG

ACT

CCA

CTC

TCC

TCA

CCT

GTC

ACC

CTT

GGA

CAG

CCG

GCC

TCC

ATC

TCC

TGC

1

A23

25

GAA

ATT

GTG

TTG

ACG

CAG

TCT

CCA

GGC

ACC

CTG

TCT

TTG

TCT

CCA

GGG

GAA

AGA

GCC

ACC

CTC

TCC

TGC

!

A27

GAA

ATT

GTG

TTG

ACG

CAG

TCT

CCA

GCC

ACC

CTG

TCT

TTG

TCT

CCA

GGG

GAA

AGA

GCC

ACC

CTC

TCC

TGC

1

All

GAA

ATA

GTG

ATG

ACG

CAG

TCT

CCA

GCC

ACC

CTG

TCT

30

GTG

TCT

CCA

GGG

GAA

AGA

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Table 10 Lambda FR1 GLG sequences

o

CAG

TCT

GTG

CTG

ACT

CAG

CCA

CCC

TCG

GTG

TCT GAA

GCC

ccc

AGG

CAG

AGG

GTC

ACC

ATC

TCC

TGT

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5

cag

tct

gtg

ctg

acG

cag

ccG

ccc

tcA

gtg

tct gGG

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gcc

CCA

Ggg

cag

agg

gtc

acc

ate

tee

tgC

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ι—H tJ

cag

tct

gtg

ctg

act

cag

cca

ccc

tcA

geg

tct gGG

CM rs/*)

Acc

ccc

Ggg

cag

agg

gtc

acc

ate

teT

tgt

! le

cag

tct

gtg

ctg

act

cag

cca

ccc

tcA

gCg

tct gGG

o CM

o

Acc

ccc

Ggg

cag

agg

gtc

acc

ate

teT

tgt

- ig

cag

tct

gtg

Ttg

acG

cag

ccG

ccc

tcA

gtg

tct gCG

gcc

ccA

GgA

cag

aAg

gtc

acc

ate

tee

tgC

! lb

! VL2

CAG	TCT	GCC	CTG	ACT	CAG	CCT	CCC	TCC	GCG	TCC GGG
TCT	CCT	GGA	CAG	TCA	GTC	ACC	ATC	TCC	TGC	! 2c
cag	tct	gcc	ctg	act	cag	cct	eGe	tcA	gTg	tee ggg
tct	cct	gga	cag	tea	gtc	acc	ate	tee	tgc!	! 2e
cag	tct	gcc	ctg	act	cag	cct	Gcc	tee	gTg	teT ggg
tct	cct	gga	cag	teG	Ate	acc	ate	tee	tgc	! 2a2
cag	tct	gee	ctg	act	cag	cct	ccc	tee	gTg	tec ggg
tct	cct	gga	cag	tea	gtc	acc	ate	tee	tgc	! 2d
cag	tct	gcc	ctg	act	cag	cct	Gcc	tee	gTg	teT ggg
tct	cct	gga	cag	teG	Ate	acc	ate	tee	tgc	ί 2b2

! VL3

TCC	TAT	GAG	CTG	ACT	CAG	CCA	CCC	TCA	GTG	TCC GTG
TCC	CCA	GGA	CAG	ACA	GCC	AGC	ATC	ACC	TGC!	! 3r
tee	tat	gag	ctg	act	cag	cca	cTc	tea	gtg	tcA gtg
Gcc	cTG	gga	cag	acG	gcc	agG	atT	acc	tgT	! 3j
tee	tat	gag	ctg	acA	cag	cca	ccc	teG	gtg	tcA gtg
tee	cca	gga	caA	acG	gcc	agG	ate	acc	tgc!	! 3p
tee	tat	gag	ctg	acA	cag	cca	ccc	t cG	gtg	tcA gtg
tee	cTa	gga	cag	aTG	gcc	agG	ate	acc	tgc	! 3a
teT	tct	gag	ctg	act	cag	GAC	ccT	GeT	gtg	teT gtg
Gcc	TTG	gga	cag	aca	gTc	agG	ate	acA	tgc	! 31

- 128 2018241075 03 Oct 2018

'.CC	tat	gTg	ctg	act	cag	cca	ccc	tea	gtg	tcA gtg
Gcc	cca	gga	Aag	acG	gcc	agG	atT	acc	tgT	! 3h
tcc	tat	gag	ctg	acA	cag	cTa	ccc	teG	gtg	tcA gtg
tcc	cca	gga	cag	aca	gcc	agG	ate	acc	tgc	! 3e
tcc	tat	gag	ctg	aTG	cag	cca	ccc	teG	gtg	tcA gtg
tcc	cca	gga	cag	acG	gcc	agG	ate	acc	tgc	! 3m
tcc	tat	gag	ctg	acA	cag	cca	Tcc	tea	gtg	tcA gtg
tcT	ccG	gga	cag	aca	gcc	agG	ate	acc	tgc	! V2-19

VL4

1

CTG

CCT

GTG

CTG

ACT

CAG

CCC

CCG

TCT

GCA

TCT

GCC

TTG

CTG

GGA

GCC

TCG

ATC

AAG

CTC

ACC

TGC

I

4c

cAg

cct

gtg

ctg

act

caA

TcA

TeC

tet

geC

tet

geT

tee

ctg

gga

Tcc

teg

Gtc

aag

etc

acc

tgc

t

4a

cAg

eTt

gtg

ctg

act

caA

TeG

ccC

tet

geC

tet

gcc

i

tee

ctg

gga

gcc

teg

Gtc

aag

etc

acc

tgc

t

4b

! VL5

CAG

CCT

GTG

CTG

ACT

CAG

CCA

CCT

TCC

TCC

TCC

GCA

TCT

CCT

GGA

GAA

TCC

GCC

AGA

CTC

ACC

TGC

I

5e

cag

Get

gtg

ctg

act

cag

ccG

Get

tcc

CTc

tcT

gca

)

tet

cct

gga

gCa

tcA

gcc

agT

etc

acc

tgc

I

5c

cag

cct

gtg

ctg

act

cag

cca

Tet

tcc

CAT

tcT

gca

tet

Tet

gga

gCa

tcA

gTc

aga

etc

acc

tgc

1

5b

! VL6

AAT

TTT

ATG

CTG

ACT

CAG

CCC

CAC

TCT

GTG

TCG

GAG

5

! VL7

TCT

CCG

GGG

AAG

ACG

GTA

ACC

ATC

TCC

TGC

t

6a

CAG

ACT

GTG

GTG

ACT

CAG

GAG

CCC

TCA

CTG

ACT

GTG

TCC

CCA

GGA

GGG

ACA

GTC

ACT

CTC

ACC

TGT

t

7a

cag

Get

gtg

gtg

act

cag

gag

ccc

tea

ctg

act

gtg

3

! VL8

tcc

cca

gga

ggg

aca

gtc

act

etc

acc

tgt

t

7b

CAG

ACT

GTG

GTG

ACC

CAG

GAG

CCA

TCG

TTC

TCA

. GTG

TCC

CCT

GGA

GGG

ACA

GTC

ACA

CTC

ACT

TGT

j

8a

- 129 2018241075 03 Oct 2018 ! VL9 ! VL10 5

CAG CCT GTG CTG ACT CAG CCA CCT TCT GCA

TCC CTG GGA GCC TCG GTC ACA CTC ACC TGC

CAG GCA GGG CTG ACT CAG CCA CCC TCG GTG

GGC TTG AGA CAG ACC GCC ACA CTC ACC TGC

TCA GCC ! 9a

TCC AAG ! 10a

2018241075 03 Oct 2018

	- 130 -
Table 11 RERSs found in human lambda FR.1 GLGs
! There	are 31	lambda GLGs
Mlyl	NnnnnnGACTC			25
1:	6	3:	6	4:	6	6:	6	7 :	6	8:	6
9:	6	10:	6	11:	6	12:	6	15:	6	16:	6
20:	6	21:	6	22:	6	23:	6	23:	50	24:	6
25:	6	25:	50	26:	6	27:	6	28 :	6	30:	6
31:	6
There are 23	hits	at	base#	6
_ I» _	GAGTCNNNNNn				1
26:	34
Mwol	GCNNNNNnngc				20
, 1:	9	2:	9	3:	9	4:	9	11:	9	11:	56
12:	9	13:	9	14:	9	16:	9	17:	9	18:	9
19:	9	20:	9	23:	9	24:	9	25:	9	26:	9
30:	9	31:	9
There are 19	hits	at	base*	9
1 Hinfl Gantc					27
1:	12	3:	12	4:	12	6:	12	7 :	12	8:	12
9:	12	10:	12	11:	12	12:	12	15:	12	16:	12
20:	12	21:	12	22:	12	23:	12	23:	46	23:	56
24:	12	25:	12	25:	56	26:	12	26:	34	27:	12
i 28:	12	30:	12	31:	12
There are 23	hits	at	base)	12
Plel	gactc					25
1:	12	3:	12	4:	12	6:	12	7:	12	8:	12
9:	12	10:	12	11:	12	12:	12	15:	12	16:	12
) 20:	12	21:	12	22:	12	23:	12	23:	56	24:	12
25:	12	25:	56	26:	12	27:	12	28:	12	30:	12
31:	12

There are 23 hits at base# 12 gagtc

- 131 2018241075 03 Oct 2018

	26: 34
	Ddel Ctnag				32
	1: 14	2: 24	3:	14	3:	24	4 :	14	4 :	24
5	5: 24	6: 14	7:	14	7:	24	8 :	14	9:	14
	10: 14	11: 14	11:	24	12:	14	12:	24	15:	5
	15: 14	16: 14	16:	24	19:	24	20:	14	23:	14
	24: 14	25: 14	26:	14	27:	14	28:	14	29:	30
	30: 14	31: 14
0	There are	21 hits	at	base#	14
	BsaJI Ccnngg			38
	1: 23	1: 40	2:	39	2:	40	3:	39	3:	40
	4: 39	4: 40	5:	39	11:	39	12:	38	12:	39
5	13: 23	13: 39	14:	23	14:	39	15:	38	16:	39
	17: 23	17: 39	18:	23	18:	39	21:	38	21:	39
	21: 47	22: 38	22:	39	22:	47	26:	40	27:	39
	28: 39	29: 14	29:	39	30:	38	30:	39	30:	47
	31: 23	31: 32
0	There are	17 hits	at	base#	39
	There are	5 hits	at	base#	38
	There are	5 hits	at	base#	40	Makes cleavage ragged.
	Mnll cctc				35
	1: 23	2: 23	3:	23	4:	23	5:	23	6:	19
5	6: 23	7: 19	8:	23	9:	19	9:	23	10:	23
	11: 23	13: 23	14:	23	16:	23	17:	23	18:	23
	19: 23	20: 47	21:	23	21:	29	21:	47	22:	23
	22: 29	22: 35	22:	47	23:	26	23:	29	24:	27
	27: 23	28: 23	30:	35	30:	47	31:	23
0	There are	21 hits	at	base#	23
	There are	3 hits	at	base#	19
	There are	3 hits	at	base#	29
	There are	1 hits	at	base#	26
	There are	1 hits	at	base#	27	These could	make	cleavage
5	gagg					7

- 132 2018241075 03 Oct 2018

1: 48 29: 44	2: 48	3:	48	4:	48	27:	44	28:	44
BssKI Nccngg			39
1: 40	2: 39	3:	39	3:	40	4 :	39	4:	40
5: 39	6: 31	6:	39	7:	31	7:	39	8:	39
9: 31	9: 39	10:	39	11:	39	12:	38	12:	52
13: 39	13: 52	14:	52	16:	39	16:	52	17:	39
17: 52	18: 39	18:	52	19:	39	19:	52	21:	38
22: 38	23: 39	24 :	39	26:	39	27:	39	28:	39
29: 14	29: 39	30:	38
There	are 21 hits	at	base#	39
There	are 4 hits	at	base#	38
There	are 3 hits	at	base#	31
There	are 3 hits	at	base#	40	Ragged
BstNI CCwgg			30
1: 41	2: 40	5:	40	6:	40	7:	40	8:	40
9: 40	10: 40	11:	40	12:	39	12:	53	13:	40
13: 53	14: 53	16:	40	16:	53	17:	40	17:	53
18: 40	18: 53	19:	53	21:	39	22:	39	23:	40
24: 40	27: 40	28:	40	29:	15	29:	40	30:	39
There	are 17 hits	at	base#	40
There	are 7 hits	at	base#	53
There	are 4 hits	at	base#	39
There	are 1 hits	at	base#	41	Ragged
PspGI ccwgg			30
1: 41	2: 40	5:	40	6:	40	7:	40	8:	40
9: 40	10: 40	11:	40	12:	39	12:	53	13:	40
13: 53	14: 53	16:	40	16:	53	17:	40	17:	53
18: 40	18: 53	19:	53	21:	39	22:	39	23:	40
24: 40	27: 40	28:	40	29:	15	29:	40	30:	39
There	are 17 hits	at	base#	40
There	are 7 hits	at	base#	53

- 133 2018241075 03 Oct 2018

There are 4 hits at base# 39 There are 1 hits at base# 41

	ScrFI	CCngg	39
5	1:	41	2:	40	3:	40	3: 41	4:	40	4:	41
	5:	40	6:	32	6:	40	7: 32	7:	40	8:	40
	9:	32	9:	40	10:	40	11: 40	12:	39	12:	53
	13:	40	13:	53	14 :	53	16: 40	16:	53	17:	40
	17:	53	18:	40	18:	53	19: 40	19:	53	21:	39
0	22:	39	23:	40	24:	40	26: 40	27:	40	28:	40
	29:	15	29:	40	30:	39
	There are	21	hits	at	base#	40
	There are	4	hits	at	base#	39
	There are	3	hits	at	base#	41
5
	Maelll gtnac				16
	1:	52	2:	52	3:	52	4: 52	5:	52	6:	52
	7:	52	9:	52	26:	52	27: 10	27:	52	28:	10
	28:	52	29:	10	29:	52	30: 52
0	There are	13	hits	at	base#	52
	Tsp45I gtsac				15
	1:	52	2:	52	3:	52	4: 52	5:	52	6:	52
	7:	52	9:	52	27:	10	27: 52	28 :	10	28:	52
5	29:	10	29:	52	30:	52
	There are	12	hits	at	based	52
	HphI	tcacc				26
	1:	53	2:	53	3:	53	4: 53	5:	53	6:	53
0	7:	53	8:	53	9:	53	10: 53	11:	59	13:	59
	14:	59	17:	59	18:	59	19: 59	20:	59	21:	59
	22:	59	23:	59	24:	59	25: 59	27:	59	28:	59

30: 59 31: 59

There are 16 hits at base# 59

241075 03 Oct 2018

- 134 There are 10 hits at base# 53

BspMI ACCTGCNNNNn 14

11:	61	13: 61	14: 61	17:	61	18: 61	19: 61
20:	61	21: 61	22: 61	23:	61	24: 61	25: 61
30:	61	31: 61
There	are 14 hits	at base#	61	Goes	into	CDR1

O

CM

- 135 2018241075 03 Oct 2018

Table 12: Matches to URE FR3 adapters in 7 9 human HC. A. List of Heavy-chains genes sampled

	AF008566 AF035043	AF103367 AF103368	HSA235674 HSA235673	HSU94417 HSU94418	S83240 SABVH369
5	AF103026		AF103369		HSA240559	HSU96389	SADEIGVH
	afl03033		AF103370		HSCB201	HSU96391	SAH2IGVH
	AF103061		afl03371		HSIGGVHC	HSU96392	SDA3IGVH
	Afl03072		AF103372		HSU44791	HSU96395	SIGVHTTD
	afl03078		AF158381		HSU44793	HSZ93849	SUK4IGVH
L0	AF103099		E05213			HSU82771	HSZ93850
	AF103102		E05886			HSU82949	HSZ93851
	AF103103		E05887			HSU82950	HSZ93853
	AF103174		HSA235661		HSU82952	HSZ93855
	AF103186		HSA235664		HSU82961	HSZ93857
L5	afl03187		HSA235660		HSU86522	HSZ93860
	AF103195		HSA235659		HSU86523	HSZ93863
	afl03277		HSA235678		HSU92452	MCOMFRAA
	afl03286		HSA235677		HSU94412	MCOMFRVA
	AF103309		HSA235676		HSU94415	S82745
20	afl03343		HSA235675		HSU94416	S82764
	Table 12B.	Testing all	distinct	GLGs :	from bases 89.1	to 93.2 o:
	the heavy	variable domain
	Id	Nb	0	1	2	3	4			SEQ ID
	NO:
25	1	38	15	11	10	0	2	Seql	gtgtattactgtgc	25
	2	19	7	6	4	2	0	Seq2	gtAtattactgtgc	26
	3	1	0	0	1	0	0	Seq3	gtgtattactgtAA	27
	4	7	1	5	1	0	0	Seq4	gtgtattactgtAc	28
	5	0	0	0	0	0	0	Seq5	Ttgtattactgtgc	29
30	6	0	0	0	0	0	0	Seq6	TtgtatCactgtgc	30
	7	3	1	0	1	1	0	Seq7	ACAtattactgtgc	31
	8	2	0	2	0	0	0	Seq8	ACgtattactgtgc	32
	9	9	2	2	4	1	0	Seo9	ATqtattactqtqc	33
	Group		26	26	21	4	2
35	Cumulative		26	52	73	77	79

Table 12C Most important URE recognition seqs in FR3 Heavy

	1	VHSzyl	GTGtattactgtgc	(ON	SHC103)	(SEQ	ID	NO:25)
	2	VHSzy2	GTAtattactgtgc	(ON	SHC323)	(SEQ	ID	NO:26)
	3	VHSzy4	GTGtattactgtac	(ON	SHC349)	(SEQ	ID	NO:28)
40	4	VHSzy9	ATGtattactgtgc	(ON~	SHC5a)	(SEQ	ID	NO:33)

Number of sequences.......... 7 9

Number of bases.............. 29143

Table 12D, testing 79 human HC V genes with four probes

Number of sequences.......... 7 9

Number of bases.............. 29143

- 136 2018241075 03 Oct 2018

Number of mismatches

	Id	Best	0	1	2	3	4	5
5	1	39	15	11	10	1	2	0 Seql gtgtattactgtgc (SEQ ID NO:25)
	2	22	7	6	5	3	0	1 Seg2 gtAtattactgtgc (SEQ ID NO:26)
	3	7	1	5	1	0	0	0 Seg4 gtgtattactgtAc (SEQ ID NO:28)
	4	11	2	4	4	1	0	0 Seq9 ATgtattactqtqc (SEO ID NO:33)
	Group		25	26	20	5	2
0	Cumulative	25	51	71	76	78

One sequence has five mismatches with sequences 2, 4, and 9; it is scored as best for 2.

Id is the number of the adapter.

Best is the number of sequence for which the identified .5 adapter was the best available.

The rest of the table shows how well the sequences match the adapters. For example, there are 10 sequences that match VHSzyl(Id=l) with 2 mismatches and are worse for all other adapters. In this sample, 90% come within 2 bases of one of the four adapters.

- 137 2018241075 03 Oct 2018

Table 13

The following list of enzymes was taken from http://rebase . neb.com/cqi-bin/asvmmlist.

I have removed the enzymes that a) cut within the recognition, b) cut on both sides of the recognition, or c) have fewer than 2 bases between recognition and closest cut site.

REBASE Enzymes

04/13/2001

10	Type II restriction enzymes with	asymmetric recognition	sequences :
	Enzymes	Recognition Sequence		Isoschizomers	Suppliers
	Aarl	CACCTGCNNNNANNNN		-	y
	Acelll	CAGCTCNNNNNNNANNNN		-	-
	Bbr7I	GAAGACNNNNNNNANNNN		-	-
15	Bbvl	GCAGCNNNNNNNNANNNN			y
	BbvII	GAAGACNNANNNN
	Bce83I	CTTGAGNNNNNNNNNNNNNN	_NNA	-	-
	BceAI	ACGGCNNNNNNNNNNNNANN		-	y
	BcefI	ACGGCNNNNNNNNNNNNAN		-	-
20	BciVI	GTATCCNNNNN NA		Bful	y
	Bfil	ACTGGGNNNN NA		BmrI	y
	BinI	GGATCNNNNAN
	BscAI	GCATCNNNNTNN		-	-
	BseRI	GAGGAGNNNNNNNN NNA		-	y
25	BsmFI	GGGACNNNNNNNNNN A NNNN		BspLUllIII	y
	BspMI	ACCTGCNNNNANNNN		Acc36I	y
	Ecil	GGCGGANNNNNNNNN NNA		-	Ύ
	Eco57I	CTGAAGNNNNNNNNNNNNNN	_NNA	BspKT5I	y
	Faul	CCCGCNNNNANN		BstFZ438I	y
30	FokI	GGATGNNNNNNNNNANNNN		BstPZ418I	y
	Gsul	CTGGAGNNNNNNNNNNNNNN	_NNA	-	y
	Hgal	GACGCNNNNNANNNNN		-	y
	HphI	GGTGANNNNNNN NA		AsuHPI	y
	MboII	GAAGANNNNNNN NA		-	y
35	Mlyl	GAGTCNNNNNA		Schl	y
	Mmel	TCCRACNNNNNNNNNNNNNNNNNN	NNA	-
	Mnll	CCTCNNNNNN NA			y
	Plel	GAGTCNNNNAN		PpsI	y
	RleAI	CCCACANNNNNNNNN NNNA		-	-
40	SfaNI	GCATCNNNNNANNNN		BspST5I	y
	SspD5I	GGTGANNNNNNNNA		-	-
	Sthl32I	CCCGNNNNANNNN		-	-
	StsI	GGATGNNNNNNNNNN A NNNN		-	-
	TaqII	GACCGANNNNNNNNN NNA,	CACCCANNNNNNNNN NNA	-
45	Tthlllll	CAARCANNNNNNNNN NNA		-	-
	UbaPI	CGAACG		-	-

The notation is ^Λ means cut the upper strand and _ means cut the lower strand. If the upper and lower strand are cut at the same place, then only ^Λ appears.

138

2018241075 03 Oct 2018

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- 139 2018241075 03 Oct 2018

Table 15: Use ofT&UE as Universal Restriction Enzyme

FokI - for dsDNA, | represents sites of cleavage sites of cleavage

5'-cacGGATGtg—nnnnnnn|nnnnnnn-3'(SEQ ID N0:15)

3'-qtqCCTACac--nnnnnnnnnnnInnn-5'(SEQ ID N0:16)

RECOG

NITion of FokI

Case I

5.. .gtgItatt-actgtgc. . Substrate ....-3' (SEQ ID N0:17) ί 0 3' -cac-ataa I tqacacq—i qtGTAGGcac\

5’- caCATCCgtg/(SEQ ID NO:18)

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5'-...gtgtattIagac-tgc..Substrate....-3' (SEQ ID NO:19) L5 i—cacataa-tctaIacq-5 ' /gtgCCTACac \cacGGATGtg-3'(SEQ ID NO:20)

Case III (Case I rotated 180 degrees) /gtgCCTACac-5' \cacGGATGta-, qtqtcttIacaq-tcc-3' Adapter (SEQ ID NO:21)

3.. .cacagaa-tgtcIagg.. substrate....-5' (SEQ ID NO:22)

Case IV (Case II rotated 180 degrees)

3'- gtGTAGGcac\ (SEQ ID NO:23) i—caCATCCgtg/

5'-gag|tctc-actqaqc

Substrate 3'-...ctc-agagItgactcg...-5'(SEQ ID NO:24)

Improved FokI adapters

FokI - for dsDNA, | represents sites of cleavage 30 Case I

Stem 11, loop 5, stem 11, recognition 17

5'-... catgtg|tatt-actgtgc.. Substrate... . - 3'

3’-qtacac-ataaItqacacq—i _rT—, qtGTAGGca cG T 5'- caCATCCgtgc C Li”pJ

- 140 2018241075 03 Oct 2018

Case II

Stem 10, loop 5, stem 10, recognition 18

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3' -...cacagaa-tgtcIaggtaagac.. substrate....-5' .5 Case IV (Case II rotated 180 degrees)

Stem 11, loop 4, stem 11, recognition 17 r^Ti

3'- gtGTAGGcacc T j—caCATCCqtqq T !0 5'-atcaaq I tctc-actqaqc ^LT^J

Substrate 3’- ...tagctc-agagItgactcg...-5'

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I sites of cleavage 5 ' -cacGAGGAGnnnnnnnnnn I nnnnn-3 ' (5 3'-qtqctcctcnnnnnnnn|nnnnnnn-5'

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NITion of BseRI

Stem 11, loop 5, stem 11, recognition 19

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C GCTGAGGAGTC-J

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141

2018241075 03 Oct 2018

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- 146 2018241075 03 Oct 2018

What happens in the top strand:

I ) site of cleavage in the upper strand (VL133-2a2*) 5'-g tet cct g | ga cag teg ate ,

(VL133-31*) 5'-g gee ttg g | ga cag aca gtc [

(VL133-2c*) 5'-g tet cct g) ga cag tea gtc .0 (VL133-lc*) 5'-g gee cca g | gg cag agg gtc

The following Extenders and Bridges all encode the AA sequence of 2a2 for codons 1 · 1 (ON_LamExl33) 5'-ccTcTgAcTgAgT gcA cAg ι ! 2 3 4 5 6 7 8 9 10 11 12

AGt geT TtA acC caA ccG geT AGT gtT AGC ggT15 >0

14 15 teC ccG g ! 2a2 >5 (ON_LamBl-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg ι ! 2 3 4 5 6 7 8 9 10 11 12

AGt geT TtA acC caA ccG geT AGT gtT AGC ggT13 14 15 teC ccG g ga cag teg at-3'! 2a2 N.B. the actual seq is the reverse complement of the one shown.

(ON_LamB2-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg 35

3 4 5 6 7 8 9 10 11 12

AGt geT TtA acC caA ccG geT AGT gtT AGC ggT13 14 15 teC ccG g ga cag aca gt-3'! 31 N.B. the actual seq is die reverse complement of the one shown.

(ON_LairiB3-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg t ! 2 3 4 5 6 7 8 9 10 11 12

AGt geT TtA acC caA ccG geT AGT gtT AGC ggTI ! 13 14 15 teC ccG g ga cag tea gt -3'! 2c N.B. the actual seq is the reverse complement of the one shown.

(ON LamB4-133) [RC] 5'-ccTcTgAcTgAgT gcA cAg 50

- 147 75 03 Oct 2018 ι

I	2	3	4 5	6	7 8	9	10 11	12
|	AGt	gcT	TtA acC	caA	ccG gcT	AGT	gtT AGC	ggT-s
5 !	13	14	15
	tcC	ccG	g gg cag	agg	gt-3' !	lc	N.B. the	actual seq is the

reverse complement of the one shown.

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- 148 2018241075 03 Oct 2018

	Table 19:	Cleavage of 75	human light	chains .
	Enzvrae	Recognition*	Nch	Ns	Planned location of
	Afel	AGCgct	0	0
	Aflll	Cttaag	0	0	HC FR3
5	Age I	Accggt	0	0
	Ascl	GGcgcgcc	0	0	After LC
	Bglll	Agatct	0	0
	BsiWI	Cgtacg	0	0
	BspDI	ATcgat	0	0
.0	BssHII	Gcgcgc	0	0
	BstBI	TTcgaa	0	0
	DralTI	CACNNNgtg	0	0
	Eagl	Cggccg	0	0
	Fsel	GGCCGGcc	0	0
.5	FspI	TGCgca	0	0
	Hpal	GTTaac	0	0
	Mfel	Caattg	0	0	HC FR1
	Mlul	Acgcgt	0	0
	Neo I	Ccatgg	0	0	Heavy chain signal
>0	Nhel	Gctagc	0	0	HC/anchor linker
	Notl	GCggccgc	0	0	In linker after HC
	Nrul	TCGcga	0	0
	Pacl	TTAATtaa	0	0
	Pmel	GTTTaaac	0	0
>5	Pmll	CACgtg	0	0
	Pvul	CGATcg	0	0
	SacII	CCGCgg	0	0
	Sail	Gtcgac	0	0
	Sfil	GGCCNNNNnggcc	0	0	Heavy Chain signal
30	Sgfl	GCGATcgc	0	0
	SnaBI	TACgta	0	0
	Stul	AGGcct	0	0
	Xbal	Tctaga	0	0	HC FR3
	Aatll	GACGTc	1	1
35	Acll	AAcgtt	1	1
	Asel	ATtaat	1	1
	Bsml	GAATGCN	1	1
	BspEI	Tccgga	1	1	HC FR1
	BstXI	CCANNNNNn tgg	1	1	HC PR2
10	Drdl	GACNNNNnngtc	1	1
	Hindlll	Aagctt	1	1
	Pci I	Acatgt	1	1
	Sapl	gaagagc	1	1
	Seal	AGTact	1	1
15	SexAI	Accwggt	1	1
	Spel	Actagt	1	1
	Tlil	Ctcgag	1	1
	XhoI	Ctcgag	1	1
	Bcgl	cgannnnnntgc	2	2
50	BlpI	GCtnagc	2	2
	BssSI	Ctcgtg	2	2
	BstAPI	GCANNNNntgc	2	2
	EspI	GCtnagc	2	2
	KasI	Ggcgcc	2	2
55	PflMI	CCANNNNntgg	2	2
	XmnI	GAANNnnttc	2	2
	ApaLl	Gtgcac	3	3	LC signal seq

- 149 2018241075 03 Oct 2018

Nael	GCCggc	3	3
	NgoMI	Gccggc	3	3
	PvuII	CAGctg	3	3
	RsrII	CGgwccg	3	3
5	BsrBI	GAGcgg	4	4
	BsrDI	GCAATGNNn	4	4
	BstZ17I	GTAtac	4	4
	EcoRI	Gaattc	4	4
	SphI	GCATGc	4	4
10	SspI	AATatt	4	4
	AccI	GTmkac .	5	5
	Bell	Tgatca	5	5
	BsmBI	Nnnnnngagacg	5	5
	BsrGI	Tgtaca	5	5
15	Dral	TTTaaa	6	6
	Ndel	CAtatg	6	6
	Swal	ATTTaaat	6	6
	BamHI	Ggatcc	7	7
	Sacl	GAGCTc	7	7
20	BciVI	GTATCCNNNNNN	8	8
	BsaBI	GATNNnnatc	8	8
	Nsil	ATGCAt	8	8
	Bspl20l	Gggccc	9	9
	Apal	GGGCCc	9	9
25	PspOOMI	Gggccc	9	9
	BspHI	Tcatga	9	11
	EcoRV	GATatc	9	9
	AhdI	GACNNNnngtc	11	11
	Bbsl	GAAGAC	11	14
30	Psil	TTAtaa	12	12
	Bsal	GGTCTCNnnnn	13	15
	Xmal	Cccggg	13	14
	Aval	Cycgrg	14	16
	Bgll	GCCNNNNnggc	14	17
35	AlwNI	CAGNNNctg	16	16
	BspMI	ACCTGC	17	19
	Xcml	CCANNNNNnnnntgg	17	26
	BstEII	Ggtnacc	19	22
	Sse8387l	CCTGCAgg	20	20
40	Avril	Cctagg	22	22
	Hindi	GTYrac	22	22
	Bsgl	GTGCAG	27	29
	Mscl	TGGcca	30	34
	BseRI	NNnnnnnnnnctcctc	32	35
45	Bsu36l	CCtnagg	35	37
	Pstl	CTGCAg	35	40
	Ecil	nnnnnnnnntccgcc	38	40
	PpuMI	RGgwccy	41	50
	Styl	Ccwwgg	44	73
50	Eco0109I	RGgnccy	46	70
	Acc65I	Ggtacc	50	51
	Kpnl	GGTACc	50	51
	Bpml	ctccag	53	82
	Avail	Ggwcc	71	124
55	* cleavage	occurs in the top	strand
	that cut palindromic sequences,	the

HC FR4

CHI

CHI

HC FR4 after the last upper-case base. For REs lower strand is cut at the symmetrical site.

- 150 2018241075 03 Oct 2018

Table 20: Cleavage of 79 human heavy chains

Enzvme	Recoanition	Nch	Ns	Planned location of site
	Afel	AGCgct	0	0
	Aflll	Cttaag	0	0	HC FR3
5	Ascl	GGcgcgcc	0	0	After LC
	BsiWI	Cgtacg	0	0
	BspDI	ATcgat	0	0
	BssHII	Gcgcgc	0	0
	Fsel	GGCCGGcc	0	0
0	Hpal	GTTaac	0	0
	Nhel	Getage	0	0	HC Linker
	Notl	GCggccgc	0	0	In linker, HC/anchor
	Nrul	TCGcga	0	0
	Nsil	ATGCAt	0	0
5	Pacl	TTAATtaa	0	0
	Pcil	Acatgt	0	0
	Pmel	GTTTaaac	0	0
	Pvul	CGATcg	0	0
	RsrII	CGgwccg	0	0
0	Sapl	gaagagc	0	0
	Sfil	GGCCNNNNnggcc	0	0	HC signal seq
	Sgfl	GCGATcgc	0	0
	Swal	ATTTaaat	0	0
	Acll	AAcgtt	1	1
5	Age I	Accggt	1	1
	Asel	ATtaat	1	1
	Avril	Cctagg	1	1
	BsmI	GAATGCN	1	1
	BsrBI	GAGcgg	1	1
0	BsrDI	GCAATGNNn	1	1
	Dral	TTTaaa	1	1
	FspI	TGCgca	1	1
	Hindlll	Aagctt	1	1
	Mfel	Caattg	1	1	HC FRl
5	Nael	GCCggc	1	1
	NgoMI	Gccggc	1	1
	Spel	Actagt	1	1
	Acc65I	Ggtacc	2	2
	BstBI	TTcgaa	2	2
0	Kpnl	GGTACc	2	2
	Mlul	Acgcgt	2	2
	Neo I	Ccatgg	2	2	In HC signal seq
	Ndel	CAtatg	2	2	HC FR4
	Pmll	CACgtg	2	2
5	Xcml	CCANNNNNnnnntgg	2	2
	Bcgl	cgannnnnntgc	3	3
	Bell	Tgatca	3	3
	Bgll	GCCNNNNnggc	3	3
	BsaBI	GATNNnnatc	3	3
0	BsrGI	Tgtaca	3	3
	SnaBI	TACgta	3	3
	Sse8387I	CCTGCAgg	3	3
	ApaLI	Gtgcac	4	4	LC Signal/FRl
	BspHI	Tcatga	4	4
5	BssSI	Ctcgtg	4	4
	Psil	TTAtaa	4	5

- 151 2018241075 03 Oct 2018

SphI	GCATGc	4	4
AhdI	GACNNNnngtc	5	5
BspEI	Tccgga	5	5	HC FR1
Mscl	TGGcca	5	5
Sacl	GAGCTc	5	5
Seal	AGTact	5	5
SexAI	Accwggt	5	6
SspI	AATatt	5	5
Tlil	Ctcgag	5	5
Xhol	Ctegag	5	5
Bbsl	GAAGAC	7	8
BstAPI	GCANNNNntgc	7	8
BstZ17I	GTAtac	7	7
EcoRV	GATatc	7	7
EcoRI	Gaattc	8	8
BlpI	GCtnagc	9	9
Bsu36I	CCtnagg	9	9
Drain	CACNNNgtg	9	9
EspI	GCtnagc	9	9
Stul	AGGcct	9	13
Xbal	Tetaga	9	9	HC FR3
Bspl20I	Gggccc	10	11	CHI
Apal	GGGCCc	10	11	CHI
PspOOMI	Gggccc	10	11
BciVI	GTATCCNNNNNN	11	11
Sail	Gtcgac	11	12
Drdl	GACNNNNnngtc	12	12
KasI	Ggegee	12	12
Xmal	Cccggg	12	14
Bglll	Agatet	14	14
Hindi	GTYrac	16	18
BamHI	Ggatcc	17	17
PflMI	CCANNNNntgg	17	18
BsntBI	Nnnnnngagacg	18	21
BstXI	CCANNNNNntgg	18	19	HC FR2
Xmnl	GAANNnnttc	18	18
SacII	CCGCgg	19	19
Pstl	CTGCAg	20	24
PvuII	CAGetg	20	22
Aval	Cycgrg	21	24
Eagl	Cggccg	21	22
Aatll	GACGTc	22	22
BspMI	ACCTGC	27	33
AccI	GTmkac	30	43
Styl	Ccwwgg	36	49
AlwNI	CAGNNNctg	38	44
Bsal	GGTCTCNnnnn	38	44
PpuMI	RGgwccy	43	46
Bsgl	GTGCAG	44	54
BseRI	NNnnnnnnnnctcctc	48	60
Ecil	nnnnnnnnntccgcc	52	57
BstEII	Ggtnacc	54	61	HC Fr4, 47/79 have one
EcoO!09l	RGgnccy	54	86
Bpml	ctccag	60	121
Avail	Ggwee	71	140

- 152 2018241075 03 Oct 2018

Table 21: MALIA3, annotated ! MALIA3 9532 bases

1

aat

get

act

act

att

agt

aga att gat

gcc

acc

ttt

tea

get

ege

gcc

5

! gene ii continued

49

cca

aat

gaa

aat

ata

get

aaa cag gtt

att

gac

cat

ttg

cga

aat

gta

97

tct

aat

ggt

caa

act

aaa

tct act cgt

teg

cag

aat

tgg

gaa

tea

act

145

gtt

aca

tgg

aat

gaa

act

tec aga cac

cgt

act

tta

gtt

gca

tat

tta

193

aaa

cat

gtt

gag

eta

cag

cac cag att

cag

caa

tta

age

tct

aag

cca

0

241

tcc

gca

aaa

atg

acc

tct

tat caa aag

gag

caa

tta

aag

gta

etc

tct

289

aat

cct

gac

ctg

ttg

gag

ttt get tec

ggt

ctg

gtt

ege

ttt

gaa

get

337

cga

att

aaa

acg

cga

tat

ttg aag tct

ttc

ggg

ctt

cct

ctt

aat

ctt

385

ttt

gat

gca

ate

ege

ttt

get tct gac

tat

aat

agt

cag

ggt

aaa

gac

433

ctg

att

ttt

gat

tta

tgg

tea ttc teg

ttt

tct

gaa

ctg

ttt

aaa

gca

5

481

ttt

gag

ggg

gat

tea

ATG

aat att tat

gac

gat

tec

gca

gta

ttg

gac

1

rbs:

Start gene x, ii continues

529

get

ate

cag

tct

aaa

cat

ttt act att

acc

ccc

tct

ggc

aaa

act

tct

577

ttt

gca

aaa

gcc

tct

ege

tat ttt ggt

ttt

tat

cgt

cgt

ctg

gta

aac

625

gag

ggt

tat

gat

agt

gtt

get ctt act

atg

cct

cgt

aat

tec

ttt

tgg

0

673

cgt

tat

gta

tct

gca

tta

gtt gaa tgt

ggt

att

cct

aaa

tct

caa

ctg

721

atg

aat

ctt

tct

acc

tgt

aat aat gtt

gtt

ccg

tta

gtt

cgt

ttt

att

769

aac

gta

gat

ttt

tct

tec

caa cgt cct

gac

tgg

tat

aat

gag

cca

gtt

817

ctt

aaa

ate

gca

TAA

!

End

X &

II

5

832

ggtaattca ca

1

Ml

E5

Q10

T15

843

ATG

att

aaa

gtt

gaa

att

aaa cca tct

caa

gee

caa

ttt

act

act

cgt

0

1

Start gene V

1

S17

S20

P25

E30

891

tct

ggt

gtt

tct

cgt

cag

ggc aag cct

tat

tea

ctg

aat

gag

cag

ctt

1

V35

E4 0

V4 5

5

939

tgt

tac

gtt

gat

ttg

ggt

aat gaa tat

ccg

gtt

ctt

gtc

aag

att

act

1

D50

A55

L60

987

Ctt

gat

gaa

ggt

cag

cca

gcc tat geg

cct

ggt

cTG

TAC

Acc

gtt

cat

BsrGI.

0

1 1035 l 1

L65 ctg tec

tct ttc

aaa P85

V7 0 gtt

ggt K87

cag ttc ggt end of V

S75 tec

ctt

atg

att gac

R80 cgt

5

1083 j

ctg

ege

etc

gtt

ccg

get

aag

TAA

C

1108

ATG

gag

cag

gtc

geg

gat

ttc

gac

aca att

tat

cag

geg

atg

Start gene VII

0	1150	ata	caa	ate	tee gtt gta ctt tgt ttc geg ctt	ggt	ata ate
				VII and IX overlap. ..... S2 V3 L4 V5		S10
	1192	get	ggg	ggt	caa agA TGA gt gtt tta gtg tat End VII	tct	ttc gcc tct ttc
5					I start IX
		L13		W15	G20		T25

1242 tta ggt tgg tgc ctt cgt agt ggc att acg tat ttt acc cgt tta atg gaa

- 153 2018241075 03 Oct 2018

1293 act tcc tc

.... stop of IX, IX and VIII overlap by four bases 1301 ATG aaa aag tet tta gtc etc aaa gee tet gta gee gtt get acc etc

Start signal sequence of viii.

1349	gtt	ccg atg	ctg	tet	ttc	get	get gag ggt mature VIII	gac gat --->	ccc gca	aaa	gcg
1397	gee	ttt aac	tcc	ctg	caa	gee	tea gcg acc	gaa tat	ate ggt	tat	gcg
1445	tgg	gcg atg	gtt	gtt	gtc	att
1466	gtc	ggc gca	act	ate	ggt	ate	aag ctg ttt	aag
1499	aaa	ttc acc	teg	aaa	gca	! 1515

........... -35

1517 age tga taaaccgat acaattaaag gctccttttg

..... -10

1552 gagccttttt ttttGGAGAt ttt ! S.D. underlined

1575

caac

<— M GTG

K aaa

III K aaa

signal L L tta tta

sequence

I att

P cct

L tta

v gtt !

! 1611

---->

F ttc

A gca

V

P

F

Y

S H

S

A

Q

1612

gtt

cct

ttc

tat

tet cac

aGT

gcA

Cag

tCT

ApaLI...

	1642	GTC	GTG	ACG	CAG	CCG	CCC	TCA	GTG	TCT
30		AGG	GTC	ACC	ATC	TCC	TGC	ACT	GGG	AGC
	1	BstEII...
	1729	GGT	TAT	GAT	GTA	CAC	TGG	TAC	CAG	CAG
	1777	CTC	CTC	ATC	TAT	GGT	AAC	AGC	AAT	CGG
	1825	TTC	TCT	GGC	TCC	AAG	TCT	GGC	ACC	TCA
35	1870	GGG	CTC	CAG	GCT	GAG	GAT	GAG	GCT	GAT
	1900	TAC	TGC	CAG	TCC	TAT	GAC	AGC	AGC	CTG
	1930	GGC	CTT	TAT	GTC	TTC	GGA	ACT	GGG	ACC
	1969	CTA	GGT	CAG	CCC	AAG	GCC	AAC	CCC	ACT
40	2002	CTG	TTC	CCG	CCC	TCC	TCT	GAG	GAG	CTC
	2050	GTG	TGT	CTG	ATC	AGT	GAC	TTC	TAC	CCG
	2098	AAG	GCA	GAT	AGC	AGC	CCC	GTC	AAG	GCG
	2146	TCC	AAA	CAA	AGC	AAC	AAC	AAG	TAC	GCG
	2194	ACG	CCT	GAG	CAG	TGG	AAG	TCC	CAC	AGA
45	2242	CAT	GAA	GGG	AGC	ACC	GTG	GAG	AAG	ACA
	2290	TAA	TAA	ACCG CCTCCACCGG	GCGCGCCAAT
	1						Ascl.
	I	PelB signal
50	I	M	K	Y	L	L	P	T	A	A
	2343	ATG	AAA	TAC	CTA	TTG	CCT	ACG	GCA	GCC
	1	16	17	18	19	20		21	22
	1	A	A	Q	P	A		M	A
55	2388	qcG GCC cag ccG GCC		atq	qcc

A

GGG	GCC	CCA	GGG	CAG
AGC	TCC	AAC	ATC	GGG	GCA
CTT	CCA	GGA	ACA	GCC	CCC
CCC	TCA	GGG	GTC	CCT	GAC
GCC	TCC	CTG	GCC	ATC	ACT
TAT
AGT
AAG	GTC	ACC	GTC
BstEII...
GTC	ACT
CAA	GCC	AAC	AAG	GCC	ACA
GGA	GCT	GTG	ACA	GTG	GCC
GGA	GTG	GAG	ACC	ACC	ACA
GCC	AGC	AGC	TAT	CTG	AGC
AGC	TAC	AGC	TGC	CAG	GTC
GTG	GCC	CCT	ACA	GAA	TGT

CGA

CTG

TCA

TCTATTTCAA GGAGACAGTC ATA

->

L

Sfil.

NgoMI...(1/2)

Neol..

- 154 2018241075 03 Oct 2018

FR1(DP47/V3-23)--------------23 24 25 26 27 28 29 30

1

E

V

Q

L

L

E

s

G

5

2409

gaa1gtt|CAA ί TTG1tta|gag|tet1ggt I

t

1

Mfel

1

ITD 1

• r i\J. — — -

1

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

0

1

G

G

L

V

Q

P

G

G

S

L

R

L

S

C

A

2433

I ggc

iggt

I ett

gtt |

cag

cct 3

ggtIggt

1 tet

tta I

cgt

ett

tet

1 tgc

Igct |

PP 1

CDR1 53

—

ΕΈ2-

1

46

47

48

49

50

51

52

54

55

56

57

58

59

60

5

1

A

S

G

F

T

F

S

S

Y

A

M

S

W

V

R

2478

I get

ITCC

I GGA

ttc I

act

ttc I

tetItCG

|TAC|

Get|atg

tet

tgg

1gttIcgC|

!

I BspEI

1 BsiWII

1BstXI

...CDR2

0

1

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

1

Q

A

P

G

K

G

L

E

W

V

S

A

I

S

G

2523

1CAaI get|ccT

GGt!

aaa

ggti

ttgI gag

1 tgg

gtt |

tet|get

1 ate

1 tet

iggt I

1

BstXI

I

5

1

CDR2

|___

FR3---

1

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

1

S

G

G

S

T

Y

Y

A

D

S

V

K

G

R

F

2568 1

Itctlggt

l ggc

agt |

act

tac

tat

I get|gac

tee

gtt

aaa

i ggt

1 ege

1 ttc |

0

l

1

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

1

T

I

S

R

D

N

S

K

N

T

L

Y

L

Q

M

2613

1 act

I ate

|TCT

AGA |

gac

aac1tet1aag

Iaat|act

etc

tac

1 ttg

1 cag

|atg|

5

1 1

I Xbal

0

1 I 2658 1

E I\J -- 106 107 108

109 R AGg| 1

110 A get

111 E gag

112 D gac

113 T aCT 1

114 A 1 GCA Pstl

115 116

117 Y tat

118 C tgc

' 1 119 120

N I aac

S L agC|TTAI lAflll

V Gtc 1

Y tac |

A get

K aaa 1

1

..CDR3..

-FR4-

i

121

122 123

124

125

126

127

128

129

130

131

132

133

134

135

5

1

D

Y E

G

T

G

Y

A

F

D

I

W

G

Q

G

2703

1 gac

tat Igaa|

ggtl

act

ggt|tat

get

IttcIgaCIATA|TGg|ggt|caa

ggti

1

1

Ndel

1(1/4)

1

-FR4

—>

0

i

136

137 138

139

140

141

142

t

T

Μ V

T

V

S

S

2748

I act

atGIGTCI

ACCI

gtc

tet

agt

BstEII I ! From BstEII onwards, pV323 is same as pCESl, except as noted.

'5 i BstEII sites may occur in light chains; not likely to be unique in final ! vector.

- 155 2018241075 03 Oct 2018

	1	143	144	145	146 147 148	149	150	151	152
	1	A	S	T	K G P	S	V	F	P
	2769	gcc	tcc	acc	aaG GGC CCa	teg	GTC	TTC	ccc
5	t				Bspl20I.		Bbsl		(2/2)

Apal....

153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 LAPS SKSTS GGTAAL

2799 ctg gca ccC TCC TCc aag age acc tet ggg ggc aca gcg gcc ctg BseRI. . . (2/2)

1

168

169

170

171

172

173

174

175

176

177

178

179

180

181

182

1

G

C

L

V

K

D

Y

F

P

E

P

V

T

V

S

15

2844

ggc

tgc

ctg

GTC

AAG

GAC

TAC

TTC

CCc

gaA

CCG

GTg

aeg

gtg

teg

1

Age I.

1

183

184

185

186

187

188

189

190

191

192

193

194

195

196

197

!

w

N

S

G

A

L

T

S

G

V

H

T

F

P

A

20

2889

tgg

aac

tea

GGC

GCC

ctg

acc

age

ggc

gtc

cac

acc

ttc

ccg

get

1

Kasl

(1/4)

1

198

199

200

201

202

203

204

205

206

207

208

209

210

211

212

V

L

Q

S

S

G

L

Y

S

L

S

S

V

V

T

25

2934

gtc

eta

cag

tet

age

GGa

etc

tac

tcc

etc

age

age

gta

gtg

acc

1

(Bsu36I

...)(knocked

out)

I

213

214

215

216

217

218

219

220

221

222

223

224

225

226

227

1

V

P

S

S

S

L

G

T

Q

T

Y

I

C

N

V

30

2979

gtg

ccC

tet

tet

age

tTG

Ggc

acc

cag

acc

tac

ate

tgc

aac

gtg

1

(BstXI..

. )N

B. destruction

of BstXI

& Bpml

1

228

229

230

231

232

233

234

235

236

237

238

239

240

241

242

t

N

H

K

P

S

N

T

K

V

D

K

K

V

E

P

35

3024 t

aat

cac

aag

CCC

age

aac

acc

aag

gtg

gac

aag

aaa

gtt

gag

ccc

1

243

244

245

1

K

S

C

A

A

A

H

H

H

H

H

H

S

A

3069

aaa

tet

tgt

GCG

GCC

GCt

cat

cac

cac

cat

cat

cac

tet

get

40

1

Notl...

1

E

Q

K

L

I

S

E

E

D

L

N

G

A

A

3111

gaa

caa

aaa

etc

ate

tea

gaa

gag

gat

ctg

aat

ggt

gcc

gca

45

1

1

D

I

N

D

D

R

M

A ί

G

A

3153

GAT

ATC

aac

gat

gat

cgt

atg

get AGC

ggc

gee

sites .

rEK cleavage site. EcoRV..

Nhel..

Kasl...

Domain 1 -----------------------------------AETVESCLA

3183 get gaa act gtt gaa agt tgt tta gca

KPHTEISF 3210 aaa ccc cat aca gaa aat tea ttt

TNVW KDDKT 3234 aCT AAC GTC TGG AAA GAC GAC AAA Act

- 156 2018241075 03 Oct 2018 .5 :o :5 ιο iO

1

L

D

R

Y

A

N

Y

E

G

C

L

w

N

A

T

G

V

3261

tta

gat

cgt

tac

get

aac

tat

gag

ggt

tgt

ctg

tgG AAT BsmI

GCt

aca

ggc

gtt

V

V

C

T

G

D

E

T

Q

c

Y

G

T

W

V

P

I

3312 1 1 3363

gta G ggg

gtt L ctt

tgt A get

act I ate

ggt P cct

GAC E gaa

GAA N aat

ACT

CAG

TGT

TAC

GGT ACA

TGG

GTT

cct

att

! LI linker ---
! E	G	G	G	s	E	G	G	G	s
3384 gag 1	ggt	ggt	ggc	tct	gag	ggt	ggc	ggt	tct
! E	G	G	G	s	E	G	G	G	T
3414 gag	ggt	ggc	ggt	tct	gag	ggt	ggc	ggt	act

t ! Domain 2

3444

aaa

cct

cct

gag

tac

ggt

gat

aca

cct

att

ccg

ggc

tat

act

tat

ate

aac

3495

cct

etc

gac

ggc

act

tat

ccg

cct

ggt

act

gag

caa

aac

ccc

get

aat

cct

3546

aat

cct

tct

ctt

GAG GAG BseRI

tct

cag

cct

ctt

aat

act

ttc

atg

ttt

cag

aat

3597

aat

agg

ttc

ega

aat

agg

cag

ggg

gca

tta

act

gtt

tat

aeg

ggc

act

3645

gtt

act

caa

ggc

act

gac

ccc

gtt

aaa

act

tat

tac

cag

tac

act

cct

3693

gta

tea

tea

aaa

gcc

atg

tat

gac

get

tac

tgg

aac

ggt

aaa

ttc

AGA

AlwNI

3741	GAC TGc AlwNI	get ttc cat	tct	ggc ttt	aat	gaa	gat	cca ttc gtt	tgt gaa
3789	tat caa	ggc caa teg	tct	gac ctg	cct	caa	cct	cct gtc aat	get

3834 ggc ggc ggc tct ! start L2 ----------

3846 ggt ggt ggt tct
3858	ggt	ggc	ggc	tct
3870	gag	ggt	ggt	ggc	tct	gag	ggt	ggc	ggt	tct
3900	gag	ggt	ggc	ggc	tct	gag	gga	ggc	ggt	tee
3930	ggt	ggt	ggc	tct	ggt		! end	. L2

-U -J s

G

D

F

D

Y

E

K

M

A

N

A

N

K

G

A

3945

tee

ggt

gat

ttt

gat

tat

gaa

aag

atg

gca

aac

get

aat

aag

ggg

get

M

T

E

N

A

D

E

N

A

L

Q

S

D

A

K

G

3993

atg

acc

gaa

aat

gcc

gat

gaa

aac

geg

eta

cag

tct

gac

get

aaa

ggc

K

L

D

S

V

A

T

D

Y

G

A

A

I

D

G

F

4041

aaa

ctt

gat

tct

gtc

get

act

gat

tac

ggt

get

get

ate

gat

ggt

ttc

I

G

D

V

S

G

L

A

N

G

N

G

A

T

G

D

4089

att

ggt

gac

gtt

tee

ggc

ctt

get

aat

ggt

aat

ggt

get

act

ggt

gat

F

A

G

S

N

s

Q

M

A

Q

V

G

D

G

D

' N

4137

ttt

get

ggc

tct

aat

tee

caa

atg

get

caa

gtc

ggt

gac

ggt

gat

aat

S

P

L

M

N

N

F

R

Q

Y

L

P

S

L

P

Q

4185

tea

cct

tta

atg

aat

aat

ttc

cgt

caa

tat

tta

cct

tee

etc

cct

caa

)5

- 157 2018241075 03 Oct 2018

4233

S teg

V gtt

E gaa

c tgt

R ege

P cct

F ttt

V gtc

F ttt

S age

A get

G ggt

K aaa

P cca

Y tat

E gaa

4281

F ttt

s tet

I att

D gat

C tgt

D gac

K aaa

I ata

N aac

L tta

F ttc

R cgt End

Domain

3

4317

G V F A F ggt gtc ttt geg ttt start transmembrane

L L ett tta segment

Y tat

V gtt

A gee

T acc

F ttt

M atg

Y tat

V gta

F14 ttt

4365

S tet

T aeg

F ttt

A get

N aac

I ata

L ctg

4386

R cgt

N aat

K aag

E gag

S tet

TAA

ί stop

of iii

Intracellular anchor.

Ml

P2 V

L

L5

G

I

P

L

L10

L

R

F

L

G15

4404 tc ATG

cca gtt

ett

ttg

ggt

att

ccg

tta

tta

ttg

cgt

ttc

etc

ggt

Start VI

4451

ttc

ett

ctg

gta

act

ttg

ttc

ggc

tat

ctg

ett

act

ttt

ett

aaa

aag

4499

ggc

ttc

ggt

aag

ata

get

att

get

att

tea

ttg

ttt

ett

get

ett

att

4547

att

ggg

ett

aac

tea

att

ett

gtg

ggt

tat

etc

tet

gat

att

age

get

4595

caa

tta

ccc

tet

gac

ttt

gtt

cag

ggt

gtt

cag

tta

att

etc

ccg

tet

4643

aat

geg

ett

ccc

tgt

ttt

tat

gtt

att

etc

tet

gta

aag

get

get

att

4691

ttc

att

ttt

gac

gtt

aaa

caa

aaa

ate

gtt

tet

tat

ttg

gat

tgg

gat

!

! Ml A2 V3 F5 L10 G13

4739 aaa TAA t ATG get gtt tat ttt gta act ggc aaa tta ggc tet gga ! end VI Start gene I

35

1 I 4785 I

14 K aag

15 T aeg

16 L etc

17 V gtt

18 S age

19 V gtt

20 G ggt

21 K aag

22 I att

23 Q cag

24 D gat

25 K aaa

26 I att

27 V gta

28 A get

1

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

40

1

G

C

K

I

A

T

N

L

D

L

R

L

Q

N

L

4830 !

ggg

tgc

aaa

ata

gca

act

aat

ett

gat

tta

agg

ett

caa

aac

etc

1

44

45

46

47

48

49

50

51

52

53

54

55

56

57

58

1

P

Q

V

G

R

F

A

K

T

P

R

V

L

R

I

45

4875 1

ccg

caa

gtc

ggg

agg

ttc

get

aaa

aeg

cct

ege

gtt

ett

aga

ata

1

59

60

61

62

63

64

65

66

67

68

69

70

71

72

7 3

1

P

D

K

P

S

I

S

D

L

L

A

I

G

R

G

4920

ccg

gat

aag

cct

tet

ata

tet

gat

ttg

ett

get

att

ggg

ege

ggt

50

1

1

74

75

76

77

78

79

80

81

82

83

84

85

86

87

88

1

N

D

S

Y

D

E

N

K

N

G

L

L

V

L

D

4965 I

aat

gat

tee

tac

gat

gaa

aat

aaa

aac

ggc

ttg

ett

gtt

etc

gat

55

ί

89

90

91

92

93

94

95

96

97

98

99

100

101

102

103

I

E

C

G

T

W

F

N

T

R

S

W

N

D

K

E

5010

gag

tgc

ggt

act

tgg

ttt

aat

acc

cgt

tet

tgg

aat

gat

aag

gaa

- 158 2018241075 03 Oct 2018

ί 1 5055 (

104 R aga

105 Q cag

106 P ccg

107 I att

108 I att

109 D gat

110 W tgg

111 F ttt

112 L eta

113 H cat

114 A get

115 R cgt

116 K aaa

117 L tta

118 G gga

5

1

119

120

121

122

123

124

125

126

127

128

129

130

131

132

133

1

W

D

I

I

F

L

V

Q

D

L

S

I

V

D

K

5100 j

tgg

gat

att

att

ttt

ctt

gtt

cag

gac

tta

tet

att

gtt

gat

aaa

1

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

0

t

Q

A

R

S

A

L

A

E

H

V

V

Y

C

R

R

5145 f

cag

geg

cgt

tet

gca

tta

get

gaa

cat

gtt

gtt

tat

tgt

cgt

cgt

1

149

150

151

152

153

154

155

156

157

158

159

160

161

162

163

1

L

D

R

I

T

L

P

F

V

G

T

L

Y

S

L

.5

5190 |

ctg

gac

aga

att

act

tta

cct

ttt

gtc

ggt

act

tta

tat

tet

ctt

J

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

{

I

T

G

S

K

M

P

L

P

K

L

H

V

G

V

:o

5235 |

att

act

ggc

teg

aaa

atg

cct

ctg

cct

aaa

tta

cat

gtt

ggc

gtt

1

179

180

181

182

183

184

185

186

187

188

189

190

191

192

193

1

V

K

Y

G

D

S

Q

L

S

P

T

V

E

R

W

5280 I

gtt

aaa

tat

ggc

gat

tet

caa

tta

age

cct

act

gtt

gag

cgt

tgg

.’5

1

194

195

196

197

198

199

200

201

202

203

204

205

206

207

208

1

L

Y

T

G

K

N

L

Y

N

A

Y

D

T

K

Q

5325 |

ctt

tat

act

ggt

aag

aat

ttg

tat

aac

gca

tat

gat

act

aaa

cag

I

209

210

211

212

213

214

215

216

217

218

219

220

221

222

223

10

I

A

F

S

S

N

Y

D

S

G

V

Y

S

Y

L

T

5370 |

get

ttt

tet

agt

aat

tat

gat

tcc

ggt

gtt

tat

tet

tat

tta

aeg

I

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

1

P

Y

L

S

H

G

R

Y

F

K

P

L

N

L

G

15

5415 (

cct

tat

tta

tea

cac

ggt

egg

tat

ttc

aaa

cca

tta

aat

tta

ggt

1

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

I

Q

K

M

K

L

T

K

I

Y

L

K

K

F

S

R

10

5460 1

cag

aag

atg

aaa

tta

act

aaa

ata

tat

ttg

aaa

aag

ttt

tet

ege

1

254

255

256

257

258

259

260

261

262

263

264

265

266

267

268

1

V

L

C

L

A

I

G

F

A

S

A

F

T

Y

S

5505 ί

gtt

ctt

tgt

ctt

geg

att

gga

ttt

gca

tea

gca

ttt

aca

tat

agt

15

1

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

1

Y

I

T

Q

P

K

P

E

V

K

K

V

V

S

Q

5550 l

tat

ata

acc

caa

cct

aag

ccg

gag

gtt

aaa

aag

gta

gtc

tet

cag

1

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

50

1

T

Y

D

F

D

K

F

T

I

D

S

S

Q

R

L

5595 I

acc

tat

gat

ttt

gat

aaa

ttc

act

att

gac

tet

tet

cag

cgt

ctt

1

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

1

N

L

S

Y

R

Y

V

F

K

D

S

K

G

K

L

55

5640 1

aat

eta

age

tat

ege

tat

gtt

ttc

aag

gat

tet

aag

gga

aaa

TTA Pacl

I

- 159 2018241075 03 Oct 2018

314 315 316 317 318 319 INS DDL

320

Q

321 322 323 324 K Q G Y

325 326 327 328 S L T Y

5685 ATT Pacl	AAt	age	gac	gat	tta	cag	aag	caa	ggt	tat	tea	etc	aca	tat
329	330	331	332	333	334	335	336	337	338	339	340	341	342	343

ilDLCTVS IKKGNSNE iv Ml K

5730 att gat tta tgt act gtt tcc att aaa aaa ggt aat tea aAT Gaa

Start IV

	1 ! i ! iv	344 I L3	345 V L	346 K N5	347 C V	348 349
N 17	.End of I
N	F V10
15	5775	att	gtt	aaa	tgt	aat	TAA T	TTT GTT

IV continued.....

5800 5848 5896

ttc aat ggc

ttg aat gaa

atg teg tcc

ttt cct gtt

gtt ctg att

tea ege gtt

tea gat tet

tet ttt ccc

tet gta gat

ttt act gta

get tgg aaa

cag tat ggt

gta tea act

att aag gtt

gaa caa act

atg tea gta

20

5944

tat

tea

tet

gac

gtt

aaa

cct

gaa

aat

eta

ege

aat

ttc

ttt

att

tet

5992

gtt

tta

cgt

get

aat

aat

ttt

gat

atg

gtt

ggt

tea

att

cct

tcc

ata

6040

att

cag

aag

tat

aat

cca

aac

aat

cag

gat

tat

att

gat

gaa

ttg

cca

6088

tea

tet

gat

aat

cag

gaa

tat

gat

gat

aat

tcc

get

cct

tet

ggt

ggt

6136

ttc

ttt

gtt

ccg

caa

aat

gat

aat

gtt

act

caa

act

ttt

aaa

att

aat

25

6184

aac

gtt

egg

gca

aag

gat

tta

ata

ega

gtt

gtc

gaa

ttg

ttt

gta

aag

6232

tet

aat

act

tet

aaa

tcc

tea

aat

gta

tta

tet

att

gac

ggc

tet

aat

6280

eta

tta

gtt

gtt

TCT

gca

cct

aaa

gat

att

tta

gat

aac

ett

cct

caa

ApaLI removed

6328

ttc

ett

tet

act

gtt

gat

ttg

cca

act

gac

cag

ata

ttg

att

gag

ggt

30

6376

ttg

ata

ttt

gag

gtt

cag

caa

ggt

gat

get

tta

gat

ttt

tea

ttt

get

6424

get

ggc

tet

cag

cgt

ggc

act

gtt

gca

ggc

ggt

gtt

aat

act

gac

ege

6472

etc

acc

tet

gtt

tta

tet

tet

get

ggt

ggt

teg

ttc

ggt

att

ttt

aat

6520

ggc

gat

gtt

tta

ggg

eta

tea

gtt

ege

gca

tta

aag

act

aat

age

cat

6568

tea

aaa

ata

ttg

tet

gtg

cca

cgt

att

ett

aeg

ett

tea

ggt

cag

aag

35

6616

ggt

tet

ate

tet

gtT

GGC

CAg

aat

gtc

cct

ttt

att

act

ggt

cgt

gtg

Mscl_

6664 6712 6760

act caa ggt

ggt aat aat

gaa gta att

tet ggt gtt

gee att ctg

aat tcc gat

gta atg att

aat age acc

aat gtt age

cca ttt aag

ttt cct gee

cag gtt gat

aeg gca agt

att atg ttg

gag get agt

cgt ggc tet

40

6808

tet

act

cag

gca

agt

gat

gtt

att

act

aat

caa

aga

agt

att

get

aca

6856

aeg

gtt

aat

ttg

cgt

gat

gga

cag

act

ett

tta

etc

ggt

ggc

etc

act

6904

gat

tat

aaa

aac

act

tet

caa

gat

tet

ggc

gta

ccg

ttc

ctg

tet

aaa

6952

ate

cct

tta

ate

ggc

etc

ctg

ttt

age

tee

ege

tet

gat

tcc

aac

gag

7000

gaa

age

aeg

tta

tac

gtg

etc

gtc

aaa

gca

acc

ata

gta

ege

gee

ctg

45

7048

TAG

cggcgcatt

End IV

7060 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct

7120 gcccgctcct ttcgctttct tcccttcctt tctcgccacg

7180 agetetaaat cgggggctcc ctttagggtt ccgatttagt

7240 caaaaaactt gatttgggtg atggttCACG TAGTGggcca

Drain_

7300 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga Drdl_

7360 aacactcaac cctatctcgg getattettt tgatttataa

7420 accaccatca aacaggattt tcgcctgctg gggcaaacca

7480 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc

PvuII.

acacttgcca ttcGCCGGCt

NgoMI_ getttaegge tcgccctgat ctcttgttcc gggattttgc gcgtggaccg cCGTCTCact

BsmBI.

gcgccctagc ttccccgtca acctcgaccc agacggtttt aaactggaac egatttegga cttgctgcaa ggtgaaaaga

7540 aaaaccaccc tGGATCC BamHI

AAGCTT Hindlll (½)

- 160 2018241075 03 Oct 2018 ! Insert carrying bla gene

7563 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc

7600 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct ! BciVI

7660 gataaatgct tcaataatat tgaaaaAGGA AGAgt ! RBS.?...

! Start bla gene

7695 ATG agt att caa cat ttc cgt gtc gcc ctt att ccc ttt ttt gcg gca ttt 7746 tgc ctt cct gtt ttt get cac cca gaa aeg ctg gtg aaa gta aaa gat get

7797 gaa gat cag ttg ggC gCA CGA Gtg ggt tac ate gaa ctg gat etc aac age ! BssSI...

! ApaLl removed

7848 ggt aag ate ctt gag agt ttt ege ccc gaa gaa cgt ttt cca atg atg age 7899 act ttt aaa gtt ctg eta tgt cat aca eta tta tee cgt att gac gcc ggg

7950 caa gaG CAA CTC GGT CGc egg gcg egg tat tet cag aat gac ttg gtt gAG ! BegI_ Seal

8001 TAC Tea cca gtc aca gaa aag cat ctt aeg gat ggc atg aca gta aga gaa ! Scal_

8052 tta tgc agt get gcc ata acc atg agt gat aac act gcg gcc aac tta ctt

8103 ctg aca aCG ATC Gga gga ccg aag gag eta acc get ttt ttg cac aac atg ! Pvul_

8154 ggg gat cat gta act ege ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc

8205 ata cca aac gac gag cgt gac acc aeg atg cct gta gca atg cca aca aeg

8256 tTG CGC Aaa eta tta act ggc gaa eta ctt act eta get tee egg caa caa ! FspI....

ι

8307 tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ctt ctg ege teg 8358 GCC ctt ccG GCt ggc tgg ttt att get gat aaa tet gga gcc ggt gag cgt ! Bgll_

8409 gGG TCT Cgc ggt ate att gca gca ctg ggg cca gat ggt aag ccc tee cgt ! Bsal_

8460 ate gta gtt ate tac aeG ACg ggg aGT Cag gca act atg gat gaa ega aat ! AhdI_

8511 aga cag ate get gag ata ggt gcc tea ctg att aag cat tgg TAA ctgt ! stop

8560 cagaccaagt ttactcatat ataetttaga ttgatttaaa acttcatttt taatttaaaa

8620 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt

8680 cgttccactg tacgtaagac cccc

8704 AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct ! Hindlll Sail..

! (2/2) Hindi

8740 ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgegatett ι

8790 CCTGAGG ! Bsu36I_

8797 ccgat actgtcgtcg tcccctcaaa ctggcagatg

8832 cacggttacg atgcgcccat ctacaccaac gtaacctatc ccattacggt caatccgccg

8892 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc

8952 tggetacagg aaggccagac gegaattatt tttgatggcg ttcctattgg ttaaaaaatg <0 9012 agctgattta acaaaaattt aaegegaatt ttaacaaaat attaacgttt acaATTTAAA ! Swal...

9072 Tatttgetta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat

RBS?

9131

ATG att gac

atg

eta

gtt

tta

ega

tta

ccg

ttc

ate gat

tet

ctt

gtt

tgc

Start gene '

II

9182

tee aga etc

tea

ggc

aat

gac

ctg

ata

gcc

ttt

gtA GAT

CTc

tea

aaa

ata

Bgll I

9233

get acc etc

tee

ggc

atg

aat

tta

tea

get

aga

aeg gtt

gaa

tat

cat

att

»5

- 161 75 03 Oct 2018

9284

gat

ggt

gat

ttg

act

gtc

tec

ggc

ett

tet

cac

cct

ttt

gaa

tet

tta

cct

9335

aca

cat

tac

tea

ggc

att

gca

ttt

aaa

ata

tat

gag

ggt

tet

aaa

aat

ttt

9386

tat

cct

tgc

gtt

gaa

ata

aag

get

tet

ccc

gca

aaa

gta

tta

cag

ggt

cat

9437

aat

gtt

ttt

ggt

aca

acc

gat

tta

get

tta

tgc

tet

gag

get

tta

ttg

ett

9488

aat

ttt

get

aat

tet

ttg

cct

tgc

ctg

tat

gat

tta

ttg

gat

gtt

! 9532

gene

II

continues

Ο τ—Η

CM

OO ο

CM

- 162 2018241075 03 Oct 2018

Table 21B: Sequence of MALIA3, condensed

LOCUS MALIA3 9532 CIRCULAR

ORIGIN

AATGCTACTA CTATTAGTAG AATTGATGCC ACCTTTTCAG CTCGCGCCCC AAATGAAAAT

61 ATAGCTAAAC AGGTTATTGA CCATTTGCGA AATGTATCTA ATGGTCAAAC TAAATCTACT

121 CGTTCGCAGA ATTGGGAATC AACTGTTACA TGGAATGAAA CTTCCAGACA CCGTACTTTA

181 GTTGCATATT TAAAACATGT TGAGCTACAG CACCAGATTC AGCAATTAAG CTCTAAGCCA

241 TCCGCAAAAA TGACCTCTTA TCAAAAGGAG CAATTAAAGG TACTCTCTAA TCCTGACCTG

361 TCTTTCGGGC TTCCTCTTAA TCTTTTTGAT GCAATCCGCT TTGCTTCTGA CTATAATAGT

421 CAGGGTAAAG ACCTGATTTT TGATTTATGG TCATTCTCGT TTTCTGAACT GTTTAAAGCA

481 TTTGAGGGGG ATTCAATGAA TATTTATGAC GATTCCGCAG TATTGGACGC TATCCAGTCT

541 AAACATTTTA CTATTACCCC CTCTGGCAAA ACTTCTTTTG CAAAAGCCTC TCGCTATTTT

601 GGTTTTTATC GTCGTCTGGT AAACGAGGGT TATGATAGTG TTGCTCTTAC TATGCCTCGT

661 AATTCCTTTT GGCGTTATGT ATCTGCATTA GTTGAATGTG GTATTCCTAA ATCTCAACTG

721 ATGAATCTTT CTACCTGTAA TAATGTTGTT CCGTTAGTTC GTTTTATTAA CGTAGATTTT

781 TCTTCCCAAC GTCCTGACTG GTATAATGAG CCAGTTCTTA AAATCGCATA AGGTAATTCA

841 CAATGATTAA AGTTGAAATT AAACCATCTC AAGCCCAATT TACTACTCGT TCTGGTGTTT

901 CTCGTCAGGG CAAGCCTTAT TCACTGAATG AGCAGCTTTG TTACGTTGAT TTGGGTAATG

961 AATATCCGGT TCTTGTCAAG ATTACTCTTG ATGAAGGTCA GCCAGCCTAT GCGCCTGGTC

1021 TGTACACCGT TCATCTGTCC TCTTTCAAAG TTGGTCAGTT CGGTTCCCTT ATGATTGACC

1081 GTCTGCGCCT CGTTCCGGCT AAGTAACATG GAGCAGGTCG CGGATTTCGA CACAATTTAT

1141 CAGGCGATGA TACAAATCTC CGTTGTACTT TGTTTCGCGC TTGGTATAAT CGCTGGGGGT

1201 CAAAGATGAG TGTTTTAGTG TATTCTTTCG CCTCTTTCGT TTTAGGTTGG TGCCTTCGTA

1261 GTGGCATTAC GTATTTTACC CGTTTAATGG AAACTTCCTC ATGAAAAAGT CTTTAGTCCT

1321 CAAAGCCTCT GTAGCCGTTG CTACCCTCGT TCCGATGCTG TCTTTCGCTG CTGAGGGTGA

1381 CGATCCCGCA AAAGCGGCCT TTAACTCCCT GCAAGCCTCA GCGACCGAAT ATATCGGTTA

1441 TGCGTGGGCG ATGGTTGTTG TCATTGTCGG CGCAACTATC GGTATCAAGC TGTTTAAGAA

1501 ATTCACCTCG AAAGCAAGCT GATAAACCGA TACAATTAAA GGCTCCTTTT GGAGCCTTTT

1561 TTTTTGGAGA TTTTCAACGT GAAAAAATTA TTATTCGCAA TTCCTTTAGT TGTTCCTTTC ;0 1621 TATTCTCACA GTGCACAGTC TGTCGTGACG CAGCCGCCCT CAGTGTCTGG GGCCCCAGGG

1681 CAGAGGGTCA CCATCTCCTG CACTGGGAGC AGCTCCAACA TCGGGGCAGG TTATGATGTA

1741 CACTGGTACC AGCAGCTTCC AGGAACAGCC CCCAAACTCC TCATCTATGG TAACAGCAAT

1801 CGGCCCTCAG GGGTCCCTGA CCGATTCTCT GGCTCCAAGT CTGGCACCTC AGCCTCCCTG

1861 GCCATCACTG GGCTCCAGGC TGAGGATGAG GCTGATTATT ACTGCCAGTC CTATGACAGC

1921 AGCCTGAGTG GCCTTTATGT CTTCGGAACT GGGACCAAGG TCACCGTCCT AGGTCAGCCC

1981 AAGGCCAACC CCACTGTCAC TCTGTTCCCG CCCTCCTCTG AGGAGCTCCA AGCCAACAAG

2041 GCCACACTAG TGTGTCTGAT CAGTGACTTC TACCCGGGAG CTGTGACAGT GGCCTGGAAG

2101 GCAGATAGCA GCCCCGTCAA GGCGGGAGTG GAGACCACCA CACCCTCCAA ACAAAGCAAC

2161 AACAAGTACG CGGCCAGCAG CTATCTGAGC CTGACGCCTG AGCAGTGGAA GTCCCACAGA :0 2221 AGCTACAGCT GCCAGGTCAC GCATGAAGGG AGCACCGTGG AGAAGACAGT GGCCCCTACA

2281 GAATGTTCAT AATAAACCGC CTCCACCGGG CGCGCCAATT CTATTTCAAG GAGACAGTCA

2341 TAATGAAATA CCTATTGCCT ACGGCAGCCG CTGGATTGTT ATTACTCGCG GCCCAGCCGG

2401 CCATGGCCGA AGTTCAATTG TTAGAGTCTG GTGGCGGTCT TGTTCAGCCT GGTGGTTCTT

2461 TACGTCTTTC TTGCGCTGCT TCCGGATTCA CTTTCTCTTC GTACGCTATG TCTTGGGTTC :5 2521 GCCAAGCTCC TGGTAAAGGT TTGGAGTGGG TTTCTGCTAT CTCTGGTTCT GGTGGCAGTA

2581 CTTACTATGC TGACTCCGTT AAAGGTCGCT TCACTATCTC TAGAGACAAC TCTAAGAATA

2641 CTCTCTACTT GCAGATGAAC AGCTTAAGGG CTGAGGACAC TGCAGTCTAC TATTGCGCTA

2701 AAGACTATGA AGGTACTGGT TATGCTTTCG ACATATGGGG TCAAGGTACT ATGGTCACCG

2761 TCTCTAGTGC CTCCACCAAG GGCCCATCGG TCTTCCCCCT GGCACCCTCC TCCAAGAGCA >0 2821 CCTCTGGGGG CACAGCGGCC CTGGGCTGCC TGGTCAAGGA CTACTTCCCC GAACCGGTGA

2881 CGGTGTCGTG GAACTCAGGC GCCCTGACCA GCGGCGTCCA CACCTTCCCG GCTGTCCTAC

2941 AGTCTAGCGG ACTCTACTCC CTCAGCAGCG TAGTGACCGT GCCCTCTTCT AGCTTGGGCA

3001 CCCAGACCTA CATCTGCAAC GTGAATCACA AGCCCAGCAA CACCAAGGTG GACAAGAAAG

3061 TTGAGCCCAA ATCTTGTGCG GCCGCTCATC ACCACCATCA TCACTCTGCT GAACAAAAAC >5 3121 TCATCTCAGA AGAGGATCTG AATGGTGCCG CAGATATCAA CGATGATCGT ATGGCTGGCG

3181 CCGCTGAAAC TGTTGAAAGT TGTTTAGCAA AACCCCATAC AGAAAATTCA TTTACTAACG

3241 TCTGGAAAGA CGACAAAACT TTAGATCGTT ACGCTAACTA TGAGGGTTGT CTGTGGAATG

163

CTACAGGCGT

TTGGGCTTGC

GCGGTTCTGA

ATACTTATAT

ATCCTAATCC

GGTTCCGAAA

ACCCCGTTAA

ACTGGAACGG

TTTGTGAATA

GCTCTGGTGG

AGGGTGGCGG

ATGAAAAGAT

TACAGTCTGA

ATGGTTTCAT

CTGGCTCTAA

ATTTCCGTCA

GCGCTGGTAA

TCTTTGCGTT

TACTGCGTAA

TTTCCTCGGT

CTTCGGTAAG

AATTCTTGTG

TGTTCAGTTA

GGCTGCTATT

ATAATATGGC

TTGGTAAGAT

GGCTTCAAAA

CGGATAAGCC

AAAATAAAAA

GGAATGATAA

GGGATATTAT

TAGCTGAACA

CTTTATATTC

TTAAATATGG

ATTTGTATAA

ATTCTTATTT

AGAAGATGAA

TTGGATTTGC

AGGTAGTCTC

ATCTAAGCTA

TACAGAAGCA

GTAATTCAAA

TCTTCTTTTG

TATTCAAAGC

GTATATTCAT

GCTAATAATT

AATCAGGATT

GCTCCTTCTG

AATAACGTTC

TCTAAATCCT

AAAGATATTT

ATATTGATTG

GCTGCTGGCT

GTTTTATCTT

GTTCGCGCAT

CTTTCAGGTC

GTGACTGGTG

GGTATTTCCA

TGTAGTTTGT

TATCCCTGAA

GGGTGGCGGT

CAACCCTCTC

TTCTCTTGAG

TAGGCAGGGG

AACTTATTAC

TAAATTCAGA

TCAAGGCCAA

TGGTTCTGGT

CTCTGAGGGA

GGCAAACGCT

CGCTAAAGGC

TGGTGACGTT

TTCCCAAATG

ATATTTACCT

ACCATATGAA

TCTTTTATAT

TAAGGAGTCT

TTCCTTCTGG

ATAGCTATTG

GGTTATCTCT

ATTCTCCCGT

TTCATTTTTG

TGTTTATTTT

TCAGGATAAA

CCTCCCGCAA

TTCTATATCT

CGGCTTGCTT

GGAAAGACAG

TTTTCTTGTT

TGTTGTTTAT

TCTTATTACT

CGATTCTCAA

CGCATATGAT

AACGCCTTAT

ATTAACTAAA

ATCAGCATTT

TCAGACCTAT

TCGCTATGTT

AGGTTATTCA

TGAAATTGTT

CTCAGGTAAT

AATCAGGCGA

CTGACGTTAA

TTGATATGGT

ATATTGATGA

GTGGTTTCTT

GGGCAAAGGA

CAAATGTATT

TAGATAACCT

AGGGTTTGAT

CTCAGCGTGG

CTGCTGGTGG

TAAAGACTAA

AGAAGGGTTC

AATCTGCCAA

TGAGCGTTTT

ACTGGTGACG AATGAGGGTG ACTAAACCTC GACGGCACTT GAGTCTCAGC GCATTAACTG CAGTACACTC GACTGCGCTT TCGTCTGACC GGCGGCTCTG GGCGGTTCCG AATAAGGGGG AAACTTGATT TCCGGCCTTG GCTCAAGTCG TCCCTCCCTC TTTTCTATTG GTTGCCACCT TAATCATGCC TAACTTTGTT CTATTTCATT CTGATATTAG CTAATGCGCT ACGTTAAACA GTAACTGGCA ATTGTAGCTG GTCGGGAGGT GATTTGCTTG GTTCTCGATG CCGATTATTG CAGGACTTAT TGTCGTCGTC GGCTCGAAAA TTAAGCCCTA ACTAAACAGG TTATCACACG ATATATTTGA ACATATAGTT GATTTTGATA TTCAAGGATT CTCACATATA AAATGTAATT TGAAATGAAT ATCCGTTATT ACCTGAAAAT TGGTTCAATT ATTGCCATCA TGTTCCGCAA TTTAATACGA ATCTATTGAC TCCTCAATTC ATTTGAGGTT CACTGTTGCA TTCGTTCGGT TAGCCATTCA TATCTCTGTT TGTAAATAAT TCCTGTTGCA

AAACTCAGTG

GTGGCTCTGA

CTGAGTACGG

ATCCGCCTGG

CTCTTAATAC

TTTATACGGG

CTGTATCATC

TCCATTCTGG

TGCCTCAACC

AGGGTGGTGG

GTGGTGGCTC

CTATGACCGA

CTGTCGCTAC

CTAATGGTAA

GTGACGGTGA

AATCGGTTGA

ATTGTGACAA

TTATGTATGT

AGTTCTTTTG

CGGCTATCTG

GTTTCTTGCT

CGCTCAATTA

TCCCTGTTTT

AAAAATCGTT

AATTAGGCTC

GGTGCAAAAT

TCGCTAAAAC

CTATTGGGCG

AGTGCGGTAC

ATTGGTTTCT

CTATTGTTGA

TGGACAGAAT

TGCCTCTGCC

CTGTTGAGCG

CTTTTTCTAG

GTCGGTATTT

AAAAGTTTTC

ATATAACCCA

AATTCACTAT

CTAAGGGAAA

TTGATTTATG

AATTTTGTTT

AATTCGCCTC

GTTTCTCCCG

CTACGCAATT

CCTTCCATAA

TCTGATAATC

AATGATAATG

GTTGTCGAAT

GGCTCTAATC

CTTTCTACTG

CAGCAAGGTG

GGCGGTGTTA atttttaatg

AAAATATTGT

GGCCAGAATG

CCATTTCAGA

ATGGCTGGCG

TTACGGTACA

GGGTGGCGGT

TGATACACCT

TACTGAGCAA

TTTCATGTTT

CACTGTTACT

AAAAGCCATG

CTTTAATGAA

TCCTGTCAAT

CTCTGAGGGT

TGGTTCCGGT

AAATGCCGAT

TGATTACGGT

TGGTGCTACT

TAATTCACCT

ATGTCGCCCT

AATAAACTTA

ATTTTCTACG

GGTATTCCGT

CTTACTTTTC

CTTATTATTG

CCCTCTGACT

TATGTTATTC

TCTTATTTGG

TGGAAAGACG

AGCAACTAAT

GCCTCGCGTT

CGGTAATGAT

TTGGTTTAAT

ACATGCTCGT

TAAACAGGCG

TACTTTACCT

TAAATTACAT

TTGGCTTTAT

TAATTATGAT

CAAACCATTA

TCGCGTTCTT

ACCTAAGCCG

TGACTCTTCT

ATTAATTAAT

TACTGTTTCC

TCTTGATGTT

TGCGCGATTT

ATGTAAAAGG

TCTTTATTTC

TTCAGAAGTA

AGGAATATGA

TTACTCAAAC

TGTTTGTAAA

TATTAGTTGT

TTGATTTGCC

ATGCTTTAGA

ATACTGACCG

GCGATGTTTT

CTGTGCCACG

TCCCTTTTAT

CGATTGAGCG

GTAATATTGT

TGGGTTCCTA

TCTGAGGGTG

ATTCCGGGCT

AACCCCGCTA

CAGAATAATA

CAAGGCACTG

TATGACGCTT

GATCCATTCG

GCTGGCGGCG

GGCGGTTCTG

GATTTTGATT

GAAAACGCGC

GCTGCTATCG

GGTGATTTTG

TTAATGAATA

TTTGTCTTTA

TTCCGTGGTG

TTTGCTAACA

TATTATTGCG

TTAAAAAGGG

GGCTTAACTC

TTGTTCAGGG

TCTCTGTAAA

ATTGGGATAA

CTCGTTAGCG

CTTGATTTAA

CTTAGAATAC

TCCTACGATG

ACCCGTTCTT

AAATTAGGAT

CGTTCTGCAT

TTTGTCGGTA

GTTGGCGTTG

ACTGGTAAGA

TCCGGTGTTT

AATTTAGGTC

TGTCTTGCGA

GAGGTTAAAA

CAGCGTCTTA

AGCGACGATT

ATTAAAAAAG

TGTTTCATCA

TGTAACTTGG

TACTGTTACT

TGTTTTACGT

TAATCCAAAC

TGATAATTCC

TTTTAAAATT

GTCTAATACT

TTCTGCACCT

AACTGACCAG

TTTTTCATTT

CCTCACCTCT

AGGGCTATCA

TATTCTTACG

TACTGGTCGT

TCAAAATGTA

TCTGGATATT

- 164 2018241075 03 Oct 2018 :5 ίθ

6781

6841

6901

6961

7021

7081

7141

7201

7261

7321

7381

7441

7501

7561

7621

7681

7741

7801

7861

7921

7981

8041

8101

8161

8221

8281

8341

8401

8461

8521

8581

8641

8701

8761

8821

8881

8941

9001

9061

9121

9181

9241

9301

9361

9421

9481

ACCAGCAAGG CCGATAGTTT GAGTTCTTCT AGAAGTATTG CTACAACGGT TAATTTGCGT ACTGATTATA AAAACACTTC TCAAGATTCT ATCGGCCTCC TGTTTAGCTC CCGCTCTGAT GTCAAAGCAA CCATAGTACG CGCCCTGTAG TACGCGCAGC GTGACCGCTA CACTTGCCAG CCCTTCCTTT CTCGCCACGT TCGCCGGCTT TTTAGGGTTC CGATTTAGTG CTTTACGGCA TGGTTCACGT AGTGGGCCAT CGCCCTGATA CACGTTCTTT AATAGTGGAC TCTTGTTCCA CTATTCTTTT GATTTATAAG GGATTTTGCC CGCCTGCTGG GGCAAACCAG CGTGGACCGC AAGGGCAATC AGCTGTTGCC CGTCTCACTG TTGCAGGTGG CACTTTTCGG GGAAATGTGC TACATTCAAA TATGTATCCG CTCATGAGAC GAAAAAGGAA GAGTATGAGT ATTCAACATT CATTTTGCCT TCCTGTTTTT GCTCACCCAG ATCAGTTGGG CGCACGAGTG GGTTACATCG AGAGTTTTCG CCCCGAAGAA CGTTTTCCAA ATACACTATT ATCCCGTATT GACGCCGGGC CTCAGAATGA CTTGGTTGAG TACTCACCAG CAGTAAGAGA ATTATGCAGT GCTGCCATAA TTCTGACAAC GATCGGAGGA CCGAAGGAGC ATGTAACTCG CCTTGATCGT TGGGAACCGG GTGACACCAC GATGCCTGTA GCAATGCCAA TACTTACTCT AGCTTCCCGG CAACAATTAA GACCACTTCT GCGCTCGGCC CTTCCGGCTG GTGAGCGTGG GTCTCGCGGT ATCATTGCAG TCGTAGTTAT CTACACGACG GGGAGTCAGG CTGAGATAGG TGCCTCACTG ATTAAGCATT TACTTTAGAT TGATTTAAAA CTTCATTTTT TTGATAATCT CATGACCAAA ATCCCTTAAC CCCAAGCTTG TCGACTGAAT GGCGAATGGC TGCCGGAAAG CTGGCTGGAG TGCGATCTTC ACTGGCAGAT GCACGGTTAC GATGCGCCCA TCAATCCGCC GTTTGTTCCC ACGGAGAATC TTGATGAAAG CTGGCTACAG GAAGGCCAGA GTTAAAAAAT GAGCTGATTT AACAAAAATT TACAATTTAA ATATTTGCTT ATACAATCTT CGGGGTACAT ATGATTGACA TGCTAGTTTT CTCCAGACTC TCAGGCAATG ACCTGATAGC CTCCGGCATG AATTTATCAG CTAGAACGGT CTCCGGCCTT TCTCACCCTT TTGAATCTTT AATATATGAG GGTTCTAAAA ATTTTTATCC AGTATTACAG GGTCATAATG TTTTTGGTAC ATTGCTTAAT TTTGCTAATT CTTTGCCTTG

ACTCAGGCAA GTGATGTTAT TACTAATCAA GATGGACAGA CTCTTTTACT CGGTGGCCTC GGCGTACCGT TCCTGTCTAA AATCCCTTTA TCCAACGAGG AAAGCACGTT ATACGTGCTC CGGCGCATTA AGCGCGGCGG GTGTGGTGGT CGCCCTAGCG CCCGCTCCTT TCGCTTTCTT tccccgTcaa gctctaaatc gggggctccc CCTCGACCCC AAAAAACTTG ATTTGGGTGA GACGGTTTTT CGCCCTTTGA CGTTGGAGTC AACTGGAACA ACACTCAACC CTATCTCGGG GATTTCGGAA CCACCATCAA ACAGGATTTT TTGCTGCAAC TCTCTCAGGG CCAGGCGGTG GTGAAAAGAA AAACCACCCT GGATCCAAGC GCGGAACCCC TATTTGTTTA TTTTTCTAAA AATAACCCTG ATAAATGCTT CAATAATATT TCCGTGTCGC CCTTATTCCC TTTTTTGCGG AAACGCTGGT GAAAGTAAAA GATGCTGAAG AACTGGATCT CAACAGCGGT AAGATCCTTG TGATGAGCAC TTTTAAAGTT CTGCTATGTC AAGAGCAACT CGGTCGCCGG GCGCGGTATT TCACAGAAAA GCATCTTACG GATGGCATGA CCATGAGTGA TAACACTGCG GCCAACTTAC TAACCGCTTT TTTGCACAAC ATGGGGGATC AGCTGAATGA AGCCATACCA AACGACGAGC CAACGTTGCG CAAACTATTA ACTGGCGAAC TAGACTGGAT GGAGGCGGAT AAAGTTGCAG GCTGGTTTAT TGCTGATAAA TCTGGAGCCG CACTGGGGCC AGATGGTAAG CCCTCCCGTA CAACTATGGA TGAACGAAAT AGACAGATCG GGTAACTGTC AGACCAAGTT TACTCATATA AATTTAAAAG GATCTAGGTG AAGATCCTTT GTGAGTTTTC GTTCCACTGT ACGTAAGACC GCTTTGCCTG GTTTCCGGCA CCAGAAGCGG CTGAGGCCGA TACTGTCGTC GTCCCCTCAA TCTACACCAA CGTAACCTAT CCCATTACGG CGACGGGTTG TTACTCGCTC ACATTTAATG CGCGAATTAT TTTTGATGGC GTTCCTATTG TAACGCGAAT TTTAACAAAA TATTAACGTT CCTGTTTTTG GGGCTTTTCT GATTATCAAC ACGATTACCG TTCATCGATT CTCTTGTTTG CTTTGTAGAT CTCTCAAAAA TAGCTACCCT TGAATATCAT ATTGATGGTG ATTTGACTGT ACCTACACAT TACTCAGGCA TTGCATTTAA TTGCGTTGAA ATAAAGGCTT CTCCCGCAAA AACCGATTTA GCTTTATGCT CTGAGGCTTT CCTGTATGAT TTATTGGATG TT

165

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2018241075 03 Oct 2018

- 168

Table 25: h3401-h2 captured Via CJ with BsmAI ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 'SAQDIQMTQSPATLS aGT GCA Caa gac ate cag atg acc cag tet cca gcc acc ctg tet ! ApaLl... a gcc acc ! L25,L6,L20,L2,L16,All ! Extender.................................Bridge...

! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 'VSPGERATLSCRASQ . 0 gtg tet cca ggg gaa agg gcc acc etc tee tgc agg gcc agt cag ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 ,'SVSNNLAWYQQKPGQ agt gtt agt aac aac tta gcc tgg tac cag cag aaa cct ggc cag .5 ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 iVPRLLIYGASTRATD gtt ccc agg etc etc ate tat ggt gca tee acc agg gcc act gat !0 ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 ilPARFSGSGSGTDFT ate cca gcc agg ttc agt ggc agt ggg tet ggg aca gac ttc act ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 iLTISRLEPEDFAVYY etc acc ate age aga ctg gag cct gaa gat ttt gca gtg tat tac ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 iCQRYGSSPGWTFGQG tgt cag egg tat ggt age tea ccg ggg tgg aeg ttc ggc caa ggg ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 .'TKVEIKRTVAAPSVF acc aag gtg gaa ate aaa ega act gtg get gca cca tet gtc ttc

S5 ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 ilFPPSDEQLKSGTAS ate ttc ccg cca tet gat gag cag ttg aaa tet gga act gcc tet ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 iVVCLLNNFYPREAKV gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 iQWKVDNALQSGNSQE cag tgg aag gtg gat aac gcc etc caa teg ggt aac tee cag gag ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 iSVTEQDSKDSTYSLS > 0 agt gtc aca gag cag gac age aag gac age acc tac age etc age

- 169 1075 03 Oct 2018 ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 1STLTLSKADYEKHKV age acc ctg aeg ctg age aaa gca gac tac gag aaa cac aaa gtc ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210

1YACEVTHQGLSSPVT tac gcc tgc gaa gtc acc cat cag ggc ctg age teg cct gtc aca ! 211 212 213 214 215 216 217 218 219 220 221 222 223 10 'KSFNKGECKGEFA aag age ttc aac aaa gga gag tgt aag ggc gaa ttc gc.....

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- 170 2018241075 03 Oct 2018

Table 26: h3401-d8 KAPPA captured with CJ and Bsmfid ! 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1SAQDIQMTQSPATLS aGT GCA Caa gac ate cag atg acc cag tet cct gcc acc ctg tet ! ApaLI...Extender.........................g gcc acc ! L25,L6,L20,L2,L16,All ! A GCC ACC CTG TCT ! L2 ! 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

IVSPGERATLSCRASQ gtg tet cca ggt gaa aga gcc acc etc tcc tgc agg gcc agt cag ! GTG TCT CCA GGG GAA AGA GCC ACC CTC TCC TGC ! L2 ! 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45

1NLLSNLAWYQQKPGQ aat ett etc age aac tta gcc tgg tac cag cag aaa cct ggc cag ! 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 1APRLLIYGASTGAIG get ccc agg etc etc ate tat ggt get tcc acc ggg gcc att ggt ! 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 1IPARFSGSGSGTEFT ate cca gcc agg ttc agt ggc agt ggg tet ggg aca gag ttc act ! 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 1LT1SSLQSEDFAVYF etc acc ate age age ctg cag tet gaa gat ttt gca gtg tat ttc ! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105

1CQQYGTSPPTFGGGT tgt cag cag tat ggt acc tea ccg ccc act ttc ggc gga ggg acc ! 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120

1KVEIKRTVAAPSVFI aag gtg gag ate aaa cga act gtg get gca cca tet gtc ttc ate ! 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 1FPPSDEQLKSGTASV ttc ccg cca tet gat gag cag ttg aaa tet gga act gcc tet gtt ! 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 1VCPLNNFYPREAKVQ gtg tgc ccg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag ! 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 1WKVDNALQSGNSQES tgg aag gtg gat aac gcc etc caa teg ggt aac tcc cag gag agt ! 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

1VTEQDNKDSTYSLSS gtc aca gag cag gac aac aag gac age acc tac age etc age age

- 171 2018241075 03 Oct 2018 ! 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 1TLTLSKVDYEKHEVY acc ctg acg ctg age aaa gta gac tac gag aaa cac gaa gtc tac ! 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210

1ACEVTHQGLSSPVTK gee tgc gaa gtc acc cat cag ggc ett age teg ccc gtc acg aag

1211 212 213 214 215 216 217 218 219 220 221 222 223 10 1SFNRGECKKEFV age ttc aac agg gga gag tgt aag aaa gaa ttc gtt t

- 172 2018241075 03 Oct 2018

L0

L5

Table 27: V3-23 VH framework with variegated codons shown

18 19 20 21 22 A Q P A M A

5'-ctg tet gaa cG GCC cag ccG GCC atg gcc 29 3'-gac aga ctt gc egg gtc ggc egg tac egg

Scab.........Sfil.............

NgoMl...

Ncol....

FRl(DP47/V3-23)-----------23 24 25 26 27 28 29 30 EVQLLESG gaa|gtt|CAA|TTG|tta|gag|tct|ggt| 53 ctt|caa|gtt|aac|aat|ctc|aga)cca) | Mfel |

32 33 34 35 36 37 38 39 40 41 42 43 44 45 GGLVQPGGSLRLSCA lggclggt|ctt|gtt|cag|cctlggt|ggt|tct|tta|cgt|ctt|tct|tgc|gct| 98 iccg|ccalgaa)caa|gtc|gga|cca|cca|aga|aat|gca|gaa|aga|acg|cga

Sites to be varied—> *** *** *** —-FR1------------>|...CDR1................1—FR2--46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ASGFTFSSYAMSWVR |cga|agg|cct|aag|tga|aag|aga]agclatg|cga|tac|aga$cc|caa|gcg| | BspEl | | BsiWI| |BstXI.

Sites to be varies—> *** *** ♦**

------FR2-------------------->j...CDR2.........

62 63 64 65 66 67 68 69 70 71 72 73 74 75 QAPGKGLEWVSAISG |gtt|cga|gga|cca|ttt|ccalaac|ctc|acc|caa|agaicga|tagjagajcca| .BstXI I

188 #**

.....CDR2............................................1—FR3—

77 78 79 80 81 82 83 84 85 86 87 88 89 90 SGGSTYYADSVKGRF ltct|ggt|ggclagtlact|tac|tat|gct|gac|tcc|gtt|aaa|ggt|cgc[ttcl 233 |aga|cca|ccg|tca|tga|atg|ata|cga,ctgjagg|caa|ttt|cca|gcg|aag| ! ----FR3------------------------------! 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 ! TISRDNSKNTLYLQM

0 |act|atc|TCT|AGA|gac|aac|tct|aag|aat|act|ctc|tac|ttg|cag|atg| 278 ! |tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtcjtac| ! | Xbal |

143

FR3------------------------------->|

106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 NSLRAEDTAVYYCAK laaclagC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tatltgclgctlaaal 323 |ttg|tcg|aat|tcc|cga|ctc|ctg|tga|cgt|cag|atg|ata|acg|cga|ttt|

- 173 2018241075 03 Oct 2018

I Aflll | | Pstl I

.......CDR3.................|—FR4------------------121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 DYEGTGYAFDIWGQG |gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA!TGg|ggt|caa|ggtl 368 |ctg|ata|ctt|cca|tga|cca|atafcga|aag|ctg|tat|acc|cca[gtt|cca| | Ndel |

----------FR4-------->|

136 137 138 139 140 141 142 T Μ V T V S S |act|atG|GTC|ACC|gtc|tct|agt- 389 |tga|tac|cag|tgg|cag|aga|tca| BstEIl | ! 143 144 145 146 147 148 149 150 151 152 ! ASTKGPSVFP gcc tcc acc aaG GGC CCa teg GTC TTC ccc-3' 419 20! egg agg tgg ttc ccg ggt age cag aag ggg-5’ ! Bspl20I. BbsI...(2/2) ! Apal....

(SFPRMET) 5'-ctg tet gaa cG GCC cag ccG-3' (TOPFR1 A) 5'-ctg tet gaa cG GCC cag ccG GCC atg gccgaa|gtt|CAA|TTG|tta|gag|tct|ggt||ggc|ggt|ctt|gtt|cag|cct|ggt|ggt|tct|tta-3' (BOTFRIB) 3'-caa|gtc|gga|cca|cca[aga|aatjgca|gaa|aga|acg|cga||cga|agg|cct|aag|tga|aag-5'! bottom strand (BOTFR2) 3'-acc|caa|gcg||gtt|cga|gga|cca|ttt|cca|aac|ctc|acc|caa|agaj-S’! bottom strand (BOTFR3) 3'- a|cga|ctg|agg|caa|ttt|cca|gcg|aag||tga|tag|aga|tct|ctg|ttg|aga|ttc|tta|tga|gag|atg|aac|gtc|tac||ttg|tcg|aat|tcc|cga|ctc|ctg|tga-5’ (F06) 5'-gC|TTA|AGg|gct|gag|gac|aCT|GCA|Gtc|tac|tat|tgc|gct|aaa||gac|tat|gaa|ggt|act|ggt|tat|gct|ttc|gaC|ATA|TGg|ggt|c-3' (BOTFR4) 3'-cga|aaglctg|tat|acc|cca|gtt|cca||tga|tac|cag|tgg|cag|aga|tcaegg agg tgg ttc ccg ggt age cag aag ggg-5'! bottom strand (BOTPRCPRIM) 3'-gg ttc ccg ggt age cag aag ggg-5’ !

! CDR1 diversity (ON-vgCl) 5’-|gct|TCCIGGA|ttc|act|ttc|tct|<l>|TACI<l>|atg|<l>| ! CDR1...................6859

5 ltgg|gtt|cgClCAalgct|ccT[GG-3' t

!<1> stands for an equimolar mix of {ADEFGHIKLMNPQRSTVWY}; no C ! (this is not a sequence) ! CDR2 diversity (ON-vgC2) 5'-ggt|ttg|gag|tgg|gtt|tct|<2>|atc|<2>|<3>!! CDR2............

|tct|ggt|ggc|<l>|act|<l>|tat|gct|gac|tcc|gtt|aaa|gg-3’

'. CDR2................................................

! <1> is an equimolar mixture of {ADEFGHIKLMNPQRSTVWY}; no C ! <2> is an equimolar mixture of {YRWVGS}; no ACDEFHIKLMNPQT

2018241075 03 Oct 2018

- 174 ! <3> is an equimolar mixture of {PS}; no ACDEFGHIKLMNQRTVWY

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5758 gta ttt teg aeg ttt get aac ata ctg cgt aat aag gag tet taa

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2018241075 03 Oct 2018 r«Λ

—- r- m σ\ «λ tj- v> *r> \o sO Ό Ό sO Ό

LO

O LO rH r—<

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CN <N

2018241075 03 Oct 2018

- 192

Table 30: Oligonucleotides used to clone CDR1/2 diversity All sequences are 5' to 3'.

1) ON_CDlBsp, 30 bases 5

AccTcAcTggcTTccggA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

TTcAcTTTcTcT 0 19 20 21 22 23 24 25 26 27 28 29 30

2) ON_Brl2,42 bases

AgAAAcccAcTccAAAcc 5 I 2 3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18

TTTAccAggAgcTTggcg

20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

A A c c c A 37 38 39 40 41 42

3) ON_CD2Xba, 51 bases ggAAggcAgTgATcTAgA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 gATAgTgAAgcgAccTTT 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36

AAcggAgTcAgcATA 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 ‘ ·5 4) ONBotXba, 23 bases ggAAggcAgTgATcTAgA 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 g A T A g 19 20 21 22 23

- 193 2018241075 03 Oct 2018

Table 31: Bridge/Extender Oligonucleotides

ON_LamlaB7 (rc) .........................GTGCTGACTCAGCCACCCTC.

ON_Lam2 aB7 (rc) ........................GCCCTGACTCAGCCTGCCTC.

ON_Lam31B7 (rc) .......................GAGCTGACTCAGG.ACCCTGC

ON_Lam3rB7 (rc) GAGCTGACTCAGCCACCCTC.

ON_LamHflcBrg (rc) CCTCGACAGCGAAGTGCACAGAGCGTCTTGACTCAGCC.......

ON_LamHfIcExt CCTCGACAGCGAAGTGCACAGAGCGTCTTG...............

ON_LamHf2b2Brg(rc) CCTCGACAGCGAAGTGCACAGAGCGCTTTGACTCAGCC.......

ON_LamHf2b2Ext CCTCGACAGCGAAGTGCACAGAGCGCTTTG...............

ON_LamHf2dBrg(rc) CCTCGACAGCTAAGTGCACAGAGCGCTTTGACTCAGCC.......

ON_LamHf2dExt CCTCGACAGCGAAGTGCACAGAGCGCTTTG...............

ON_LamHf31Brg(rc) CCTCGACAGCGAAGTGCACAGAGCGAATTGACTCAGCC.......

ON_LamHf3lExt CCTCGACAGCGAAGTGCACAGAGCGAATTG...............

ON_LamHf3rBrg(rc) CCTCGACAGCGAAGTGCACAGTACGAATTGACTCAGCC.......

0N_LamHf3rExt CCTCGACAGCGAAGTGCACAGTACGAATTG...............

ON_lamPlePCR CCTCGACAGCGAAGTGCACAG........................

Consensus

- 194 2018241075 03 Oct 2018

Adapters (8)

H43HF3.l?02#l 5'-cc gtg tat tac tgt gcg aga g-3'

Table 32: Oligonucleotides used to make SSDNA locally double-stranded

H43.77.97.l-03#2	5' -ct
H43.77.97.323#22	5 ’ -cc
H43.77.97.330#23	5*-eg
H43.77.97.439#44	5’-eg
H43.77.97.551#48	5 ’ -cc

gtg	tat	tac	tgt	gcg	aga
gtg	t at	t ac	t gt	gcg	a§a
gtg	tat	tac	tgt	gcg	a|a
gtg	tat	tac	tgt	gcg	aga
|tg	tat	tac	tgt	gcg	aga

do

- 195 2018241075 03 Oct 2018

Table 33: Bridge/extender pairs

Bridges (2)

H43.XABrl

5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagatgaacagCTTtAGgg ctgaggacaCTGCAGtctactattgtgcgaga-3'

H43.XABr2

5'ggtgtagtgaTCTAGtgacaactctaagaatactctctacttgcagatgaacagCTTtAGgg LO ctgaggacaCTGCAGtctactattgtgcgaaa-3'

Extender

H43.XAExt 'ATAgTAgAcTgcAgTgTccTcAgcccTTAAgcTgTTcATcTgcAAgTAgAgAgTATTcTTAg L5 AgTTgTcTcTAgATcAcTAcAcc-3'

2018241075 03 Oct 2018

- 196

Table 34: PCR primers

Primers

H43.XAPCR2 gactgggTgTAgTgATcTAg

Hucmnest cttttctttgttgccgttggggtg

- 197 2018241075 03 Oct 2018

Table 35: PCR program for amplification of heavy chain CDR3 DNA

95	degrees	C	5 minutes
95	degrees	C	20 seconds
60	degrees	C	30 seconds
72	degrees	C	1 minute
72	degrees	c	7 minutes
4	degrees <	c	hold

repeat 20x

Reagents (100 10 Template lOx PCR buffer Taq dNTPs MgCl2

H43.XAPCR2-biotin

Hucmnest ul reaction):

5ul ligation mix lx

5U

200 uM each 2mM

400 nM 200 nM

- 198 2018241075 03 Oct 2018

Table 36: Annotated sequence of CJR DY3F7(CJR-A05) 10251 bases

! Non-cutters
'.Bell Tgatca	BsiWI	Cgtacg	BssSI	Cacgag
!BstZ17I GTAtac	Btrl	CACgtg	EcoRV	GATatc
!FseI GGCCGGcc	Hpal	GTTaac	Mlul	Acgcgt
!PmeI GTTTaaac	Pmll	CACgtg	PpuMI	RGgwccy
!RsrII CGgwccg	Sapl	GCTCTTC	SexAI	Accwggt
!SgfI GCGATcgc	SgrAI	CRccggyg	SphI	GCATGc
!StuI AGGcct	Xmal	Cccggg

cutters

5	! Enzymes that cut from 1 to 4 I	times and	other	featur
	!End of genes II and X		829
	!Start gene V		843
	!BsrGI Tgtaca	1	1021
0	!BspMI Nnnnnnnnngcaggt	3	1104	5997	9183
	!-- ACCTGCNNNNn	1	2281
	!End of gene V		1106
	! Start gene VII-		1108
	iBsaBI GATNNnnatc	2	1149	3967
5	!Start gene IX		1208
	!End gene VII		1211
	iSnaBI TACgta	2	1268	7133
	iBspHI Tcatga	3	1299	6085	7093
	‘Start gene VIII		1301
• 0	!End gene IX		1304
	!End gene VIII		1522
	!Start gene III		1578
	!EagI Cggccg	2	1630	8905
	iXbal Tctaga	2	1643	8436
;5	!KasI Ggcgcc	4	1650	8724	9039
	iBsml GAATGCN	2	1769	9065
	!BseRI GAGGAGNNNNNNNNNN	2	2031	8516
	!-- NNnnnnnnnnctcctc	2	7603	8623
	'.AlwNI CAGNNNctg	3	2210	8072	8182
:0	iBspDI ATcgat	2	2520	9883
	!NdeI CAtatg	3	2716	3796	9847
	!End gene III		2846
	!Start gene VI		2848
	!AfeI AGCgct	1	3032
:5	!End gene VI		3187
	!Start gene I		3189
	iEarl CTCTTCNnnn	2	4067	9274
	!-- Nnnnngaagag	2	6126	8953
	!Pacl TTAATtaa	1	4125
.0	!Start gene IV		4213
	!End gene I		4235
	!BsmFI Nnnnnnnnnnnnnnngtccc	2	5068	9515
	!MscI TGGcca	3	5073	7597	9160
	iPsil TTAtaa	2	5349	5837
55	!End gene IV		5493
	!Start ori		5494
	iNgoMIV Gccggc	3	5606	8213	9315
	iBanll GRGCYc	4	5636	8080	8606
	! Drain CACNNNgtg	1	5709
50	!Drdl GACNNNNnngtc	1	5752
	iAval Cycgrg	2	5818	7240

9120

- 199 2018241075 03 Oct 2018

PvuII CAGctg	1	5953
BsmBI CGTCTCNnnnn	3	5964	8585	9271
End ori region		5993
BamHI Ggatcc	1	5994
Hindlll Aagctt	3	6000	7147	7384
BciVI GTATCCNNNNNN	1	6077
Start bla		6138
Eco57I CTGAAG	2	6238	7716
Spel Actagt	1	6257
Bcgl gcannnnnntcg	1	6398
Seal AGTact	1	6442
Pvul CGATcg	1	6553
FspI TGCgca	1	6700
Bgll GCCNNNNnggc	3	6801	8208	8976
Bsal GGTCTCNnnnn	1	6853
AhdI GACNNNnngtc	1	6920
EamllO5I GACNNNnngtc	1	6920
End bla		6998
AccI GTmkac	2	7153	8048
HincII GTYrac	1	7153
Sail Gtcgac	1	7153
Xhol Ctcgag	1	7240
Start PlacZ region		7246
End PlacZ region		7381
PflMI CCANNNNntgg	1	7382
RBS1		7405
start M13-iii signal seq for	LC	7418
ApaLI Gtgcac	1	7470
end M13-iii signal seq		7471
Start light chain kappa L20:	JK1	7472
PflFI GACNnngtc	3	7489	8705	9099
Sbfl CCTGCAgg	1	7542
Pstl CTGCAg	1	7543
Kpnl GGTACc	1	7581
Xcml CCANNNNNnnnntgg	2	7585	9215
Nsil ATGCAt	2	7626	9503
Bsgl ctgcac	1	7809
Bbsl gtette	2	7820	8616
BlpI GCtnagc	1	8017
EspI GCtnagc	1	8017
Eco0109I RGgnccy	2	8073	8605
Ecll36I GAGctc	1	8080
Sacl GAGCTc	1	8080
End light chain		8122
Ascl GGcgcgcc	1	8126
BssHII Gegege	1	8127
RBS2		8147
Sfil GGCCNNNNnggcc	1	8207
Neol Ccatgg	1	8218
Start 3-23, FR1		8226
Mfel Caattg	1	8232
BspEI Teegga	1	8298
Start CDR1		8316
Statt FR2		8331
BstXI CCANNNNNntgg	2	8339	8812
EcoNI CCTNNnnnagg	2	8346	8675
Start FR3		8373
Xbal Tetaga	2	8436	1643
Aflll Cttaag	1	8480
Start CDR3		8520
Aat 11 GACGTc	1	8556

- 200 2018241075 03 Oct 2018

!Start FR4	8562
!PshAI GACNNnngtc	2	8573
•BstEII Ggtnacc	1	8579
!Start CHI		8595
!ApaI GGGCCc	1	8606
!Bspl20I Gggccc	1	8606
! PspOMI Gggccc	1	8606
!AgeI Accggt	1	8699
!Bsu36I CCtnagg	2	8770
!End of CHI		8903
!NotI GCggccgc	1	8904
•Start His6 tag		8913
'Start cMyc tag		8931
!Amber codon		8982
!NheI Getage	1	8985
•Start M13 III Domain 3		8997
!NruI TCGcga	1	9106
!BstBI TTcgaa	1	9197
!EcoRI Gaattc	1	9200
!XcmI CCANNNNNnnnntgg	1	9215
!BstAPI GCANNNNntgc	1	9337
!SacII CCGCgg	1	9365
! End Illstump anchor		9455
!AvrII Cctagg	1	9462
! trp terminator		9470
!SwaI ATTTaaat	1	9784
iStart gene II		9850
•Balll Aaatet	1	9936

9231

9509

I----------------------------------------------------------------------

0

1

aat

get

act

act

att

agt

aga

att

gat

gee

acc

ttt

tea

get

ege

gee

! gene .

ii continued

49

cca

aat

gaa

aat

ata

get

aaa

cag

gtt

att

gac

cat

ttg

ega

aat

gta

97

tet

aat

ggt

caa

act

aaa

tet

act

cgt

teg

cag

aat

tgg

gaa

tea

act

145

gtt

aTa

tgg

aat

gaa

act

tee

aga

cac

cgt

act

tta

gtt

gca

tat

tta

5

193

aaa

cat

gtt

gag

eta

cag

caT

TaT

att

cag

caa

tta

age

tet

aag

cca

241

tcc

gca

aaa

atg

acc

tet

tat

caa

aag

gag

caa

tta

aag

gta

etc

tet

289

aat

cct

gac

ctg

ttg

gag

ttt

get

tcc

ggt

ctg

gtt

ege

ttt

gaa

get

337

ega

att

aaa

aeg

ega

tat

ttg

aag

tet

ttc

ggg

ctt

cct

Ctt

aat

ctt

385

ttt

gat

gca

ate

ege

ttt

get

tet

gac

tat

aat

agt

cag

ggt

aaa

gac

0

433

ctg

att

ttt

gat

tta

tgg

tea

ttc

teg

ttt

tet

gaa

ctg

ttt

aaa

gca

481

ttt

gag

ggg

gat

tea

ATG

aat

att

tat

gac

gat

tcc

gca

gta

ttg

gac

Start gene x, ii continues

529 577

get ttt

ate gca

cag aaa

tet aaa gee tet

cat ege

ttt tat

act ttt

att ggt

acc ttt

ccc tet

ggc cgt

aaa act ctg gta

tet aac

tat

cgt

5

625

gag

ggt

tat

gat

agt

gtt

get

ctt

act

atg

cct

cgt

aat

tcc

ttt

tgg

673

cgt

tat

gta

tet

gca

tta

gtt

gaa

tgt

ggt

att

cct

aaa

tet

caa

ctg

721

atg

aat

ctt

tet

acc

tgt

aat

aat

gtt

gtt

ccg

tta

gtt

cgt

ttt

att

769

aac

gta

gat

ttt

tet

tee

caa

cgt

cct

gac

tgg

tat

aat

gag

cca

gtt

817

ctt

aaa

ate

gca

TAA

0

1

End

X &

II

832 1

ggtaattca ca

1

Ml

E5

Q10

T15

843

ATG

att

aaa

gtt

gaa

att

aaa

cca

tet

caa

gee

caa

ttt

act

act

cgt

5

1 1

Start gene V

I

S17

S20

P25

E30

891 1

tet

ggt

gtt

tet

cgt

cag

ggc

aag

cct

tat

tea

ctg

aat

gag

cag

ctt

0

1

V35

E40

V4 5

939

tgt

tac

gtt

gat

ttg

ggt

aat

gaa

tat

ccg

gtt

ctt

gtc

aag

att

act

- 201 2018241075 03 Oct 2018

987 1035 1083

ett L65 ctg ctg

D50 gat tcc ege

gaa tet etc

ggt ttc gtt

cag aaa P85 ccg

cca V70 gtt get

A55 gee ggt K87 aag

tat cag end TAA

geg cct ttc ggt of V C

ggt S75 tee

L60 cTG TAC Ace gtt

cat R80 cgt

BsrGI...

gac

ett

atg

att

1108

ATG

gag

cag

gtc

geg

gat

ttc

gac

aca

att

tat

cag

geg

atg

Start gene

VII

1150

ata

caa

ate

tec

gtt

gta

ett

tgt

ttc

geg

ett

ggt

ata

ate

VII and IX overlap.

..... S2	V3	L4	V5	S10
1192 get ggg ggt caa agA TGA gt	gtt	tta	gtg tat	tet ttT gee tet ttc

End VII I start IX

1242 1

L13 tta

ggt

W15 tgg tgc

ett

cgt

agt

G20 ggc

att

aeg tat

ttt

T25 acc

cgt

tta

atg

1293 I

act

tee

tc

1

. stop of IX

, IX

anc

VIII overlap by four bases

1301

ATG

aaa

aag tet

tta

gtc

etc

aaa

gee

tet gta

gee

gtt

get

acc

etc

1 1

Start signal sequence of

viii .

1349

gtt

ccg

atg ctg

tet

ttc

get

get

gag

ggt gac

gat

ccc

gca

aaa

geg

1

mature VIII ---

>

1397

gee

ttt

aac tec

ctg

caa

gee

tea

geg

acc gaa

tat

ate

ggt

tat

geg

1445

tgg

geg

atg gtt

gtt

gtc

att

1466

gtc

ggc

gca act

ate

ggt

ate

aag

ctg

ttt aag

I •40 ! bases 1499-1539 are probable promoter for iii

1499 aaa ttc acc teg aaa gca ! 1515 ! ........... -35 . .

I

1517 age tga taaaccgat acaattaaag gctccttttg ! ..... -10

I

1552 gagccttttt ttt GGAGAt ttt ! S.D. uppercase, there may be 9 Ts

1574

caac

<— M GTG

K aaa

III K aaa

signal

sequence ------------------------

------> F ttc ! 1620

L tta

L tta

F ttc

A gca

I att

P cct

L tta

V gtt

V gtt

P cct

Y

S

G

A

A

E

s

H

L

D

G

A

1620

tat

tet

ggc

gCG

GCC

Gaa

tea

caT

CTA

GAc

ggc

gee

Eagl...

Xbal..

Domain 1

1656

A get

E gaa

T act

V gtt

E gaa

S agt

C tgt

L tta

A gca

K

s

H

T

E

I

S

F

T

N

V

W

K

D

D

K

T

1683

aaA

Tec

cat

aca

gaa

aat

tea

ttt

aCT

AAC

GTC

TGG

AAA

GAC

GAC

AAA

ACt

L

D

R

Y

A

N

Y

E

G

S

L

w

N

A

T

G

V

1734

tta

gat

cgt

tac

get

aac

tat

gag

ggC

tgt

ctg

tgG

AAT

GCt

aca

ggc

gtt

- 202 2018241075 03 Oct 2018 :5

SO >0 )5

BsmI....

V

V

C

T

G

D

E

T

Q

C

Y

G

T

W

V

P

I

1785

gta

gtt

tgt

act

ggt

GAC

GAA

ACT

CAG

TGT

TAC

GGT

ACA

TGG

GTT

cct

att

G

L

A

I

P

E

N

1836

ggg

ett

get

ate

cct

gaa

aat

! Ll linker	G	G	s	E	G	G	G	s
! E	G
1857 gag	ggt	ggt	ggc	tet	gag	ggt	ggc	ggt	tet
! E	G	G	G	s	E	G	G	G	T
1887 gag	ggt	ggc	ggt	tet	gag	ggt	ggc	ggt	act

I

Domain 2

1917

aaa

cct

cct

gag

tac

ggt

gat

aca

cct

att

ccg

ggc

tat

act

tat

ate

aac

1968

cct

etc

gac

ggc

act

tat

ccg

cct

ggt

act

gag

caa

aac

ccc

get

aat

cct

2019

aat

cct

tet

ett

GAG GAG BseRI..

tet

cag

cct

ett

aat

act

ttc

atg

ttt

cag

aat

2070

aat

agg

ttc

ega

aat

agg

cag

ggg

gca

tta

act

gtt

tat

aeg

ggc

act

2118

gtt

act

caa

ggc

act

gac

ccc

gtt

aaa

act

tat

tac

cag

tac

act

cct

2166

gta

tea

tea

aaa

gee

atg

tat

gac

get

tac

tgg

aac

ggt

aaa

ttc

AGA

AlwNI

2214

GAC TGc AlwNI

get

ttc

cat

tet

ggc

ttt

aat

gaG

gat

TTa

ttT

gtt

tgt gaa

2262

tat caa

ggc

caa

teg

tet

gac

ctg

cct

caa

cct

cct

gtc

aat

get

2307 ggc ggc ggc tet ! start L2 --------------------------------------------------------2319 ggt ggt ggt tet 2331 ggt ggc ggc tet

2343 gag ggt ggt ggc tet gag gga ggc ggt tcc 2373 ggt ggt ggc tet ggt ! end L2 ! Many published sequences of M13-derived phage have a longer linker ! than shown here by repeats of the EGGGS motif two more times.

Domain 3

G ggt

D gat

F ttt

D gat

Y tat

E gaa

K aag

M atg

A gca

N aac

A get

N aat

K aag

G ggg

A get

2388

S tcc

M

T

E

N

A

D

E

N

A

L

Q

S

D

A

K

G

2436

atg

acc

gaa

aat

gee

gat

gaa

aac

geg

eta

cag

tet

gac

get

aaa

ggc

K

L

D

S

V

A

T

D

Y

G

A

A

M

D

G

F

2484

aaa

ett

gat

tet

gtc

get

act

gat

tac

ggt

get

get

ate

gat

ggt

ttc

I

G

D

V

S

G

L

A

N

G

N

G

A

T

G

D

2532

att

ggt

gac

gtt

tcc

ggc

ett

get

aat

ggt

aat

ggt

get

act

ggt

gat

F

A

G

s

N

s

Q

M

A

Q

V

G

D

G

D

N

2580

ttt

get

ggc

tet

aat

tcc

caa

atg

get

caa

gtc

ggt

gac

ggt

gat

aat

S

P

L

M

N

N

F

R

Q

Y

L

P

S

L

P

Q

2628

tea

cct

tta

atg

aat

aat

ttc

cgt

caa

tat

tta

cct

tcc

etc

cct

caa

S

V

E

C

R

P

F

V

F

G

A

G

K

P

Y

E

2676

teg

gtt

gaa

tgt

ege

cct

ttt

gtc

ttt

Ggc

get

ggt

aaa

cca

tat

gaa

F

S

I

D

C

D

K

I

N

L

F

R

2018241075 03 Oct 2018

- 203

2724 ttt tet att gat tgt gac aaa ata aac tta ttc cgt

End Domain 3

GVFAFLLYVATFMYV F140 2760 ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat gta ttt start transmembrane segment

S T F A N IL 2808 tet aeg ttt get aac ata ctg

R N Κ E S

2829 cgt aat aag gag tet TAA Intracellular anchor.

stop of iii

Ml

P2

V

L

L5

G

I

P

L

L10

L

R

F

L

G15

2847

tc

ATG

cca

gtt

ctt

ttg

ggt

att

ccg

tta

tta

ttg

cgt

ttc

etc

ggt

Start VI

2894

ttc

Ctt

ctg

gta

act

ttg

ttc

ggc

tat

ctg

ctt

act

ttt

ctt

aaa

aag

2942

ggc

ttc

ggt

aag

ata

get

att

get

att

tea

ttg

ttt

ctt

get

ctt

att

2990

att

ggg

ctt

aac

tea

att

ctt

gtg

ggt

tat

etc

tet

gat

att

age

get

3038

caa

tta

ccc

tet

gac

ttt

gtt

cag

ggt

gtt

cag

tta

att

etc

ccg

tet

3086

aat

gcg

ctt

ccc

tgt

ttt

tat

gtt

att

etc

tet

gta

aag

get

get

att

3134

ttc

att

ttt

gac

gtt

aaa

caa

aaa

ate

gtt

tet

tat

ttg

gat

tgg

gat

Ml A2 V3 F5 L10 G13

3182 aaa TAA t ATG get gtt tat ttt gta act ggc aaa tta ggc tet gga end VI Start gene I

KTLVSVGKIQDKIVA 3228 aag aeg etc gtt age gtt ggt aag att cag gat aaa att gta get

GCKIATNLDLRLQNL 3273 ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa aac etc

PQVGRFAKTPRVLRI 3318 ccg caa gtc ggg agg ttc get aaa aeg cct cgc gtt ctt aga ata

PDKPS ISDLLAIGRG 3363 ccg gat aag cct tet ata tet gat ttg ctt get att ggg cgc ggt

NDSYDENKNGLLVLD 3408 aat gat tee tac gat gaa aat aaa aac ggc ttg ctt gtt etc gat

ECGTW FNTRSWNDKE 3453 gag tgc ggt act tgg ttt aat acc cgt tet tgg aat gat aag gaa

R Q P I I DWFLHARKLG 3498 aga cag ccg att att gat tgg ttt eta cat get cgt aaa tta gga

WDIIFLVQDLSIVDK 3543 tgg gat att att ttt ctt gtt cag gac tta tet att gtt gat aaa

QARSA L A Ε H VVYCRR 3588 cag gcg cgt tet gca tta get gaa cat gtt gtt tat tgt cgt cgt

LDRIT LPFVGTLYSL 3633 ctg gac aga att act tta cct ttt gtc ggt act tta tat tet ctt

ITGSKMPLPKLHVGV

3678 att act ggc teg aaa atg cct ctg cct aaa tta cat gtt ggc gtt

OO

204

Ο

CM

Μ—»

Ο ο

m ο

.0 .5 :θ

VKYGDSQLSPTVERW 3723 gtt aaa tat ggc gat tet caa tta age cct act gtt gag cgt tgg

LYTGKNLYNAY DTKQ 3768 ett tat act ggt aag aat ttg tat aac gca tat gat act aaa cag

AFSSNYDSGVYSYLT 3813 get ttt tet agt aat tat gat tee ggt gtt tat tet tat tta aeg

PYLSHGRYFKPLNLG 3858 cct tat tta tea cac ggt egg tat ttc aaa cca tta aat tta ggt

QKMKLTKIYLKKFSR 3903 cag aag atg aaa tta act aaa ata tat ttg aaa aag ttt tet ege

VLCLAIGFASAFTYS 3948 gtt ett tgt ett geg att gga ttt gca tea gca ttt aca tat agt

YITQPKPEVKKVVSQ 3993 tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc tet cag

TYDFDKFTIDS S Q R L 4038 acc tat gat ttt gat aaa ttc act att gac tet tet cag cgt ett

NLSYRYVFKDSKGKL 4083 aat eta age tat ege tat gtt ttc aag gat tet aag gga aaa TTA

Pacl

INSDDLQKQGYSLTY 4128 ATT AAt age gac gat tta cag aag caa ggt tat tea etc aca tat

Pacl ilDLCTVSIKKGNSNE iv Ml K

4173 att gat tta tgt act gtt tee att aaa aaa ggt aat tea aAT Gaa

Start IV i I V K C N .End of I iv L3 L N5 V 17 N F V10

4218 att gtt aaa tgt aat TAA T TTT GTT IV continued.....

4243

ttc

ttg

atg

ttt

gtt

tea

tea

tet

tet

ttt

get

cag

gta

att

gaa

atg

4291

aat

aat

teg

cct

ctg

ege

gat

ttt

gta

act

tgg

tat

tea

aag

caa

tea

4339

ggc

gaa

tee

gtt

att

gtt

tet

CCC

gat

gta

aaa

ggt

act

gtt

act

gta

4387

tat

tea

tet

gac

gtt

aaa

cct

gaa

aat

eta

ege

aat

ttc

ttt

att

tet

4435

gtt

tta

cgt

gcA

aat

aat

ttt

gat

atg

gtA

ggt

teT

aAC

cct

tee

atT

4483

att

cag

aag

tat

aat

cca

aac

aat

cag

gat

tat

att

gat

gaa

ttg

cca

4531

tea

tet

gat

aat

cag

gaa

tat

gat

gat

aat

tee

get

cct

tet

ggt

ggt

4579

ttc

ttt

gtt

ccg

caa

aat

gat

aat

gtt

act

caa

act

ttt

aaa

att

aat

4 627

aac

gtt

egg

gca

aag

gat

tta

ata

ega

gtt

gtc

gaa

ttg

ttt

gta

aag

4675

tet

aat

act

tet

aaa

tee

tea

aat

gta

tta

tet

att

gac

ggc

tet

aat

4723

eta

tta

gtt

gtt

agt

geT

cct

aaa

gat

att

tta

gat

aac

ett

cct

caa

4771

ttc

ett

tcA

act

gtt

gat

ttg

cca

act

gac

cag

ata

ttg

att

gag

ggt

4819

ttg

ata

ttt

gag

gtt

cag

caa

ggt

gat

get

tta

gat

ttt

tea

ttt

get

4867

get

ggc

tet

cag

cgt

ggc

act

gtt

gca

ggc

ggt

gtt

aat

act

gac

ege

4915

etc

acc

tet

gtt

tta

tet

tet

get

ggt

ggt

teg

ttc

ggt

att

ttt

aat

4963

ggc

gat

gtt

tta

ggg

eta

tea

gtt

ege

gca

tta

aag

act

aat

age

cat

5011

tea

aaa

ata

ttg

tet

gtg

cca

cgt

att

ett

aeg

ett

tea

ggt

cag

aag

5059

ggt

tet

ate

tet

gtT GGC Mscl..

CAg

aat

gtc

cct

ttt

att

act

ggt

cgt

gtg

- 205 2018241075 03 Oct 2018

5107

act

ggt

gaa

tct

gee

aat

gta

aat

aat

cca

ttt

cag

aeg

att

gag

cgt

5155

caa

aat

gta

ggt

att

tee

atg

age

gtt

ttt

cct

gtt

gca

atg

get

ggc

5203

ggt

aat

att

gtt

ctg

gat

att

acc

age

aag

gee

gat

agt

ttg

agt

tct

5251

tct

act

cag

gca

agt

gat

gtt

att

act

aat

caa

aga

agt

att

get

aca

5299

aeg

gtt

aat

ttg

cgt

gat

gga

cag

act

ett

tta

etc

ggt

ggc

etc

act

5347

gat

tat

aaa

aac

act

tct

caG

gat

tct

ggc

gta

ccg

ttc

ctg

tct

aaa

5395

ate

cct

tta

ate

ggc

etc

ctg

ttt

age

tee

ege

tct

gat

teT

aac

gag

5443

gaa

age

aeg

tta

tac

gtg

etc

gtc

aaa

gca

acc

ata

gta

ege

gee

ctg

5491 TAG cggcgcatt End IV

5503 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc

5563 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcGCCGGCt ttccccgtca

NgoMI.

5623 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc

5683 caaaaaactt gatttgggtg atggttCACG TAGTGggcca tcgccctgat agacggtttt

Drain....

5743 tcgccctttG ACGTTGGAGT Ccacgttctt taatagtgga ctcttgttcc aaactggaac Drdl..........

5803 aacactcaac cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga

5863 accaccatca aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa

5923 ctctctcagg gccaggcggt gaagggcaat CAGCTGttgc cCGTCTCact ggtgaaaaga

PvuII. BsmBI.

5983 aaaaccaccc tGGATCC AAGCTT

BamHI Hindlll (1/2)

Insert carrying bla gene

6006 gcaggtg gcacttttcg gggaaatgtg cgcggaaccc

6043 ctatttgttt atttttctaa atacattcaa atatGTATCC gctcatgaga caataaccct BciVI

6103 gataaatgct tcaataatat tgaaaaAGGA AGAgt

RBS.?...

Start bla gene

6138

ATG

agt

att

caa

cat

ttc

cgt

gtc

gee

ett

att

CCC

ttt

ttt

geg

gca

ttt

6189

tgc

ett

cct

gtt

ttt

get

cac

cca

gaa

aeg

ctg

gtg

aaa

gta

aaa

gat

get

6240

gaa

gat

cag

ttg

ggc

gcA

CTA

GTg

ggt

tac

ate

gaa

ctg

gat

etc

aac

age

Spel....

ApaLI & BssSI Removed . 40

6291

ggt

aag ate

ett

gag

agt

ttt

ege

CCC

gaa

gaa

cgt

ttt

cca

atg

atg

age

6342

act

ttt aaa

gtt

ctg

eta

tgt

GGC

GeG

Gta

tta

tee

cgt

att

gac

gee

ggg

6393

caa

gaG CAA

CTC

GGT

CGc

cgC

ATA

cAC

tat

tct

cag

aat

gac

ttg

gtt

gAG

BegI..

Seal

6444

TAC

Tea cca

gtc

aca

gaa

aag

cat

ett

aeg

gat

ggc

atg

aca

gta

aga

gaa

Seal.

6495

tta

tgc

agt

get

gee

ata

acc

atg

agt

gat

aac

act

geg

gee

aac

tta

ett

6546

ctg

aca

aCG

ATC

Gga

gga

ccg

aag

gag

eta

acc

get

ttt

ttg

cac

aac

atg

Pvul...

, .

6597

ggg

gat

cat

gta

act

ege

ett

gat

cgt

tgg

gaa

ccg

gag

ctg

aat

gaa

gee

6648

ata

cca

aac

gac

gag

cgt

gac

acc

aeg

atg

cct

gta

gca

atg

Gca

aca

aeg

6699

tTG

CGC

Aaa

eta

tta

act

ggc

gaa

eta

ett

act

eta

get

tee

egg

caa

caa

FspI. . . .

6750

tta ata

gac

tgg

atg

gag

geg

gat

aaa

gtt

gca

gga

cca

ett

ctg

ege

teg

6801

GCC ett Bgll....

ccG

GCt

ggc

tgg

ttt

att

get

gat

aaa

tct

gga

gee

ggt

gag

cgt

6852

gGG TCT Bsal. . .

Cgc

ggt

ate

att

gca

gca

ctg

ggg

cca

gat

ggt

aag

CCC

tee

cgt

6903

ate gta

gtt

ate

tac

aeG ACg AhdI.

ggg

aGT

Cag

gca

act

atg

gat

gaa

ega

aat

6954

aga cag

ate

get

gag

ata

ggt

gee

tea

ctg

att

aag

cat

tgg

TAA

ctgt

stop

7003 cagaccaagt ttactcatat ataetttaga ttgatttaaa acttcatttt taatttaaaa 7063 ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt

- 206 2018241075 03 Oct 2018

7123

7147

7183 cgttccactg tacgtaagac cccc

AAGCTT GTCGAC tgaa tggcgaatgg cgctttgcct

Hindlll Sail..

(2/2) Hindi ggtttccggc accagaagcg gtgccggaaa gctggctgga gtgcgatctt

Start of Fab-display cassette, the Fab DSR-A05, selected for binding to a protein antigen.

.0

7233 CCTGAcG xBsu36I

CTCGAG XhoI..

! PlacZ promoter is in the following block ι .5 7246 cgcaacgc aattaatgtg agttagctca

7274 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg

7324 tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca

7374 tgattacgCC AagcttTGGa gccttttttt tggagatttt caac ! PflMI.......

'0 ! Hind3. (there are 3) ! Gene iii signal sequence:

1 M

2 K

3 K

4 L

5 L

6 F

7 8 A I

9 P

10 L

11 V

12 V

13 P

14 F

15 Y

7418

gtg

aaa

aaa

tta

tta

ttc

gca att

cct

tta

gtt

gtt

cct

ttc

tat

16

17

18

Start light

chain (L20:

JKl)

s

H

S

A

Q

D

I Q

M

T

Q

s

P

A

7463

tct

cac

aGT

GCA

Caa

qac

ate caq

atq

acc

caq

tct

cca

gcc

i ApaLI...

!0 ! Sequence supplied by extender............

7505

T acc

L : ctg

S I tct

L ttg

S

P

G

E

R

A

T

L

S

C

R

A

S

Q

G

7517

tct

cca

ggg

gaa

aga

gcc

acc

etc

tee

tgc

agg

gcc

agt

cag

Ggt

V

S

s

Y

L

A

w

Y

Q

Q

K

P

G

Q

A

7562

gtt

age

age

tac

tta

gcc

tgg

tac

cag

cag

aaa

cct

ggc

cag

get

P

R

L

L

I

Y

D

A

s

s

R

A

T

G

I

7607

CCC

agg

etc

etc

ate

tat

gAt

gca

tcc

aAc

agg

gcc

act

ggc

ate

P

A

R

F

S

G

s

G

P

G

T

D

F

T

L

7652

cca

gCc

agg

ttc

agt

ggc

agt

ggg

Cct

ggg

aca

gac

ttc

act

etc

T

I

s

S

L

E

P

E

D

F

A

V

Y

Y

C

7697

acc

ate

age

agC

ctA

gag

cct

gaa

gat

ttt

gca

gtT

tat

tac

tgt

Q

Q

R

S

W

H

P

w

T

F

G

Q

G

T

R

7742

cag

cag

CGt

aAc

tgg

cat

ccg

tgg

ACG

TTC

GGC

CAA

GGG

ACC

AAG

V

E

I

K

R

T

V

A

A

P

S

V

F

I

F

7787

gtg

gaa

ate

aaa

ega

act

gtg

gCT GCA Bsgl. . .

Cca

tct

gtc

ttc

ate

ttc

P

P

S

D

E

Q

L

K

S

G

T

A

S

V

V

7832

ccg

cca

tct

gat

gag

cag

ttg

aaa

tct

gga

act

gcc

tct

gtt

gtg

C

L

L

N

N

F

Y

P

R

E

A

K

V

Q

w

7877

tgc

ctg

ctg

aat

aac

ttc

tat

CCC

aga

gag

gcc

aaa

gta

cag

tgg

- 207 2018241075 03 Oct 2018

I

i 7922

K aag

V gtg

D gat

N aac

A gcc

L etc

Q caa

S teg

G ggt

N aac

S tee

Q cag

E gag

s agt

V gtc

5

1

T

E

R

D

s

K

D

S

T

Y

S

L

s

S

T

7967

aca

gag

egg

gac

age

aag

gac

age

acc

tac

age

etc

age

age

acc

1

L

T

L

S

K

A

D

Y

E

K

H

K

V

Y

A

8012

ctg

acG

CTG

AGC

aaa

gca

gac

tac

gag

aaa

cac

aaa

gtc

tac

gcc

10

1

EspI.

J

C

E

V

T

H

Q

G

L

s

S

P

V

T

K

S

8057

tgc

gaa

gtc

acc

cat

cag

ggc

ctG

AGC

TCg

ccc

gtc

aca

aag

age

Sacl.. . .

15

1 1 8102 1

F ttc

N aac

R agg

G gga

E gag

C tgt

taa

taa

8126

GGCGCG CCaattctat ttcaaGGAGA cagtcata

20

1

Ascl

RBS2

-

1

PelB signal

sequence---

(22

codons)--

--->

!

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

r

M

K

Y

L

L

P

T

A

A

A

G

L

L

L

L

25

8160 t

atg

aaa

tac

eta

ttg

cct

aeg

gca

gee

get

gga

ttg

tta

tta

etc

η τ π

04- -i VU

PP 1

. . .

s ignal--

oiarl Vn

f J- 1\ X

1

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

1

A

A

Q

P

A

M

A

E

V

Q

L

L

E

S

G

30

8205

geG

GCC

cag

ccG

GCC

atg

gee

gaa

gtt

CAA

TTG

tta

gag

tet

ggt

1

Sfil..

Mfel

Ncol. . . .

35

J 1 8250 t

31 G ggc

32 G ggt

33 L ctt

34 V gtt

35 Q cag

36 P cct

37 G ggt

38 G ggt

39 S tet

40 L tta

41 R cgt

42 L Ctt

43 S tet

44 C tgc

45 A get

PT? Ί — -

• . . r i\l

CLJlkX

t

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

40

1

A

s

G

F

T

F

s

T

Y

E

M

R

W

V

R

8295

get

TCC

GGA

ttc

act

ttc

tet

act

tac

gag

atg

cgt

tgg

gtt

cgC

J 1

BspEl..

BstXI

1

FR2----

--->

CDR2 —-

---->

45

1

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

1

<2

A

P

G

K

G

L

E

W

V

S

Y

I

A

P

8340

CAa

get

ccT

GGt

aaa

ggt

ttg

gag

tgg

gtt

tet

tat

ate

get

cct

! BstXI.

RD

ΡΠΡ O

• . dJi\Z

I

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

1

S

G

G

D

T

A

Y

A

D

S

V

K

G

R

F

8385 1

tet

ggt

ggc

gat

act

get

tat

get

gac

tee

gtt

aaa

ggt

cgc

ttc

55

1

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

f

T

I

s

R

D

N

S

K

N

T

L

Y

L

Q

M

8430

act

ate

TCT

AGA

qae

aac

tet

aaq

aat

act

etc

tac

ttCL

caq

atq

I

Xbal

{

Supc

>liec

1 by extender----

FR3

- 208 2018241075 03 Oct 2018 .0

L5 >0

8475

8520

8565

8595

8640

8685

8730

8775

Bsu36I

8820

8865

8910 ..Notl

8955

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

N

S

L

R

A

E

D

T

A

V

Y

Y

C

A

R

aac

aqC

TTA AGo

qct

qaq

qae

act

qca

qtc

tac

tat

tgt

gcg

agg

Aflll.

. . .

from

exisncisr

CDR3

FR4-

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

R

L

D

G

Y

I

S

Y

Y

Y

G

M

D

V

W

agg

etc

gat

ggc

tat

att

tcc

tac

tac

tac

ggt

atg

GAC

GTC

tgg

Aatll..

136	137	138	139	140	141	142	143	144	145
G	Q	G	T	T	V	T	V	. S	S
ggc	caa	ggg	acc	acG	GTC	ACC	gtc	tea	age

BstEII...

CHI

of

IgGl-

—

—

->

A

S

T

K

G

P

S

V

F

P

L

A

P

S

S

gcc

tcc

acc

aag

ggc

cca

teg

gtc

ttc

ccc

ctg

gca

ccc

tcc

tcc

K

S

T

S

G

G

T

A

A

L

G

C

L

V

K

aag

age

acc

tet

ggg

ggc

aca

gcg

gcc

ctg

ggc

tgc

ctg

gtc

aag

D

Y

F

P

E

P

V

T

V

S

w

N

S

G

A

gac

tac

ttc

ccc

gaa

ccg

gtg

aeg

gtg

teg

tgg

aac

tea

ggc

gcc

L

T

S

G

V

H

T

F

P

A

V

L

Q

S

S

ctg

acc

age

ggc

gtc

cac

acc

ttc

ccg

get

gtc

eta

cag

tee

TCA

Bsu36I....

G

L

Y

S

L

S

S

V

V

T

V

P

s

S

S

GGa

etc

tac

tcc

etc

age

age

gta

gtg

acc

gtg

ccc

tcc

age

age

L

G

T

Q

T

Y

I

C

N

V

N

H

K

P

S

ttg

ggc

acc

cag

acc

tac

ate

tgc

aac

gtg

aat

cac

aag

ccc

age

N

T

K

V

D

K

K

V

E

P

K

S

C

A

A

aac

acc

aag

gtg

gac

aag

aaa

gtt

gag

ccc

aaa

tet

tgt

GCG

GCC

Notl......

AHHHHHHGAAEQKLI GCa cat cat cat cac cat cac ggg gcc gca gaa caa aaa etc ate

H6 tag................. Myc-Tag........................

SEEDLNGAAqASSA tea gaa gag gat ctg aat ggg gcc gca tag GCT AGC tet get Myc-Tag.................... ... Nhel. . .

Amber

III'stump

Domain 3 of III ------------------------------------------------------SG DFDYEKMA NANKG A 8997 agt ggc gac ttc gac tac gag aaa atg get aat gcc aac aaa GGC GCC tcc tttttag acttggt !W.T

Kasl. . . (2/4)

MTENADENAL QSDAK G

- 209 2018241075 03 Oct 2018

9045 1

atG

ACT c

GAG a

AAC t

GCT c

GAC t

GAG a

aat c

get g

ttg c a

caa g

age tet

gat c

gee t

aag a

ggt c

1

K

L

D

S

V

A

T

D

Y

G

A

A

1

D

G

F

5

9093

aag

tta

gac

age

gTC

GCG

Acc

gac

tat

GGC

GCC

gee

ATC

GAc

ggc

ttt

1

a

c t

t

tet

t

t

t

c

t

t

t

t

t

c

1

Nrul.. .

Kasl

(3/4)

1

1

G

D

V

s

G

L

A

N

G

N

G

A

T

G

D

10

9141

ate

ggc

gat

gtc

agt

ggt

tTG

GCC

Aac

ggc

aac

gga

gee

acc

gga

gac

1

t

t

c

t

tcc

c

c t

t

t

t

t

t

t

t

t

t

1

Mscl..

.(3/3)

1

F

A

G

S

N

s

Q

M

A

Q

V

G

D

G

D

N

15

9189

ttc

GCA

GGT

teG

AAT

TCt

cag

atg

geC

CAG

GTT

GGA

GAT

GGg

gac

aac

I

t

t

c

t

c

a

t

a

c

t

c

t

t

t

I

BspMI..

(2/2)

Xcml..

EcoRI...

20

9237

S agt tea

P ccg t

L ett t a

M atg

N aac t

N aac t

F ttt c

R aga c t

Q cag a

Y tac t

L ett t a

P ccg t

s tet c

L ett c

P ccg t

Q cag a

S

V

E

C

R

P

F

V

F

S

A

G

K

P

Y

E

25

9285

agt

gtc

gag

tgc

cgt

cca

ttc

gtt

ttc

tet

gee

ggc

aag

cct

tac

gag

teg

t

a

t

c

t

t

c

t

age

t

t

a

a

t

a

F

S

1

D

c

D

K

I

N

L

F

R

9333

ttc

aGC

Ate

gac

TGC

gat

aag

ate

aat

ett

ttc

CGC

30

t

tet

t

t

t

c

a

a

c

t a

c

t

! W

T.

BstAPI

SacII

. . .

End

Domain

3

G

V

F

A

F

L

L

Y

V

A

T

F

M

Y

V

F

35

9369

GGc

gtt

ttc

get

ttc

ttg

eta

tac

gtc

get

act

ttc

atg

tac

gtt

ttc

t

c

t

g

t

c t

t a

t

t

c

c

t

t

a

t

start transmembrane

segment

S

T

F

A

N

1

L

R

N

K

E

S

40

9417

aGC

ACT

TTC

GCC

AAT

ATT

TTA

Cgc aac aaa gaa age

tet

g

t

t

c

a

c g

t

t

g

g tet !W.T.

Intracellular

anchor.

9453 tag tga tet CCT AGG ! Avril..

I

9468 aag ccc gee taa tga geg ggc ttt ttt ttt et ggt ! I Trp terminator I !

! End Fab cassette

I

9503 ATGCAT CCTGAGG ccgat actgtcgtcg tcccctcaaa ctggcagatg ! Nsil.. Bsu361.(3/3)

9551 cacggttacg atgcgcccat ctacaccaac gtgacctatc ccattacggt caatccgccg

9611 tttgttccca cggagaatcc gacgggttgt tactcgctca catttaatgt tgatgaaagc

9671 tggctacagg aaggccagac gegaattatt tttgatggcg ttcctattgg ttaaaaaatg

9731 agctgattta acaaaaattt aaTgegaatt ttaacaaaat attaacgttt acaATTTAAA ! Swal.. .

9791 Tatttgetta tacaatcttc ctgtttttgg ggcttttctg attatcaacc GGGGTAcat

9850 ATG att gac atg eta gtt tta ega tta ccg ttc ate gat tet ett gtt tgc

- 210 2018241075 03 Oct 2018

Start gene II

9901

tcc

aga

etc

tea

ggc

aat

gac

ctg

ata

gee

ttt

gtA GAT Bglll.

CTc

tea

aaa

ata

9952

get

acc

etc

tec

ggc

atT

aat

tta

tea

get

aga

aeg gtt

gaa

tat

cat

att

10003

gat

ggt

gat

ttg

act

gtc

tec

ggc

ett

tet

cac

cct ttt

gaa

tet

tta

cct

10054

aca

cat

tac

tea

ggc

att

gca

ttt

aaa

ata

tat

gag gg*-

tet

aaa

aat

ttt

10105

tat

cct

tgc

gtt

gaa

ata

aag

get

tet

ccc

gca

aaa gta

tta

cag

ggt

cat

10156

aat

gtt

ttt

ggt

aca

acc

gat

tta

get

tta

tgc

tet gag

get

tta

ttg

ett

10207

aat

ttt

get

aat

tet

ttg

cct

tgc

ctg

tat

gat

tta ttg

gat

gtt

1

gene

II .

continues

------------------------End _of Table

- 211 2018241075 03 Oct 2018

Table 37: DNA seq of w.t. M13 gene iii

1579	1 2 fM K gtg aaa Signal	3 K aaa sequ	4 L tta ence	5 L tta	6 F ttc	7 A gca	8 I att	9 P cct	10 L tta
	16	17	18	19	20	21	22	23	24	25
	S	H	S	A	E	T	V	E	S	C
1624	tet	cac	tcc	get	gaa	act	gtt	gaa	agt	tgt

V

L

Signal sequencer Domain 112

V

P

F

Y

A

K

P

H

1669

1714

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

T

E

N

S

F

T

N

V

w

K

D

D

K

T

L

aca

gaa

aat

tea

ttt

act

aac

gtc

tgg

aaa

gac

gac

aaa

act

tta

Domain 1

46

47

48

49

50

51

52

53

54

55

56

57

58

59

60

D

R

Y

A

N

Y

E

G

c

L

W

N

A

T

G

gat

cgt

tac

get

aac

tat

gag

ggt

tgt

ctg

tgG

AAT

GCt

aca

ggc

BsmI....

1759

1804

1849

1894

1939

Domain 1---------------------------------------------------

61

62

63

64

65

66

67

68

69

70

71

72

73

74

75

V

V

V

C

T

G

D

E

T

Q

C

Y

G

T

W

gtt

gta

gtt

tgt

act

ggt

gac

gaa

act

cag

tgt

tac

ggt

aca

tgg

Domain 1

76

77

78

79

80

81

82

83

84

85

86

87

88

89

90

V

P

I

G

L

A

I

P

E

N

E

G

G

G

s

gtt

cct

att

ggg

ett

get

ate

cct

gaa

aat

gag

ggt

ggt

ggc

tet

Domain 1

Linker 1

91

92

93

94

95

96

97

98

99

100

101

102

103

104

105

E

G

G

G

S

E

G

G

G

S

E

G

G

G

T

gag

ggt

ggc

ggt

tet

gag

ggt

ggc

ggt

tet

gag

ggt

ggc

ggt

act

Linker 1

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

K

P

P

E

Y

G

D

T

P

I

P

G

Y

T

Y

aaa

cct

cct

gag

tac

ggt

gat

aca

cct

att

ccg

ggc

tat

act

tat

Domain 2

121

122

123

124

125

126

127

128

129

130

131

132

133

134

135

I

N

P

L

D

G

T

Y

P

P

G

T

E

Q

N

ate

aac

cct

etc

gac

ggc

act

taT

CCG

cct

ggt

act

gag

caa

aac

Ecil....

1984

Domain 2---------------------------------------------------

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

P

A

N

P

N

P

S

L

E

E

S

Q

P

L

N

ccc

get

aat

cct

aat

cct

tet

ett

GAG

GAG

tet

cag

cct

ett

aat

BseRI..

2029

Domain 2----------------------------------------------------

151 152 153

154

155

156

157

158

159

160

161

162

163

164

165

T F M

F

Q

N

N

R

F

R

N

R

Q

G

A

act ttc atg Domain 2----

ttt

cag

aat

aat

agg

ttc

ega

aat

agg

cag

ggg

gca

166 167 168 169 170 171 172 173 174 175 176 177 178 179 180

- 212 2018241075 03 Oct 2018 :5 ίο

LTVYTGTVTQGTDPV 2074 tta act gtt tat aeg ggc act gtt act caa ggc act gac ccc gtt

Domain 2----------------------------------------------------181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 KTYYQYTPVSSKAMY

2119 aaa act tat tac cag tac act cct gta tea tea aaa gee atg tat Domain 2--------------------------------------------------196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 DAYWNGKFRDCAFHS

2164 gac get tac tgg aac ggt aaa ttC AGa gaC TGc get ttc cat tet

AlwNI.......

Domain 2--------------------------------------------------211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 GFNEDPFVCEYQGQS

2209 ggc ttt aat gaG GAT CCa ttc gtt tgt gaa tat caa ggc caa teg BamHI...

Domain 2--------------------------------------------------226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 SDLPQPPVNAGGGSG

2254 tet gac ctg cct caa cct cct gtc aat get ggc ggc ggc tet ggt Domain 2------------------------------> Linker 2----------241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 GG SGGGSEGGGSEGG

2299 ggt ggt tet ggt ggc ggc tet gag ggt ggt ggc tet gag ggt ggc Linker 2--------------------------------------------------256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 GS EGGGSEGGGSGGG

2344 ggt tet gag ggt ggc ggc tet gag gga ggc ggt tcc ggt ggt ggc Linker 2--------------------------------------------------•ιο

271

272

273

274

275

276

277

278

279

280

281

282

283

284

285

S

G

S

G

D

F

D

Y

E

K

M

A

N

A

N

2389

tet

ggt

tcc

ggt

gat

ttt

gat

tat

gaa

aag

atg

gca

aac

get

aat

Linker 2>

Domain 3

286

287

288

289

290

291

292

293

294

295

296

297

298

299

300

K

G

A

M

T

E

N

A

D

E

N

A

L

Q

S

2434

aag

ggg

get

atg

acc

gaa

aat

gee

gat

gaa

aac

geg

eta

cag

tet

>0

Domain 3--------------------------------------------------301 302 303 304 305 306 307 308 309 310 311 312 313 314 315 DAKGKLDSVATDYGA

2479 gac get aaa ggc aaa ctt gat tet gtc get act gat tac ggt get Domain 3--------------------------------------------------316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 Al DGFIGDVSGLANG

2524 get ate gat ggt ttc att ggt gac gtt tcc ggc ctt get aat ggt Domain 3--------------------------------------------------331 332 333 334 335 336 337 338 339 340 341 342 343 344 345

NGA TGDFAGSNSQMA

2569 aat ggt get act ggt gat ttt get ggc tet aat tcc caa atg get

Domain 3--------------------------------------------------50

- 213 2018241075 03 Oct 2018

346 Q

347 V

348 G

349 D

350 G

351 D

352 N

353 S

354 P

355 L

356 M

357 N

358 N

359 F

360 R

2614

caa

gtc

ggt

gac

ggt

gat

aat

tea

cct

tta

atg

aat

aat

ttc

cgt

T-S *

UOlUci-LIl

—J

361

362

363

364

365

366

367

368

369

370

371

372

373

374

375

Q

Y

L

P

S

L

P

Q

S

V

E

C

R

P

F

2659

caa

tat

tta

cct

tee

etc

cct

caa

teg

gtt

gaa

tgt

ege

cct

ttt

! Domain 3--------------------------------------------------10 !

! 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 ! VFSAGKPYEFFIDCD

2704 gtc ttt age get ggt aaa cca tat gaa ttt tet att gat tgt gac ! Domain 3--------------------------------------------------15 !

! 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 ! KINLFRGVFAFLLYV

2749 aaa ata aac tta ttc cgt ggt gtc ttt gcg ttt ett tta tat gtt ! Domain 3--------------> Transmembrane segment-------------20 !

! 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 ! ATFMYVFSTFANILR

2794 gcc acc ttt atg tat gta ttt tet aeg ttt get aac ata ctg cgt ! Transmembrane segment---------------------------------> ICA— !

! 421 422 423 424 425 ! N Κ E S

2839 aat aag gag tet taa ! 2853 ί ICA-----------> ICA = intracellular anchor !

i------------------End of Table----------------------------------2018241075 03 Oct 2018

214 Table 38: Whole mature III anchor M13-III derived anchor with recoded DNA

3

AAA

GCG gcc gca

Notl......

	1		4	5	6	7	8	9	10	11	12	13	14	15	16	17
0	1		H	H	H	H	H	H	G	A	A	E	Q	K	L	I
		10	cat	cat .	cat	cac	cat	cac	ggg	gcc	gca	gaa	caa	aaa	etc	ate
	1		18	19	20	21	22	23	24	25	26	27	28	29
	1		S	E	E	D	L	N	G	A	A		A	S
5		52	tea	gaa	gag	gat	ctg	aat	ggg	gee	gca	Tag	GCT	AGC
	1												Nhel
			30 31	32	33 34	35	36	37	38	39
			D I	N	D D	R	M	A	S	T
0		88	GAT ATC aac aat qat cqt atq	get	tet	act

(ON_G37bot) [RC] 5'-c aac gat gat cgt atg gcG CAt Get gcc gag aca g-3' EcoRV..

Enterokinase cleavage site.

Start mature III (recoded) Domain 1 40 41 42 43

A Ε T V IgcCIgaG|acA|gtC |

--->

;5

118

E

S

C

L

A

K

W.T.

P

H

T

54

Ε N

S

F

T

N

130 |gaaITCC|tgC|CTG|GCCIAaG|ccT|caC|acT|gaGIaat | AGT|ttC|aCA|Aat| agt tta a a Mscl....

tea c ! W.

60

V W

62 K D

D

65

K T

L

D

R

70

Y A

N

73

Y E

175 |gtg|TGGIaaGIgaT|gaT|aaG|acC|CtT|gAT|CGA| TaT|gcC|aaT|taC|gaA| tta t c BspDI...

g ! W.T

75 76 77 78 79 80 81

GCLWNATG

V

84

V V

C

87

T G

D

220 |ggC|tgCITtA|tggIaat|gcCI ACC|GGC|GtCIgtTIgtC| TGCIACG|ggC|gaT|

E

T teg

Q

C

Y

G t a SgrAI.

T t t Bsgl..

c ! W.T

W

V

P

I

100 101 102 103

65 |gaGIacA|caA|tgC11aT|ggC|ACG|TGg|gtGIccGI atA| gGC|TTA|GCC|atA I get Blpl...

W.T

Domain 1-----> Linker 1---------------->

104 105 106 107 108 109 110 111 112 113 114 115 116 117 PENEGGGSEGGG SE 310 | ccGIgaG|aaC|gaA|ggCIggC|ggTIAGCIgaAlggCIggT | ggC |AGC|gaA|ggC| t a t g t t ctct g t c ttet g t ! W.T

118

G t a

Pmll..

Linker 1-> Domain 2->

- 215 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

GGSEGGGTKPPEYGD

355 I ggT | GGA I TCC I gaA | ggA | ggT I ggA | acC I aaG | ccG I ccG | gaA I taT | ggC I gaC I ett gtet t at tg c tt! W.T

BamHI..(2/2)

134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 TPIPGYTYINPLDGT

00 IacT|ccGI atA|CCT|GGT|taC|acC11aCIatT|aaTIccG|TtAIgaT | ggA I acC I att gett t cctcc c ct! W.T

SexAI....

2018241075 03 Oct 2018

LO

149 150

151

152

153

154

155

156

157

158

159

160

161

162

163

Y P

P

G

T

E

Q

N

P

A

N

P

N

P

S

445 |taCIccT1ccG1ggC1acC|gaA|caG|aaT|

ccT|geC i

I aaCI

ccG [

[aaCIccA!

[AGC

T G

t

t

t

g

a

c

c

t

t

t

t

t

tet

Hindlll...

164

165

166

167

168

169

170

171

172

173

174

175

176

177

178

L

E

E

S

Q

P

L

N

T

F

M

F

Q

N

N

490 |TTA|gaA|

gaA|AGC1caA|

ccGITtAI

aaC I

acC |

ttT |

atgIttC[

[caA|aaCI

aaC I

c t

G

G

tet

g

t

c t

t

t

c

t

g

t

t

Hindlll.

179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 RFRNRQGALTVYTGT

535 I CgT | ttT | AgG | aaC | CgT | caA | gGT | GCT | CtT | acC I gTG I TAC | AcT | ggA I acC I ag cca tag g g ata t t t g c t! W.T

HgiAI... BsrGI...

194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 VTQGTDPVKTYYQYT

580 | gtCIacCIcaGIGGTI ACC|gaT|ccT|gtC|aaG|acC|taC|taT|caA|taTIacCI ttactcctattcgct! W.T

Kpnl...

209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 PVSSKAMYDAYWNGK

625 | ccG | gtCITCGIAGtIaaGIgeT|atgItaC|gaTIgeCItaT(tgg|aaT| ggC(aaG|

t Bs

a al. . Xh

a tea ol. . . .

a

c

t

c

t

c

c

t

a !

W.T

224

225

226

227

228

229

230

231

232

233

234

235

236

237

238

F

R

D

C

A

F

H

S

G

F

N

E

D

P

F

670

IttTICgTI

gaT|

tgTIgeC|

ttT|

caC |

AGC |

ggTIttCI

aaCIgaa1

gac |

CCt|

ttT|

C

A a

C

c

t

c

t

tet

c

t

t

G

T

a

c !

W.T

239

240

241

242

243

244

245

246

247

248

249

250

251

252

253

V

C

E

Y

Q

G

Q

S

S

D

L

P

Q

P

P

715

Igtci

tgC|

gaG i taC|

caG I

ggTi

caG |

AGT |

AGC |

gaTITtAI

ccG|caGIccAlCCG I

t

t

a

t

a

c

a

teg

tet

c

c g

t

a

t

t !

W.T

Drdl.

Agel...

Domain 2-

--->

Linker

2--

--->

254

255

256

257

258

259

' 260

261

262

263

264

265

266

267

268

V

N

A

G

G

G

S

G

G

G

S

G

G

G

S

760

1GTT|AAC

IgcG

IggT

IggTIggTIAGC

: 1ggC|ggA|ggC

IAGC|ggC

IggT

IggT|AGC|

c

t

t

c

c

c

: tet

t

t

t

tet

t

c

c

: tet

! W.

Age I.....

Hpal...

- 216 2018241075 03 Oct 2018

Hindi.

Linker 2

Domain

269

270

271

272

273

274

275

276

277

278

279

280

281

282

283

5

E

G

G

G

S

E

G

G

G

S

G

G

G

S

G

805

1 gaA

ggCI

ggA

ggTIAGCI

gaA

ggAIggTIggC

AGC |

ggA

ggc

ggT

AGC

ggCI

g

t

t

c

tet

g

t

c

t

tet

g

t

c

tet

t !

t

-Domain 3

0

284

285

286

287

288

289

290

291

292

293

294

295

296

297

298

S

G

D

F

D

Y

E

K

M

A

N

A

N

K

G

850

I AGf

ggC|

gacIttc

gac|tac|gag

aaa

atg

get|aat

gee|aac

aaa

GGC |

tcc

t

t

t

t

t

a

g

a

c

t

t

g

g !

W

5

KasI..

-

299

300

301

302

303

304

305

306

307

308

309

310

311

312

313

A

M

T

E

N

A

D

E

N

A

L

Q

S

D

A

895

I GCC

atg |

act

gag 1aac|

get

gac

gaGIAAT|GCA

ctg

caa

agt

gat IgCC1

t

c

a

t

c

t

a

C

g

a

g

tet

c

t !

w

0

KasI

. . . .

BsmI.

Styl.

-

314

315

316

317

318

319

320

321

322

323

324

325

326

327

328

K

G

K

L

D

S

V

A.

T

D

Y

G

A

A

I

940

I AAG|GGt|aag|tta1gac|age 1gTC

GCc

Aca

gac

tat

ggT|GCt

gee

ate |

5

a

c

a

c t

t

tet

t

t

t

c

t

1

w

Styl

PflFI..

329

330

331

332

333

334

335

336

337

338

339

340

341

342

343

D

G

F

I

G

D

V

S

G

L

A

N

G

N

G

0

985

1 gac

ggc

tttI ate

ggc|gat

gtc

agt|ggt1ctg

get

aac

ggc

aac

gga |

t

t

c

t

t

c

t

tec

c

c t

t

t

t

t !

w

344

345

346

347

348

349

350

351

352

353

A

T

G

D

F

A

G

S

N

S

5

1030

igee

acc

gga

gac

ttc

GCA

IGGTItcG

|AAT

|TCt

t

t

t

t

t

t

c

t

c

! W

T.

BstBI...

EcoRI...

0 ! 1

354 Q cag a

355 M atg

356 A geC t X

357 Q CAG a cml.

358 V GTT c

BspMI..

361 G GGg t

362 D gac t

363 N aac t

ί W.T.

359 G GGA t

360 D GAT c

5

1 1060 1 1

J

364

365

366

367

368

369

370

371

372

373

374

375

376

377

378

379

f

S

P

L

M

N

N

F

R

Q

Y

L

P

S

L

P

Q

1090

agt

ccg

ett

atg

aac

aac

ttt

aga

cag

tac

ett

ccg

tet

ett

ccg

cag

0

!

tea

t

t a

t

t

c

c t

a

t

t a

t

c

c

t

a

I

380

381

382

383

384

385

386

387

388

389

390

391

392

393

394

395

1

S

V

E

C

R

P

F

V

F

S

A

G

K

P

Y

E

1138

agt

gtc

gag

tgc

cgt

cca

ttc

gtt

ttc

tet

gee

ggc

aag

cct

tac

gag

5

1 1

teg

t

a

t

c

t

t

c

t

age

t

t

a

a

t

a

	uuiudiu . 396 397	3 398	399	400	401	402	403	404	405	406	407
	F S	I	D	C	D	K	I	N	L	F	R
	1186 f . c aGC	Ate	gac	TGC	gat	aag	ate	aat	ett	ttc	CGC
	t tet	t	t	t	c	a	a	c	t a		t

- 217 2018241075 03 Oct 2018

BstAPI........ SacII...

transmembrane segment------------->

408

409

410

411

412

413

414

415 416 417 418

419 420

421

422

423

G

V

F

A

F

L

L

Y V A T

F M

Y

V

F

1222

GGc

gtt

ttc

get

ttc

ttg

eta

tac gtc get act

ttc atg

tac

gtt

ttc

t

c

t

g

t

c t

t a

t t c c

t

t

a

t

424

425

426

427

428

429

430

431 432 433 434 435

S

T

F

A .

N

I

L

R N K

E S

1270

aGC

ACT

TTC

GCC

AAT

ATT

TTA

Cgc aac aaa gaa age

tet

g

t

t

c

a

c g

ttg

g tet !

W.T.

Intracellular

anchor.

1306

tag

tga

tet

CCT

AGG

Avril..

1321

aag

ccc

gcc

taa

tga

geg

ggc

ttt ttt ttt et

ggt

I Trp terminator

End Fab cassette

---------------------------- End of Table

- 218 2018241075 03 Oct 2018 .0

L5

Table 39: ONs to make deletions in III ! ONs for use with Nhel

N (ON_G29bot) 5'-c gTT gAT ATc gcT Age cTA Tgc-3' ! this is the reverse complement of 5'-gca tag get age gat ate aac g-3' i Nhel... scab.........

(ON_G104top) 5'-gIataIggc|tta|gcTIaGC|ccg|gag 1aacIgaaIgg-3' ! Scab..........Nhel... 104 105 106 107 108 (ON_G236top) 5·-c|tttIcac i age j ggt|ttc|GCT|AGC|gac j cct i ttt|gtc j tgc-3' ! Nhel... 236 237 238 239 240 (ON_G236tCS) 5'-cIttt1cacI age|ggt|ttc|GCT|AGC|gacIcct I ttt|gtc | Agc! Nhel... 236 237 238 239 240 gag|tacIcag|ggt|c-3 ' >0 >5 ! ONs for use with SphI G CAT Gc (ON_X37bot) 5'-gAc TgT cTc ggc Age ATg ege cAT Aeg ATc ATc gTT g-3* !

! NDDRMAHA ! (ON_X37bot) = [RC] 5'-c aac gat gat cgt atg qcG CAt Get gcc gag aca gtc-3' ! SphI....Scab...........

(ON_X104top) (ON_X236top) ι

(ON_X236tCS)

5'-g|gtG ccg|ataIggcIttG1 CAT|GCa|ccg|gag | aac|gaa|ggScab.................SphI.... 104 105 106 107 108

5'-cItttIcacI age|ggt|ttGICaT|gCa|gacIcct| ttt|gtc|tgc-3'

SphI.... 236 237 238 239 240 5'-c|ttt|cac|age|ggtIttG1CaT|gCa|gacIcct | ttt |gtc|AgcNhel... 236 237 238 239 240 gag|tac|cag|ggtIc-3'

- 219 2018241075 03 Oct 2018

Table 40: Phage titers and enrichments of a selections with a DY3F31-based human Fab library

	Input (total cfu)	Output (total cfu)	Output/input ratio
Rl-ox selected on phOx-BSA	4,5 x 1012	3,4 x 105	7,5 x 10'8
R2-Strep selected on Strep-beads	9,2 x 1012	3 x 108	3,3 x IO’5

- 220 2018241075 03 Oct 2018

Table 41: Frequency of ELISA positives in DY3F31-based Fab libraries

	Anti-M13 HRP	9E10/RAM- HRP	Anti-CK/CL Gar-HRP
R2-ox (with IPTG induction)	18/44	10/44	10/44
R2-ox (without IPTG)	13/44	ND	ND
R3-strep (with IPTG)	39/44	38/44	36/44
R3-strep (without IPTG)	33/44	ND	ND

2018241075 03 Oct 2018

Claims (22)

1. - 221 1. A method for cleaving single-stranded nucleic acid sequences ar a desired location, the method comprising the steps of:

   (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

   the contacting and the cleaving steps being performed

   20 at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur

   25 at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
2. 2. A method for cleaving single-stranded nucleic acid sequences at a desired location, the

   30 method comprising the steps of:

   2018241075 03 Oct 2018

   - 222 (i) contacting the nucleic acid with a partially double-stranded oligonucleotide,

   Si-iiy jlc —o Lj.tcyiun ux cut;

   oligonucleotide being functionally 5 complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and

   10 (ii) cleaving the nucleic acid solely at the restriction endonuclease recognition site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

   15 the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the

   20 two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
3. 3. In a method for displaying a member of a

   25 diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the family, the improvement being characterized in that the displayed peptide, polypeptide or protein is encoded at

   30 least in part by a nucleic acid that has been cleaved

   2018241075 03 Oct 2018

   - 223 at a desired location by a method comprising the steps of:

   ( jl } — u-Oii LaC L my the nucleic acid witn a single-stranded oligonucleotide, the

   5 oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction

   10 endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the

   15 complementation of the nucleic acid and the oligonucleotide;

   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the

   20 oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction

   25 endonuclease that is acti-ve at the chosen temperature.
4. 4 . In a method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the

   30 family, the improvement being characterized in that the displayed peptide, polypeptide or protein is encoded by

   2018241075 03 Oct 2018

   - 224 a DNA sequence comprising a nucleic acid that has been cleaved at a desired location by (i) contacting the nucleic acid with a partially double-stranded oligonucleotide,
5. 5 the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide

   10 having a restriction endonuclease recognition site; and (ii) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site formed by the complementation

   15 of the nucleic acid and the single-stranded region of the oligonucleotide;

   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the

   20 oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction

   25 endonuclease that is active at the chosen temperature.

   5. A method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the

   30 family, the method comprising the steps of:

   2018241075 03 Oct 2018

   - 225 (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;

   (ii) rendering the nucleic acids single5 stranded;

   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:

   (a) contacting the nucleic acid with a

   10 single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement

   15 in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at

   20 the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

   the contacting and the cleaving steps being performed at a temperature sufficient to maintain 25 the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature; and

   2018241075 03 Oct 2018

   - 226 (iv) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by Lhe cleaved nucleic acids on the surface of the genetic package and collectively displaying at

   5 least a portion of the diversity of the family.
6. 6. A method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a portion of the diversity of the

   10 family, the method comprising the steps of:

   (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse family;

   (ii) rendering the nucleic acids single15 stranded;

   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:

   (a) contacting the nucleic acid with a

   20 partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the

   25 double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and (b) cleaving the nucleic acid solely at the restriction endonuclease recognition

   30 cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

   2018241075 03 Oct 2018

   - 227 the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nueieie acid in substantially single-stranded form, the oligonucleotide being functionally

   5 complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a

   10 cleavage endonuclease that is active at the chosen temperature; and (iv) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of

   15 the genetic package and collectively displaying at least a portion of the diversity of the family.
7. 7. In a method for expressing a member of a diverse family of peptides, polypeptides or proteins and collectively expressing at least a part of the 20 diversity of the family, the improvement being characterized in that the expressed peptide, polypeptide or protein is encoded at least in part by a nucleic acid that has been cleaved at a desired location by a method comprising the steps of:

   25 (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on

   2018241075 03 Oct 2018

   - 228 restriction results in cleavage of the nucleic acid at the desired location; and (ii) ^iedviny the nucleic acid solely at the recognition site formed by the

   5 complementation of the nucleic acid and the oligonucleotide;

   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the

   10 oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction

   15 endonuclease that is active at the chosen temperature.
8. 8. In a method for expressing a member of a diverse family of peptides, polypeptides or proteins and collectively expressing at least a part of the diversity of the family, the improvement being 20 characterized in that the expressed peptide, polypeptide or protein is encoded by a DNA sequence comprising a nucleic acid that has been cleaved at a desired location by (i) contacting the nucleic acid with a 25 partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide

   2018241075 03 Oct 2018

   - 229 having a restriction endonuclease recognition site; and (ii) cleaving one nucleic acid solely at the restriction endonuclease recognition

   5 cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

   the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic

   10 acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location,

   15 and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
9. 9. A method for expressing a member of a diverse family of peptides, polypeptides or proteins and collectively expressing at least a part of the

   20 diversity of the family, the method comprising the steps of:

   (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;

   25 (ii) rendering the nucleic acids singlestranded;

   (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:

   (a) contacting the nucleic acid with a single-stranded oligonucleotide, the

   2018241075 03 Oct 2018

   - 230 oligonucleotide being functionally complementary to the nucleic acid in the region m wnicn cleavage is desired and including a sequence that with its complement

   5 in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at 10 the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

   the contacting and the cleaving steps being performed at a temperature sufficient to maintain

   15 the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the

   20 chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature; and (iv) expressing a member of the family of

   25 peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids and collectively expressing at least a portion of the diversity of the family.
10. 10. A method for expressing a member of a

    30 diverse family of peptides, polypeptides or proteins and collectively expressing at least a portion of the

    2018241075 03 Oct 2018

    - 231 diversity of the family, the method comprising the steps of:

    (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse

    5 family;

    (ii) rendering the nucleic acids singlestranded;

    (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the

    10 steps of:

    (a) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally

    15 complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and

    20 (b) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

    25 the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large

    30 enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a

    2018241075 03 Oct 2018

    - 232 cleavage endonuclease that is active at the chosen temperature; and (iv) expressing a member of the family of peptides, polypeptides or proteins coded, at least in

    5 part, by the cleaved nucleic acids and collectively expressing at least a portion of the diversity of the family.
11. 11. A library comprising a collection of genetic packages that display a member of a diverse

    10 family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family, the library being produced using the methods of claims 3, 4, 5 or 6.
12. 12. A library comprising a collection of

    15 genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the family, the displayed peptides, polypeptides or proteins being encoded by DNA sequences comprising at least in part 20 sequences produced by cleaving single-stranded nucleic acid sequences at a desired location by a method comprising the steps of:

    (i) contacting the nucleic acid with a single-stranded oligonucleotide, the 25 oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on

    2018241075 03 Oct 2018

    - 233 restriction results in cleavage of the nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the

    5 complementation of the nucleic acid and the oligonucleotide;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the

    10 oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction

    15 endonuclease that is active at the chosen temperature.
13. 13. A library comprising a collection of genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and that collectively display at least a portion of the 20 diversity of the family of the displayed peptides, polypeptides or proteins being encoded by DNA sequences comprising at least in part sequences produced by cleaving single-stranded nucleic acid sequences at a desired location by a method comprising the steps of:

    25 (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide

    2018241075 03 Oct 2018

    - 234 having a restriction endonuclease recognition site; and (ii) cleaving tne nucleic acia solely at the restriction endonuclease recognition

    5 cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic

    10 acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location,

    15 and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
14. 14. A library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively comprising at least a

    20 portion of the diversity of the family, the library being produced using the methods of claims 7, 8, 9 or 10.
15. 15. A library comprising a collection of members of a diverse family of peptides, polypeptides

    25 or proteins and collectively comprising at least a portion of diversity of the family, the peptides, polypeptides or proteins being encoded by DNA sequences comprising at least in part sequences produced by cleaving single-stranded nucleic acid sequences at a

    30 desired location by a method comprising the steps of:

    2018241075 03 Oct 2018

    - 235 (i) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the

    5 region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the

    10 nucleic acid at the desired location; and (ii) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

    15 the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the

    20 two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.
16. 16. A library comprising a collection of

    25 members of a diverse family of peptides, polypeptides or proteins and collectively comprising at least a portion of the diversity of the family, the peptides, polypeptides or proteins being encoded by DNA sequences comprising at least in part sequences produced by

    30 cleaving single-stranded nucleic acid sequences at a desired location by a method comprising the steps of:

    2018241075 03 Oct 2018

    - 236 (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally

    5 complementary to the nucleic acid in the region in which cleavage is desired, and the double-stranded region of the oligonucleotide having a restriction endonuclease recognition site; and

    10 (ii) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site formed by the complementation of the nucleic acid and the single-stranded region of the oligonucleotide;

    15 the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the

    20 two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

    25 17. A library of claims 11, 12 or 13 wherein the genetic packages are selected from the group of phage, phagemid or yeast.

    18. A library of claims 17 wherein the genetic packages are selected are phage or phagemid.

    30 19. The methods or libraries according claims 2, 4, 6, 8, 10, 13 or 16 wherein in the

    - 237 2018241075 03 Oct 2018 restriction endonuclease recognition site is for a

    Type II-S restriction endonuclease.

    20. The methods or libraries according to claims 1 to 19, wherein the nucleic acid is cDNA.

    5 21. The methods or libraries according to any one of claims 1 to 20, wherein the nucleic acids encode at least a portion of an immunoglobulin.

    22. The methods or libraries according to claim 21, wherein the immunoglobulin comprises a Fab or

    10 single chain Fv.

    23. The methods or libraries according to claim 21 or 22, wherein the immunoglobulin comprises at least portion of a heavy chain.

    24. The method or libraries according to

    15 claim 23, wherein the heavy chain is IgM, IgG, IgA, IgE or IgD.

    25. The methods or libraries according to claim 23 or 24, wherein at least a portion of the heavy chain is human.

    20 26. The methods or libraries according to claim 21 or 22, wherein the immunoglobulin comprises at least a portion of FR1.

    27. The methods or libraries according to claim 26, wherein at least a portion of the FR1 is

    25 human.

    - 238 2018241075 03 Oct 2018

    28. The methods or libraries according to claim 21 or 22, wherein the immunoglobulin .comprises at least a portion of a light chain.

    5 29. The methods or libraries according to claim 28, wherein at least a portion of the light chain is human.

    30. The methods or libraries according to any one of claims 1 to 16, wherein the nucleic acid

    10 sequences are at least in part derived from patients suffering from at least one autoimmune disease and/or cancer.

    31. The methods or libraries according to claim 30, wherein the autoimmune disease is selected

    15 from the group comprising lupus, erythematosus, systemic sclerosis, rheumatoid arthritis, antiphosolipid syndrome or vasculitis.

    32. The methods or libraries according to claim 30, wherein the nucleic acids are at least in

    20 part isolated from the group comprising peripheral blood cells, bone marrow cells spleen cells or lymph node cells.

    33. The methods according to claim 5, 6, 9 or 10 further comprising at least one nucleic acid

    25 amplification step between one or more of steps (i) and (ii), steps (ii) and (iii) or between steps (iii) and (iv) .

    2018241075 03 Oct 2018

    - 239 34. The method according to claim 33, wherein amplification primers for the amplification step are functionally complementary to a constant region of the nucleic acids.

    5 35. The method according to claim 34, wherein the constant region is genetically constant in the nucleic acids.

    36. The method according to claim 35, wherein the genetically constant region is a part of

    10 the genome of immunoglobulin genes selected from the group of IgM, IgG, IgA, IgE or IgD.

    37. The method according to claim 34, wherein the constant region is exogenous to the nucleic acids .

    15 38. The methods according to claim 33, wherein the amplification step uses geneRACE™.

    39. The methods or libraries according to any one of claims 1 to 16, wherein the chosen temperature is between 37°C and 75°C

    20 40. The methods or libraries according to claim 39, wherein the chosen temperature is between 45°C and 75°C.

    41. The methods or libraries according to claim 40, wherein the chosen temperature is between

    25 50°C and 60°C.

    2018241075 03 Oct 2018

    - 240 42. The methods or libraries according to claim 41, wherein the chosen temperature is between 55°C and 60°C.

    43. The methods or libraries according to 5 claim 1, 3, 5, 7, 9, 12 or 15, wherein the length of the single-stranded oligonucleotide is between 17 and 30 bases.

    44. The methods or libraries according to claim 43, wherein the length of the single-stranded

    10 oligonucleotide is between 18 and 24 bases.

    45. The methods or libraries according to claim 1, 3, 5, 7, 9, 12 or 15, wherein the restriction endonuclease is selected from the group comprising Waelll, Tsp45I, HphI, BsaJI, Alul, BlpI, Ddel, Bglll,

    15 MslI, BsiEI, Eael, Eagl, Haelll, BstACI, HpyCH4III, fiinfl, Mlyl, Plel, Mnll, flpyCH4V, BsmAI, Bpml, Xmnl, or Sacl.

    46. The methods'or libraries according to claim 45, wherein the restriction endonuclease is

    20 selected from the group comprising Bst4CI, Taal, HpyCH4III, BlpI, BpyCH4V or MslI.

    47. The methods or libraries according to claim 2, 4, 6, 8, 10, 13 or 16, wherein the length of the single-stranded region of the partially double25 stranded oligonucleotide is between 14 and 22 bases.

    48. The methods or libraries according to claim 47, wherein the length of the single-stranded

    2018241075 03 Oct 2018

    - 241 region of the partially double-stranded oligonucleotide is between 14 and 17 bases.

    49. The methods or libraries according to claim 47, wherein the length of the single-stranded

    5 region of the oligonucleotide is between 18 and 20 bases.

    50. The methods or libraries according to claim 2, 4, 6, 8, 10, 13 or 16, wherein the length of the double-stranded region of the partially double10 stranded oligonucleotide is between 10 and 14 base pairs formed by a stem and its palindrome.

    51. The methods or libraries according to claim 50 wherein, the partially double-stranded oligonucleotide comprises a loop of 3 to 8 bases

    15 between the stem and the palindrome.

    52. The methods or libraries according to claim 19 wherein the Type II-S restriction endonuclease is selected from the group comprising AarICAC, Acelll, Bbr7l, Bbvl, BbvII, Bce83l, BceAI, BcefI, BciVI, Bfil,

    20 BinI, BscAI, BseRI, BsmFI, BspMI, Ecil, Eco57l, Faul, FokI, Gsul, Hgal, HphI, MboII, Mlyl, Mmel, Mnll, Plel, RleAI, SfaNI, SspD5I, Sthl32I, StsI, TaqII, Tthlllll, or UbaPI.

    53. The methods or libraries according to

    25 claim 52, wherein the Type II-S restriction endonuclease is FokI.

    2018241075 03 Oct 2018

    - 242 54. A method for preparing single-stranded nucleic acids, the method comprising the steps of:

    (i) contacting a single-stranded nucleic acid sequence that has been cleaved with a

    5 restriction endonuclease with a partially double-stranded oligonucleotide, the singlestranded region of the oligonucleotide being functionally complementary to the nucleic acids in the region that remains after

    10 cleavage, the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into proper and original reading frame for expression and containing a

    15 restriction endonuclease recognition site 5' of those sequences; and (ii) cleaving the partially doublestranded oligonucleotide sequence solely at the restriction endonuclease recognition site

    20 contained within the double-stranded region of the partially double-stranded oligonucleotide.

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic

    25 acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location,

    30 and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

    2018241075 03 Oct 2018

    - 243 55. The method according to claim 54, wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 2 and 15 bases.

    5 56. The method according to claim 55, wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 7 and 10 bases.

    57. The method according to claim 54,

    10 wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is between 12 and 100 base pairs.

    58. The method according to claim 57, wherein the length of the double-stranded portion of

    15 the partially double-stranded oligonucleotide is between 20 and 100 base pairs.

    59. A method for preparing a library comprising a collection of genetic packages that display a member of a diverse family of peptides,

    20 polypeptides or proteins and that collectively display at least a portion of the family comprising the steps:

    (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;

    25 (ii) rendering the nucleic acids singlestranded;

    (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:

    2018241075 03 Oct 2018

    - 244 (a) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the

    5 region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the

    10 nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

    15 the contacting and the cleaving steps being performed at a temperature sufficient to maintain· the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large

    20 enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the

    25 chosen temperature;

    (iv) contacting the nucleic acid with a partially double-stranded oligonucleotide, the singlestranded region of the oligonucleotide being functionally complementary to the nucleic acids in the

    30 region that remains after the cleavage in step (iii) has been effected, and the double-stranded region of the oligonucleotide including any sequences necessary to return the sequences that remain after cleavage into

    2018241075 03 Oct 2018

    - 245 proper and original reading frame for display and containing a restriction endonuclease recognition site 0' or tnose sequences tftat is airrerent rrom the restriction site used in step (iii); and

    5 (v) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site contained within the double-stranded region of the partially double-stranded oligonucleotide;

    the contacting and the cleaving steps being 10 performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to

    15 associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and

    20 (vi) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of the genetic package and collectively displaying at least a portion of the diversity of the family.

    25 60. A method for preparing a library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively comprising at least a portion of the family comprising the steps:

    30 (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;

    2018241075 03 Oct 2018

    - 246 (ii) rendering the nucleic acids singlestranded;

    (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the

    5 steps of:

    (a) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the

    10 region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the

    15 nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

    20 the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large

    25 enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the

    30 chosen temperature;

    (iv) contacting the nucleic acid with a partially double-stranded oligonucleotide, the singlestranded region of the oligonucleotide being

    2018241075 03 Oct 2018

    - 247 functionally complementary to the nucleic acids in the region that remains after the cleavage in step (iii) has been effected, and the double-stranded region of the oligonucleotide including any sequence necessary to

    5 return the sequences that remain after cleavage into proper and original reading frame for expression and containing a restriction endonuclease recognition site 5' of those sequences that is different from the restriction site used in step (iii); and

    10 (v) cleaving the nucleic acid solely at the restriction endonuclease recognition cleavage site contained within the double-stranded region of the partially double-stranded oligonucleotide;

    the contacting and the cleaving steps being

    15 performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to

    20 associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and

    25 (vi) expressing a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids and collectively expressing at least a portion of the diversity of the family.

    30 61. The methods according to claim 59 or 60, further comprising at least one nucleic acid amplification step between one or more of steps (i) and

    2018241075 03 Oct 2018

    - 248 (ii), steps (ii) and (iii), steps (iii) and (iv) and steps (iv) and (v).

    62. A library comprising a collection of genetic packages that display a member of a diverse

    5 family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family, the library being produced using the methods of claims 59 or 61.

    63. A library comprising a collection of 10 members of a diverse family of peptides, polypeptides or proteins and collectively comprise at least a portion of the diversity of the family, the library being produced using the methods of claims 60 or 61.

    64. The methods and libraries according to 15 any one of claim 59 to 63, wherein the members of the library encode immunoglobulins.

    65. The method and libraries according to claim 64, wherein the double-stranded region of the oligonucleotide encodes at least a part of a framework

    20 sequence of an immunoglobulin.

    66. The method and libraries according to claim 65, wherein the framework sequence comprises framework 1 of an antibody.

    67. The method and libraries according to 25 claim 66, wherein the framework sequence comprises framework 1 of a variable domain of a light chain.

    2018241075 03 Oct 2018

    - 249 68. The method and libraries according to claim 66, wherein the framework sequence comprises framework 1 of a variable domain of a heavy chain.

    69. The method and libraries according to 5 claim 65, wherein the framework sequence comprises framework 3 of an antibody.

    70. The method and libraries according to claim 69, wherein the framework sequence comprises framework 3 of a variable domain of a light chain.

    10 71. The method and libraries according to claim 69, wherein the framework sequence is framework 3 of a variable domain of a heavy chain.

    72. The method and libraries according to claim 66, wherein the 5' primer is complementary to a

    15 region outside framework 1.

    73. The method according to claim 61, wherein amplification primers for the amplification step are functionally complementary to a constant region of the nucleic acids.

    20 74. The method according to claim 73, wherein the constant region is genetically constant in the nucleic acids .

    75. The method according to claim 74, wherein the genetically constant region is part of the

    25 genome of immunoglobulin genes selected from the group of IgM, IgG, IgA, IgE or IgD.

    - 250 2018241075 03 Oct 2018

    76. The method according to claim 73, wherein the constant region is exogenous to the nucleic acids.

    77. The methods according to claim 61,

    5 wherein the amplification step uses geneRACE™.

    78. A vector comprising:

    (i) a DNA seguence encoding an antibody variable region linked to a version of PHI anchor which does not mediate infection of

    10 phage particles; and (ii) wild-type gene III.

    79. The vector according to claim 78, wherein the DNA encodes a Fab.

    80. The vector according to claim 78,

    15 wherein the DNA encodes heavy chain VHCHl.

    81. The vector according to claim 80, wherein the heavy chain VHCHl is linked to trpIII.

    82. The vector according to claim 78, wherein the DNA encodes light chain VLCL.

    20 83. The vector according to claim 82, wherein the light chain VLCL is linked to trpIII.

    84. The vector according to claim 78, wherein the DNA encodes scFv.

    2018241075 03 Oct 2018

    - 251 85. The vector according to claim 84, wherein the scFv is VL-VH.

    86. The vector according to claim 84, wherein the scFv is VH-VL.

    5 87. The vector according to claim 78, wherein the DNA sequence encoding an antibody variable region linked to a version of PHI anchor further comprises an inducible promoter.

    88. The vector according to claim 87,

    10 wherein the inducible promoter regulates expression of the DNA sequence encoding an antibody variable region linked to a version of PHI anchor.

    89. The vector according to claim 78, wherein the DNA sequence encoding an antibody variable

    15 region linked to a version of PHI anchor further comprises an amber stop codon.

    90. The vector according to claim 89, wherein the DNA encoding the amber stop codon is located between the antibody variable region and the

    20 version of pill.

    91. The vector according to any one of claims 78 to 90 wherein the vector is phage or phagemid.

    92. A method for producing a population of 25 immunoglobulin genes that comprises steps of:

    2018241075 03 Oct 2018

    - 252 (i) introducing synthetic diversity into at least one of CDR1 or CDR2 of those genes; and (ii) combining the diversity from

    5 step (i) with CDR3 diversity captured from B cells. 93. The method according to claim 92, wherein synthetic diversity is introduced into both CDR1 and CDR2.

    10 94. A method for producing a library of immunoglobulin genes that comprises (i) introducing synthetic diversity into at least one of CDR1 or CDR2 of those genes; and

    15 (ii) combining the diversity from step (i) with CDR3 diversity captured from B cells.

    95. The method according to claim 94, wherein synthetic diversity is introduced into both

    20 CDR1 and CDR2.

    96. A library of immunoglobulins that comprise members with at least one variable domain in which at least one of CDR1 and CDR2 contain synthetic diversity and CDR3 diversity is captured from B cells.

    25 97. A library according to claim 96, where both CDR1 and CDR2 contain synthetic diversity.

    - 253 98. The vector according to claim 78, wherein the version of PHI anchor is characterized by

    a.wild type amino acid sequence and is encoded by a non-wild type degenerate DNA sequence to a very high

    5 extent.

    2018241075 03 Oct 2018

    99. In a method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a part of the diversity of the family, the improvement being characterized in that the displayed peptide, polypeptide or protein is encoded by a DNA sequence comprising a nucleic acid that has been cleaved at a desired location by (i) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid at its 5' terminal and (ii) cleaving the nucleic acid solely at a restriction endonuclease cleavage site located in the double-stranded region of the oligonucleotide or amplifying the nucleic acid using a primer at least in part functionally complementary to at least a part of the double-stranded region of the oligonucleotide, the primer also introducing on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence solely at that site;

    2018241075 03 Oct 2018

    - 254 the contacting and the cleaving steps being performed 3 73^^333^3^3 337^*3333^7 73 P¹ ? 3nI^-73 τ η 1^-h(=> γιπγΊ pH Γ acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the

    5 nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

    10 100. A method for displaying a member of a diverse family of peptides, polypeptides or proteins on the surface of a genetic package and collectively displaying at least a portion of the diversity of the family, the method comprising the steps of:

    15 (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse family;

    (ii) rendering the nucleic acids singlestranded;

    20 (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:

    (a) contacting the nucleic acid with a partially double-stranded oligonucleotide,

    25 the single-stranded region of the oligonucleotide being functionally complementary to the nucleic acid at its 5' terminal region; and (b) cleaving the nucleic acid solely at

    30 a restriction endonuclease cleavage site located in the double-stranded region of the oligonucleotide or amplifying the nucleic

    2018241075 03 Oct 2018

    - 255 acid using a primer at least in part f Unct iof¹?. 1 J ^v r-nmnl ernpnts rv fn At least A nart of the double-stranded region of the oligonucleotide, the primer also introducing

    5 on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence solely at that site;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain

    10 the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the

    15 chosen temperature and at the desired location, and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and (iv) displaying a member of the family of

    20 peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of the genetic package and collectively displaying at least a portion of the diversity of the family.

    101. In a method for expressing a member of a

    25 diverse family of peptides, polypeptides or proteins and collectively expressing at least a part of the diversity of the family, the improvement being characterized in that the expressed peptide, polypeptide or protein is encoded by a DNA sequence

    30 comprising a nucleic acid that has been cleaved at a desired location by

    2018241075 03 Oct 2018

    - 256 (i) contacting the nucleic acid with a partiallv double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally

    5 complementary to the nucleic acid at its 5' terminal region; and (ii) cleaving the nucleic acid solely at the restriction endonuclease cleavage site located in the double-stranded region of the

    10 oligonucleotide or amplifying the nucleic acid using a primer at least in part functionally complementary to at least a part of the double-stranded region of the oligonucleotide, the primer also introducing

    15 on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence solely at that site;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic

    20 acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location,

    25 and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature.

    102. A method for expressing a member of a diverse family of peptides, polypeptides or proteins and collectively expressing at least a portion of the

    2018241075 03 Oct 2018

    - 257 diversity of the family, the method comprising the sterns nf:

    (i) preparing a collection of nucleic acids that code, at least in part, for members of the diverse

    5 family;

    (ii) rendering the nucleic acids singlestranded;

    (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the

    10 steps of:

    (a) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single-stranded region of the oligonucleotide being functionally

    15 complementary to the nucleic acid at its 5' terminal region; and (b) cleaving the nucleic acid solely at a restriction endonuclease cleavage site located in the double-stranded region of the

    20 nucleotide; or amplifying the nucleic acid using a primer at least in part functionally complementary to at least a part of the double-stranded region of the oligonucleotide, the primer also introducing

    25 on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence solely at that site;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large

    2018241075 03 Oct 2018

    - 258 enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a

    5 cleavage endonuclease that is active at the chosen temperature; and (iv) expressing a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids and collectively

    10 expressing at least a portion of the diversity of the family.

    103. A method for preparing a library comprising a collection of genetic packages that display a member of a diverse family of peptides,

    15 polypeptides or proteins and that collectively display at least a portion of the family comprising the steps:

    (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;

    20 (ii) rendering the nucleic acids singlestranded;

    (iii) cleaving the single-stranded nucleic acids at a desired location by a method comprising the steps of:

    25 (a) contacting the nucleic acid with a single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement in the nucleic acid forms a restriction endonuclease recognition site that on

    2018241075 03 Oct 2018

    - 259 restriction results in cleavage of the nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at the recognition site formed by the

    5 complementation of the nucleic acid and the oligonucleotide;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded

    10 form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location,

    15 and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature;

    (iv) contacting the nucleic acid with a partially double-stranded oligonucleotide, the single20 stranded region of the oligonucleotide being functionally complementary to the nucleic acids in the 5' terminal region that remains after the cleavage in step (iii) has been effected, and the double-stranded region of the oligonucleotide including any sequences

    25 necessary to return the sequences that remain after cleavaqe into proper and original reading frame for display; and (v) cleaving the nucleic acid solely at a restriction endonuclease cleavage site contained within

    30 the double-stranded region of the partially doublestranded oligonucleotide, the site being different from that used in step (iii) or amplifying the nucleic acid using a primer at least in part functionally

    2018241075 03 Oct 2018

    - 260 complementary to at least a part of the double-stranded χ~χχ x* 4 χχ>-χ x-s £ 4-Vvx-x xx Ί 4 xx v> λ -i /-< 1 -ί Jr. +- Η o r.r ί mor a Ί c: r\ introducing on amplification an endonuclease cleavage site and cleaving the amplified nucleic acid sequence

    5 solely at that site;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally

    10 complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location, and the restriction being carried out using a

    15 cleavage endonuclease that is active at the chosen temperature; and (vi) displaying a member of the family of peptides, polypeptides or proteins coded, at least in part, by the cleaved nucleic acids on the surface of

    20 the genetic package and collectively displaying at least a portion of the diversity of the family.

    104. A method for preparing a library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively

    25 comprising at least a portion of the family comprising the steps:

    (i) preparing a collection of nucleic acids that code at least in part for members of the diverse family;

    30 (ii) rendering the nucleic acids singlestranded;

    2018241075 03 Oct 2018

    - 261 (iii) cleaving the single-stranded nucleic acids at a desirad location by a method comprising the steps of:

    (a) contacting the nucleic acid with a

    5 single-stranded oligonucleotide, the oligonucleotide being functionally complementary to the nucleic acid in the region in which cleavage is desired and including a sequence that with its complement

    10 in the nucleic acid forms a restriction endonuclease recognition site that on restriction results in cleavage of the nucleic acid at the desired location; and (b) cleaving the nucleic acid solely at

    15 the recognition site formed by the complementation of the nucleic acid and the oligonucleotide;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain

    20 the nucleic acid in substantially single-stranded form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the

    25 chosen temperature and at the desired location, and the cleavage being carried out using a restriction endonuclease that is active at the chosen temperature;

    (iv) contacting the nucleic acid with a

    30 partially double-stranded oligonucleotide, the singlestranded region of the oligonucleotide being functionally complementary to the nucleic acids in the 5' terminal region that remains after the cleavage in

    2018241075 03 Oct 2018

    - 262 step (iii) has been effected, and the double-stranded recrion of the oligonucleotide includinu any seauence necessary to return the sequences that remain after cleavage into proper and original reading frame for

    5 expression; and (v) cleaving the nucleic acid solely at a restriction endonuclease cleavage site contained within the double-stranded region of the partially doublestranded oligonucleotide, the site being different from

    10 that used in step (iii) or amplifying the nucleic acid using a primer at least in part functionally complementary to at least a part of the double-stranded region of the oligonucleotide, the primer introducing on amplification an endonuclease cleavage site and

    15 cleaving the amplified nucleic acid seguence solely at that site;

    the contacting and the cleaving steps being performed at a temperature sufficient to maintain the nucleic acid in substantially single-stranded

    20 form, the oligonucleotide being functionally complementary to the nucleic acid over a large enough region to allow the two strands to associate such that cleavage may occur at the chosen temperature and at the desired location,

    25 and the restriction being carried out using a cleavage endonuclease that is active at the chosen temperature; and (vi) expressing a member of the family of peptides, polypeptides or proteins coded, at least in

    30 part, by the cleaved nucleic acids and collectively expressing at least a portion of the diversity of the family.

    2018241075 03 Oct 2018

    - 263 105. A library of immunoglobins comprising members having at least one variable domain in which one or both of the CDR 1 and CDR 2 have synthetic diversity and the CDR 3 has diversity captured from

    5 B-Cells.

    106. The library according to claim 104, wherein a first variable domain has synthetic diversity in CDR 1 and CDR 2 and has diversity in CDR 3 captured from B-cells and a second variable domain has diversity

    10 captured from B-cells.

    107. The library according to claim 104 or 105, wherein the variable domain is selected from the group of VH or VL.

    108. A method for cleaving a nucleic acid 15 sequence at a desired location, the method comprising the steps of:

    (i) contacting a single-stranded nucleic acid sequence with a partially doublestranded oligonucleotide, the single-stranded

    20 region of the oligonucleotide being functionally complementary to the 5' terminal region of the nucleic acid sequence, the double-stranded region of the oligonucleotide including any sequences necessary to return

    25 the sequence in the single-stranded nucleic acid sequence into proper and original reading frame for expression; and (ii) cleaving the partially doublestranded oligonucleotide-single-stranded

    30 nucleic acid combination solely at a

    2018241075 03 Oct 2018

    - 264 restriction endonuclease cleavage site contained within the double-stranded oligonucleotide or amplifying the combination using a primer at least in part functionally

    5 complementary to at least part of the doublestranded region of the oligonucleotide, the primer introducing during amplification an endonuclease cleavage site and cleaving the amplified sequence solely at the site.

    10 109. The method according to claim 108, wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 2 and 15 bases.

    110. The method according to claim 109,

    15 wherein the length of the single-stranded portion of the partially double-stranded oligonucleotide is between 7 and 10 bases.

    111. The method according to claim 108, wherein the length of the double-stranded portion of

    20 the partially double-stranded oligonucleotide is between 12 and 100 base pairs.

    112. The method according to claim 111, wherein the length of the double-stranded portion of the partially double-stranded oligonucleotide is

    25 between 20 and 100 base pairs.

    113. The methods according to any one of claims 99 to 104 and 108, further comprising at least

    2018241075 03 Oct 2018

    - 265 one nucleic acid amplification step between one or more of steps (i) and (ii), steps (ii) and (iii), steps (iii) and (iv) and steps (iv) and (v).

    114. A library comprising a collection of 5 genetic packages that display a member of a diverse family of peptides, polypeptides or proteins and collectively display at least a portion of the diversity of the family, the library being produced using the methods of claims 99, 100, 103 or 113.

    10 115. A library comprising a collection of members of a diverse family of peptides, polypeptides or proteins and collectively comprise at least a portion of the diversity of the family, the library being produced using the methods of claims 101, 102,

    15 104 or 113.

    116. The methods and libraries according to any one of claims 99 to 104 or 113, wherein the members of the library encode immunoglobulins.

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    2018241075 03 Oct 2018

    SEQUENCE LISTING

    <110> LADNER, ROBERT C. COHEN, EDWARD H. NASTRI, HORACIO G. ROOKEY, KRISTIN L. HOET, RENE ' HOOGENBOOM, HENDRICUS R. J. M. <120> NOVEL METHODS OF CONSTRUCTING LIBRARIES COMPRISING DISPLAYED AND/OR EXPRESSED MEMBERS OF A DIVERSE FAMILY OF PEPTIDES, POLYPEPTIDES OR PROTEINS AND THE NOVEL LIBRARIES <130> DYAX/002 CIP2 <140> <141> 10/045,674 2001-10-25 <150> <151> 06/198,069 2000-04-17 <150> <151> 09/837,306 2001-04-17 <160> 635 <170> PatentIn Ver. 2.1 <210> <211> <212> <213> 1 17 DNA . Artificial Sequence

    <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide

    <400> 1 catgtgtatt actgtgc 17 <210> 2 <211> 44

    <212> <213> DNA Artificial Sequence

    <220> <223> Description of Artificial Sequence: Synthetic ' oligonucleotide

    <400> 2 cacatccgtg cttcttgcac ggatgtggca cagtaataca catg 44 <210> 3 <211> 18

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 3 gtgtattaga ctgctgcc . 18 <210> 4 <211> 43 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 4 ggcagcagtc taatacacca catccgtgtt cttcacggat gtg 43 <210> 5 <211> 47 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 5 cacatccgtg tttgttacac ggatgtggtg tcttacagtc cattctg 47 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 6 cagaatggac tgtaagacac 20 <210> 7 <211> 43 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 7 atcgagtctc actgagccac atccgtggtt ttccacggat gtg 43

    2018241075 03 Oct 2018 <210> 8 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide <400> 8 gctcagtgag actcgat <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide

    Synthetic

    Synthetic <220>

    <221> modified_base <222> (10) . . (24) <223> A, T, C, G, other or unknown <400> 9 cacgaggagn nnnnnnnnnn nnnn <210> 10 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide

    Synthetic <400> 10 atgaccgaat tgctacaag <210> 11 <211> 46 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <400> 11 gactcctcag cttcttgctg aggagtcctt gtagcaattc ggtcat 46 <210> 12 <211> 6 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: 6 His tag <400> 12

    His His His His His His 1 5 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6).,(10) <223> A, T, C, G, other or unknown <400> 13 gtctcnnnnn · 10 <210> 14 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(6) <223> A, T, C, G, other or unknown <400> 14 nnnnnngaga c <210> 15 <211> 24 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (11) .. (24) <223> A, T, C, G, other or unknown <400> 15 cacggatgtg nnnnnnnnnn nrinn <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide

    Synthetic <220>

    <221> modified_base <222> (1)..(14) <223> A, T, C, G, other or unknown <400> 16 nnnnnnnnnn nnnncacatc cgtg <210> 17 <211> 14 · <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide

    Synthetic <400> 17 gtgtattact gtgc <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 18 cacatccgtg cacggatgtg gcacagtaat acac

    2018241075 03 Oct 2018 <210> 19 <211> 14 <212> DNA <213> Artificial Sequence .

    <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 19 gtgtattaga ctgc 14 <210> 20 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 20 gcagtctaat acaccacatc cgtgcacgga tgtg 34 <210> 21 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 21 cacatccgtg cacggatgtg gtgtcttaca gtcc <210> 22 <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 22 ggactgtaag acac <210> 23 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 23 gagtctcact gagccacatc cgtgcacgga tgtg 34

    2018241075 03 Oct 2018 <210> 24 .

    <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 24 gctcagtgag actc 14 <210> 25 <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 25 gtgtattact gtgc <210> 26 <211> 14 .

    <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic ’ oligonucleotide <400> 26 gtatattact gtgc 14 <210> 27 <211> 14 · <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 27 gtgtattact gtaa 14

    2018241075 03 Oct 2018

    <210> 28 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 28 gtgtattact gtac <210> 29 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 29 ttgtattact gtgc <210> 30 <211> 14 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 30

    ttgtatcact gtgc

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic <210> 31 <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide <400> 31 acatattact gtgc

    Sequence: Synthetic <210> 32 <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <400> 32 acgtattact gtgc <210> 33 <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    <400> 33 atgtattact gtgc <210> 34 <211> 101 <212> DNA <213> Homo sapiens <400> 34 agggtcacca tgaccaggga ctgagatctg acgacacggc <210> 35 <211> 98 <212> DNA <213> Homo sapiens <400> 35 agagtcacca ttaccaggga agatctgaag acacggctgt <210> 36 <211> 98 <212> DNA <213> Homo sapiens <400> 36 agagtcacca tgaccaggaa agatctgagg acacggccgt <210> 37 <211> 98 <212> DNA <213> Homo sapiens

    101

    2018241075 03 Oct 2018

    <400> 37 agagtcacca tgaccacaga cacatccacg gtattactgt agcacagcct gcgagaga acatggagct gaggagcctg 60 98 agatctgacg acacggccgt <210> 38 <211> 98 <212> DNA <213> Homo sapiens <400> 38 agagtcacca tgaccgagga cacatctaca gacacagcct acatggagct gagcagcctg 60 agatctgagg acacggccgt gtattactgt gcaacaga 98 <210> 39 <211> 98 <212> DNA <213> Homo sapiens <400> 39 agagtcacca ttaccaggga caggtctatg agcacagcct acatggagct gagcagcctg 60 agatctgagg acacagccat gtattactgt gcaagata 98 <210> 40 <211> 98 <212> DNA <213> Homo sapiens <400> 40 agagtcacca tgaccaggga cacgtccacg agcacagtct acatggagct gagcagcctg 60 agatctgagg acacggccgt gtattactgt gcgagaga 98 <210> 41 <211> 98 <212> DNA <213> Homo sapiens <400> 41 agagtcacca ttaccaggga catgtccaca agcacagcct acatggagct gagcagcctg 60 agatccgagg acacggccgt gtattactgt gcggcaga 98 <210> 42 <211> 98 <212> DNA <213> Homo sapiens <400> 42 agagtcacga ttaccgcgga cgaatccacg agcacagcct acatggagct gagcagcctg 60 agatctgagg acacggccgt gtattactgt gcgagaga 98

    <210> 43 <211> 98

    2018241075 03 Oct 2018

    <212> DNA <213> Homo <400> 43 agagtcacga agatctgagg sapiens agcacagcct gcgagaga acatggagct gagcagcctg 60 98 ttaccgcgga acacggccgt caaatccacg gtattactgt <210> 44 <211> 98 <212> DNA <213> Homo sapiens <400> 44 agagtcacca taaccgcgga cacgtctaca gacacagcct acatggagct gagcagcctg 60 agatctgagg acacggccgt gtattactgt gcaacaga 98 <210> 45 <211> 100 <212> DNA <213> Homo sapiens <400> 45 aggctcacca tcaccaagga cacctccaaa aaccaggtgg tccttacaat gaccaacatg 60 gaccctgtgg acacagccac atattactgt gcacacagac 100 <210> 46 <211> 100 <212> DNA <213> Homo sapiens <400> 46 aggctcacca tctccaagga cacctccaaa agccaggtgg tccttaccat gaccaacatg 60 gaccctgtgg acacagccac atattactgt gcacggatac 100 <210> 47 <211> 100 <212> DNA <213> Homo sapiens <400> 47 aggctcacca tctccaagga cacctccaaa aaccaggtgg tccttacaat gaccaacatg 60 gaccctgtgg acacagccac gtattactgt gcacggatac 100 <210> 48 <211> 98 <212> DNA <213> Homo sapiens <400> 48 cgattcacca tctccagaga caacgccaag aactcactgt atctgcaaat gaacagcctg 60 agagccgagg acacggctgt gtattactgt gcgagaga 98

    2018241075 03 Oct 2018

    <210> 49 <211> 100 <212> DNA <213> Homo <400> 49 cgattcacca agagctgagg sapiens tctccagaga acacggcctt caacgccaag gtattactgt aactccctgt gcaaaagata atctgcaaat gaacagtctg <210> 50 <211> 98 <212> DNA <213> Homo sapiens <400> 50 cgattcacca tctccaggga caacgccaag aactcactgt atctgcaaat gaacagcctg agagccgagg acacggccgt gtattactgt gcgagaga <210> 51 <211> 98 <212> DNA <213> Homo sapiens <400> 51 cgattcacca tctccagaga aaatgccaag aactccttgt atcttcaaat gaacagcctg agagccgggg acacggctgt gtattactgt gcaagaga <210> 52 <211> 98 <212> DNA <213> Homo sapiens <400> 52 agattcacca tctcaagaga tgattcaaaa aacacgctgt atctgcaaat gaacagcctg aaaaccgagg acacagccgt gtattactgt accacaga <210> 53 <211> 98 <212> DNA <213> Homo sapiens <400> 53 cgattcacca tctccagaga caacgccaag aactccctgt atctgcaaat gaacagtctg agagccgagg acacggcctt gtatcactgt gcgagaga <210> 54 <211> 98 <212> DNA <213> Homo sapiens <400> 54 cgattcacca tctccagaga caacgccaag aactcactgt atctgcaaat gaacagcctg agagccgagg acacggctgt gtattactgt gcgagaga

    2018241075 03 Oct 2018

    <210> 55 <211> 98 <212> DNA <213> Homo <400> 55 cggttcacca agagccgagg sapiens tctccagaga acacggccgt caattccaag atattactgt aacacgctgt gcgaaaga <210> 56 <211> 98 <212> DNA <213> Homo sapiens <400> 56 cgattcacca tctccagaga caattccaag aacacgctgt agagctgagg acacggctgt gtattactgt gcgaaaga <210> 57 <211> 98 <212> DNA <213> Homo sapiens <400> 57 cgattcacca tctccagaga caattccaag aacacgctgt agagctgagg acacggctgt gtattactgt gcgagaga <210> 58 <211> 98 <212> DNA <213> Homo sapiens <400> 58 cgattcacca tctccagaga caattccaag aacacgctgt agagctgagg acacggctgt gtattactgt gcgaaaga <210> 59 <211> 98 <212> DNA <213> Homo sapiens <400> 59 cgattcacca tctccagaga caattccaag aacacgctgt agagccgagg acacggctgt gtattactgt gcgagaga <210> 60 <211> 100 <212> DNA <213> Homo sapiens

    atctgcaaat atctgcaaat atctgcaaat atctgcaaat atctgcaaat gaacagcctg gaacagcctg gaacagcctg gaacagcctg gaacagcctg

    2018241075 03 Oct 2018

    <400> 60 cgattcacca tctccagaga caacagcaaa aactccctgt atctgcaaat agaactgagg acaccgcctt gtattactgt gcaaaagata gaacagtctg 60 100 <210> 61 <211> 98 <212> DNA <213> Homo sapiens <400> 61 cgattcacca tctccagaga caatgccaag aactcactgt atctgcaaat gaacagcctg 60 agagacgagg acacggctgt gtattactgt gcgagaga 98 <210> 62 <211> 98 <212> DNA <213> Homo sapiens <400> 62 agattcacca tctcaagaga tggttccaaa agcatcgcct atctgcaaat gaacagcctg 60 aaaaccgagg acacagccgt gtattactgt actagaga 98 <210> 63 <211> 98 <212> DNA <213> Homo sapiens <400> 63 cgattcacca tctccagaga caattccaag aacacgctgt atcttcaaat gaacagcctg 60 agagccgagg acacggccgt gtattactgt gcgagaga 98 <210> 64 <211> 98 <212> DNA <213> Homo sapiens <400> 64 agattcacca tctccagaga caattccaag aacacgctgt atcttcaaat gggcagcctg 60 agagctgagg acatggctgt gtattactgt gcgagaga 98 <210> 65 <211> 98 <212> DNA <213> Homo sapiens <400> 65 agattcacca tctccagaga caattccaag aacacgctgt atcttcaaat gaacagcctg 60 agagctgagg acacggctgt gtattactgt gcgagaga 98

    <210> 66 <211> 98

    2018241075 03 Oct 2018

    <212> DNA <213> Homo <400> 66 agattcacca aaaaccgagg sapiens tctcaagaga acacggccgt tgattcaaag gtattactgt aactcactgt gctagaga atctgcaaat gaacagcctg <210> 67 <211> 98 <212> DNA <213> Homo sapiens <400> 67 aggttcacca tctccagaga tgattcaaag aacacggcgt atctgcaaat gaacagcctg aaaaccgagg acacggccgt gtattactgt actagaca <210> 68 <211> 98 <212> DNA <213> Homo sapiens <400> 68 cgattcacca tctccagaga caacgccaag aacacgctgt atctgcaaat gaacagtctg agagccgagg acacggctgt gtattactgt gcaagaga <210> 69 <211> 98 <212> DNA <213> Homo sapiens <400> 69 agattcacca tctccagaga caattccaag aacacgctgc atcttcaaat gaacagcctg agagctgagg acacggctgt gtattactgt aagaaaga <210> 70 <211> 98 <212> DNA <213> Homo sapiens <400> 70 cgagtcacca tatcagtaga caagtccaag aaccagttct ccctgaagct gagctctgtg accgccgcgg acacggccgt gtattactgt gcgagaga <210> 71 <211> 98 <212> DNA <213> Homo sapiens <400> 71 cgagtcacca tgtcagtaga cacgtccaag aaccagttct ccctgaagct gagctctgtg accgccgtgg acacggccgt gtattactgt gcgagaaa

    2018241075 03 Oct 2018

    <210> 72 <211> 98 <212> DNA <213> Homo <400> 72 cgagttacca actgccgcgg sapiens tatcagtaga acacggccgt cacgtctaag aaccagttct ccctgaagct gagctctgtg gtattactgt gcgagaga <210> 73 <211> 98 <212> DNA <213> Homo sapiens <400> 73 cgagtcacca tatcagtaga caggtccaag aaccagttct ccctgaagct gagctctgtg accgccgcgg acacggccgt gtattactgt gccagaga <210> 74 <211> 98 <212> DNA <213> Homo sapiens <400> 74 cgagttacca tatcagtaga cacgtccaag aaccagttct ccctgaagct gagctctgtg actgccgcag acacggccgt gtattactgt gccagaga <210> 75 <211> 98 <212> DNA <213> Homo sapiens <400> 75 cgagttacca tatcagtaga cacgtctaag aaccagttct ccctgaagct gagctctgtg actgccgcgg acacggccgt gtattactgt gcgagaga <210> 76 <211> 98 <212> DNA <213> Homo sapiens <400> 76 cgagtcacca tatcagtaga cacgtccaag aaccagttct ccctgaagct gagctctgtg accgccgcgg acacggctgt gtattactgt gcgagaga <210> 77 <211> 98 <212> DNA <213> Homo sapiens <400> 77 cgagtcacca tatccgtaga cacgtccaag aaccagttct ccctgaagct gagctctgtg accgccgcag acacggctgt gtattactgt gcgagaca

    2018241075 03 Oct 2018

    <210> 78 <211> 98 <212> DNA <213> Homo <400> 78 cgagtcacca accgctgcgg sapiens tatcagtaga acacggccgt cacgtccaag gtattactgt aaccagttct gcgagaga <210> 79 <211> 98 <212> DNA <213> Homo sapiens <400> 79 cgagtcacca tatcagtaga cacgtccaag aaccagttct accgctgcgg acacggccgt gtattactgt gcgagaga <210> 80 <211> 98 <212> DNA <213> Homo sapiens <400> 80 cgagtcacca tatcagtaga cacgtccaag aaccagttct accgccgcag acacggccgt gtattactgt gcgagaga <210> 81 <211> 98 <212> DNA <213> Homo sapiens <400> 81 caggtcacca tctcagccga caagtccatc agcaccgcct aaggcctcgg acaccgccat gtattactgt gcgagaca <210> 82 <211> 96 <212> DNA <213> Homo sapiens <400> 82 cacgtcacca tctcagctga caagtccatc agcactgcct aaggcctcgg acaccgccat gtattactgt gcgaga <210> 83 <211> 98 <212> DNA <213> Homo sapiens

    ccctgaagct gagctctgtg ccctgaagct gagctctgtg ccctgaagct gagctctgtg acctgcagtg gagcagcctg acctgcagtg gagcagcctg

    2018241075 03 Oct 2018

    <400> 83 cgaataacca tcaacccaga cacatccaag aaccagttct ccctgcagct actcccgagg acacggctgt gtattactgt gcaagaga <210> 84 <211> 98 <212> DNA <213> Homo sapiens <400> 84 cggtttgtct tctccttgga cacctctgtc agcacggcat atctgcagat aaggctgagg acactgccgt gtattactgt gcgagaga <210> 85 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified base <222> (3) . . (9) <223> A, T, C, G, other or unknown <400> 85 gcnnnnnnng c <210> 86 <211> 10 <212> DNA <213> Artificial Sequence

    <220>

    <223> Description of Artificial Sequence: oligonucleotide

    Synthetic <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 86 caynnnnrtg <210> 87 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic

    2018241075 03 Oct 2018 oligonucleotide <220>

    <221> modified_base <222> (6) .. (11) <223> A, T, C, G, other or unknown <400> 87 gagtcnnnnn n <210> 88 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(6) <223> A, T, C, G, other or unknown <400> 88 nnnnnngaga c <210> 89 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 89 gaannnnttc <210> 90 <211> 90 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic 3-23 FR3 nucleotide sequence

    2018241075 03 Oct 2018

    <220> <221> <222> CDS (1) · · (90) <220> <221> modified base <222> (3) <223> A, T, C or G <220> <221> modified base <222> (9) <223> A, T, C or G <220> <221> modified base <222> (12) <223> A, T, C or G <220> <221> modified base <222> (21) <223> A, T, C or G <220> <221> modified base <222> (30) <223> A, T, C or G <220> <221> modified base <222> (36) <223> A, T, C or G <220> <221> modified base <222> (51) <223> A, T, C or G <220> <221> modified base <222> (57) <223> A, T, C or G <220> <221> modified base <222> (60) <223> A, T, C or G <220> <221> modified base <222> (69) <223> A, T, C or G <220> <221> modified base <222> (72) <223> A, T, C or G

    2018241075 03 Oct 2018 <220>

    <221> modified_base <222> (75) <223> A, T, C or G <220>

    <221> modified_base <222> (78) <223> A, T, C or G <220>

    <221> modified_base <222> (87) <223> A, T, C or G <400> 90

    acn Thr 1 ath Ile wsn Ser mgn Arg gay Asp 5 aay Asn wsn Ser aar Lys aay Asn acn Thr 10 ytn Leu tay Tyr ttn Leu car Gln atg Met 15 aay Asn 48 wsn ttr mgn gen gar gay acn gen gtn tay tay tgy gen aar 90 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25 30

    <210> 91 <211> 30 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic 3-23 FR3 protein sequence <400> 91

    Thr 1 Ile Ser Arg Asp 5 Asn Ser Lys Asn Thr 10 Leu Tyr Leu Gln Met Asn 15 Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys 20 25 30

    <210> 92 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 92 agttctccct gcagctgaac tc

    2018241075 03 Oct 2018

    <210> 93 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 93

    cactgtatct gcaaatgaac ag

    Sequence: Synthetic

    <210> 94 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 94

    ccctgtatct gcaaatgaac ag

    Sequence: Synthetic

    <210> 95 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 95

    ccgcctacct gcagtggagc ag

    Sequence: Synthetic

    <210> 96 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 96 cgctgtatct gcaaatgaac ag

    Sequence: Synthetic <210> 97 <211> 22 <212> DNA <213> Artificial Sequence <220>

    2018241075 03 Oct 2018

    23 <223> Description of Artificial probe . Sequence : Synthetic <400> 97 cggcatatct gcagatctgc ag 22 <210> 98 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe Sequence : Synthetic <400> 98 cggcgtatct gcaaatgaac ag 22 <210> 99 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe Sequence : Synthetic <400> 99 ctgcctacct gcagtggagc ag 22

    <210> 100 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe Sequence : Synthetic <400> 100 tcgcctatct gcaaatgaac ag 22 <210> 101 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic

    oligonucleotide <400> 101 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaacagctta 60 agg 63

    2018241075 03 Oct 2018

    <210> 102 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 102 caagtagaga gtattettag agttgtctct agaettagtg aagcg

    <210> 103 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 103 cgcttcacta agtctagaga caactctaag aatactctct aettgeaget

    <210> 104 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 104

    cgcttcacta agtctagaga caactctaag aatactctct acttgcaaat gaac 54 <210> 105 <211> 54 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 105 cgcttcacta agtctagaga caactctaag aatactctct acttgcagtg gage 54 <210> 106 <211> 21 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial <400> 106 cgcttcacta agtctagaga c

    <210> 107 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 107

    acatggagct gagcagcctg ag

    Sequence: Synthetic

    Sequence: Primer

    <210> 108 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 108

    acatggagct gagcaggctg ag

    Sequence: Synthetic

    <210> 109 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 109

    acatggagct gaggagcctg ag

    Sequence: Synthetic <210> 110 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 110 acctgcagtg gagcagcctg aa

    2018241075 03 Oct 2018

    <210> 111 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 111 atctgcaaat gaacagcctg aa

    Sequence: Synthetic

    <210> 112 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 112 atctgcaaat gaacagcctg ag

    Sequence: Synthetic

    <210> 113 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 113 atctgcaaat gaacagtctg ag

    Sequence: Synthetic <210> 114 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial probe <400> 114 atctgcagat ctgcagccta aa

    Sequence: Synthetic <210> 115 <211> 22 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 115 atcttcaaat gaacagcctg ag <210> 116 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 116 atcttcaaat gggcagcctg ag <210> 117 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 117 ccctgaagct gagctctgtg ac <210> 118 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence probe <400> 118 ccctgcagct gaactctgtg ac

    Synthetic <210> 119 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe

    2018241075 03 Oct 2018 <400> 119 tccttacaat gaccaacatg ga <210> 120 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 120 tccttaccat gaccaacatg ga .

    <210> 121 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 121 acatggagct gagcagcctg ag <210> 122 <211> 22 <212> DNA · <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 122 ccctgaagct gagctctgtg ac <210> 123 <211> 54 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 123 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaac <210> 124 <211> 60

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 124 cgcttcactc agtctagaga taacagtaaa aatactttgt acttgcagct gagcagcctg 60 <210> 125 <211> 60 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 125 cgcttcactc agtctagaga taacagtaaa aatactttgt acttgcagct gagctctgtg 60

    <210> 126 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 126 tcagctgcaa gtacaaagta tttttactgt tatctctaga ctgagtgaag

    <210> 127 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 127

    cgcttcactc agtctagaga taac 24 <210> 128 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <400> 128 ccgtgtatta ctgtgcgaga ga

    <210> 129 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 129 ctgtgtatta ctgtgcgaga ga <210> 130 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 130 ccgtgtatta ctgtgcgaga gg <210> 131 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 131 ccgtgtatta ctgtgcaaca ga <210> 132 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 132

    ccatgtatta ctgtgcaaga ta

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    2018241075 03 Oct 2018

    <210> 133 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 133

    ccgtgtatta ctgtgcggca ga

    Sequence: Synthetic

    <210> 134 <211> 22 <212> DNA ' <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 134 ccacatatta ctgtgcacac ag <210> 135 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 135 ccacatatta ctgtgcacgg at <210> 136 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 136

    ccacgtatta ctgtgcacgg at

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic <210> 137 <211> 22 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial oligonucleotide <400> 137 ccttgtatta ctgtgcaaaa ga

    <210> 138 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 138 ctgtgtatta ctgtgcaaga ga <210> 139 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 139 ccgtgtatta ctgtaccaca ga <210> 140 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 140 ccttgtatca ctgtgcgaga ga <210> 141 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 141

    ccgtatatta ctgtgcgaaa ga

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    22'

    2018241075 03 Oct 2018 <210> 142 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 142 ctgtgtatta ctgtgcgaaa ga 22 <210> 143 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 143 ccgtgtatta ctgtactaga ga 22

    <210> 144 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 144 ccgtgtatta ctgtgctaga ga 22 <210> 145 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic

    oligonucleotide <400> 145 ccgtgtatta ctgtactaga ca 22 <210> 146 <211> 22 <212> DNA <213> Artificial Sequence oo ©

    <N <220>

    <223> Description of Artificial oligonucleotide <400> 146 ctgtgtatta ctgtaagaaa ga

    2018241075 03 Oct

    Sequence: Synthetic

    <210> 147 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 147 ccgtgtatta ctgtgcgaga aa

    Sequence: Synthetic

    <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 148 ccgtgtatta ctgtgccaga ga

    Sequence: Synthetic

    <210> 149 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 149 ctgtgtatta ctgtgcgaga ca

    Sequence: Synthetic <210> 150 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide

    Sequence: Synthetic

    2018241075 03 Oct 2018 <400> 150 ccatgtatta ctgtgcgaga ca

    <210> 151 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 151 ccatgtatta ctgtgcgaga <210> 152 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 152 ccgtgtatta ctgtgcgaga g <210> 153 <211> 21 <212> DNA . <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 153 ctgtgtatta ctgtgcgaga g <210> 154 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 154

    ccgtgtatta ctgtgcgaga g

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic <210> 155 <211> 21

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide <400> 155 ccgtatatta ctgtgcgaaa g

    <210> 156 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 156 ctgtgtatta ctgtgcgaaa g

    Sequence: Synthetic

    Sequence: Synthetic

    <210> 157 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 157 ctgtgtatta ctgtgcgaga c

    Sequence: Synthetic

    <210> 158 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 158 ccatgtatta ctgtgcgaga c

    Sequence: Synthetic <210> 159 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide

    Sequence: Synthetic <400> 159 ccatgtatta ctgtgcgaga 20

    2018241075 03 Oct 2018 <210> 160 <211> 94 <212> DNA <213> Artificial Sequence .

    <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 160 ggtgtagtga tctagtgaca actctaagaa tactctctac ttgcagatga acagctttag 60 ggctgaggac actgcagtct actattgtgc gaga 94 <210> 161 <211> 94 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 161 ggtgtagtga tctagtgaca actctaagaa tactctctac ttgcagatga acagctttag 60 ggctgaggac actgcagtct actattgtgc gaaa 94 <210> 162 <211> 85 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 162 atagtagact gcagtgtcct cagcccttaa gctgttcatc tgcaagtaga gagtattctt 60 agagttgtct ctagatcact acacc 85 <210> 163 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 163 ggtgtagtga tctagagaca ac

    2018241075 03 Oct 2018

    <210> 164 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 164 ggtgtagtga aacagcttta gggctgagga cactgcagtc tactattgtg cgaga

    <210> 165 <211> 55 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 165

    ggtgtagtga aacagcttta gggctgagga cactgcagtc tactattgtg cgaaa 55 <210> 166 <211> 46 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 166 atagtagact gcagtgtcct cagcccttaa gctgtttcac tacacc 46 <210> 167 <211> 46 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 167 ggtgtagtga aacagcttaa gggctgagga cactgcagtc tactat <210> 168 <211> 26 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    <220> <223> Description of Artificial oligonucleotide <400> 168 ggtgtagtga aacagcttaa gggctg <210> 169 <211> 22 <212> DNA <213> Artificial Sequence <220> Sequence : Synthetic 26 <223> Description of Artificial probe Sequence : Synthetic

    <400> 169 agttctccct gcagctgaac tc 22 <210> 170 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 170 cactgtatct gcaaatgaac ag <210> 171 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 171 ccctgtatct gcaaatgaac ag <210> 172 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 172 ccgcctacct gcagtggagc ag 22

    2018241075 03 Oct 2018 <210> 173 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 173 cgctgtatct gcaaatgaac ag <210> 174 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 174 cggcatatct gcagatctgc ag 22 <210> 175 <211> 22 <212> DNA .

    <213> Artificial Sequence

    <220> <223> Description of Artificial probe Sequence : Synthetic <400> 175 cggcgtatct gcaaatgaac ag 22 <210> 176 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe Sequence : Synthetic <400> 176

    ctgcctacct gcagtggagc ag 22 <210> 177 <211> 22

    2018241075 03 Oct 2018

    <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe Sequence : Synthetic <400> 177 tcgcctatct gcaaatgaac ag 22 <210> 178 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 178 acatggagct gagcagcctg ag 22 <210> 179 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 179 acatggagct gagcaggctg ag 22

    <210> 180 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 180 acatggagct gaggagcctg ag 22 <210> 181 <211> 22 <212> DNA <213> Artificial Sequence

    <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <400> 181 acctgcagtg gagcagcctg aa

    <210> 182 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 182 atctgcaaat gaacagcctg aa <210> 183 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 183 atctgcaaat gaacagcctg ag <210> 184 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 184 atctgcaaat gaacagtctg ag <210> 185 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 185

    atctgcagat ctgcagccta aa

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    2018241075 03 Oct 2018

    <210> 186 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 186 atcttcaaat gaacagcctg ag <210> 187 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 187 atcttcaaat gggcagcctg ag <210> 188 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 188

    ccctgaagct gagctctgtg ac

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic •<210> 189 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide <400> 189 ccctgcagct gaactctgtg ac

    Sequence: Synthetic <210> 190 <211> 22 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial oligonucleotide <400> 190 tccttacaat gaccaacatg ga

    <210> 191 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 191 tccttaccat gaccaacatg ga

    Sequence: Synthetic

    <210> 192 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 192 ccgtgtatta ctgtgcgaga ga

    Sequence: Synthetic

    <210> 193 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 193 ctgtgtatta ctgtgcgaga ga

    Sequence: Synthetic <210> 194 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide

    Sequence: Synthetic

    Sequence: Synthetic <400> 194 ccgtgtatta ctgtgcgaga gg 22

    2018241075 03 Oct 2018

    <210> 195 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 195 ccgtgtatta ctgtgcaaca ga <210> 196 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 196 ccatgtatta ctgtgcaaga ta <210> 197 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 197

    ccgtgtatta ctgtgcggca ga

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic <210> 198 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide <400> 198 ccacatatta ctgtgcacac ag

    Sequence: Synthetic <210> 199 <211> 22 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 199 ccacatatta ctgtgcacgg at <210> 200 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic ’ oligonucleotide <400> 200 ccacgtatta ctgtgcacgg at 22 <210> 201 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 201 ccttgtatta ctgtgcaaaa ga 22 <210> 202 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 202 ctgtgtatta ctgtgcaaga ga 22 <210> 203 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <400> 203 ccgtgtatta ctgtaccaca ga <210> 204 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic ' oligonucleotide <400> 204 ccttgtatca ctgtgcgaga ga 22 <210> 205 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 205 ccgtatatta ctgtgcgaaa ga 22 <210> 206 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 206 ctgtgtatta ctgtgcgaaa ga 22 <210> 207 <211> 22 <212> DNA <213> Artificial Sequence <220> · <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 207 ccgtgtatta ctgtactaga ga 22 <210> 208 <211> 22

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide <400> 208 ccgtgtatta ctgtgctaga ga

    <210> 209 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 209 ccgtgtatta ctgtactaga ca

    Sequence: Synthetic

    Sequence: Synthetic

    <210> 210 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 210 ctgtgtatta ctgtaagaaa ga

    Sequence: Synthetic

    <210> 211 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 211 ccgtgtatta ctgtgcgaga aa

    Sequence: Synthetic <210> 212 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide

    Sequence: Synthetic

    2018241075 03 Oct 2018

    <400> 212 ccgtgtatta ctgtgccaga ga <210> 213 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 213 ctgtgtatta ctgtgcgaga ca <210> 214 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 214

    ccatgtatta ctgtgcgaga ca

    Sequence: Synthetic

    Sequence: Synthetic <210> 215 <211> 22 · <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    <400> 215 ccatgtatta ctgtgcgaga aa 22 <210> 216 <211> 90 <212> DNA <213> Homo sapiens <400> 216 caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60 tcctgcaagg cttctggata caccttcacc 90

    <210> 217 <211> 90 <212> DNA <213> Homo sapiens

    2018241075 03 Oct 2018 <400> 217

    caggtccagc tcctgcaagg ttgtgcagtc cttctggata tggggctgag caccttcact gtgaagaagc ctggggcctc agtgaaggtt <210> 218 <211> 90 <212> DNA <213> Homo sapiens <400> 218 caggtgcagc tcctgcaagg tggtgcagtc cttctggata tggggctgag caccttcacc gtgaagaagc ctggggcctc agtgaaggtc <210> 219 <211> 90 <212> DNA <213> Homo sapiens <400> 219 caggttcagc tcctgcaagg tggtgcagtc cttctggtta tggagctgag cacctttacc gtgaagaagc ctggggcctc agtgaaggtc <210> 220 <211> 90 <212> DNA <213> Homo sapiens <400> 220 caggtccagc tcctgcaagg tggtacagtc tttccggata tggggctgag caccctcact gtgaagaagc ctggggcctc agtgaaggtc <210> 221 <211> 90 <212> DNA <213> Homo sapiens <400> 221 cagatgcagc tcctgcaagg tggtgcagtc cttccggata tggggctgag caccttcacc gtgaagaaga ctgggtcctc agtgaaggtt <210> 222 <211> 90 <212> DNA <213> Homo sapiens <400> 222 caggtgcagc tcctgcaagg tggtgcagtc catctggata tggggctgag caccttcacc gtgaagaagc ctggggcctc agtgaaggtt

    <210> 223 <211> 90

    2018241075 03 Oct 2018 <212> DNA <213> Homo sapiens <400> 223

    caaatgcagc tcctgcaagg tggtgcagtc cttctggatt tgggcctgag cacctttact gtgaagaagc ctgggacctc agtgaaggtc <210> 224 <211> 90 <212> DNA <213> Homo sapiens <400> 224 caggtgcagc tcctgcaagg tggtgcagtc cttctggagg tggggctgag caccttcagc gtgaagaagc ctgggtcctc ggtgaaggtc <210> 225 <211> 90 <212> DNA <213> Homo sapiens <400> 225 caggtgcagc tcctgcaagg tggtgcagtc cttctggagg tggggctgag caccttcagc gtgaagaagc ctgggtcctc ggtgaaggtc <210> 226 <211> 90 <212> DNA <213> Homo sapiens <400> 226 gaggtccagc tcctgcaagg tggtacagtc tttctggata tggggctgag caccttcacc gtgaagaagc ctggggctac agtgaaaatc <210> 227 <211> 90 <212> DNA <213> Homo sapiens <400> 227 cagatcacct acctgcacct tgaaggagtc tctctgggtt tggtcctacg ctcactcagc ctggtgaaac ccacacagac cctcacgctg <210> 228 <211> 90 <212> DNA <213> Homo sapiens <400> 228 caggtcacct acctgcaccg tgaaggagtc tctctgggtt tggtcctgtg ctcactcagc ctggtgaaac ccacagagac cctcacgctg

    2018241075 03 Oct 2018

    <210> 229 <211> 90 <212> DNA <213> Homo <400> 229 caggtcacct acctgcacct sapiens tgaaggagtc tctctgggtt <210> 230 <211> 90 <212> DNA <213> Homo sapiens <400> 230 gaggtgcagc tggtggagtc tcctgtgcag cctctggatt <210> 231 <211> 90 <212> DNA <213> Homo sapiens <400> 231 gaagtgcagc tggtggagtc tcctgtgcag cctctggatt <210> 232 <211> 90 <212> DNA <213> Homo sapiens <400> 232 caggtgcagc tggtggagtc tcctgtgcag cctctggatt <210> 233 <211> 90 <212> DNA <213> Homo sapiens <400> 233 gaggtgcagc tggtggagtc tcctgtgcag cctctggatt <210> 234 <211> 90 <212> DNA <213> Homo sapiens <400> 234 gaggtgcagc tggtggagtc tcctgtgcag cctctggatt

    tggtcctgcg ctcactcagc tgggggaggc cacctttagt tgggggaggc cacctttgat tgggggaggc caccttcagt tgggggaggc caccttcagt ctggtgaaac ccacacagac cctcacactg 60 90 ttggtccagc ctggggggtc cctgagactc 60 90 ttggtacagc ctggcaggtc cctgagactc 60 90 ttggtcaagc ctggagggtc cctgagactc 60 90 ttggtacagc ctggggggtc cctgagactc 60 90 ttggtaaagc ctggggggtc ccttagactc 60 90 tgggggaggc cactttcagt gtggtacggc ctggggggtc cctgagactc 60

    2018241075 03 Oct 2018

    <210> 235 <211> 90 <212> DNA <213> Homo sapiens <400> 235 gaggtgcagc tggtggagtc tgggggaggt tcctgtgcag cctctggatt cacctttgat <210> 236 <211> 90 <212> DNA <213> Homo sapiens <400> 236 gaggtgcagc tggtggagtc tgggggaggc tcctgtgcag cctctggatt caccttcagt <210> 237 <211> 90 <212> DNA <213> Homo sapiens <400> 237 gaggtgcagc tgttggagtc tgggggaggc tcctgtgcag cctctggatt cacctttagc <210> 238 <211> 90 <212> DNA <213> Homo sapiens <400> 238 caggtgcagc tggtggagtc tgggggaggc tcctgtgcag cctctggatt caccttcagt <210> 239 <211> 90 <212> DNA <213> Homo sapiens <400> 239 caggtgcagc tggtggagtc tgggggaggc tcctgtgcag cctctggatt caccttcagt <210> 240 <211> 90 <212> DNA <213> Homo sapiens

    ctggtcaagc ctggggggtc cctgagactc 60 90 ttggtacagc ctggggggtc cctgagactc 60 90 gtggtccagc ctgggaggtc cctgagactc 60 90 gtggtccagc ctgggaggtc cctgagactc 60 90

    2018241075 03 Oct 2018

    <400> 240 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt 90 <210> 241 <211> 90 <212> DNA <213> Homo sapiens <400> 241 caggtgcagc tggtggagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60 tcctgtgcag cgtctggatt caccttcagt 90 <210> 242 <211> 90 <212> DNA <213> Homo sapiens <400> 242 gaagtgcagc tggtggagtc tgggggagtc gtggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt cacctttgat 90 <210> 243 <211> 90 <212> DNA <213> Homo sapiens <400> 243 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc 60 tcctgtgcag cctctggatt caccttcagt 90 <210> 244 <211> 90 <212> DNA <213> Homo sapiens <400> 244 gaggtgcagc tggtggagtc tgggggaggc ttggtacagc cagggcggtc cctgagactc 60 tcctgtacag cttctggatt cacctttggt 90 <210> 245 <211> 90 <212> DNA <213> Homo sapiens <400> 245 gaggtgcagc tggtggagac tggaggaggc ttgatccagc ctggggggtc cctgagactc 60 tcctgtgcag cctctgggtt caccgtcagt 90

    <210> 246 <211> 90 <212> DNA

    2018241075 03 Oct 2018

    <213> Homo <400> 246 gaggtgcagc tcctgtgcag sapiens ttggtccagc ctggggggtc cctgagactc tggtggagtc cctctggatt tgggggaggc caccttcagt <210> 247 <211> 90 <212> DNA <213> Homo sapiens <400> 247 gaggtgcagc tcctgtgcag tggtggagtc cctctggatt tgggggaggc caccgtcagt ttggtccagc ctggggggtc cctgagactc <210> 248 <211> 90 <212> DNA <213> Homo sapiens <400> 248 gaggtgcagc tcctgtgcag tggtggagtc cctctggatt tgggggaggc caccttcagt ttggtccagc ctggagggtc cctgagactc <210> 249 <211> 90 <212> DNA <213> Homo sapiens <400> 249 gaggtgcagc tcctgtgcag tggtggagtc cctctgggtt tgggggaggc caccttcagt ttggtccagc ctggggggtc cctgaaactc <210> 250 <211> 90 <212> DNA <213> Homo sapiens <400> 250 gaggtgcagc tcctgtgcag tggtggagtc cctctggatt cgggggaggc caccttcagt ttagttcagc ctggggggtc cctgagactc <210> 251 <211> 90 <212> DNA <213> Homo sapiens <400> 251 gaggtgcagc tcctgtgcag tggtggagtc cctctggatt tcggggagtc caccgtcagt ttggtacagc ctggggggtc cctgagactc

    2018241075 03 Oct 2018

    <210> <211> <212> <213> 252 90 DNA Homo sapiens <400> 252 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc acctgcgctg tctctggtgg ctccatcagc

    cttcggggac

    <210> 253 <211> 90 <212> DNA <213> Homo sapiens

    <400> 253 caggtgcagc acctgcgctg tgcaggagtc gggcccagga tctctggtta ctccatcagc ctggtgaagc cttcggacac cctgtccctc cctgtccctc <210> 254 <211> 90 <212> DNA <213> Homo sapiens <400> 254 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc 90 <210> 255 <211> 90 <212> DNA .

    <213> Homo sapiens <400> 255 cagctgcagc tgcaggagtc cggctcagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcgctg tctctggtgg ctccatcagc 90 <210> 256 <211> 90 <212> DNA <213> Homo sapiens <400> 256 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc 90 <210> 257 <211> 90 <212> DNA <213> Homo sapiens <400> 257 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcacagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc 90

    2018241075 03 Oct 2018 <210> 258 <211> 90 <212> DNA <213> Homo sapiens <400> 258

    caggtgcagc tacagcagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60 90 acctgcgctg tctatggtgg gtccttcagt <210> 259 <211> 90 <212> DNA <213> Homo sapiens <400> 259 cagctgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagc 90 <210> 260 <211> 90 <212> DNA <213> Homo sapiens <400> 260 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccatcagt 90 <210> 261 <211> 90 <212> DNA <213> Homo sapiens <400> 261 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcactg tctctggtgg ctccgtcagc 90 <210> 262 <211> 90 <212> DNA <213> Homo sapiens <400> 262 caggtgcagc tgcaggagtc gggcccagga ctggtgaagc cttcggagac cctgtccctc 60 acctgcgctg tctctggtta ctccatcagc 90 <210> 263 <211> 90 <212> DNA <213> Homo sapiens

    2018241075 03 Oct 2018

    <400> 263 gaggtgcagc tcctgtaagg tggtgcagtc gttctggata tggagcagag cagctttacc gtgaaaaagc ccggggagtc tctgaagatc 60 90 <210> 264 <211> 90 <212> DNA <213> Homo sapiens <400> 264 gaagtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaggatc 60 tcctgtaagg gttctggata cagctttacc 90 <210> 265 <211> 90 <212> DNA <213> Homo sapiens <400> 265 caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60 acctgtgcca tctccgggga cagtgtctct 90 <210> 266 <211> 90 <212> DNA <213> Homo sapiens <400> 266 caggtgcagc tggtgcaatc tgggtctgag ttgaagaagc ctggggcctc agtgaaggtt 60 tcctgcaagg cttctggata caccttcact 90 <210> 267 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of . Artificial Sequence: Synthetic

    oligonucleotide <400> 267 ccgtgtatta ctgtgcgaga ga <210> 268 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide οο ο

    CM <400> 268 ctgtgtatta ctgtgcgaga ga

    2018241075 03 Oct

    <210> 269 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 269 ccgtgtatta ctgtgcgaga gg <210> 270 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 270 ccgtatatta ctgtgcgaaa ga <210> 271 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 271 ctgtgtatta ctgtgcgaaa ga <210> 272 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 272

    ctgtgtatta ctgtgcgaga ca

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic <210> 273 <211> 22

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 273 ccatgtatta ctgtgcgaga ca <210> 274 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    <400> 274 ccatgtatta ctgtgcgaga <210> 275 <211> 69 <212> DNA <213> Homo sapiens <400> 275 gacatccaga tgacccagtc atcacttgc <210> 276 <211> 69 <212> DNA <213> Homo sapiens <400> 276 gacatccaga tgacccagtc atcacttgc <210> 277 <211> 69 <212> DNA <213> Homo sapiens <400> 277 gacatccaga tgacccagtc atcacttgc <210> 278 <211> 69 <212> DNA <213> Homo sapiens

    2018241075 03 Oct 2018

    <400> 278 gacatccaga atcacttgc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 69 <210> 279 <211> 69 <212> DNA <213> Homo sapiens <400> 279 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgc 69 <210> 280 <211> 69 <212> DNA <213> Homo sapiens <400> 280 gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgc 69 <210> 281 <211> 69 <212> DNA <213> Homo sapiens <400> 281 aacatccaga tgacccagtc tccatctgcc atgtctgcat ctgtaggaga cagagtcacc 60 atcacttgt 69 <210> 282 <211> 69 <212> DNA <213> Homo sapiens <400> 282 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgt 69 <210> 283 <211> 69 <212> DNA <213> Homo sapiens <400> 283 gacatccaga tgacccagtc tccatcctca ctgtctgcat ctgtaggaga cagagtcacc 60 atcacttgt 69

    <210> 284 <211> 69

    2018241075 03 Oct 2018 <212> DNA <213> Homo sapiens <400> 284

    gccatccagt atcacttgc tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc <210> 285 <211> 69 <212> DNA <213> Homo sapiens <400> 285 gccatccagt tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc

    atcacttgc

    <210> 286 <211> 69 <212> DNA <213> Homo sapiens <400> 286 gacatccaga atcacttgt tgacccagtc tccatcttcc gtgtctgcat ctgtaggaga cagagtcacc <210> 287 <211> 69 <212> DNA <213> Homo sapiens <400> 287 gacatccaga atcacttgt tgacccagtc tccatcttct gtgtctgcat ctgtaggaga cagagtcacc

    <210> <211> <212> <213> 288 69 DNA Homo sapiens <400> 288 gacatccagt tgacccagtc tccatccttc ctgtctgcat ctgtaggaga cagagtcacc atcacttgc

    <210> <211> <212> <213> 289 69 DNA Homo sapiens <400> 289 gccatccgga tgacccagtc tccattctcc ctgtctgcat ctgtaggaga cagagtcacc atcacttgc

    tgacccagtc tccatcctca ttctctgcat ctacaggaga cagagtcacc

    2018241075 03 Oct 2018 <210> 290 <211> 69 <212> DNA <213> Homo <400> 290 gccatccgga atcacttgt <210> 291 <211> 69 <212> DNA <213> Homo <400> 291 gtcatctgga atcagttgt <210> 292 <211> 69 <212> DNA <213> Homo <400> 292 gccatccaga atcacttgc <210> 293 <211> 69 <212> DNA <213> Homo <400> 293 gacatccaga atcacttgc <210> 294 <211> 69 <212> DNA <213> Homo <400> 294 gatattgtga atctcctgc sapiens sapiens tgacccagtc tccatcctta ctctctgcat ctacaggaga sapiens tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga sapiens tgacccagtc tccttccacc ctgtctgcat ctgtaggaga sapiens tgacccagac tccactctcc ctgcccgtca cccctggaga cagagtcacc cagagtcacc cagagtcacc gccggcctcc

    <210> 295 <211> 69 <212> DNA <213> Homo <400> 295

    sapiens gatattgtga tgacccagac tccactctcc ctgcccgtca cccctggaga gccggcctcc atctcctgc

    2018241075 03 Oct 2018 <210> 296 <211> 69 <212> DNA <213> Homo sapiens <400> 296 gatgttgtga tgactcagtc tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 atctcctgc 69

    <210> <211> <212> <213> 297 69 DNA Homo sapiens <400> 297 gatgttgtga tgactcagtc atctcctgc

    tccactctcc ctgcccgtca cccttggaca gccggcctcc 60 69

    <210> <211> <212> <213> 298 69 DNA Homo sapiens <400> 298 gatattgtga tgacccagac atctcctgc

    tccactctct ctgtccgtca cccctggaca gccggcctcc 60 69

    <210> <211> <212> <213> 299 69 DNA Homo sapiens <400> 299 gatattgtga tgacccagac atctcctgc

    tccactctct ctgtccgtca cccctggaca gccggcctcc 60 69

    <210> <211> <212> <213> 300 69 DNA Homo sapiens <400> 300 gatattgtga tgactcagtc atctcctgc

    tccactctcc ctgcccgtca cccctggaga gccggcctcc 60 69 <210> 301 <211> 69 <212> DNA <213> Homo sapiens

    2018241075 03 Oct 2018

    <400> 301 gatattgtga tgactcagtc atctcctgc <210> 302 <211> 69 <212> DNA <213> Homo sapiens

    tccactctcc ctgcccgtca <400> 302 gatattgtga tgacccagac atctcctgc

    <210> <211> <212> <213> 303 69 DNA Homo sapiens <400> 303 gaaattgtgt tgacgcagtc ctctcctgc

    tccaggcacc ctgtctttgt

    <210> <211> <212> <213> 304 69 DNA Homo sapiens <400> 304 gaaattgtgt tgacgcagtc ctctcctgc

    tccagccacc ctgtctttgt

    <210> <211> <212> <213> 305 69 DNA Homo sapiens <400> 305 gaaatagtga tgacgcagtc ctctcctgc

    tccagccacc ctgtctgtgt

    <210> <211> <212> <213> 306 69 DNA Homo sapiens <400> 306 gaaatagtga tgacgcagtc ctctcctgc

    tccagccacc ctgtctgtgt <210> 307 <211> 69 cccctggaga gccggcctcc tccactctcc tcacctgtca cccttggaca gccggcctcc ctccagggga aagagccacc ctccagggga ctccagggga ctccagggga aagagccacc aagagccacc aagagccacc

    2018241075 03 Oct 2018 <212> DNA <213> Homo sapiens <400> 307

    gaaattgtgt ctctcctgc tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 69 <210> 308 <211> 69 <212> DNA <213> Homo sapiens <400> 308 gaaattgtgt ctctcctgc tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 69 <210> 309 <211> 69 <212> DNA <213> Homo sapiens <400> 309 gaaattgtaa ctctcctgc tgacacagtc tccagccacc ctgtctttgt ctccagggga aagagccacc 60 69 <210> 310 <211> 69 <212> DNA <213> Homo sapiens <400> 310 gacatcgtga atcaactgc tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60 69 <210> 311 <211> 69 <212> DNA <213> Homo sapiens <400> 311 gaaacgacac atctcctgc tcacgcagtc tccagcattc atgtcagcga ctccaggaga caaagtcaac 60 69 <210> 312 <211> 69 <212> DNA <213> Homo sapiens <400> 312 gaaattgtgc atcacctgc tgactcagtc tccagacttt cagtctgtga ctccaaagga gaaagtcacc 60 69

    cagtctgtga ctccaaagga gaaagtcacc 60

    2018241075 03 Oct 2018 <210> 313 <211> 69 <212> DNA <213> Homo sapiens <400> 313 gaaattgtgc tgactcagtc tccagacttt atcacctgc

    <210> <211> <212> <213> 314 69 DNA Homo sapiens <400> 314 gatgttgtga tgacacagtc tccagctttc atcacctgc <210> 315 <211> 66 <212> DNA <213> Homo sapiens <400> 315 cagtctgtgc tgactcagcc accctcggtg tcctgt <210> 316 <211> 66 <212> DNA <213> Homo sapiens <400> 316 cagtctgtgc tgacgcagcc gccctcagtg tcctgc <210> 317 <211> 66 <212> DNA <213> Homo sapiens <400> 317 cagtctgtgc tgactcagcc accctcagcg tcttgt <210> 318 <211> 66 <212> DNA <213> Homo sapiens <400> 318 cagtctgtgc tgactcagcc accctcagcg tcttgt

    ctctctgtga ctccagggga gaaagtcacc 60 69 tctgaagccc ccaggcagag ggtcaccatc 60 66 tctggggccc cagggcagag ggtcaccatc 60 66 tctgggaccc ccgggcagag ggtcaccatc 60 66 tctgggaccc ccgggcagag ggtcaccatc 60 66 gccctcagtg tctgcggccc caggacagaa ggtcaccatc

    2018241075 03 Oct 2018 <210> 319 <211> 66

    <212> <213> DNA Homo sapiens <400> 319 cagtctgtgt tgacgcagcc tcctgc <210> 320 <211> 66 <212> DNA <213> Homo sapiens <400> 320 cagtctgccc tgactcagcc tcctgc <210> 321 <211> 66 <212> DNA <213> Homo sapiens <400> 321 cagtctgccc tgactcagcc tcctgc <210> 322 <211> 66 <212> DNA <213> Homo sapiens

    tccctccgcg tccgggtctc ctggacagtc agtcaccatc tcgctcagtg tccgggtctc ctggacagtc agtcaccatc <400> 322 cagtctgccc tgactcagcc tcctgc <210> 323 <211> 66 <212> DNA <213> Homo sapiens <400> 323 cagtctgccc tgactcagcc tcctgc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc tccctccgtg tccgggtctc ctggacagtc agtcaccatc

    <210> 324 <211> 66 <212> DNA <213> Homo

    <400> 324

    2018241075 03 Oct 2018

    cagtctgccc tcctgc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60 66 <210> 325 <211> 66 <212> DNA <213> Homo sapiens <400> 325 tcctatgagc acctgc tgactcagcc accctcagtg tccgtgtccc caggacagac agccagcatc 60 66 <210> 326 <211> 66 <212> DNA <213> Homo sapiens <400> 326 tcctatgagc acctgt tgactcagcc actctcagtg tcagtggccc tgggacagac ggccaggatt 60 66 <210> 327 <211> 66 <212> DNA <213> Homo sapiens <400> 327 tcctatgagc acctgc tgacacagcc accctcggtg tcagtgtccc caggacaaac ggccaggatc 60 66 <210> 328 <211> 66 <212> DNA <213> Homo sapiens <400> 328 tcctatgagc acctgc tgacacagcc accctcggtg tcagtgtccc taggacagat ggccaggatc 60 66 <210> 329 <211> 66 <212> DNA <213> Homo sapiens <400> 329 tcttctgagc acatgc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60 66 <210> 330 <211> 66 <212> DNA <213> Homo sapiens

    2018241075 03 Oct 2018

    <400> 330 tcctatgtgc acctgt tgactcagcc accctcagtg tcagtggccc caggaaagac ggccaggatt 60 66 <210> 331 <211> 66 <212> DNA <213> Homo sapiens <400> 331 tcctatgagc tgacacagct accctcggtg tcagtgtccc caggacagac agccaggatc 60 acctgc 66 <210> 332 <211> 66 <212> DNA <213> Homo sapiens <400> 332 tcctatgagc tgatgcagcc accctcggtg tcagtgtccc caggacagac ggccaggatc 60 acctgc 66 <210> 333 <211> 66 <212> DNA <213> Homo sapiens <400> 333 tcctatgagc tgacacagcc atcctcagtg tcagtgtctc cgggacagac agccaggatc 60 acctgc 66 <210> 334 <211> 66 <212> DNA <213> Homo sapiens <400> 334 ctgcctgtgc tgactcagcc cccgtctgca tctgccttgc tgggagcctc gatcaagctc 60 acctgc 66 <210> 335 <211> 66 <212> DNA <213> Homo sapiens <400> 335 cagcctgtgc tgactcaatc atcctctgcc tctgcttccc tgggatcctc ggtcaagctc 60 acctgc 66

    <210> 336 <211> 66

    2018241075 03 Oct 2018

    <212> DNA <213> Homo <400> 336 cagcttgtgc acctgc sapiens tgggagcctc ggtcaagctc 60 66 tgactcaatc gccctctgcc tctgcctccc <210> 337 <211> 66 <212> DNA <213> Homo sapiens <400> 337 cagcctgtgc acctgc tgactcagcc accttcctcc tccgcatctc ctggagaatc cgccagactc 60 66 <210> 338 <211> 66 <212> DNA <213> Homo sapiens <400> 338 caggctgtgc acctgc tgactcagcc ggcttccctc tctgcatctc ctggagcatc agccagtctc 60 66 <210> 339 <211> 66 <212> DNA <213> Homo sapiens <400> 339 cagcctgtgc acctgc tgactcagcc atcttcccat tctgcatctt ctggagcatc agtcagactc 60 66 <210> 340 <211> 66 <212> DNA <213> Homo sapiens <400> 340 aattttatgc tcctgc tgactcagcc ccactctgtg tcggagtctc cggggaagac ggtaaccatc 60 66 <210> 341 <211> 66 <212> DNA <213> Homo sapiens <400> 341 cagactgtgg acctgt tgactcagga gccctcactg actgtgtccc caggagggac agtcactctc 60 66

    2018241075 03 Oct 2018

    <210> 342 <211> 66 <212> DNA <213> Homo sapiens <400> 342 caggctgtgg tgactcagga gccctcactg actgtgtccc caggagggac acctgt <210> 343 <211> 66 <212> DNA <213> Homo sapiens <400> 343 cagactgtgg tgacccagga gccatcgttc tcagtgtccc ctggagggac acttgt <210> 344 <211> 66 <212> DNA <213> Homo sapiens <400> 344 cagcctgtgc tgactcagcc accttctgca tcagcctccc tgggagcctc acctgc <210> 345 <211> 66 <212> DNA <213> Homo sapiens <400> 345 caggcagggc tgactcagcc accctcggtg tccaagggct tgagacagac acctgc <210> 346 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified base <222> (1) · • (6) <223> A, T, . C, G, other or unknown <400> 346

    nnnnnngact c

    2018241075 03 Oct 2018 <210> 347 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6) .. (11) <223> A, T, C, G, other or unknown <400> 347 gagtcnnnnn n 11 <210> 348 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (3)..(9) <223> A, T, C, G, other or unknown <400> 348 · gcnnnnnnng c 11 <210> 349 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown · <400> 349 acctgcnnnn n 11 <210> 350 <211> 25 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 350 cacatccgtg ttgttcacgg atgtg 25 <210> 351 ' <211> 88 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 351 aatagtagac tgcagtgtcc tcagccctta agctgttcat ctgcaagtag agagtattct 60 tagagttgtc tctagactta gtgaagcg 88 <210> 352 <211> 88 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 352 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaacagctta 60 agggctgagg acactgcagt ctactatt 88 <210> 353 <211> 95 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 353 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaacagctta 60 agggctgagg acactgcagt ctactattgt gcgag 95 <210> 354 <211> 95 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 354 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaacagctta 60 agggctgagg acactgcagt ctactattgt acgag 95 <210> 355 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 355 cgcttcacta agtctagaga caac <210> 356 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (8)..(15) <223> A, T, C, G, other or unknown <400> 356 cacctgcnnn nnnnn <210> 357 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (17) <223> A, T, C, G, other or unknown <400> 357 cagctcnnnn nnnnnnn

    2018241075 03 Oct 2018 <210> 358 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (17) <223> A, T, C, G, other or unknown <400> 358 gaagacnnnn nnnnnnn 17 <210> 359 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(17) <223> A, T, C, G, other or unknown <400> 359 gcagcnnnnn nnnnnnn 17 <210> 360 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide · <220>

    <221> modified_base <222> (7)..(12) <223> A, T, C, G, other or unknown <400> 360 gaagacnnnn nn <210> 361 <211> 22 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (22) <223> A, T, C, G, other or unknown <400> 361 cttgagnnnn nnnnnnnnnn nn 22 <210> 362 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(19) <223> A, T, C, G, other or unknown <400> 362 acggcnnnnn nnnnnnnnn 19 <210> 363 <211> 18 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(18) <223> A, T, C, G, other or unknown <400> 363 acggcnnnnn nnnnnnnn 18 <210> 364 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <220>

    <221> modified_base <222> (7)..(12) <223> A, T, C, G, other or unknown <400> 364 gtatccnnnn nn <210> 365 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 365 actgggnnnn n <210> 366 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(10) <223> A, T, C, G, other or unknown <400> 366 ggatcnnnnn <210> 367 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(11)

    2018241075 03 Oct 2018 <223> A, T, C, G, other or unknown <400> 367 gcatcnnnnn n <210> 368 <211> 16 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(16) <223> A, T, C, G, other or unknown <400> 368 gaggagnnnn nnnnnn <210> 369 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(19) <223> A, T, C, G, other or unknown <400> 369 gggacnnnnn nnnnnnnnn <210> 370 <211> 14 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(14) <223> A, T, C, G, other or unknown <400> 370 acctgcnnnn nnnn

    2018241075 03 Oct 2018 <210> 371 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (17) <223> A, T, C, G, other or unknown <400> 371 ggcggannnn nnnnnnn ' 17 <210> 372 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(22) <223> A, T, G, G, other or unknown <400> 372 ctgaagnnnn nnnnnnnnnn nn 22 <210> 373 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(11) <223> A, T, C, G, other or unknown <400> 373 cccgcnnnnn n 11 <210> 374 <211> 18

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6) .. (18) <223> A, T, C, G, other or unknown <400> 374 ggatgnnnnn nnnnnnnn 18 <210> 375 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(22) <223> A, T, C, G, other or unknown <400> 375 ctggagnnnn nnnnnnnnnn nn 22 <210> 376 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6) .. (15) <223> A, T, C, G, other or unknown <400> 376 gacgcnnnnn nnnnn 15 <210> 377 <211> 13 <212> DNA <213> Artificial Sequence <220>

    2018241075 03 Oct 2018 <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(13) <223> A, T, C, G, other or unknown <400> 377 .

    ggtgannnnn nnn <210> 378 <211> 13 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(13) <223> A, T, C, G, other or unknown <400> 378 gaagannnnn nnn <210> 379 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6) .. (10) <223> A, T, C, G, other or unknown <400> 379 gagtcnnnnn <210> 380 <211> 26 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018 <220>

    <221> modified_base <222> (7) .. (26) <223> A, T, C, G, other or unknown <400> 380 tccracnnnn nnnnnnnnnn nnnnnn <210> 381 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (5) .. (11) <223> A, T, C, G, other or unknown <400> 381 cctcnnnnnn n <210> 382 <211> 10 <212> DNA <213> Artificial Sequence <220> · <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (6)..(10) <223> A, T, C, G, other or unknown <400> 382 gagtcnnnnn <210> 383 <211> 18 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(18) <223> A, T, C, G, other or unknown

    2018241075 03 Oct 2018

    <400> 383 cccacannnn nnnnnnnn <210> 384 <211> 14 <212> DNA <213> Artificial Sequence . <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (6)..(14) <223> A, T, C, G, other or unknown <400> 384 gcatcnnnnn nnnn <210> 385 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> · <221> modified base <222> (6)..(13) <223> A, T, C, G, other or unknown <400> 385 ggtgannnnn nnn <210> 386 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified base <222> (5) .. (12) <223> A, T, C, G, other or unknown <400> 386

    cccgnnnnnn nn 12

    2018241075 03 Oct 2018 <210> 387 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide .

    <220>

    <221> modified_base <222> (6)..(19) <223> A, T, C, G, other or unknown <400> 387 ggatgnnnnn nnnnnnnnn 19 <210> 388 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(17) <223> A, T, C, G, other or unknown <400> 388 gaccgannnn nnnnnnn 17 <210> 389 <211> 17 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(17) <223> A, T, C, G, other or unknown ' <400> 389 cacccannnn nnnnnnn <210> 390 <211> 17 <212> DNA

    2018241075 03 Oct 2018 <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (17) .

    <223> A, T,. C, G, other or unknown <400> 390 caarcannnn nnnnnnn 17

    <210> 391 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 391 gctgtgtatt actgtgcgag

    Sequence: Synthetic

    <210> 392 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial probe <400> 392 gccgtgtatt actgtgcgag

    Sequence: Synthetic <210> 393 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 393 gccgtatatt actgtgcgag <210> 394 <211> 20 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 394 gccgtgtatt actgtacgag 20 <210> 395 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic probe <400> 395 gccatgtatt actgtgcgag <210> 396 <211> 25 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 396 cacatccgtg ttgttcacgg atgtg <210> 397 <211> 88 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 397 aatagtagac tgcagtgtcc tcagccctta agctgttcat ctgcaagtag agagtattct 60 tagagttgtc tctagactta gtgaagcg 88 <210> 398 <211> 95 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 398 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaacagctta 60 agggctgagg acactgcagt ctactattgt gcgag 95

    2018241075 03 Oct 2018

    <210> 399 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 399 cgcttcacta agtctagaga caac <210> 400 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 400 cacatccgtg ttgttcacgg atgtgggagg atggagactg ggtc <210> 401 <211> 44 · <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 401 cacatccgtg ttgttcacgg atgtgggaga gtggagactg agtc <210> 402 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 402

    cacatccgtg ttgttcacgg atgtgggtgc ctggagactg cgtc 44 <210> 403

    2018241075 03 Oct 2018 <211> 44 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 403 cacatccgtg ttgttcacgg atgtgggtgg ctggagactg cgtc 44 <210> 404 <211> 34 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 404 cctctactct tgtcacagtg cacaagacat ccag <210> 405 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 405 cctctactct tgtcacagtg <210> 406 <211> 44 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 406 ggaggatgga ctggatgtct tgtgcactgt gacaagagta gagg <210> 407 <211> 44 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 407 ggagagtgga ctggatgtct tgtgcactgt gacaagagta gagg 44

    2018241075 03 Oct 2018

    <210> 408 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 408 ggtgcctgga ctggatgtct tgtgcactgt gacaagagta gagg

    <210> 409 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 409 ggtggctgga ctggatgtct tgtgcactgt gacaagagta gagg

    <210> 410 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 410 cacatccgtg ttgttcacgg atgtggatcg actgtccagg agac

    <210> 411 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 411

    cacatccgtg ttgttcacgg atgtggactg tctgtcccaa ggcc 44

    2018241075 03 Oct 2018

    <210> 412 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 412 cacatccgtg ttgttcacgg atgtggactg actgtccagg agac <210> 413 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 413 cacatccgtg ttgttcacgg atgtggaccc tctgccctgg ggcc <210> 414 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 414

    cctctgactg agtgcacaga gtgctttaac ccaaccggct agtgttagcg gttccccgg 59 <210> 415 <211> 69 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 415 cctctgactg agtgcacaga gtgctttaac ccaaccggct agtgttagcg gttccccggg 60 acagtcgat 69 <210> 416 <211> 69 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 416 cctctgactg agtgcacaga gtgctttaac ccaaccggct agtgttagcg gttccccggg 60 acagacagt 69 <210> 417 <211> 69 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 417 cctctgactg agtgcacaga gtgctttaac ccaaccggct agtgttagcg gttccccggg 60 acagtcagt 69 <210> 418 <211> 70 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 418 · cctctgactg agtgcacaga gtgctttaac ccaaccggct agtgttagcg gtstccccgg 60 ggcagagggt 70 <210> 419 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 419 cctctgactg agtgcacaga gtgc 24 <210> 420 <211> 13 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018

    <220> <221> modified base <222> (5)..(9) <223> A, T, C, G, other or unknown <400> 420 ggccnnnnng gcc

    <210> 421 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(12) <223> A, T, C, G, other or unknown <400> 421 ccannnnnnn nntgg <210> 422 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> .

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown <400> 422 cgannnnnnt gc <210> 423 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8)

    2018241075 03 Oct 2018 <223> A, T, C, G, other or unknown <400> 423 gccnnnnngg c <210> 424 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 424 gatnnnnatc <210> 425 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 425 gacnnnnngt c <210> 426 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4) .. (8) <223> A, T, C, G, other or unknown <400> 426 gcannnnntg c

    2018241075 03 Oct 2018 <210> 427 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (12) <223> A, T, C, G, other or unknown <400> 427 gtatccnnnn nn 12 <210> 428 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, G, G, other or unknown <400> 428 gacnnnnnng tc 12 <210> 429 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base ' <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 429 ccannnnntg g 11 <210> 430 <211> 12

    2018241075 03 Oct 2018 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base .

    <222> (1)..(6) <223> A, T, C, G, other or unknown <400> 430 nnnnnngaga eg 12 <210> 431 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown <400> 431 ccannnnnnt gg 12 <210> 432 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 432 gaannnnttc 10 <210> 433 <211> 11 <212> DNA <213> Artificial Sequence <220>

    2018241075 03 Oct 2018 <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 433 .

    ggtctcnnnn n <210> 434 <211> 16 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(10) <223> A, T, C, G, other or unknown <400> 434 nnnnnnnnnn ctcctc <210> 435 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(9) <223> A, T, C, G, other or unknown <400> 435 nnnnnnnnnt ccgcc 15 <210> 436 · <211> 13 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    2018241075 03 Oct 2018 <221> modified_base <222> (5) . . (9) <223> A, T, C, G, other or unknown <400> 436 ggccnnnnng gcc <210> 437 .

    <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown <400> 437 ccannnnnnt gg <210> 438 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown <400> 438 gacnnnnnng tc <210> 439 <211> 12 ' <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide .

    <220>

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown

    2018241075 03 Oct 2018

    <400> 439 cgannnnnnt gc <210> 440 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic

    oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 440 gcannnnntg c 11 <210> 441 <211> 11 <212> DNA <213> Artificial Sequence ' <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown ' <400> 441 ccannnnntg g 11 <210> 442 <211> 10 <212> DNA <213> Artificial Sequence <220> .

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 442 gaannnnttc

    2018241075 03 Oct 2018

    100 <210> 443 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(6) <223> A, T, C, G, other or unknown <400> 443 nnnnnngaga eg <210> 444 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (12) <223> A, T, C, G, other or unknown <400> 444 gtatccnnnn nn <210> 445 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, ”, C, G, other or unknown <400> 445 gacnnnnngt c <210> 446 <211> 11 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    101 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown .

    <400> 446 ggtctcnnnn n 11 <210> 447 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 447 gccnnnnngg c 11 <210> 448 · <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(12) <223> A, T, C, G, other or unknown <400> 448 ccannnnnnn nntgg 15 <210> 449 <211> 16 <212> DNA <213> Artificial Sequence <220> .

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018

    102 <220>

    <221> modified_base <222> (1)..(10) <223> A, T, C, G, other or unknown <400> 449 nnnnnnnnnn ctcctc 16 <210> 450 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(9) <223> A, T, C, G, other or unknown <400> 450 nnnnnnnnnt ccgcc <210> 451 <211> 9532 <212> DNA <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3 nucleotide sequence <220>

    <221> CDS <222> (1579).. (1638) <220>

    <221> CDS <222> (2343)..(3443) <220>

    <221> CDS <222> (3945)..(4400) <220>

    <221> CDS <222> (4406) . . (4450) <220>

    <221> CDS <222> (4746)..(5789) <400> 451

    2018241075 03 Oct 2018

    103 aatgctacta ctattagtag aattgatgee accttttcag ctcgcgcccc aaatgaaaat 60 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120 cgttcgcaga attgggaatc aactgttaca tggaatgaaa cttccagaca ccgtacttta 180 gttgeatatt taaaacatgt tgagctacag caccagattc agcaattaag ctctaagcca 240 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300 ttggagtttg cttccggtct ggttcgcttt gaagetegaa ttaaaacgcg atatttgaag 360 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420 cagggtaaag acctgatttt tgatttatgg teattetegt tttctgaact gtttaaagca 480 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggaege tatccagtct 540 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600 ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660 aattcctttt ggcgttatgt atetgeatta gttgaatgtg gtattcctaa atctcaactg 720 atgaatettt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960 aatatccggt tettgteaag attactcttg atgaaggtea gccagcctat gcgcctggtc 1020 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg eggatttega cacaatttat 1140 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200 caaagatgag tgttttagtg tattettteg cctctttcgt tttaggttgg tgccttcgta 1260 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320 caaagcctct gtagccgttg ctaccctcgt teegatgetg tctttcgctg ctgagggtga 1380 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440 tgcgtgggcg atggttgttg teattgtegg cgcaactatc ggtatcaagc tgtttaagaa 1-500 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560

    tttttggaga ttttcaac gtg aaa aaa tta tta ttc gca att cct tta gtt 1611

    Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val

    15 10

    1658 gtt cct ttc tat tet cac agt gca cag tctgtcgtga cgcagccgcc

    Val Pro Phe Tyr Ser His Ser Ala Gln

    2018241075 03 Oct 2018

    104

    15 20

    ctcagtgtct ggggccccag ggcagagggt caccatctcc tgcactggga gcagctccaa 1718 catcggggca ggttatgatg tacactggta ccagcagctt ccaggaacag cccccaaact 1778 cctcatctat ggtaacagca atcggccctc aggggtccct gaccgattct ctggctccaa 1838 gtctggcacc tcagcctccc tggccatcac tgggctccag gctgaggatg aggetgatta 1898 ttactgccag tcctatgaca gcagcctgag tggcctttat gtcttcggaa ctgggaccaa 1958 ggtcaccgtc ctaggtcagc ccaaggccaa ccccactgtc actctgttcc cgccctcctc 2018 tgaggagctc caagccaaca aggccacact agtgtgtctg atcagtgact tctacccggg 2078 agctgtgaca gtggcctgga aggcagatag cagccccgtc aaggcgggag tggagaccac 2138 cacaccctcc aaacaaagca acaacaagta cgcggccagc agctatctga gcctgacgcc 2198 tgagcagtgg aagtcccaca gaagctacag ctgccaggtc acgcatgaag ggagcaccgt 2258 ggagaagaca gtggccccta cagaatgttc ataataaacc gcctccaccg ggcgcgccaa 2318 ttctatttca aggagacagt cata atg aaa tac eta Met Lys Tyr Leu ttg cct aeg Leu Pro Thr gca gcc Ala Ala 2369

    get Ala 30 gga Gly ttg Leu tta Leu tta Leu etc Leu 35 geg Ala gcc Ala cag Gln ccg Pro gcc atg gcc Ala gaa Glu gtt Val caa Gln 45 2417 Ala 40 Met ttg tta gag tet ggt ggc ggt ett gtt cag cct ggt ggt tet tta cgt 2465 Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg 50 55 60 ett tet tgc get get tcc gga ttc act ttc tet teg tac get atg tet 2513 Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser 65 70 75 tgg gtt ege caa get cct ggt aaa ggt ttg gag tgg gtt tet get ate 2561 Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile 80 85 90 tet ggt tet ggt ggc agt act tac tat get gac tcc gtt aaa ggt ege 2609 Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg 95 100 105 ttc act ate tet aga gac aac tet aag aat act etc tac ttg cag atg 2657 Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met 110 115 120 125 aac age tta agg get gag gac act gca gtc tac tat tgc get aaa gac 2705 Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp 130 135 140 tat gaa ggt act ggt tat get ttc gac ata tgg ggt caa ggt act atg 2753 Tyr Glu Gly Thr Gly Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met

    2018241075 03 Oct 2018

    105

    145 150 155

    gtc Val acc Thr gtc Val 160 tet Ser agt Ser gcc Ala tcc Ser acc Thr 165 aag Lys ggc Gly cca Pro teg Ser gtc Val 170 ttc Phe CCC Pro ctg Leu 2801 gca CCC tcc tcc aag age acc tet ggg ggc aca gcg gcc ctg ggc tgc 2849 Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys 175 180 185 ctg gtc aag gac tac ttc CCC gaa ccg gtg aeg gtg teg tgg aac tea 2897 Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser 190 195 200 205 ggc gcc ctg acc age ggc gtc cac acc ttc ccg get gtc eta cag tet 2945 Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser 210 215 220 age gga etc tac tcc etc age age gta gtg acc gtg CCC tet tet age 2993 Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser 225 230 235 ttg ggc acc cag acc tac ate tgc aac gtg aat cac aag CCC age aac 3041 Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn 240 245 250 acc aag gtg gac aag aaa gtt gag CCC aaa tet tgt gcg gcc get cat 3089 Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Ala Ala Ala His 255 260 265 cac cac cat cat cac tet get gaa caa aaa etc ate tea gaa gag gat 3137 His His His His His Ser Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp 270 275 280 285 ctg aat ggt gcc gca gat ate aac gat gat cgt atg get ggc gcc get 3185 Leu Asn Gly Ala Ala Asp Ile Asn Asp Asp Arg Met Ala Gly Ala Ala 290 295 300 gaa act gtt gaa agt tgt tta gca aaa CCC cat aca gaa aat tea ttt 3233 Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe 305 310 315 act aac gtc tgg aaa gac gac aaa act tta gat cgt tac get aac tat 3281 Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr 320 325 330 gag ggt tgt ctg tgg aat get aca ggc gtt gta gtt tgt act ggt gac 3329 Glu Gly Cys Leu Trp Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp 335 340 345 gaa act cag tgt tac ggt aca tgg gtt cct att ggg ett get ate cct 3377 Glu Thr Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro 350 355 360 365 gaa aat gag ggt ggt ggc tet gag ggt ggc ggt tet gag ggt ggc ggt 3425 Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 370 375 380

    106 tct gag ggt ggc ggt act aaacctcctg agtacggtga tacacctatt 3473

    Ser Glu Gly Gly Gly Thr

    385

    2018241075 03 Oct 2018

    ccgggctata cttatatcaa ccctctcgac ggcacttatc cgcctggtac tgagcaaaac 3533 cccgctaatc ctaatccttc tcttgaggag tctcagcctc ttaatacttt catgtttcag 3593 aataataggt tccgaaatag gcagggggca ttaactgttt atacgggcac tgttactcaa 3653 ggcactgacc ccgttaaaac ttattaccag tacactcctg tatcatcaaa agccatgtat 3713 gacgcttact ggaacggtaa attcagagac tgcgctttcc attctggctt taatgaagat 3773 ccattcgttt gtgaatatca aggccaatcg tctgacctgc ctcaacctcc tgtcaatgct 3833 ggcggcggct ctggtggtgg ttctggtggc ggctctgagg gtggtggctc tgagggtggc 3893 ggttctgagg gtggcggctc tgagggaggc ggttccggtg gtggctctgg t tcc ggt 3950

    Ser Gly

    gat Asp 390 ttt Phe gat Asp tat Tyr gaa Glu aag Lys 395 atg Met gca Ala aac Asn get Ala aat Asn 400 aag Lys ggg Gly get Ala atg Met acc Thr 405 3998 gaa aat gcc gat gaa aac geg eta cag tct gac get aaa ggc aaa ctt 4046 Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu 410 415 420 gat tct gtc get act gat tac ggt get get ate gat ggt ttc att ggt 4094 Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly 425 430 435 gac gtt tcc ggc ctt get aat ggt aat ggt get act ggt gat ttt get 4142 Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala 440 445 450 ggc tct aat tcc caa atg get caa gtc ggt gac ggt gat aat tea cct 4190 Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro 455 460 465 tta atg aat aat ttc cgt caa tat tta cct tcc etc cct caa teg gtt 4238 Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val 470 475 480 485 gaa tgt cgc cct ttt gtc ttt age get ggt aaa cca tat gaa ttt tct 4286 Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser 490 495 500 att gat tgt gac aaa ata aac tta ttc cgt ggt gtc ttt geg ttt ctt 4334 Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu 505 510 515 tta tat gtt gcc acc ttt atg tat gta ttt tct aeg ttt get aac ata 4382 Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile 520 525 530

    ctg cgt aat aag gag tct taatc atg cca gtt ctt ttg ggt att ccg tta 4432

    2018241075 03 Oct 2018

    107

    Leu Arg Asn Lys Glu Ser Met Pro Val Leu Leu Gly Ile Pro Leu

    535 540 545 tta ttg cgt ttc etc ggt ttccttctgg taactttgtt eggetatetg 4480

    Leu Leu Arg Phe Leu Gly

    550 cttacttttc ttaaaaaggg etteggtaag atagetattg ctatttcatt gtttcttgct 4540 ettattattg ggcttaactc aattcttgtg ggttatctct ctgatattag cgctcaatta 4600 ccctctgact ttgttcaggg tgttcagtta attctcccgt etaatgeget tccctgtttt 4660 tatgttattc tctctgtaaa ggctgctatt ttcatttttg acgttaaaca aaaaatcgtt 4720

    tcttatttgg attgggataa ataat atg get Met Ala gtt Val tat Tyr ttt Phe gta Val 560 act Thr ggc Gly aaa Lys 4772 555 tta ggc tet gga aag aeg etc gtt age gtt ggt aag att cag gat aaa 4820 Leu Gly Ser Gly Lys Thr Leu Val Ser Val Gly Lys Ile Gln Asp Lys 565 570 575 att gta get ggg tgc aaa ata gca act aat ctt gat tta agg ctt caa 4868 Ile Val Ala Gly Cys Lys Ile Ala Thr Asn Leu Asp Leu Arg Leu Gln 580 585 590 595 aac etc ccg caa gtc ggg agg ttc get aaa aeg cct cgc gtt ctt aga 4916 Asn Leu Pro Gln Val Gly Arg Phe Ala Lys Thr Pro Arg Val Leu Arg 600 605 610 ata ccg gat aag cct tet ata tet gat ttg ctt get att ggg cgc ggt 4964 Ile Pro Asp Lys Pro Ser Ile Ser Asp Leu Leu Ala Ile Gly Arg Gly 615 620 625 aat gat tee tac gat gaa aat aaa aac ggc ttg ctt gtt etc gat gag 5012 Asn Asp Ser Tyr Asp Glu Asn Lys Asn Gly Leu Leu Val Leu Asp Glu 630 635 640 tgc ggt act tgg ttt aat acc cgt tet tgg aat gat aag gaa aga cag 5060 Cys Gly Thr Trp Phe Asn Thr Arg Ser Trp Asn Asp Lys Glu Arg Gln 645 650 655 ccg att att gat tgg ttt eta cat get cgt aaa tta gga tgg gat att 5108 Pro Ile Ile Asp Trp Phe Leu His Ala Arg Lys Leu Gly Trp Asp Ile 660 665 670 675 att ttt ctt gtt cag gac tta tet att gtt gat aaa cag gcg cgt tet 5156 Ile Phe Leu Val Gln Asp Leu Ser Ile Val Asp Lys Gln Ala Arg Ser 680 685 690 gca tta get gaa cat gtt gtt tat tgt cgt cgt ctg gac aga att act 5204 Ala Leu Ala Glu His Val Val Tyr Cys Arg Arg Leu Asp Arg Ile Thr 695 700 705 tta cct ttt gtc ggt act tta tat tet ctt att act ggc teg aaa atg 5252 Leu Pro Phe Val Gly Thr Leu Tyr Ser Leu Ile Thr Gly Ser Lys Met 710 715 720

    2018241075 03 Oct 2018

    108

    cct Pro ctg Leu 725 cct Pro aaa Lys tta Leu cat His gtt Val 730 ggc Gly gtt Val gtt Val aaa Lys tat Tyr 735 ggc Gly gat Asp tet Ser caa Gln 5300 tta age cct act gtt gag cgt tgg ett tat act ggt aag aat ttg tat 5348 Leu Ser Pro Thr Val Glu Arg Trp Leu Tyr Thr Gly Lys Asn Leu Tyr 740 745 750 755 aac gca tat gat act aaa cag get ttt tet agt aat tat gat tee ggt 5396 Asn Ala Tyr Asp Thr Lys Gln Ala Phe Ser Ser Asn Tyr Asp Ser Gly 760 765 770 gtt tat tet tat tta aeg cct tat tta tea cac ggt egg tat ttc aaa 5444 Val Tyr Ser Tyr Leu Thr Pro Tyr Leu Ser His Gly Arg Tyr Phe Lys 775 780 785 cca tta aat tta ggt cag aag atg aaa tta act aaa ata tat ttg aaa 5492 Pro Leu Asn Leu Gly Gln Lys Met Lys Leu Thr Lys lie Tyr Leu Lys 790 795 800 aag ttt tet ege gtt ett tgt ett geg att gga ttt gca tea gca ttt 5540 Lys Phe Ser Arg Val Leu Cys Leu Ala Ile Gly Phe Ala Ser Ala Phe 805 810 815 aca tat agt tat ata acc caa cct aag ccg gag gtt aaa aag gta gtc 5588 Thr Tyr Ser Tyr Ile Thr Gln Pro Lys Pro Glu Val Lys Lys Val Val 820 825 830 835 tet cag acc tat gat ttt gat aaa ttc act att gac tet tet cag cgt 5636 Ser Gln Thr Tyr Asp Phe Asp Lys Phe Thr Ile Asp Ser Ser Gln Arg 840 845 850 ett aat eta age tat ege tat gtt ttc aag gat tet aag gga aaa tta 5684 Leu Asn Leu Ser Tyr Arg Tyr Val Phe Lys Asp Ser Lys Gly Lys Leu 855 860 865 att aat age gac gat tta cag aag caa ggt tat tea etc aca tat att 5732 Ile Asn Ser Asp Asp Leu Gln Lys Gln Gly Tyr Ser Leu Thr Tyr Ile 870 875 880 gat tta tgt act gtt tee att aaa aaa ggt aat tea aat gaa att gtt 5780 Asp Leu Cys Thr Val Ser Ile Lys Lys Gly Asn Ser Asn Glu Ile Val 885 890 895

    aaa tgt aat Lys Cys Asn 900 taattttgtt ttcttgatgt ttgtttcatc atcttctttt 5829 gctcaggtaa ttgaaatgaa taattcgcct ctgcgcgatt ttgtaacttg gtattcaaag 5889 caatcaggcg aatccgttat tgtttctccc gatgtaaaag gtactgttac tgtatattca 5949 tctgacgtta aacctgaaaa tctacgcaat ttctttattt ctgttttacg tgctaataat 6009 tttgatatgg ttggttcaat tccttccata attcagaagt ataatccaaa caatcaggat 6069 tatattgatg aattgccatc atctgataat caggaatatg atgataattc cgctccttct 6129

    2018241075 03 Oct 2018

    109

    ggtggtttct ttgttccgca aaatgataat gttactcaaa cttttaaaat taataacgtt 6189 cgggcaaagg atttaatacg agttgtcgaa ttgtttgtaa agtctaatac ttctaaatcc 6249 tcaaatgtat tatctattga cggctctaat ctattagttg tttctgcacc taaagatatt 6309 ttagataacc ttcctcaatt cctttctact gttgatttgc caactgacca gatattgatt 6369 gagggtttga tatttgaggt tcagcaaggt gatgctttag atttttcatt tgctgctggc 6429 tctcagcgtg gcactgttgc aggcggtgtt aatactgacc gcctcacctc tgttttatct 6489 tctgctggtg gttcgttcgg tatttttaat ggcgatgttt tagggctatc agttcgcgca 6549 ttaaagacta atagccattc aaaaatattg tctgtgccac gtattcttac gctttcaggt 6609 cagaagggtt ctatctctgt tggccagaat gtccctttta ttactggtcg tgtgactggt 6669 gaatctgcca atgtaaataa tccatttcag acgattgagc gtcaaaatgt aggtatttcc 6729 atgagcgttt ttcctgttgc aatggctggc ggtaatattg ttctggatat taccagcaag 6789 gccgatagtt tgagttcttc tactcaggca agtgatgtta ttactaatca aagaagtatt 6849 gctacaacgg ttaatttgcg tgatggacag actcttttac tcggtggcct cactgattat 6909 aaaaacactt ctcaagattc tggcgtaccg ttcctgtcta aaatcccttt aatcggcctc 6969 ctgtttagct cccgctctga ttccaacgag gaaagcacgt tatacgtgct cgtcaaagca 7029 accatagtac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag 7089 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt 7149 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt 7209 ccgatttagt gctttacggc acctcgaccc caaaaaactt gatttgggtg atggttcacg 7269 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt 7329 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg gctattcttt 7389 tgatttataa gggattttgc cgatttcgga accaccatca aacaggattt tcgcctgctg 7449 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat 7509 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggatccaag cttgcaggtg 7569 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa 7629 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga 7689 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc 7749 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg 7809 gcgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc 7869

    2018241075 03 Oct 2018

    110

    gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt catacactat 7929 tatcccgtat tgacgccggg caagagcaac tcggtcgccg ggcgcggtat tctcagaatg 7989 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag 8049 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa 8109 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc 8169 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca 8229 cgatgcctgt agcaatgcca acaacgttgc gcaaactatt aactggcgaa ctacttactc 8289 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc 8349 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg 8409 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta 8469 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag 8529 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga 8589 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc 8649 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg tacgtaagac ccccaagctt 8709 gtcgactgaa tggcgaatgg cgctttgcct ggtttccggc accagaagcg gtgccggaaa 8769 gctggctgga gtgcgatctt cctgaggccg atactgtcgt cgtcccctca aactggcaga 8829 tgcacggtta cgatgcgccc atctacacca acgtaaccta tcccattacg gtcaatccgc 8889 cgtttgttcc cacggagaat ccgacgggtt gttactcgct cacatttaat gttgatgaaa 8949 gctggctaca ggaaggccag acgcgaatta tttttgatgg cgttcctatt ggttaaaaaa 9009 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaattta 9069 aatatttgct tatacaatct tcctgttttt ggggcttttc tgattatcaa ccggggtaca 9129 tatgattgac atgctagttt tacgattacc gttcatcgat tctcttgttt gctccagact 9189 ctcaggcaat gacctgatag cctttgtaga tctctcaaaa atagctaccc tctccggcat 9249 gaatttatca gctagaacgg ttgaatatca tattgatggt gatttgactg tctccggcct 9309 ttctcaccct tttgaatctt tacctacaca ttactcaggc attgcattta aaatatatga 9369 gggttctaaa aatttttatc cttgcgttga aataaaggct tctcccgcaa aagtattaca 9429 gggtcataat gtttttggta caaccgattt agctttatgc tctgaggctt tattgcttaa 9489

    ttttgctaat tctttgcctt gcctgtatga tttattggat gtt

    9532

    2018241075 03 Oct 2018

    111 <210> 452 <211> 20 <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3 peptide sequence <400> 452

    Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser 1 5 10 15

    His Ser Ala Gln 20 <210> 453 <211> 367 <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3 protein sequence <400> 453

    Met Lys Tyr Leu Leu Pro Thr Ala 1 5

    Ala Gln Pro Ala Met Ala Glu Val 20

    Leu Val Gln Pro Gly Gly Ser Leu 35 40

    Phe Thr Phe Ser Ser Tyr Ala Met 50 55

    Lys Gly Leu Glu Trp Val Ser Ala 65 70

    Tyr Tyr Ala Asp Ser Val Lys Gly 85

    Ser Lys Asn Thr Leu Tyr Leu Gln 100

    Thr Ala Val Tyr Tyr Cys Ala Lys 115 120

    Phe Asp Ile Trp Gly Gln Gly Thr 130 135

    Thr Lys Gly Pro Ser Val Phe Pro 145 150

    Ser Gly Gly Thr Ala Ala Leu Gly

    Ala Ala Gly Leu Leu Leu Leu Ala 10 15

    Gln Leu Leu Glu Ser Gly Gly Gly 25 30

    Arg Leu Ser Cys Ala Ala Ser Gly 45

    Ser Trp Val Arg Gln Ala Pro Gly 60

    Ile Ser Gly Ser Gly Gly Ser Thr 75 80

    Arg Phe Thr Ile Ser Arg Asp Asn 90 95

    Met Asn Ser Leu Arg Ala Glu Asp 105 110

    Asp Tyr Glu Gly Thr Gly Tyr Ala 125

    Met Val Thr Val Ser Ser Ala Ser 140

    Leu Ala Pro Ser Ser Lys Ser Thr 155 160

    Cys Leu Val Lys Asp Tyr Phe Pro

    2018241075 03 Oct 2018

    112

    165 170 175 Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val 180 185 190 His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser 195 200 205 Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile 210 215 220 Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val 225 230 235 240 Glu Pro Lys Ser Cys Ala Ala Ala His His His His His His Ser Ala 245 250 255 Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala Asp Ile 260 265 270 Asn Asp Asp Arg Met Ala Gly Ala Ala Glu Thr Val Glu Ser Cys Leu 275 280 285 Ala Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val Trp Lys Asp Asp 290 295 300 Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys Leu Trp Asn Ala 305 310 315 320 Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln Cys Tyr Gly Thr 325 330 335 Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu Gly Gly Gly Ser 340 345 350 Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Thr

    355 360 365 <210> 454 <211> 152 <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3 protein sequence <400> 454

    Ser Gly Asp 1 Phe Asp 5 Tyr Glu Lys Met Ala Asn Ala Asn Lys 10 Gly 15 Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly 20 25 30 Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe 35 40 45

    2018241075 03 Oct 2018

    113 Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp 50 55 60 Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn 65 70 75 80 Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln 85 90 95 Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu 100 105 110 Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala 115 120 125 Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala 130 135 140 Asn Ile Leu Arg Asn Lys Glu Ser

    145 150 <210> 455 <211> 15 <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3 peptide sequence <400> 455

    Met Pro Val Leu Leu Gly Ile Pro Leu Leu Leu Arg Phe Leu Gly 15 10 15 <210> 456 <211> 348 <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3 protein sequence <400> 456

    Met 1 Ala Val Tyr Phe 5 Val Thr Gly Lys Leu 10 Gly Ser Gly Lys Thr 15 Leu Val Ser Val Gly 20 Lys Ile Gln Asp Lys 25 Ile Val Ala Gly Cys 30 Lys Ile Ala Thr Asn 35 Leu Asp Leu Arg Leu 40 Gln Asn Leu Pro Gln 45 Val Gly Arg Phe Ala 50 Lys Thr Pro Arg Val 55 Leu Arg Ile Pro Asp 60 Lys Pro Ser Ile

    2018241075 03 Oct 2018

    Ser Asp Leu 65

    Lys Asn Gly

    Arg Ser Trp

    His Ala Arg 115

    Ser Ile Val 130

    Tyr Cys Arg 145

    Tyr Ser Leu

    Gly Val Val

    Trp Leu Tyr 195

    Ala Phe Ser 210

    Tyr Leu Ser 225

    Met Lys Leu

    Leu Ala Ile

    Pro Lys Pro 275

    Lys Phe Thr 290

    Val Phe Lys 305

    Lys Gln Gly

    Lys Lys Gly

    Leu Ala Ile Gly Arg 70

    Leu Leu Val Leu Asp 85

    Asn Asp Lys Glu Arg 100

    Lys Leu Gly Trp Asp 120

    Asp Lys Gln Ala Arg 135

    Arg Leu Asp Arg Ile 150

    Ile Thr Gly Ser Lys 165

    Lys Tyr Gly Asp Ser 180

    Thr Gly Lys Asn Leu 200

    Ser Asn Tyr Asp Ser 215

    His Gly Arg Tyr Phe 230

    Thr Lys Ile Tyr Leu 245

    Gly Phe Ala Ser Ala 260

    Glu Val Lys Lys Val 280

    Ile Asp Ser Ser Gln 295

    Asp Ser Lys Gly Lys 310

    Tyr Ser Leu Thr Tyr 325

    Asn Ser Asn Glu Ile 340

    114

    Gly Asn

    Glu Cys 90

    Gln Pro 105

    Ile Ile

    Ser Ala

    Thr Leu

    Met Pro 170

    Gln Leu 185

    Tyr Asn

    Gly Val

    Lys Pro

    Lys Lys 250

    Phe Thr 265

    Val Ser

    Arg Leu

    Leu Ile

    Ile Asp 330

    Val Lys 345

    Asp Ser 75

    Gly Thr

    Ile Ile

    Phe Leu

    Leu Ala 140

    Pro Phe 155

    Leu Pro

    Ser Pro

    Ala Tyr

    Tyr Ser 220

    Leu Asn 235

    Phe Ser

    Tyr Ser

    Gln Thr

    Asn Leu 300

    Asn Ser 315

    Leu Cys

    Cys Asn

    Tyr Asp Glu

    Trp Phe Asn 95

    Asp Trp Phe 110

    Val Gln Asp 125

    Glu His Val

    Val Gly Thr

    Lys Leu His 175

    Thr Val Glu 190

    Asp Thr Lys 205

    Tyr Leu Thr

    Leu Gly Gln

    Arg Val Leu 255

    Tyr Ile Thr 270

    Tyr Asp Phe 285

    Ser Tyr Arg

    Asp Asp Leu

    Thr Val Ser 335

    Asn

    Thr

    Leu

    Leu

    Val

    Leu

    160

    Val

    Arg

    Gln

    Pro

    Lys

    240

    Cys

    Gln

    Asp

    Tyr

    Gln

    320

    Ile <210> 457 <211> 24 <212> DNA

    2018241075 03 Oct 2018

    115 <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 457 tggaagaggc acgttctttt cttt 24 <210> 458 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 458 cttttctttg ttgccgttgg ggtg 24 <210> 459 <211> 24 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 459 acactctccc ctgttgaagc tctt 24 <210> 460 <211> 51 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 460 accgcctcca ccgggcgcgc cttattaaca ctctcccctg ttgaagctct t 51 <210> 461 <211> 23 <212> DNA <213> Artificial Sequence · <220>

    <223> Description of Artificial Sequence: Primer <400> 461 tgaacattct gtaggggcca ctg 23 <210> 462

    2018241075 03 Oct 2018

    116 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 462 agagcattct gcaggggcca ctg . 23 <210> 463 <211> 50 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 463 accgcctcca ccgggcgcgc cttattatga acattctgta ggggccactg 50 <210> 464 <211> 50 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 464 accgcctcca ccgggcgcgc cttattaaga gcattctgca ggggccactg 50 <210> 465 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 465 cgactggagc acgaggacac tga 23 <210> 466 <211> 26 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 466 ggacactgac atggactgaa ggagta

    2018241075 03 Oct 2018

    117

    <210> 4 67 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 467 gggaggatgg agactgggtc

    <210> 468 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 468 gggaagatgg agactgggtc

    <210> 469 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 469 gggagagtgg agactgagtc

    <210> 470 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 470 gggtgcctgg agactgcgtc

    <210> 471 <211> 20 <212> DNA <213> Artificial

    2018241075 03 Oct 2018

    118 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    <400> 471 gggtggctgg agactgcgtc <210> 472 . <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 472 gggaggatgg agactgggtc atctggatgt cttgtgcact gtgacagagg <210> 473 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 473 gggaagatgg agactgggtc atctggatgt cttgtgcact gtgacagagg

    <210> 474 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 474 gggagagtgg agactgggtc atctggatgt cttgtgcact gtgacagagg

    <210> 475 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 475

    gggtgcctgg agactgggtc atctggatgt cttgtgcact gtgacagagg

    2018241075 03 Oct 2018

    119 <210> 476 <211> 50 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 476 gggtggctgg agactgggtc atctggatgt cttgtgcact gtgacagagg 50

    <210> 477 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 477 gggagtctgg agactgggtc atctggatgt cttgtgcact gtgacagagg

    <210> 478 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 478

    cctctgtcac agtgcacaag acatccagat gacccagtct cc 42 <210> 479 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 479 cctctgtcac agtgcacaag ac <210> 480 <211> 24 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    120 <220>

    <223> Description of Artificial Sequence: Primer <400> 480 acactctccc ctgttgaagc tctt 24 <210> 481 <211> 668 .

    <212> DNA <213> Homo sapiens <220>

    <221> CDS <222> (1) .. (668) <400> 481 agt gca caa gac ate cag atg acc cag tet cca gcc acc ctg tet gtg 48

    Ser Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Val

    1 5 10 15 tet cca ggg gaa agg gcc acc etc tcc tgc agg gcc agt cag agt gtt 96

    Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val

    20 25 30 agt aac aac tta gcc tgg tac cag cag aaa cct ggc cag gtt ccc agg 144

    Ser Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg

    35 40 45 etc etc ate tat ggt gca tcc acc agg gcc act gat ate cca gcc agg 192

    Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg

    50 55 60 ttc agt ggc agt ggg tet ggg aca gac ttc act etc acc ate age aga 240

    Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg

    65 70 75 80 ctg gag cct gaa gat ttt gca gtg tat tac tgt cag egg tat ggt age 288

    Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Arg Tyr Gly Ser

    85 90 95 tea ccg ggg tgg aeg ttc ggc caa ggg acc aag gtg gaa ate aaa ega 336

    Ser Pro Gly Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg

    100 105 110 act gtg get gca cca tet gtc ttc ate ttc ccg cca tet gat gag cag 384

    Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln

    115 120 125 ttg aaa tet gga act gcc tet gtt gtg tgc ctg ctg aat aac ttc tat 432

    Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

    130 135 140 ccc aga gag gcc aaa gta cag tgg aag gtg gat aac gcc etc caa teg 480

    Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    145 150 155 160 ggt aac tcc cag gag agt gtc aca gag cag gac age aag gac age acc 528

    2018241075 03 Oct 2018

    121 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 tac age etc age age acc ctg aeg ctg age aaa gca gac tac gag aaa 576 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190 cac aaa gtc tac gee tgc gaa gtc acc cat cag ggc ctg age teg cct 624 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205 gtc aca aag age ttc aac aaa gga gag tgt aag ggc gaa ttc gc 668 Val Thr Lys Ser Phe Asn Lys Gly Glu Cys Lys Gly Glu Phe Ala

    210 215 220 <210> 482 <211> 223 <212> PRT <213> Homo sapiens <400> 482

    Ser 1 Ala Gln Asp Ile 5 Gln Met Thr Gln Ser 10 Pro Ala Thr Leu Ser 15 Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val 20 25 30 Ser Asn Asn Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Val Pro Arg 35 40 45 Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Asp Ile Pro Ala Arg 50 55 60 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg 65 70 75 80 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Arg Tyr Gly Ser 85 90 95 Ser Pro Gly Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 100 105 110 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 115 120 125 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 130 135 140 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 145 150 155 160 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 165 170 175 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 180 185 190

    2018241075 03 Oct 2018

    122

    His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 195 200 205

    Val Thr Lys Ser Phe Asn Lys Gly Glu Cys Lys Gly Glu Phe Ala 210 215 220 <210> 483 <211> 13 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 483 agccaccctg tet 13 <210> 484 <211> 700 <212> DNA <213> Homo sapiens <220>

    <221> CDS <222> (1) .. (699) <400> 484

    agt Ser 1 gca Ala caa Gln gac Asp ate Ile 5 cag Gln atg Met acc Thr cag Gln tet Ser 10 cct Pro gcc Ala acc Thr ctg Leu tet Ser 15 gtg Val 48 tet cca ggt gaa aga gcc acc etc tcc tgc agg gcc agt cag gtg tet 96 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Val Ser 20 25 30 cca ggg gaa aga gcc acc etc tcc tgc aat ett etc age aac tta gcc 144 Pro Gly Glu Arg Ala Thr Leu Ser Cys Asn Leu Leu Ser Asn Leu Ala 35 40 45 tgg tac cag cag aaa cct ggc cag get ccc agg etc etc ate tat ggt 192 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly 50 55 60 get tcc acc ggg gcc att ggt ate cca gcc agg ttc agt ggc agt ggg 240 Ala Ser Thr Gly Ala Ile Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly 65 70 75 80 tet ggg aca gag ttc act etc acc ate age age ctg cag tet gaa gat 288 Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 85 90 95 ttt gca gtg tat ttc tgt cag cag tat ggt acc tea ccg ccc act ttc 336 Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Thr Ser Pro Pro Thr Phe 100 105 110

    2018241075 03 Oct 2018

    123 ggc gga ggg acc aag gtg gag ate aaa ega act gtg get gca cca tet 384 Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 115 120 125 gtc ttc ate ttc ccg cca tet gat gag cag ttg aaa tet gga act gcc 432 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 130 135 140 tet gtt gtg tgc ccg ctg aat aac ttc tat ccc aga gag gcc aaa gta 480 Ser Val Val Cys Pro Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 145 150 155 160 cag tgg aag gtg gat aac gcc etc caa teg ggt aac tcc cag gag agt 528 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 165 170 175 gtc aca gag cag gac aac aag gac age acc tac age etc age age acc 576 Val Thr Glu Gln Asp Asn Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 180 185 190 ctg acg ctg age aaa gta gac tac gag aaa cac gaa gtc tac gcc tgc 624 Leu Thr Leu Ser Lys Val Asp Tyr Glu Lys His Glu Val Tyr Ala Cys 195 200 205 gaa gtc acc cat cag ggc ett age teg ccc gtc acg aag age ttc aac 672 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 210 215 220 agg gga gag tgt aag aaa gaa ttc gtt t 700 Arg Gly Glu Cys Lys Lys Glu Phe Val

    225 230 <210> 485 <211> 233 <212> PRT

    <213> Homo ; sapiens <400> 485 Ser Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Val 1 5 10 15 Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Val Ser 20 25 30 Pro Gly Glu Arg Ala Thr Leu Ser Cys Asn Leu Leu Ser Asn Leu Ala 35 40 45 Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly 50 55 60 Ala Ser Thr Gly Ala Ile Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly 65 70 75 80 Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp 85 90 95 Phe Ala Val Tyr Phe Cys Gln Gln Tyr Gly Thr Ser Pro Pro Thr Phe

    2018241075 03 Oct 2018

    124

    100

    Gly Gly Gly Thr Lys 115

    Val Phe Ile Phe Pro 130

    Ser Val Val Cys Pro 145

    Gln Trp Lys Val Asp 165

    Val Thr Glu Gln Asp 180

    Leu Thr Leu Ser Lys 195

    Glu Val Thr His Gln 210

    Arg Gly Glu Cys Lys 225

    Val Glu Ile 120

    Pro Ser Asp 135

    Leu Asn Asn 150

    Asn Ala Leu

    Asn Lys Asp

    Val Asp Tyr 200

    Gly Leu Ser 215

    Lys Glu Phe 230

    105 110

    Lys Arg Thr Val Ala Ala Pro Ser 125

    Glu Gln Leu Lys Ser Gly Thr Ala 140

    Phe Tyr Pro Arg Glu Ala Lys Val 155 160

    Gln Ser Gly Asn Ser Gln Glu Ser 170 175

    Ser Thr Tyr Ser Leu Ser Ser Thr 185 190

    Glu Lys His Glu Val Tyr Ala Cys 205

    Ser Pro Val Thr Lys Ser Phe Asn 220

    Val <210> 486 <211> 419 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic 3-23 VH nucleotide sequence <220>

    <221> CDS <222> (12)..(419) <400> 486 ctgtctgaac g gcc cag ccg gcc atg gcc gaa gtt caa ttg tta gag tet Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser

    15 10

    ggt Gly ggc Gly 15 ggt Gly ctt Leu gtt Val cag cct ggt Gly ggt Gly tet Ser tta Leu cgt Arg 25 ctt Leu tet Ser tgc Cys get Ala Gln Pro 20 get tcc gga ttc act ttc tet teg tac get atg tet tgg gtt ege caa Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln 30 35 40 45 get cct ggt aaa ggt ttg gag tgg gtt tet get ate tet ggt tet ggt Ala Pro Gly Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly 50 55 60

    146

    194

    2018241075 03 Oct 2018

    125 ggc agt act tac tat get gac tcc gtt aaa ggt cgc ttc act ate tet 242 Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser 65 70 75 aga gac aac tet aag aat act etc tac ttg cag atg aac age tta agg 290 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg 80 85 90 get gag gac act gca gtc tac tat tgc get aaa gac tat gaa ggt act 338 Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala Lys Asp Tyr Glu Gly Thr 95 100 105 ggt tat get ttc gac ata tgg ggt caa ggt act atg gtc acc gtc tet 386 Gly Tyr Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser 110 115 120 125 agt gcc tcc acc aag ggc cca teg gtc ttc ccc 419 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

    130 135 <21.0> 487 <211> 136 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence ·.: Synthetic 3-23 VP I protein i sequence <400> 487 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 1 5 10 15 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 20 25 30 Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly 35 40 45 Lys Gly Leu Glu Trp Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr 50 55 60 Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 65 70 75 80 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 85 90 95 Thr Ala Val Tyr Tyr Cys Ala Lys Asp Tyr Glu Gly Thr Gly Tyr Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser Ala Ser

    115 120 125

    Thr Lys Gly Pro Ser Val Phe Pro

    130 135

    2018241075 03 Oct 2018

    126 <210> 488 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 488 ctgtctgaac ggcccagccg 20 <210> 489 <211> 83 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 489 ctgtctgaac ggcccagccg gccatggccg aagttcaatt gttagagtct ggtggcggtc 60 ttgttcagcc tggtggttct tta 83 <210> 490 <211> 54 <212> DNA <213> Artificial Sequence <220> · <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 490 .

    gaaagtgaat ccggaagcag cgcaagaaag acgtaaagaa ccaccaggct gaac 54 <210> 491 <211> 42 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide ' <400> 491 agaaacccac tccaaacctt taccaggagc ttggcgaacc ca 42 <210> 492 <211> 94 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    127 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 492 agtgtcctca gcccttaagc tgttcatctg caagtagaga gtattcttag agttgtctct 60 agagatagtg aagcgacctt taacggagtc agca 94 <210> 493 <211> 81 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 493 gcttaagggc tgaggacact gcagtctact attgcgctaa agactatgaa ggtactggtt 60 atgctttcga catatggggt c 81 <210> 494 <211> 72 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 494 ggggaagacc gatgggccct tggtggaggc actagagacg gtgaccatag taccttgacc 60 tatgtcgaaa gc 72 <210> 495 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 495 ggggaagacc gatgggccct tgg 23 <210> 496 <211> 56 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018

    128 <220>

    <221> modified_base <222> (22).. ¢24) <223> A, T, C, G, other or unknown <220>

    <221> modified_base <222> (28)..(30) .

    <223> A, T, C, G, other or unknown <220>

    <221> modified_base <222> (34)..(36) <223> A, T, C, G, other or unknown <220>

    <223> nnn codes for any amino acid but Cys <400> 496 gcttccggat tcactttctc tnnntacnnn atgnnntggg ttcgccaagc tcctgg 56 <210> 497 <211> 68 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (19)..(21) <223> A, T, C or G <220>

    <221> modified_base <222> (25)..(30) <223> A, T, C or G <220>

    <221> modified_base <222> (40)..(42) <223> A, T, C or G <220>

    <221> modified_base · <222> (46)..(48) <223> A, T, C or G <400> 497 ggtttggagt gggtttctnn natcnnnnnn tctggtggcn nnactnnnta tgctgactcc 60 gttaaagg 68 <210> 498

    2018241075 03 Oct 2018

    129 <211> 912 <212> DNA <213> Escherichia coli <400> 498 tccggagctt cagatctgtt tgcctttttg tggggtggtg cagatcgcgt tacggagatc 60 gaccgactgc ttgagcaaaa gccacgctta actgctgatc aggcatggga tgttattcgc 120 caaaccagtc gtcaggatct taacctgagg ctttttttac ctactctgca agcagcgaca 180 tctggtttga cacagagcga tccgcgtcgt cagttggtag aaacattaac acgttgggat 240 ggcatcaatt tgcttaatga tgatggtaaa acctggcagc agccaggctc tgccatcctg 300 aacgtttggc tgaccagtat gttgaagcgt accgtagtgg ctgccgtacc tatgccattt 360 gataagtggt acagcgccag tggctacgaa acaacccagg acggcccaac tggttcgctg 420 aatataagtg ttggagcaaa aattttgtat gaggcggtgc agggagacaa atcaccaatc 480 ccacaggcgg ttgatctgtt tgctgggaaa ccacagcagg aggttgtgtt ggctgcgctg 540 gaagatacct gggagactct ttccaaacgc tatggcaata atgtgagtaa ctggaaaaca 600 cctgcaatgg ccttaacgtt ccgggcaaat aatttctttg gtgtaccgca ggccgcagcg 660 gaagaaacgc gtcatcaggc ggagtatcaa aaccgtggaa cagaaaacga tatgattgtt 720 ttctcaccaa cgacaagcga tcgtcctgtg cttgcctggg atgtggtcgc acccggtcag 780 agtgggttta ttgctcccga tggaacagtt gataagcact atgaagatca gctgaaaatg 840 tacgaaaatt ttggccgtaa gtcgctctgg ttaacgaagc aggatgtgga ggcgcataag 900 gagtcgtcta ga 912 <210> 499 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 499 gatnnnnatc 10 <210> 500 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(15) <223> A, T, C, G, other or unknown <400> 500 nnnnnnnnnn nnnnngtccc 20

    2018241075 03 Oct 2018

    130 <210> 501 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide .

    <220>

    <221> modified_base <222> (4).. (8) <223> A, T, C, G, other or unknown <400> 501 gcannnnntg c

    Synthetic <210> 502 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 502 gacnnnngtc

    Synthetic <210> 503 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide <220>

    <221> modified_base <222> (1)..(7) <223> A, T, C, G, other or unknown <400> 503 nnnnnnngcg gg

    Synthetic <210> 504 <211> 12 <212> DNA oo

    131

    2018241075 03 Oct 20 <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(12) <223> A, T, C, G, other or unknown <400> 504 gtatccnnnn nn 12 <210> 505 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown <400> 505 gcannnnnnt eg 12 <210> 506 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 506 gccnnnnngg c 11 <210> 507 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic

    2018241075 03 Oct 2018

    oligonucleotide <220> <221> modified base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 507 ggtctcnnnn n

    132

    <210> 508 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence oligonucleotide <220> <221> modified base <222> (4)..(11) <223> A, T, C, G, other or unknown <400> 508 gacnnnnngt c

    Synthetic

    <210> 509 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence oligonucleotide <220> <221> modified base <222> (4) . . (8) <223> A, T, C, G, other or unknown <400> 509 gacnnnnngt c

    Synthetic <210> 510 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide <220>

    <221> modified_base

    Synthetic

    2018241075 03 Oct 2018

    133

    <222> (4) . . (9) <223> A, T, C, G, other or unknown <400> 510 gacnnnnnng tc

    <210> 511 <211> 11 .

    <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 511 ccannnnntg g 11 <210> 512 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(9) <223> A, T, C, G, other or unknown <400> 512 nnnnnnnnng caggt ’ 15 <210> 513 <211> 11 <212> DNA <213> Artificial Sequence ' <220> · <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 513 acctgcnnnn n

    2018241075 03 Oct 2018

    134 <210> 514 <211> 13 <212> DNA <213> Artificial Sequence <220> .

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (5)..(9) <223> A, T, C, G, other or unknown <400> 514 ggccnnnnng gcc <210> 515 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: oligonucleotide

    Synthetic <220>

    <221> modified_base <222> (4)..(12) <223> A, T, C, G, other or unknown <400> 515 ccannnnnnn nntgg <210> 516 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> · <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 516 cgtctcnnnn n 11 <210> 517

    2018241075 03 Oct 2018

    135 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> .

    <221> modified_base <222> (1)..(6) <223> A, T, C, G, other or unknown <400> 517 nnnnnngaga eg 12 <210> 518 <211> 16 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(10) <223> A, T, C, G, other or unknown <400> 518 nnnnnnnnnn ctcctc 16 <210> 519 <211> 16 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(16) <223> A, T, C, G, other or unknown <400> 519 gaggagnnnn nnnnnn <210> 520 <211> 11 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    136 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4) .. (8) <223> A, T, C, G, other or unknown <400> 520 cctnnnnnag g <210> 521 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4) . . (9) <223> A, T, C, G, other or unknown <400> 521 ccannnnnnt gg <210> 522 <211> 6680 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Vector pCES5 nucleotide sequence <220>

    <221> CDS <222> (201) . . (1058) <220>

    <221> CDS <222> (2269)..(2682) <220>

    <221> CDS <222> (2723)..(2866) <220>

    <221> CDS <222> (3767)..(3850) <220>

    <221> CDS

    2018241075 03 Oct 2018

    137 <222> (4198) .. (5799) <400> 522 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60 cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120 tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180 aatattgaaa aaggaagagt atg agt att caa cat ttc cgt gtc gcc ctt att 233 Met Ser Ile Gln His Phe Arg Val Ala Leu Ile

    15 10 ccc ttt ttt gcg gca ttt tgc ctt cct gtt ttt get cac cca gaa aeg 281

    Pro Phe Phe Ala Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr

    15 20 25 ctg gtg aaa gta aaa gat get gaa gat cag ttg ggt gcc ega gtg ggt 329

    Leu Val Lys Val Lys Asp Ala Glu Asp Gln Leu Gly Ala Arg Val Gly

    30 35 40 tac ate gaa ctg gat etc aac age ggt aag ate ctt gag agt ttt ege 377

    Tyr Ile Glu Leu Asp Leu Asn Ser Gly Lys Ile Leu Glu Ser Phe Arg

    45 50 55 ccc gaa gaa cgt ttt cca atg atg age act ttt aaa gtt ctg eta tgt 425

    Pro Glu Glu Arg Phe Pro Met Met Ser Thr Phe Lys Val Leu Leu Cys

    60 65 70 75 ggc gcg gta tta tcc cgt att gac gcc ggg caa gag caa etc ggt ege 473

    Gly Ala Val Leu Ser Arg Ile Asp Ala Gly Gln Glu Gln Leu Gly Arg

    80 85 90 ege ata cac tat tet cag aat gac ttg gtt gag tac tea cca gtc aca 521

    Arg Ile His Tyr Ser Gln Asn Asp Leu Val Glu Tyr Ser Pro Val Thr

    95 100 105 gaa aag cat ctt aeg gat ggc atg aca gta aga gaa tta tgc agt get 569

    Glu Lys His Leu Thr Asp Gly Met Thr Val Arg Glu Leu Cys Ser Ala

    110 115 120 gcc ata acc atg agt gat aac act gcg gcc aac tta ctt ctg aca aeg 617

    Ala Ile Thr Met Ser Asp Asn Thr Ala Ala Asn Leu Leu Leu Thr Thr

    125 130 135 ate gga gga ccg aag gag eta acc get ttt ttg cac aac atg ggg gat 665

    Ile Gly Gly Pro Lys Glu Leu Thr Ala Phe Leu His Asn Met Gly Asp

    140 145 150 155 cat gta act ege ctt gat cgt tgg gaa ccg gag ctg aat gaa gcc ata 713

    His Val Thr Arg Leu Asp Arg Trp Glu Pro Glu Leu Asn Glu Ala Ile

    160 165 170 cca aac gac gag cgt gac acc aeg atg cct gta gca atg gca aca aeg 761

    Pro Asn Asp Glu Arg Asp Thr Thr Met Pro Val Ala Met Ala Thr Thr

    175 180 185 ttg ege aaa eta tta act ggc gaa eta ctt act eta get tcc egg caa 809

    2018241075 03 Oct 2018

    138

    Leu Arg Lys 190 Leu Leu Thr Gly Glu 195 Leu Leu Thr Leu Ala 200 Ser Arg Gln caa tta ata gac tgg atg gag gcg gat aaa gtt gca gga cca ett ctg 857 Gln Leu Ile Asp Trp Met Glu Ala Asp Lys Val Ala Gly Pro Leu Leu 205 210 215 cgc teg gcc ett ccg get ggc tgg ttt att get gat aaa tet gga gcc 905 Arg Ser Ala Leu Pro Ala Gly Trp Phe Ile Ala Asp Lys Ser Gly Ala 220 225 230 235 ggt gag cgt ggg tet cgc ggt ate att gca gca ctg ggg cca gat ggt 953 Gly Glu Arg Gly Ser Arg Gly lie lie Ala Ala Leu Gly Pro Asp Gly 240 245 250 aag ccc tcc cgt ate gta gtt ate tac aeg aeg ggg agt cag gca act 1001 Lys Pro Ser Arg Ile Val Val Ile Tyr Thr Thr Gly Ser Gln Ala Thr 255 260 265 atg gat gaa cga aat aga cag ate get gag ata ggt gcc tea ctg att 1049 Met Asp Glu Arg Asn Arg Gln Ile Ala Glu Ile Gly Ala Ser Leu Ile

    270 275 280 aag cat tgg taactgtcag accaagttta ctcatatata ctttagattg 1098

    Lys His Trp

    285

    atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatetea 1158 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga 1218 tcaaaggatc ttettgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa 1278 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga 1338 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tageegtagt 1398 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt 1458 taccagtggc tgctgccagt ggegataagt cgtgtcttac cgggttggac teaagaegat 1518 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcata cagcccagct 1578 tggagcgaac gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca 1638 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag 1698 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc 1758 gccacctctg aettgagegt cgatttttgt gatgetegte aggggggcgg agcctatgga 1818 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca 1878 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag 1938 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagegg 1998

    aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct 2058

    2018241075 03 Oct 2018

    139 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt 2118 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg 2178 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc 2238 tttggagcct tttttttgga gattttcaac gtg aaa aaa tta tta ttc gca att 2292 Met Lys Lys Leu Leu Phe Ala Ile

    290

    cct Pro 295 tta Leu gtt Val gtt Val cct Pro ttc Phe 300 tat Tyr tet Ser cac His agt Ser gca Ala 305 cag Gln gtc Val caa Gln ctg Leu cag Gln 310 2340 gtc gac etc gag ate aaa cgt gga act gtg get gca cca tet gtc ttc 2388 Val Asp Leu Glu Ile Lys Arg Gly Thr Val Ala Ala Pro Ser Val Phe 315 320 325 ate ttc ccg cca tet gat gag cag ttg aaa tet gga act gcc tet gtt 2436 Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val 330 335 340 gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc aaa gta cag tgg 2484 Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp 345 350 355 aag gtg gat aac gcc etc caa teg ggt aac tcc cag gag agt gtc aca 2532 Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr 360 365 370 gag cag gac age aag gac age acc tac age etc age age acc ctg aeg 2580 Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr 375 380 385 390 ctg age aaa gca gac tac gag aaa cac aaa gtc tac gcc tgc gaa gtc 2628 Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val 395 400 405 acc cat cag ggc ctg agt tea ccg gtg aca aag age ttc aac agg gga 2676 Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly 410 415 420

    gag tgt taataaggcg cgccaattct atttcaagga gacagtcata atg aaa tac 2731 Glu Cys Met Lys Tyr

    425

    eta Leu ttg Leu cct Pro 430 aeg Thr gca Ala gcc Ala get Ala gga Gly 435 ttg Leu tta Leu tta Leu etc Leu gcg Ala 440 gee Ala cag Gln ccg Pro 2779 gcc atg gcc gaa gtt caa ttg tta gag tet ggt ggc ggt ett gtt cag 2827 Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln 445 450 455 cct ggt ggt tet tta cgt ett tet tgc get get tec gga gcttcagatc 2876 Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 460 465 470

    2018241075 03 Oct 2018

    140

    tgtttgcctt tttgtggggt ggtgcagatc gegttaegga gatcgaccga ctgcttgagc 2936 aaaagccacg cttaactgct gatcaggcat gggatgttat tcgccaaacc agtegteagg 2996 atcttaacct gaggcttttt ttacctactc tgcaagcagc gacatctggt ttgacacaga 3056 gcgatccgcg tcgtcagttg gtagaaacat taacacgttg ggatggcatc aatttgctta 3116 atgatgatgg taaaacctgg cagcagccag gctctgccat cctgaacgtt tggctgacca 3176 gtatgttgaa gcgtaccgta gtggctgccg tacctatgcc atttgataag tggtacagcg 3236 ccagtggcta cgaaacaacc caggacggcc caactggttc getgaatata agtgttggag 3296 caaaaatttt gtatgaggcg gtgcagggag acaaatcacc aatcccacag gcggttgatc 3356 tgtttgctgg gaaaccacag caggaggttg tgttggctgc gctggaagat acctgggaga 3416 ctctttccaa aegetatgge aataatgtga gtaactggaa aacacctgca atggccttaa 3476 cgttccgggc aaataatttc tttggtgtac cgcaggccgc ageggaagaa acgcgtcatc 3536 aggeggagta tcaaaaccgt ggaacagaaa aegatatgat tgttttctca ccaacgacaa 3596 gcgatcgtcc tgtgcttgcc tgggatgtgg tcgcacccgg tcagagtggg tttattgctc 3656 ccgatggaac agttgataag cactatgaag atcagctgaa aatgtacgaa aattttggcc 3716 gtaagteget ctggttaacg aagcaggatg tggaggegea taaggagteg tct aga 3772 Ser Arg

    gac aac tct aag aat act etc tac ttg cag atg aac age tta agt ctg 3820 Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Ser Leu 475 480 485 490 age att egg tcc ggg caa cat tct cca aac tgaccagacg acacaaacgg 3870 Ser Ile Arg Ser Gly Gln His Ser Pro Asn 495 500

    cttacgctaa atcccgcgca tgggatggta aagaggtggc gtctttgctg gcctggactc 3930 atcagatgaa ggccaaaaat tggcaggagt ggacacagca ggcagcgaaa caagcactga 3990 ccatcaactg gtactatgct gatgtaaacg gcaatattgg ttatgttcat actggtgctt 4050 atccagatcg tcaatcaggc catgatccgc gattacccgt tcctggtacg ggaaaatggg 4110 actggaaagg gctattgcct tttgaaatga accctaaggt gtataacccc cagaagctag 4170 cctgcggctt cggtcaccgt ctcaagc gcc tcc acc aag ggc cca teg gtc ttc 4224 Ala Ser Thr Lys Gly Pro Ser Val Phe

    505

    4272 ccc ctg gca ccc tcc tcc aag age acc tct ggg ggc aca geg gcc ctg

    Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

    510 515 520 525

    75 03 Oct 2018 ο

    (N

    OO o

    CM

    ggc tgc ctg gtc aag gac tac ttc ccc Gly Cys Leu Val Lys 530 Asp Tyr Phe Pro aac tea ggc gee ctg acc age ggc gtc Asn Ser Gly Ala 545 Leu Thr Ser Gly Val 550 cag tec tea gga etc tac tec etc age Gln Ser Ser 560 Gly Leu Tyr Ser Leu 565 Ser age age ttg ggc acc cag acc tac ate Ser Ser 575 Leu Gly Thr Gln Thr 580 Tyr Ile age aac acc aag gtg gac aag aaa gtt Ser 590 Asn Thr Lys Val Asp 595 Lys Lys Val gca cat cat cat cac cat cac ggg gee Ala His His His His 610 His His Gly Ala gaa gag gat ctg aat ggg gee gca tag Glu Glu Asp Leu 625 Asn Gly Ala Ala aaa cct cat aca gaa aat tea ttt act Lys Pro His Thr 640 Glu Asn Ser Phe Thr 645 act tta gat cgt tac get aac tat gag Thr Leu Asp 655 Arg Tyr Ala Asn Tyr 660 Glu ggc gtt gtg gtt tgt act ggt gac gaa Gly Val 670 Val Val Cys Thr Gly 675 Asp Glu gtt cct att ggg ett get ate cct gaa Val 685 Pro Ile Gly Leu Ala 690 Ile Pro Glu ggt ggc ggt tet gag ggt ggc ggt tet Gly Gly Gly Ser Glu 705 Gly Gly Gly Ser cct gag tac ggt gat aca cct att ccg Pro Glu Tyr Gly 720 Asp Thr Pro Ile Pro 725 etc gac ggc act tat ccg cct ggt act Leu Asp Gly 735 Thr Tyr Pro Pro Gly 740 Thr

    141 gaa ccg gtg aeg gtg teg tgg 4320

    Glu Pro Val Thr Val Ser Trp

    535 540 cac acc ttc ccg get gtc eta 4368

    His Thr Phe Pro Ala Val Leu

    555 age gta gtg acc gtg ccc tec 4416

    Ser Val Val Thr Val Pro Ser

    570 tgc aac gtg aat cac aag ccc 4464

    Cys Asn Val Asn His Lys Pro

    585 gag ccc aaa tet tgt geg gee 4512

    Glu Pro Lys Ser Cys Ala Ala

    600 605 gca gaa caa aaa etc ate tea 4560

    Ala Glu Gln Lys Leu Ile Ser

    615 620 act gtt gaa agt tgt·tta gca 4608 Thr Val Glu Ser Cys Leu Ala 630 635 aac gtc tgg aaa gac gac aaa 4656 Asn Val Trp Lys Asp Asp Lys

    650 ggc tgt ctg tgg aat get aca 4704

    Gly Cys Leu Trp Asn Ala Thr

    665 act cag tgt tac ggt aca tgg 4752

    Thr Gln Cys Tyr Gly Thr Trp

    680 aat gag ggt ggt ggc tet gag 4800

    Asn Glu Gly Gly Gly Ser Glu

    695 700 gag ggt ggc ggt act aaa cct 4848

    Glu Gly Gly Gly Thr Lys Pro

    710 715 ggc tat act tat ate aac cct 4896

    Gly Tyr Thr Tyr Ile Asn Pro

    730 gag caa aac ccc get aat cct 4944

    Glu Gln Asn Pro Ala Asn Pro

    745 aat cct tet ett gag gag tet cag cct ett aat act ttc atg ttt cag 4992

    Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn Thr Phe Met Phe Gln

    2018241075 03 Oct 2018

    142

    750 755 760

    aat Asn 765 aat Asn agg Arg ttc Phe ega Arg aat Asn 770 agg Arg cag Gln ggt Gly gca Ala tta Leu 775 act Thr gtt Val tat Tyr aeg Thr ggc Gly 780 5040 act gtt act caa ggc act gac ccc gtt aaa act tat tac cag tac act 5088 Thr Val Thr Gln Gly Thr Asp Pro Val Lys Thr Tyr Tyr Gln Tyr Thr 785 790 795 cct gta tea tea aaa gee atg tat gac get tac tgg aac ggt aaa ttc 5136 Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe 800 805 810 aga gac tgc get ttc cat tet ggc ttt aat gag gat cca ttc gtt tgt 5184 Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu Asp Pro Phe Val Cys 815 820 825 gaa tat caa ggc caa teg tet gac ctg cct caa cct cct gtc aat get 5232 Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro Val Asn Ala 830 835 840 ggc ggc ggc tet ggt ggt ggt tet ggt ggc ggc tet gag ggt ggc ggc 5280 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly 845 850 855 860 tet gag ggt ggc ggt tet gag ggt ggc ggc tet gag ggt ggc ggt tcc 5328 Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser 865 870 875 ggt ggc ggc tcc ggt tcc ggt gat ttt gat tat gaa aaa atg gca aac 5376 Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn 880 885 890 get aat aag ggg get atg acc gaa aat gee gat gaa aac gcg eta cag 5424 Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln 895 900 905 tet gac get aaa ggc aaa ett gat tet gtc get act gat tac ggt get 5472 Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala 910 915 920 get ate gat ggt ttc att ggt gac gtt tcc ggc ett get aat ggt aat 5520 Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn 925 930 935 940 ggt get act ggt gat ttt get ggc tet aat tcc caa atg get caa gtc 5568 Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val 945 950 955 ggt gac ggt gat aat tea cct tta atg aat aat ttc cgt caa tat tta 5616 Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu 960 965 970 cct tet ttg cct cag teg gtt gaa tgt ege cct tat gtc ttt ggc get 5664 Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Tyr Val Phe Gly Ala 975 980 985

    2018241075 03 Oct 2018

    143 ggt aaa cca tat gaa ttt tet att gat tgt gac aaa ata aac tta ttc 5712 Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe 990 995 1000 cgt ggt gtc ttt geg ttt ctt tta tat gtt gcc acc ttt atg tat gta 5760 Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val 1005 1010 1015 1020 ttt teg aeg ttt get aac ata ctg cgt aat aag gag tet taataagaat 5809 Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser

    1025 1030

    tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat 5869 cgccttgcag cacatccccc tttcgccagc tggegtaata gegaagagge ccgcaccgat 5929 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc 5989 cttacgcatc tgtgcggtat ttcacaccgc atataaattg taaacgttaa tattttgtta 6049 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc egaaategge 6109 aaaatccctt ataaatcaaa agaatagccc gagatagggt tgagtgttgt tccagtttgg 6169 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat 6229 cagggcgatg gcccactacg tgaaccatca cccaaatcaa gttttttggg gtcgaggtgc 6289 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag 6349 ccggcgaacg tggcgagaaa ggaagggaag aaagegaaag gagegggege tagggcgctg 6409 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta 6469 cagggcgcgt actatggttg ctttgacggg tgeagtetea gtacaatctg ctctgatgcc 6529 gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt 6589 ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag 6649 aggttttcac cgtcatcacc gaaaegegeg a 6680

    <210> 523 <211> 286 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Vector pCES5 protein sequence <400> 523

    Met 1 Ser Ile Gln His 5 Phe Arg Val Ala Leu 10 Ile Pro Phe Phe Ala 15 Ala Phe Cys Leu Pro Val Phe Ala His Pro Glu Thr Leu Val Lys Val Lys

    20 25 30

    2018241075 03 Oct 2018

    Asp Ala Glu Asp Gln Leu Gly Ala 35 40

    Leu Asn Ser Gly Lys Ile Leu Glu 50 55

    Pro Met Met Ser Thr Phe Lys Val 65 70

    Arg Ile Asp Ala Gly Gln Glu Gln 85

    Gln Asn Asp Leu Val Glu Tyr Ser 100

    Asp Gly Met Thr Val Arg Glu Leu 115 120

    Asp Asn Thr Ala Ala Asn Leu Leu 130 135

    Glu Leu Thr Ala Phe Leu His Asn 145 150

    Asp Arg Trp Glu Pro Glu Leu Asn 165

    Asp Thr Thr Met Pro Val Ala Met 180

    Thr Gly Glu Leu Leu Thr Leu Ala 195 · 200

    Met Glu Ala Asp Lys Val Ala Gly 210 215

    Ala Gly Trp Phe Ile Ala Asp Lys 225 230

    Arg Gly Ile Ile Ala Ala Leu Gly 245

    Val Val Ile Tyr Thr Thr Gly Ser 260

    Arg Gln Ile Ala Glu Ile Gly Ala

    275 280 <210> 524 <211> 138 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial

    144

    Arg Val Gly Tyr Ile Glu Leu Asp 45

    Ser Phe Arg Pro Glu Glu Arg Phe 60

    Leu Leu Cys Gly Ala Val Leu Ser . 75 80

    Leu Gly Arg Arg Ile His Tyr Ser 90 95

    Pro Val Thr Glu Lys His Leu Thr 105 110

    Cys Ser Ala Ala Ile Thr Met Ser 125

    Leu Thr Thr Ile Gly Gly Pro Lys 140

    Met Gly Asp His Val Thr Arg Leu 155 160

    Glu Ala Ile Pro Asn Asp Glu Arg 170 175

    Ala Thr Thr Leu Arg Lys Leu Leu 185 190

    Ser Arg Gln Gln Leu Ile Asp Trp 205

    Pro Leu Leu Arg Ser Ala Leu Pro 220

    Ser Gly Ala Gly Glu Arg Gly Ser 235 240

    Pro Asp Gly Lys Pro Ser Arg Ile 250 255

    Gln Ala Thr Met Asp Glu Arg Asn 265 270

    Ser Leu Ile Lys His Trp 285

    Sequence: Vector pCES5 protein sequence

    2018241075 03 Oct 2018

    145 <400> 524

    Met 1 Lys Lys Leu Leu 5 Phe Ala Ile Pro Leu 10 Val Val Pro Phe Tyr 15 Ser His Ser Ala Gln Val Gln Leu Gln Val Asp Leu Glu Ile Lys Arg Gly 20 25 30 Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 35 40 45 Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr 50 55 60 Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser 65 70 75 80 Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr 85 90 95 Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys 100 105 110 His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 115 120 125 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 130 135

    <210> 525 <211> 48 <212> PRT <213> Artificial . Sequence <220> <223> Description of Artificial Sequence: Vector pCES5 protein sequence <400> 525 Met Lys Tyr Leu 1 Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 5 10 15 Ala Gln Pro Ala 20 Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 25 30 Leu Val Gln Pro 35 Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 40 45

    <210> 526 <211> 28 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Vector pCES5

    2018241075 03 Oct 2018

    146 protein sequence <400> 526

    Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu 15 10 15

    Ser Leu Ser Ile Arg Ser Gly Gln His Ser Pro Asn 20 25 <210> 527 <211> 533 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Vector pCES5 protein sequence <400> 527

    Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 15 10 15

    Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30

    Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45

    Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60

    Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

    65 70 75 80

    Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

    85 90 95

    Lys Val Glu Pro Lys Ser Cys Ala Ala Ala His His His His His His 100 105 110

    Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala 115 120 125

    Ala Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu Asn Ser Phe 130 135 140

    Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr

    145 150 155 160

    Glu Gly Cys Leu Trp Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp

    165 170 175

    Glu Thr Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro 180 185 190

    Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 195 200 205

    2018241075 03 Oct 2018

    147

    Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp Thr Pro Ile 210 215 220

    Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr Pro Pro Gly

    225 230 235 240

    Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu Glu Ser Gln

    245 250 255

    Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg Asn Arg Gln 260 265 270

    Gly Ala Leu Thr Val Tyr Thr Gly Thr Val Thr Gln Gly Thr Asp Pro 275 280 285

    Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys Ala Met Tyr 290 295 300

    Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe His Ser Gly

    305 310 315 320

    Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln Ser Ser Asp

    325 330 335

    Leu Pro Gln Pro Pro Val Asn Ala Gly Gly Gly Ser Gly Gly Gly Ser 340 345 350

    Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly 355 360 365

    Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp 370 · 375 380

    Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu

    385 390 395 400

    Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp

    405 410 415

    Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp 420 425 430

    Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly 435 440 445

    Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu 450 455 460

    Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu

    465 470 475 480

    Cys Arg Pro Tyr Val Phe Gly Ala Gly Lys Pro Tyr Glu Phe Ser Ile

    485 490 495

    Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu 500 505 510

    2018241075 03 Oct 2018

    148

    Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu 515 520 525

    Arg Asn Lys Glu Ser 530

    <210> 528 <211> 30 . <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 528 acctcactgg cttccggatt cactttctct

    <210> 529 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 529 agaaacccac tccaaacctt taccaggagc ttggcgaacc ca

    <210> 530 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 530

    ggaaggcagt gatctagaga tagtgaagcg acctttaacg gagtcagcat a 51 <210> 531 <211> 23 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 531 ggaaggcagt gatctagaga tag

    2018241075 03 Oct 2018

    149 <210> 532 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 532 gtgctgactc agccaccctc 20 <210> 533 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 533 gccctgactc agcctgcctc 20

    <210> 534 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 534 gagctgactc aggaccctgc 20 <210> 535 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Synthetic

    oligonucleotide <400> 535 gagctgactc agccaccctc 20 <210> 536 <211> 38 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    150 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 536 cctcgacagc gaagtgcaca gagcgtcttg actcagcc 38 <210> 537 .

    <211> 30 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 537 cctcgacagc gaagtgcaca gagcgtcttg 30 <210> 538 <211> 38 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 538 cctcgacagc gaagtgcaca gagcgctttg actcagcc 38 <210> 539 <211> 30 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 539 cctcgacagc gaagtgcaca gagcgctttg 30 <210> 540 <211> 38 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 540 cctcgacagc taagtgcaca gagcgctttg actcagcc 38

    2018241075 03 Oct 2018

    151

    <210> 541 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 541 cctcgacagc gaagtgcaca gagcgctttg

    <210> 542 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 542 cctcgacagc gaagtgcaca gagcgaattg actcagcc

    <210> 543 <211> 30 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 543 cctcgacagc gaagtgcaca gagcgaattg

    <210> 544 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 544

    cctcgacagc gaagtgcaca gtacgaattg actcagcc <210> 545 <211> 30 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    152 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 545 cctcgacagc gaagtgcaca gtacgaattg 30 <210> 546 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 546 cctcgacagc gaagtgcaca g 21 <210> 547 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 547 ccgtgtatta ctgtgcgaga g 21 <210> 548 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 548 ctgtgtatta ctgtgcgaga g 21 <400> 549 <210> 549 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide

    2018241075 03 Oct 2018

    153 ccgtatatta ctgtgcgaaa g <210> 550 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 550 ctgtgtatta ctgtgcgaaa g <210> 551 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 551 ctgtgtatta ctgtgcgaga c <210> 552 <211> 21 <212> DNA <213> Artificial Sequence

    <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 552 ccatgtatta ctgtgcgaga c 21 <210> <211> <212> <213> 553 94 DNA Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic <400> 553

    <210> 554 <211> 94 ggtgtagtga tctagtgaca actctaagaa tactctctac ttgcagatga acagctttag 60 ggctgaggac actgcagtct actattgtgc gaga 94

    2018241075 03 Oct 2018

    154 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 554 ggtgtagtga tctagtgaca actctaagaa tactctctac ttgcagatga acagctttag 60 ggctgaggac actgcagtct actattgtgc gaaa 94 <210> 555 <211> 85 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 555 atagtagact gcagtgtcct cagcccttaa gctgttcatc tgcaagtaga gagtattctt 60

    agagttgtct ctagatcact acacc 85 <210> 556 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Primer <400> 556 gactgggtgt agtgatctag 20 <210> 557 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence : Primer <400> 557 cttttctttg ttgccgttgg ggtg 24 <210> 558 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence : Synthetic

    2018241075 03 Oct 2018 <220>

    <221> modified_base <222> (1)..(9) <223> A, T, C, G, other or unknown <400> 558 nnnnnnnnng caggt

    155 <210> 559 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)., (11) <223> A, T, C, G, other or unknown <400> 559 acctgcnnnn n <210> 560 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 560 gatnnnnatc <210> 561 <211> 16 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(16)

    2018241075 03 Oct 2018 <223> A, T, C, G, other or unknown <400> 561 gaggagnnnn nnnnnn

    156 <210> 562 <211> 16 <212> DNA .

    <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(10) <223> A, T, C, G, other or unknown <400> 562 nnnnnnnnnn ctcctc <210> 563 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)., (10) <223> A, T, C, G, other or unknown <400> 563 ctcttcnnnn <210> 564 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(5) <223> A, T, C, G, other or unknown <400> 564 nnnnngaaga g

    2018241075 03 Oct 2018

    157 <210> 565 <211> 20 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (1)..(15) <223> A, T, C, G, other or unknown <400> 565 nnnnnnnnnn nnnnngtccc 20 <210> 566 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, G, G, other or unknown <400> 566 gacnnnnnng tc 12 <210> 567 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 567 cgtctcnnnn n 11 <210> 568 <211> 12

    2018241075 03 Oct 2018

    158 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7) .. (12) <223> A, T, C, G, other or unknown <400> 568 gtatccnnnn nn 12 <210> 569 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4) .. (9) <223> A, T, C, G, other or unknown <400> 569 gcannnnnnt eg 12 <210> 570 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4) .. (8) <223> A, T, C, G, other or unknown <400> 570 gccnnnnngg c 11 <210> 571 <211> 11 <212> DNA <213> Artificial Sequence <220>

    2018241075 03 Oct 2018

    159 <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(11) <223> A, T, C, G, other or unknown <400> 571 .

    ggtctcnnnn n <210> 572 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 572 gacnnnnngt c <210> 573 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 573 gacnnnnngt c <210> 574 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    2018241075 03 Oct 2018 <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 574 ccannnnntg g

    160 <210> 575 .

    <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(12) <223> A, T, C, G, other or unknown <400> 575 ccannnnnnn nntgg <210> 576 <211> 13 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (5)..(9) <223> A, T, C, G, other or unknown <400> 576 ggccnnnnng gcc <210> 577 <211> 12 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(9) <223> A, T, C, G, other or unknown

    2018241075 03 Oct 2018

    161

    <400> 577 ccannnnnnt gg <210> 578 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic

    oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 578 cctnnnnnag g 11 <210> 579 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> ’ <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 579 gacnnnngtc 10 <210> 580 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4) .. (12) <223> A, T, C, G, other or unknown <400> 580 ccannnnnnn nntgg 15

    2018241075 03 Oct 2018

    162 <210> 581 <211> 11 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(8) <223> A, T, C, G, other or unknown <400> 581 gcannnnntg c 11 <210> 582 <211> 10251 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: CJRA05 nucleotide sequence <220>

    <221> CDS <222> (1578)..(1916) <220>

    <221> CDS <222> (2388) .. (2843) <220>

    <221> CDS <222> (2849) . . (2893) <220>

    <221> CDS <222> (3189) . . (4232) <220>

    <221> CDS <222> (7418) .. (8119) <220>

    <221> CDS · <222> (8160) .. (9452) <400> 582 aatgctacta ctattagtag aattgatgcc accttttcag ctcgcgcccc aaatgaaaat 60 atagctaaac aggttattga ccatttgcga aatgtatcta atggtcaaac taaatctact 120 cgttcgcaga attgggaatc aactgttata tggaatgaaa cttccagaca ccgtacttta 180

    2018241075 03 Oct 2018

    163 gttgcatatt taaaacatgt tgagctacag cattatattc agcaattaag ctctaagcca 240 tccgcaaaaa tgacctctta tcaaaaggag caattaaagg tactctctaa tcctgacctg 300 ttggagtttg cttccggtct ggttcgcttt gaagctcgaa ttaaaacgcg atatttgaag 360 tctttcgggc ttcctcttaa tctttttgat gcaatccgct ttgcttctga ctataatagt 420 cagggtaaag acctgatttt tgatttatgg tcattctcgt tttctgaact gtttaaagca 480 tttgaggggg attcaatgaa tatttatgac gattccgcag tattggacgc tatccagtct 540 aaacatttta ctattacccc ctctggcaaa acttcttttg caaaagcctc tcgctatttt 600 ggtttttatc gtcgtctggt aaacgagggt tatgatagtg ttgctcttac tatgcctcgt 660 aattcctttt ggcgttatgt atctgcatta gttgaatgtg gtattcctaa atctcaactg 720 atgaatcttt ctacctgtaa taatgttgtt ccgttagttc gttttattaa cgtagatttt 780 tcttcccaac gtcctgactg gtataatgag ccagttctta aaatcgcata aggtaattca 840 caatgattaa agttgaaatt aaaccatctc aagcccaatt tactactcgt tctggtgttt 900 ctcgtcaggg caagccttat tcactgaatg agcagctttg ttacgttgat ttgggtaatg 960 aatatccggt tcttgtcaag attactcttg atgaaggtca gccagcctat gcgcctggtc 1020 tgtacaccgt tcatctgtcc tctttcaaag ttggtcagtt cggttccctt atgattgacc 1080 gtctgcgcct cgttccggct aagtaacatg gagcaggtcg cggatttcga cacaatttat 1140 caggcgatga tacaaatctc cgttgtactt tgtttcgcgc ttggtataat cgctgggggt 1200 caaagatgag tgttttagtg tattcttttg cctctttcgt tttaggttgg tgccttcgta 1260 gtggcattac gtattttacc cgtttaatgg aaacttcctc atgaaaaagt ctttagtcct 1320 caaagcctct gtagccgttg ctaccctcgt tccgatgctg tctttcgctg ctgagggtga 1380 cgatcccgca aaagcggcct ttaactccct gcaagcctca gcgaccgaat atatcggtta 1440 tgcgtgggcg atggttgttg tcattgtcgg cgcaactatc ggtatcaagc tgtttaagaa 1500 attcacctcg aaagcaagct gataaaccga tacaattaaa ggctcctttt ggagcctttt 1560

    ttttggagat tttcaac gtg aaa aaa tta tta ttc gca att cct tta gtt 1610

    Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val 15 10

    gtt Val cct Pro ttc Phe tat Tyr 15 tet Ser ggc Gly gcg Ala gcc Ala gaa Glu 20 tea Ser cat His eta Leu gac Asp ggc Gly 25 gcc Ala get Ala 1658 gaa act gtt gaa agt tgt tta gca aaa tcc cat aca gaa aat tea ttt 1706 Glu Thr Val Glu Ser Cys Leu Ala Lys Ser His Thr Glu Asn Ser Phe

    30 35 40

    2018241075 03 Oct 2018

    164

    act Thr aac gtc tgg aaa gac gac aaa act tta gat Leu Asp cgt Arg 55 tac get aac Asn tat Tyr 1754 Asn 45 Val Trp Lys Asp Asp 50 Lys Thr Tyr Ala gag ggc tgt ctg tgg aat get aca ggc gtt gta gtt tgt act ggt gac 1802 Glu Gly Cys Leu Trp Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp 60 65 70 75 gaa act cag tgt tac ggt aca tgg gtt cct att ggg ett get ate cct 1850 Glu Thr Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro 80 85 90 gaa aat gag ggt ggt ggc tct gag ggt ggc ggt tct gag ggt ggc ggt 1898 Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 95 100 105 tct gag ggt ggc ggt act aaacctcctg agtacggtga tacacctatt 1946 Ser Glu Gly Gly Gly Thr 110 ccgggctata cttatatcaa ccctctcgac : ggcacttatc cgcctggtac tgagcaaaac 2006 cccgctaatc ctaatccttc tettgaggag 1 tctcagcctc ttaatacttt catgtttcag 2066 aataataggt teegaaatag gcagggggca i ttaactgttt atacgggcac tgttactcaa 2126 ggcactgacc ccgttaaaac ttattaccag 1 tacactcctg tatcatcaaa agccatgtat 2186 gacgcttact ggaacggtaa attcagagac : tgcgctttcc attctggctt taatgaggat 2246 ttatttgttt gtgaatatca aggccaatcg [ tctgacctgc ctcaacctcc tgteaatget 2306 ggcggcggct ctggtggtgg ttctggtggc : ggctctgagg gtggtggctc tgagggaggc 2366 ggttccggtg gtggctctgg t tee ggt gat ttt gat tat gaa aag atg gca 2417 Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala 115 120 aac get aat aag ggg get atg acc gaa aat gee gat gaa aac geg eta 2465 Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu 125 130 135 cag tct gac get aaa ggc aaa ett gat tct gtc get act gat tac ggt 2513 Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly 140 145 150 155 get get ate gat ggt ttc att ggt gac gtt tee ggc ett get aat ggt 2561 Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn Gly 160 165 170 aat ggt get act ggt gat ttt get ggc tct aat tee caa atg get caa 2609 Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala Gln 175 180 185 gtc ggt gac ggt gat aat tea cct tta atg aat aat ttc cgt caa tat 2657 Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr 190 195 200

    2018241075 03 Oct 2018

    165 tta cct tcc etc cct caa teg gtt gaa tgt ege cct ttt gtc ttt ggc 2705

    Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe Gly

    205 210 215 get ggt aaa cca tat gaa ttt tet att gat tgt gac aaa ata aac tta 2753

    Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu

    220 225 230 235 ttc cgt ggt gtc ttt gcg ttt ctt tta tat gtt gcc acc ttt atg tat 2801

    Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr

    240 245 250 gta ttt tet aeg ttt get aac ata ctg cgt aat aag gag tet taatc atg 2851

    Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser Met

    255 260 265 cca gtt ctt ttg ggt att ccg tta tta ttg cgt ttc etc ggt 2893

    Pro Val Leu Leu Gly Ile Pro Leu Leu Leu Arg Phe Leu Gly

    270 275 280

    ttccttctgg taactttgtt eggetatetg cttacttttc ttaaaaaggg etteggtaag 2953 atagetattg ctatttcatt gtttcttgct ettattattg ggcttaactc aattcttgtg 3013 ggttatctct ctgatattag cgctcaatta ccctctgact ttgttcaggg tgttcagtta 3073 attctcccgt etaatgeget tccctgtttt tatgttattc tctctgtaaa ggctgctatt 3133 ttcatttttg acgttaaaca aaaaatcgtt tcttatttgg attgggataa ataat atg 3191

    Met

    get Ala gtt Val tat Tyr ttt Phe 285 gta Val act Thr ggc Gly aaa Lys tta Leu 290 ggc Gly tet Ser gga Gly aag Lys aeg Thr 295 etc Leu gtt Val 3239 age gtt ggt aag att cag gat aaa att gta get ggg tgc aaa ata gca 3287 Ser Val Gly Lys Ile Gln Asp Lys Ile Val Ala Gly Cys Lys Ile Ala 300 305 310 act aat ctt gat tta agg ctt caa aac etc ccg caa gtc ggg agg ttc 3335 Thr Asn Leu Asp Leu Arg Leu Gln Asn Leu Pro Gln Val Gly Arg Phe 315 320 325 get aaa aeg cct ege gtt ctt aga ata ccg gat aag cct tet ata tet 3383 Ala Lys Thr Pro Arg Val Leu Arg Ile Pro Asp Lys Pro Ser Ile Ser 330 335 340 345 gat ttg ctt get att ggg ege ggt aat gat tcc tac gat gaa aat aaa 3431 Asp Leu Leu Ala Ile Gly Arg Gly Asn Asp Ser Tyr Asp Glu Asn Lys 350 355 360 aac ggc ttg ctt gtt etc gat gag tgc ggt act tgg ttt aat acc cgt 3479 Asn Gly Leu Leu Val Leu Asp Glu Cys Gly Thr Trp Phe Asn Thr Arg 365 370 375 tet tgg aat gat aag gaa aga cag ccg att att gat tgg ttt eta cat 3527 Ser Trp Asn Asp Lys Glu Arg Gln Pro Ile Ile Asp Trp Phe Leu His 380 385 390

    2018241075 03 Oct 2018

    166

    get Ala cgt Arg 395 aaa Lys tta Leu gga Gly tgg Trp gat Asp 400 att Ile att Ile ttt Phe ett Leu gtt Val 405 cag Gln gac Asp tta Leu tet Ser 3575 att gtt gat aaa cag geg cgt tet gca tta get gaa cat gtt gtt tat 3623 Ile Val Asp Lys Gln Ala Arg Ser Ala Leu Ala Glu His Val Val Tyr 410 415 420 425 tgt cgt cgt ctg gac aga att act tta cct ttt gtc ggt act tta tat 3671 Cys Arg Arg Leu Asp Arg Ile Thr Leu Pro Phe Val Gly Thr Leu Tyr 430 435 440 tet ett att act ggc teg aaa atg cct ctg cct aaa tta cat gtt ggc 3719 Ser Leu Ile Thr Gly Ser Lys Met Pro Leu Pro Lys Leu His Val Gly 445 450 455 gtt gtt aaa tat ggc gat tet caa tta age cct act gtt gag cgt tgg 3767 Val Val Lys Tyr Gly Asp Ser Gln Leu Ser Pro Thr Val Glu Arg Trp 460 465 470 ett tat act ggt aag aat ttg tat aac gca tat gat act aaa cag get 3815 Leu Tyr Thr Gly Lys Asn Leu Tyr Asn Ala Tyr Asp Thr Lys Gln Ala 475 480 485 ttt tet agt aat tat gat tcc ggt gtt tat tet tat tta aeg cct tat 3863 Phe Ser Ser Asn Tyr Asp Ser Gly Val Tyr Ser Tyr Leu Thr Pro Tyr 490 495 500 505 tta tea cac ggt egg tat ttc aaa cca tta aat tta ggt cag aag atg 3911 Leu Ser His Gly Arg Tyr Phe Lys Pro Leu Asn Leu Gly Gln Lys Met 510 515 520 aaa tta act aaa ata tat ttg aaa aag ttt tet cgc gtt ett tgt ett 3959 Lys Leu Thr Lys Ile Tyr Leu Lys Lys Phe Ser Arg Val Leu Cys Leu 525 530 535 geg att gga ttt gca tea gca ttt aca tat agt tat ata acc caa cct 4007 Ala Ile Gly Phe Ala Ser Ala Phe Thr Tyr Ser Tyr Ile Thr Gln Pro 540 545 550 aag ccg gag gtt aaa aag gta gtc tet cag acc tat gat ttt gat aaa 4055 Lys Pro Glu Val Lys Lys Val Val Ser Gln Thr Tyr Asp Phe Asp Lys 555 560 565 ttc act att gac tet tet cag cgt ett aat eta age tat cgc tat gtt 4103 Phe Thr Ile Asp Ser Ser Gln Arg Leu Asn Leu Ser Tyr Arg Tyr Val 570 575 580 585 ttc aag gat tet aag gga aaa tta att aat age gac gat tta cag aag 4151 Phe Lys Asp Ser Lys Gly Lys Leu lie Asn Ser Asp Asp Leu Gln Lys 590 595 600 caa ggt tat tea etc aca tat att gat tta tgt act gtt tcc att aaa 4199 Gln Gly Tyr Ser Leu Thr Tyr Ile Asp Leu Cys Thr Val Ser Ile Lys 605 610 615

    aaa ggt aat tea aat gaa att gtt aaa tgt aat taattttgtt ttcttgatgt 4252

    2018241075 03 Oct 2018

    167

    Lys Gly Asn Ser Asn Glu Ile Val Lys Cys Asn 620 625

    ttgtttcatc atcttctttt gctcaggtaa ttgaaatgaa taattcgcct ctgcgcgatt 4312 ttgtaacttg gtattcaaag caatcaggcg aatccgttat tgtttctccc gatgtaaaag 4372 gtactgttac tgtatattca tctgacgtta aacctgaaaa tctacgcaat ttctttattt 4432 ctgttttacg tgcaaataat tttgatatgg taggttctaa cccttccatt attcagaagt 4492 ataatccaaa caatcaggat tatattgatg aattgccatc atctgataat caggaatatg 4552 atgataattc cgctccttct ggtggtttct ttgttccgca aaatgataat gttactcaaa 4612 cttttaaaat taataacgtt cgggcaaagg atttaatacg agttgtcgaa ttgtttgtaa 4672 agtctaatac ttctaaatcc tcaaatgtat tatctattga cggctctaat ctattagttg 4732 ttagtgctcc taaagatatt ttagataacc ttcctcaatt cctttcaact gttgatttgc 4792 caactgacca gatattgatt gagggtttga tatttgaggt tcagcaaggt gatgctttag 4852 atttttcatt tgctgctggc tctcagcgtg gcactgttgc aggcggtgtt aatactgacc 4912 gcctcacctc tgttttatct tctgctggtg gttcgttcgg tatttttaat ggcgatgttt 4972 tagggctatc agttcgcgca ttaaagacta atagccattc aaaaatattg tctgtgccac 5032 gtattcttac gctttcaggt cagaagggtt ctatctctgt tggccagaat gtccctttta 5092 ttactggtcg tgtgactggt gaatctgcca atgtaaataa tccatttcag acgattgagc 5152 gtcaaaatgt aggtatttcc atgagcgttt ttcctgttgc aatggctggc ggtaatattg 5212 ttctggatat taccagcaag gccgatagtt tgagttcttc tactcaggca agtgatgtta 5272 ttactaatca aagaagtatt gctacaacgg ttaatttgcg tgatggacag actcttttac 5332 tcggtggcct cactgattat aaaaacactt ctcaggattc tggcgtaccg ttcctgtcta 5392 aaatcccttt aatcggcctc ctgtttagct cccgctctga ttctaacgag gaaagcacgt 5452 tatacgtgct cgtcaaagca accatagtac gcgccctgta gcggcgcatt aagcgcggcg 5512 ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc gcccgctcct 5572 ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca agctctaaat 5632 cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc caaaaaactt 5692 gatttgggtg atggttcacg tagtgggcca tcgccctgat agacggtttt tcgccctttg 5752 acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac 5812 cctatctcgg gctattcttt tgatttataa gggattttgc cgatttcgga accaccatca 5872 aacaggattt tcgcctgctg gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg 5932

    2018241075 03 Oct 2018

    168

    gccaggcggt gaagggcaat cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc 5992 tggatccaag cttgcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 6052 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatget 6112 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 6172 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 6232 agatgctgaa gatcagttgg gcgcactagt gggttacatc gaactggatc tcaacagcgg 6292 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 6352 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 6412 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 6472 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 6532 ggccaactta cttctgacaa egateggagg accgaaggag ctaaccgctt ttttgcacaa 6592 catgggggat catgtaactc geettgateg ttgggaaccg gagctgaatg aagccatacc 6652 aaaegaegag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 6712 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggegga 6772 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 6832 atctggagcc ggtgagcgtg ggtetegegg tatcattgca gcactggggc cagatggtaa 6892 gccctcccgt ategtagtta tctacacgac ggggagtcag gcaactatgg atgaaegaaa 6952 tagacagatc getgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 7012 ttactcatat ataetttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 7072 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 7132 tacgtaagac ccccaagctt gtcgactgaa tggcgaatgg cgctttgcct ggtttccggc 7192 accagaagcg gtgccggaaa gctggctgga gtgegatett cctgacgctc gagcgcaacg 7252 caattaatgt gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg 7312 ctcgtatgtt gtgtggaatt gtgageggat aacaatttca cacaggaaac agctatgacc 7372 atgattaege caagctttgg agcctttttt ttggagattt tcaac gtg , Met : aaa aaa tta Lys Lys Leu 7429

    630

    tta ttc gca att cct tta gtt gtt cct ttc tat tet cac agt gca caa 7477 Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser His Ser Ala Gln 635 640 645

    gac ate cag atg acc cag tet cca gcc acc ctg tet ttg tet cca ggg 7525

    7573

    2018241075 03 Oct 2018

    169

    Asp Ile 650 Gln Met Thr Gln Ser 655 Pro Ala Thr Leu Ser 660 Leu Ser Pro Gly gaa aga gcc acc etc tcc tgc agg gcc agt cag ggt gtt age age tac Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly Val Ser Ser Tyr 665 670 675 680 tta gcc tgg tac cag cag aaa cct ggc cag get ccc agg etc etc ate Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 685 690 695 tat gat gca tcc aac agg gcc act ggc ate cca gcc agg ttc agt ggc Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 700 705 710 agt ggg cct ggg aca gac ttc act etc acc ate age age eta gag cct Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 715 720 725 gaa gat ttt gca gtt tat tac tgt cag cag cgt aac tgg cat ccg tgg Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn Trp His Pro Trp 730 735 740 aeg ttc ggc caa ggg acc aag gtg gaa ate aaa cga act gtg get gca Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala 745 750 755 760 cca tet gtc ttc ate ttc ccg cca tet gat gag cag ttg aaa tet gga Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly 765 770 775 act gcc tet gtt gtg tgc ctg ctg aat aac ttc tat ccc aga gag gcc Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala 780 785 790 aaa gta cag tgg aag gtg gat aac gcc etc caa teg ggt aac tcc cag Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 795 800 805 gag agt gtc aca gag egg gac age aag gac age acc tac age etc age Glu Ser Val Thr Glu Arg Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 810 815 820 age acc ctg aeg ctg age aaa gca gac tac gag aaa cac aaa gtc tac Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 825 830 835 840 gcc tgc gaa gtc acc cat cag ggc ctg age teg ccc gtc aca aag age Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser 845 850 855

    7621

    7669

    7717

    7765

    7813

    7861

    7909

    7957

    8005

    8053

    8101

    8149

    8198 ttc aac agg gga gag tgt taataaggcg cgccaattct atttcaagga Phe Asn Arg Gly Glu Cys

    860 gacagtcata atg aaa tac eta ttg cct aeg gca gcc get gga ttg tta

    Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu

    865 870 875

    2018241075 03 Oct 2018

    170

    tta Leu etc Leu geg Ala gcc Ala cag Gln 880 ccg Pro gcc Ala atg Met gee Ala gaa Glu 885 gtt Val caa Gln ttg Leu tta Leu gag Glu 890 tct Ser 8246 ggt ggc ggt ett gtt cag cct ggt ggt tct tta cgt ett tct tgc get 8294 Gly Gly Gly Leu 895 Val Gln Pro Gly Gly 900 Ser Leu Arg Leu Ser 905 Cys Ala get tcc gga ttc act ttc tct act tac gag atg cgt tgg gtt ege caa 8342 Ala Ser Gly 910 Phe Thr Phe Ser Thr 915 Tyr Glu Met Arg Trp 920 Val Arg Gln get cct ggt aaa ggt ttg gag tgg gtt tct tat ate get cct tct ggt 8390 Ala Pro 925 Gly Lys Gly Leu Glu 930 Trp Val Ser Tyr Ile 935 Ala Pro Ser Gly ggc gat act get tat get gac tcc gtt aaa ggt ege ttc act ate tct 8438 Gly 940 Asp Thr Ala Tyr Ala 945 Asp Ser Val Lys Gly 950 Arg Phe Thr Ile Ser 955 aga gac aac tct aag aat act etc tac ttg cag atg aac age tta agg 8486 Arg Asp Asn Ser Lys 960 Asn Thr Leu Tyr Leu 965 Gln Met Asn Ser Leu 970 Arg get gag gac act gca gtc tac tat tgt geg agg agg etc gat ggc tat 8534 Ala Glu Asp Thr 975 Ala Val Tyr Tyr Cys 980 Ala Arg Arg Leu Asp 985 Gly Tyr att tcc tac tac tac ggt atg gac gtc tgg ggc caa ggg acc aeg gtc 8582 Ile Ser Tyr 990 Tyr Tyr Gly Met Asp 995 Val Trp Gly Gln Gly 1000 Thr Thr Val acc gtc tea age gcc tcc acc aag ggc cca teg gtc ttc CCC ctg gca 8630 Thr Val 1005 Ser Ser Ala Ser Thr 1010 Lys Gly Pro Ser Val 1015 Phe Pro Leu Ala CCC tcc tcc aag age acc tct ggg ggc aca geg gcc ctg ggc tgc ctg 8678 Pro Ser 1020 Ser Lys Ser Thr 1025 Ser Gly Gly Thr Ala 1030 Ala Leu Gly Cys Leu 1035 gtc aag gac tac ttc CCC gaa ccg gtg aeg gtg teg tgg aac tea ggc 8726 Val Lys Asp Tyr Phe 1040 Pro Glu Pro Val Thr 1045 Val Ser Trp Asn Ser 1050 Gly gcc ctg acc age ggc gtc cac acc ttc ccg get gtc eta cag tcc tea 8774 Ala Leu Thr Ser 1055 Gly Val His Thr Phe 1060 Pro Ala Val Leu Gln 1065 Ser Ser gga etc tac tcc etc age age gta gtg acc gtg CCC tcc age age ttg 8822 Gly Leu Tyr 1070 Ser Leu Ser Ser Val 1075 Val Thr Val Pro Ser 1080 Ser Ser Leu ggc acc cag acc tac ate tgc aac gtg aat cac aag CCC age aac acc 8870 Gly Thr 1085 Gln Thr Tyr Ile Cys 1090 Asn Val Asn His Lys 1095 Pro Ser Asn Thr aag gtg gac aag aaa gtt gag CCC aaa tct tgt geg gcc gca cat cat 8918

    2018241075 03 Oct 2018

    171

    Lys Val 1100 Asp Lys Lys Val 1105 Glu Pro Lys Ser Cys 1110 Ala Ala Ala His His 1115 cat cac cat cac ggg gcc gca gaa caa aaa etc ate tea gaa gag gat 8966 His His His His Gly 1120 Ala Ala Glu Gln Lys 1125 Leu Ile Ser Glu Glu 1130 Asp ctg aat ggg gcc gca tag get age tet get wsy ggy gay tty gay tay 9014 Leu Asn Gly Ala 1135 Ala Gln Ala Ser Ser 1140 Ala Ser Gly Asp Phe 1145 Asp Tyr gar aar atg get aaw gey aay aar ggs gey atg acy gar aay gey gay 9062 Glu Lys Met 1150 Ala Asn Ala Asn Lys 1155 Gly Ala Met Thr Glu 1160 Asn Ala Asp gar aay gck ytr car wsy gay gey aar ggy aar ytw gay wsy gtc gck 9110 Glu Asn 1165 Ala Leu Gln Ser Asp 1170 Ala Lys Gly Lys Leu 1175 Asp Ser Val Ala acy gay tay ggy gey gcc ate gay ggy tty aty ggy gay gtc wsy ggy 9158 Thr Asp 1180 Tyr Gly Ala Ala Ile 1185 Asp Gly Phe Ile Gly 1190 Asp Val Ser Gly 1195 ytk gey aay ggy aay ggy gey acy ggw gay tty gew ggy tek aat tcy 9206 Leu Ala Asn Gly Asn 1200 Gly Ala Thr Gly Asp 1205 Phe Ala Gly Ser Asn 1210 Ser car atg gey car gty ggw gay ggk gay aay wsw cck ytw atg aay aay 9254 Gln Met Ala Gln 1215 Val Gly Asp Gly Asp 1220 Asn Ser Pro Leu Met 1225 Asn Asn tty mgw car tay ytw cck tcy cty cck car wsk gty gar tgy egy ccw 9302 Phe Arg Gln 1230 Tyr Leu Pro Ser Leu 1235 Pro Gln Ser Val Glu 1240 Cys Arg Pro tty gty tty wsy gey ggy aar ccw tay gar tty wsy aty gay tgy gay 9350 Phe Val 1245 Phe Ser Ala Gly Lys 1250 Pro Tyr Glu Phe Ser 1255 Ile Asp Cys Asp aar atm aay ytw tty egy ggy gty tty gck tty ytk yta tay gty gey 9398 Lys Ile 1260 Asn Leu Phe Arg 1265 Gly Val Phe Ala Phe 1270 Leu Leu Tyr Val Ala 1275 acy tty atg tay gtw tty wsy ack tty gey aay atw ytr egy aay aar 9446 Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys

    1280 1285 1290 gar wsy tagtgatctc ctaggaagcc cgcctaatga gcgggctttt tttttctggt 9502 Glu Ser atgcatcctg aggccgatac tgtcgtcgtc ccctcaaact ggcagatgca cggttacgat 9562 gcgcccatct acaccaacgt gacctatccc attacggtca atccgccgtt tgttcccacg 9622 gagaatccga cgggttgtta ctcgctcaca tttaatgttg atgaaagctg gctacaggaa 9682 ggccagacgc gaattatttt tgatggcgtt cctattggtt aaaaaatgag ctgatttaac 9742

    2018241075 03 Oct 2018

    172 aaaaatttaa tgcgaatttt aacaaaatat taacgtttac aatttaaata tttgcttata 9802 caatcttcct gtttttgggg cttttctgat tatcaaccgg ggtacatatg attgacatgc 9862 tagttttacg attaccgttc atcgattctc ttgtttgctc cagactctca ggcaatgacc 9922 tgatagcctt tgtagatctc tcaaaaatag ctaccctctc cggcattaat ttatcagcta 9982 gaacggttga atatcatatt gatggtgatt tgactgtctc cggcctttct cacccttttg 10042 aatctttacc tacacattac tcaggcattg catttaaaat atatgagggt tctaaaaatt 10102 tttatccttg cgttgaaata aaggcttctc ccgcaaaagt attacagggt cataatgttt 10162 ttggtacaac cgatttagct ttatgctctg aggctttatt gcttaatttt gctaattctt 10222 tgccttgcct gtatgattta ttggatgtt 10251 <210> 583 <211> 113 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: CJRA05 protein sequence <400> 583

    Met 1 Lys Lys Leu Leu 5 Phe Ala Ile Pro Leu 10 Val Val Pro Phe Tyr 15 Ser Gly Ala Ala Glu Ser His Leu Asp Gly Ala Ala Glu Thr Val Glu Ser 20 25 30 Cys Leu Ala Lys Ser His Thr Glu Asn Ser Phe Thr Asn Val Trp Lys 35 40 45 Asp Asp Lys Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys Leu Trp 50 55 60 Asn Ala Thr Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln Cys Tyr 65 70 75 80 Gly Thr Trp Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu Gly Gly 85 90 95 Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly 100 105 110 Thr <210> 584 <211> 152 <212> PRT <213> Artificial . Sequence

    2018241075 03 Oct 2018

    173 <220>

    <223> Description of Artificial Sequence: CJRA05 protein sequence <400> 584

    Ser Gly 1 Asp Phe Asp 5 Tyr Glu Lys Met Ala 10 Asn Ala Asn Lys Gly 15 Ala Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly 20 25 30 Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe 35 40 45 Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp 50 55 60 Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn 65 70 75 80 Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln 85 90 95 Ser Val Glu Cys Arg Pro Phe Val Phe Gly Ala Gly Lys Pro Tyr Glu 100 105 110 Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala 115 120 125 Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala 130 135 140 Asn Ile Leu Arg Asn Lys Glu Ser

    145 150 <210> 585 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: CJRA05 peptide sequence <400> 585 Met Pro Val Leu Leu Gly Ile Pro Leu Leu Leu Arg Phe Leu Gly 1 5 10 15

    <210> 586 <211> 348 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: CJRA05

    2018241075 03 Oct 2018

    174 protein sequence <400> 586

    Met Ala Val Tyr Phe Val Thr Gly Lys Leu Gly Ser Gly Lys Thr Leu

    1 5 10 15

    Val Ser Val Gly Lys Ile Gln Asp Lys Ile Val Ala Gly Cys Lys Ile

    20 25 30

    Ala Thr Asn Leu Asp Leu Arg Leu Gln Asn Leu Pro Gln Val Gly Arg 35 40 45

    Phe Ala Lys Thr Pro Arg Val Leu Arg Ile Pro Asp Lys Pro Ser Ile 50 55 60

    Ser Asp Leu Leu Ala Ile Gly Arg Gly Asn Asp Ser Tyr Asp Glu Asn

    65 70 75 80

    Lys Asn Gly Leu Leu Val Leu Asp Glu Cys Gly Thr Trp Phe Asn Thr

    85 90 95

    Arg Ser Trp Asn Asp Lys Glu Arg Gln Pro Ile Ile Asp Trp Phe Leu 100 105 110

    His Ala Arg Lys Leu Gly Trp Asp Ile Ile Phe Leu Val Gln Asp Leu . 115 120 125

    Ser Ile Val Asp Lys Gln Ala Arg Ser Ala Leu Ala Glu His Val Val

    130 135 140

    Tyr Cys Arg Arg Leu Asp Arg Ile Thr Leu Pro Phe Val Gly Thr Leu

    145 150 155 160

    Tyr Ser Leu Ile Thr Gly Ser Lys Met Pro Leu Pro Lys Leu His Val

    165 170 175

    Gly Val Val Lys Tyr Gly Asp Ser Gln Leu Ser Pro Thr Val Glu Arg 180 185 190

    Trp Leu Tyr Thr Gly Lys Asn Leu Tyr Asn Ala Tyr Asp Thr Lys Gln 195 200 205

    Ala Phe Ser Ser Asn Tyr Asp Ser Gly Val Tyr Ser Tyr Leu Thr Pro 210 215 220

    Tyr Leu Ser His Gly Arg Tyr Phe Lys Pro Leu Asn Leu Gly Gln Lys

    225 230 235 240

    Met Lys Leu Thr Lys Ile Tyr Leu Lys Lys Phe Ser Arg Val Leu Cys

    245 250 255

    Leu Ala Ile Gly Phe Ala Ser Ala Phe Thr Tyr Ser Tyr Ile Thr Gln 260 265 270

    Pro Lys Pro Glu Val Lys Lys Val Val Ser Gln Thr Tyr Asp Phe Asp 275 280 285

    Lys Phe Thr Ile Asp Ser Ser Gln Arg Leu Asn Leu Ser Tyr Arg Tyr

    2018241075 03 Oct 2018

    175

    290 295 300 Val Phe Lys Asp Ser Lys Gly Lys Leu Ile Asn Ser Asp Asp Leu Gln 305 310 315 320 Lys Gln Gly Tyr Ser Leu Thr Tyr Ile Asp Leu Cys Thr Val Ser Ile 325 330 335 Lys Lys Gly Asn Ser Asn Glu Ile Val Lys Cys Asn

    340 345 <210> 587 <211> 234 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence

    protein sequence <400> 587

    Met Lys Lys Leu Leu Phe Ala Ile Pro Leu Val Val Pro Phe Tyr Ser 15 10 15

    His Ser Ala Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser 20 25 30

    Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Gly 35 40 45

    Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro 50 · 55 60

    Arg Leu Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala

    65 70 75 80

    Arg Phe Ser Gly Ser Gly Pro Gly Thr Asp Phe Thr Leu Thr Ile Ser

    85 90 95

    Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Asn 100 105 110

    Trp His Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg 115 120 125

    Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln 130 135 140

    Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr

    145 150 155 160

    Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser

    165 170 175

    Gly Asn Ser Gln Glu Ser Val Thr Glu Arg Asp Ser Lys Asp Ser Thr 180 185 190

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    176

    Tyr Ser Leu 195 Ser Ser Thr Leu Thr Leu 200 Ser Lys Ala Asp 205 Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro 210 215 220 Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

    225 230 <210> 588 <211> 431 <212> PRT

    <213> Artificial . Sequence <220> <223> Description of Artificial Sequence: CJRA05 protein sequence <400> 588 Met Lys Tyr Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala 1 5 10 15 Ala Gln Pro Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly 20 25 30 Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly 35 40 45 Phe Thr Phe Ser Thr Tyr Glu Met Arg Trp Val Arg Gln Ala Pro Gly 50 55 60 Lys Gly Leu Glu Trp Val Ser Tyr Ile Ala Pro Ser Gly Gly Asp Thr 65 70 75 80 Ala Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 85 90 95 Ser Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp 100 105 110 Thr Ala Val Tyr Tyr Cys Ala Arg Arg Leu Asp Gly Tyr Ile Ser Tyr 115 120 125 Tyr Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser 130 135 140 Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser 145 150 155 160 Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp 165 170 175 Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr 180 185 190 Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr

    195 200 205

    2018241075 03 Oct 2018

    177

    Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln 210 215 220

    Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp

    225 230 235 240

    Lys Lys Val Glu Pro Lys Ser Cys Ala Ala Ala His His His His His

    245 250 255

    His Gly Ala Ala Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly 260 265 270

    Ala Ala Gln Ala Ser Ser Ala Ser Gly Asp Phe Asp Tyr Glu Lys Met 275 280 285

    Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala Asp Glu Asn Ala 290 295 300

    Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val Ala Thr Asp Tyr

    305 310 315 320

    Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser Gly Leu Ala Asn

    325 330 335

    Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn Ser Gln Met Ala 340 345 350

    Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn Asn Phe Arg Gln 355 360 365

    Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg Pro Phe Val Phe

    370 · 375 380

    Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys Asp Lys Ile Asn

    385 390 395 400

    Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val Ala Thr Phe Met

    405 410 415

    Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn Lys Glu Ser 420 425 430 <210> 589 <211> 5 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Illustrative peptide <400> 589

    Glu Gly Gly Gly Ser

    1 5

    2018241075 03 Oct 2018

    178 <210> 590 <211> 1275 <212> DNA <213> Unknown Organism <220>

    <221> CDS <222> (1) .. (1272) <220>

    <223> Description of Unknown Organism: M13 nucleotide sequence <400> 590

    gtg Met 1 aaa Lys aaa Lys tta Leu tta Leu 5 ttc Phe gca Ala att Ile cct Pro tta Leu 10 gtt Val gtt Val cct Pro ttc Phe tat Tyr 15 tet Ser 48 cac tcc get gaa act gtt gaa agt tgt tta gca aaa ccc cat aca gaa 96 His Ser Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu 20 25 30 aat tea ttt act aac gtc tgg aaa gac gac aaa act tta gat cgt tac 144 Asn Ser Phe Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr 35 40 45 get aac tat gag ggt tgt ctg tgg aat get aca ggc gtt gta gtt tgt 192 Ala Asn Tyr Glu Gly Cys Leu Trp Asn Ala Thr Gly Val Val Val Cys 50 55 60 act ggt gac gaa act cag tgt tac ggt aca tgg gtt cct att ggg ett 240 Thr Gly Asp Glu Thr Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu 65 70 75 80 get ate cct gaa aat gag ggt ggt ggc tet gag ggt ggc ggt tet gag 288 Ala Ile Pro Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu 85 90 95 ggt ggc ggt tet gag ggt ggc ggt act aaa cct cct gag tac ggt gat 336 Gly Gly Gly Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp 100 105 110 aca cct att ccg ggc tat act tat ate aac cct etc gac ggc act tat 384 Thr Pro Ile Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr 115 120 125 ccg cct ggt act gag caa aac ccc get aat cct aat cct tet ett gag 432 Pro Pro Gly Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu 130 135 140 gag tet cag cct ett aat act ttc atg ttt cag aat aat agg ttc ega 480 Glu Ser Gln Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg 145 150 155 160 aat agg cag ggg gca tta act gtt tat aeg ggc act gtt act caa ggc 528 Asn Arg Gln Gly Ala Leu Thr Val Tyr Thr Gly Thr Val Thr Gln Gly 165 170 175

    2018241075 03 Oct 2018

    179 act gac ccc gtt aaa act tat tac cag tac act cct gta tea tea aaa

    Thr Asp Pro Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys

    180 185 190 gee atg tat gac get tac tgg aac ggt aaa ttc aga gac tgc get ttc

    Ala Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe

    195 200 205 cat tet ggc ttt aat gag gat cca ttc gtt tgt gaa tat caa ggc caa

    His Ser Gly Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln

    210 215 220 teg tet gac ctg cct caa cct cct gtc aat get ggc ggc ggc tet ggt

    Ser Ser Asp Leu Pro Gln Pro Pro Val Asn Ala Gly Gly Gly Ser Gly

    225 230 235 240 ggt ggt tet ggt ggc ggc tet gag ggt ggt ggc tet gag ggt ggc ggt

    Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly

    245 250 255 tet gag ggt ggc ggc tet gag gga ggc ggt tec ggt ggt ggc tet ggt

    Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly

    260 ' 265 270 tec ggt gat ttt gat tat gaa aag atg gca aac get aat aag ggg get

    Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala

    275 280 285 atg acc gaa aat gee gat gaa aac geg eta cag tet gac get aaa ggc

    Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly

    290 295 300 aaa ett gat tet gtc gc.t act gat tac ggt get get ate gat ggt ttc

    Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe

    305 310 315 320 att ggt gac gtt tec ggc ett get aat ggt aat ggt get act ggt gat

    Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp

    325 330 335 ttt get ggc tet aat tec caa atg get caa gtc ggt gac ggt gat aat

    Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn

    340 345 350 tea cct tta atg aat aat ttc cgt caa tat tta cct tec etc cct caa

    Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln

    355 360 365 teg gtt gaa tgt ege cct ttt gtc ttt age get ggt aaa cca tat gaa

    Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu

    370 375 380 ttt tet att gat tgt gac aaa ata aac tta ttc cgt ggt gtc ttt geg

    Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala

    385 390 395 400

    576

    624

    672

    720

    768

    816

    864

    912

    960

    1008

    1056

    1104

    1152 ttt ett tta tat gtt gee acc ttt atg tat gta ttt tet aeg ttt get 1248

    Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala

    1200

    2018241075 03 Oct 2018

    180

    405 410 415 aac ata ctg cgt aat aag gag tct taa

    Asn Ile Leu Arg Asn Lys Glu Ser 420 <210> 591 <211> 424 .

    <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: M13 protein sequence <400> 591

    Met 1 Lys Lys Leu Leu 5 Phe Ala Ile Pro Leu 10 Val Val Pro Phe Tyr 15 Ser His Ser Ala Glu Thr Val Glu Ser Cys Leu Ala Lys Pro His Thr Glu 20 25 30 Asn Ser Phe Thr Asn Val Trp Lys Asp Asp Lys Thr Leu Asp Arg Tyr 35 40 45 Ala Asn Tyr Glu Gly Cys Leu Trp Asn Ala Thr Gly Val Val Val Cys 50 55 60 Thr Gly Asp Glu Thr Gln Cys Tyr Gly Thr Trp Val Pro Ile Gly Leu 65 70 75 80 Ala Ile Pro Glu Asn Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu 85 90 95 Gly Gly Gly Ser Glu Gly Gly Gly Thr Lys Pro Pro Glu Tyr Gly Asp 100 105 110 Thr Pro Ile Pro Gly Tyr Thr Tyr Ile Asn Pro Leu Asp Gly Thr Tyr 115 120 125 Pro Pro Gly Thr Glu Gln Asn Pro Ala Asn Pro Asn Pro Ser Leu Glu 130 135 140 Glu Ser Gln Pro Leu Asn Thr Phe Met Phe Gln Asn Asn Arg Phe Arg 145 150 155 160 Asn Arg Gln Gly Ala Leu Thr Val Tyr Thr Gly Thr Val Thr Gln Gly 165 170 175 Thr Asp Pro Val Lys Thr Tyr Tyr Gln Tyr Thr Pro Val Ser Ser Lys 180 185 190 Ala Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe Arg Asp Cys Ala Phe 195 200 205 His Ser Gly Phe Asn Glu Asp Pro Phe Val Cys Glu Tyr Gln Gly Gln 210 215 220

    1275

    2018241075 03 Oct 2018

    181

    Ser Ser Asp Leu Pro Gln Pro Pro Val Asn Ala Gly Gly Gly Ser 225 230 235

    Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly

    245 250 255

    Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser

    260 265 . 270

    Ser Gly Asp Phe Asp Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly

    275 280 285

    Met Thr Glu Asn Ala Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys 290 295 300

    Lys Leu Asp Ser Val Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly 305 310 315

    Ile Gly Asp Val Ser Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly 325 330 335

    Phe Ala Gly Ser Asn Ser Gln Met Ala Gln Val Gly Asp Gly Asp 340 345 350

    Ser Pro Leu Met Asn Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro 355 360 365

    Ser Val Glu Cys Arg Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr 370 375 380

    Phe Ser Ile Asp Cys Asp Lys Ile Asn Leu Phe Arg Gly Val Phe 385 390 395

    Phe Leu Leu Tyr Val Ala Thr Phe Met Tyr Val Phe Ser Thr Phe 405 410 415

    Asn Ile Leu Arg Asn Lys Glu Ser 420 <210> 592 <211> 35 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 592 caacgatgat cgtatggcgc atgctgccga gacag

    Gly

    240

    Gly

    Gly

    Ala

    Gly

    Phe

    320

    Asp

    Asn

    Gln

    Glu

    Ala

    400

    Ala <210> 593 <211> 1355 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    182 <220>

    <223> Description of Artificial Sequence: M13-III nucleotide sequence <220>

    <221> CDS <222> (1) .. (1305) <400> 593

    gcg Ala 1 gcc Ala gca Ala cat His cat His 5 cat His cac His cat His cac His ggg Gly 10 gcc Ala gca Ala gaa Glu caa Gln aaa Lys 15 etc Leu 48 ate tea gaa gag gat ctg aat ggg gcc gca tag get age gat ate aac 96 Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala Ala Ser Asp Ile Asn 20 25 30 gat gat cgt atg get tet act gey gar acw gty gaa wsy tgy ytr gem 144 Asp Asp Arg Met Ala Ser Thr Ala Glu Thr Val Glu Ser Cys Leu Ala 35 40 45 aar ccy cay acw gar aat wsw tty acw aay gts tgg aar gay gay aar 192 Lys Pro His Thr Glu Asn Ser Phe Thr Asn Val Trp Lys Asp Asp Lys 50 55 60 acy ytw gat cgw tay gey aay tay gar ggy tgy ytr tgg aat gey a cm 240 Thr Leu Asp Arg Tyr Ala Asn Tyr Glu Gly Cys Leu Trp Asn Ala Thr 65 70 75 ggc gty gtw gty tgy ack ggy gay gar acw car tgy tay ggy acr tgg 288 Gly Val Val Val Cys Thr Gly Asp Glu Thr Gln Cys Tyr Gly Thr Trp 80 85 90 95 gtk cck atw ggs ytw gey atm cck gar aay gar ggy ggy ggy wsy gar 336 Val Pro Ile Gly Leu Ala Ile Pro Glu Asn Glu Gly Gly Gly Ser Glu 100 105 110 ggy ggy ggy wsy gar ggy ggy ggw tcy gar ggw ggy ggw acy aar cck 384 Gly Gly Gly Ser Glu Gly Gly Gly Ser Glu Gly Gly Gly Thr Lys Pro 115 120 125 cck gar tay ggy gay acw cck atw cck ggy tay acy tay aty aay cck 432 Pro Glu Tyr Gly Asp Thr Pro Ile Pro Gly Tyr Thr Tyr Ile Asn Pro 130 135 140 ytm gay ggm acy tay cck cck ggy acy gar car aay ccy gey aay cck 480 Leu Asp Gly Thr Tyr Pro Pro Gly Thr Glu Gln Asn Pro Ala Asn Pro 145 150 155 aay ccw wsy ytw gar gar wsy car cck ytw aay acy tty atg tty car 528 Asn Pro Ser Leu Glu Glu Ser Gln Pro Leu Asn Thr Phe Met Phe Gln 160 165 170 175 aay aay mgk tty mgr aay mgk car ggk gew ytw acy gtk tay ack ggm 576 Asn Asn Arg Phe Arg Asn Arg Gln Gly Ala Leu Thr Val Tyr Thr Gly 180 185 190

    2018241075 03 Oct 2018

    183

    acy gty Thr Val acy car ggy acy gay ccy Pro gty Val 200 aar Lys acy Thr tay Tyr tay Tyr car Gln 205 tay Tyr acy Thr 624 Thr Gln 195 Gly Thr Asp cck gtm ter wsw aar gey atg tay gay gey tay tgg aay ggy aar tty 672 Pro Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr Trp Asn Gly Lys Phe 210 215 220 mgw gay tgy gey tty cay wsy ggy tty aay gar gay ccw tty gty tgy 720 Arg Asp Cys Ala Phe His Ser Gly Phe Asn Glu Asp Pro Phe Val Cys 225 230 235 gar tay car ggy car wsk wsy gay ytr cck car ccw cck gty aay gck 768 Glu Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro Val Asn Ala 240 245 250 255 ggy ggy ggy wsy ggy ggw ggy wsy ggy ggy ggy wsy gar ggy ggw ggy 816 Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu Gly Gly Gly 260 265 270 wsy gar ggw ggy ggy wsy ggr ggy ggy wsy ggy wsy ggy gay tty gay 864 Ser Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly Asp Phe Asp 275 280 285 tay gar aar atg gew aay gey aay aar ggs gey atg acy gar aay gey 912 Tyr Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr Glu Asn Ala 290 295 300 gay gar aay gcr ctr car wst gay gey aar ggy aar ytw gay wsy gtc 960 Asp Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu Asp Ser Val 305 310 315 gey acw gay tay ggt get gey ate gay ggy tty aty ggy gay gty wsy 1008 Ala Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly Asp Val Ser 320 325 330 335 ggy ctk get aay ggy aay ggw gey acy ggw gay tty gew ggy tek aat 1056 Gly Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala Gly Ser Asn 340 345 350 tcy car atg gey car gty ggw gay ggk gay aay wsw cck ytw atg aay 1104 Ser Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro Leu Met Asn 355 360 365 aay tty mgw car tay ytw cck tcy cty cck car wsk gty gar tgy egy 1152 Asn Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val Glu Cys Arg 370 375 380 ccw tty gty tty wsy gey ggy aar ccw tay gar tty wsy aty gay tgy 1200 Pro Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser Ile Asp Cys 385 390 395 gay aar atm aay ytw ttc egy ggy gty tty gck tty ytk yta tay gty 1248 Asp Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu Leu Tyr Val 400 405 410 415 gey acy tty atg tay gtw tty wsy ack tty gey aay atw ytr egy aay 1296 Ala Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile Leu Arg Asn

    1345

    2018241075 03 Oct 2018

    184

    420 425 430 aar gar wsy tagtgatctc ctaggaagcc cgcctaatga gcgggctttt Lys Glu Ser tttttctggt

    1355 <210> 594 <211> 434 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial protein sequence <400> 594

    Ala Ala Ala His His His His His 1 5

    Ile Ser Glu Glu Asp Leu Asn Gly 20

    Asp Arg Met Ala Ser Thr Ala Glu 35 40

    Pro His Thr Glu Asn Ser Phe Thr

    50 55

    Leu Asp Arg Tyr Ala Asn Tyr Glu

    65 · 70

    Val Val Val Cys Thr Gly Asp Glu

    Pro Ile Gly Leu Ala Ile Pro Glu 100

    Gly Gly Ser Glu Gly Gly Gly Ser 115 120

    Glu Tyr Gly Asp Thr Pro Ile Pro 130 135

    Asp Gly Thr Tyr Pro Pro Gly Thr 145 150

    Pro Ser Leu Glu Glu Ser Gln Pro 165

    Asn Arg Phe Arg Asn Arg Gln Gly 180

    Val Thr Gln Gly Thr Asp Pro Val 195 200

    Sequence: M13-III

    His Gly Ala Ala Glu Gln Lys Leu 10 15

    Ala Ala Ala Ser Asp Ile Asn Asp 25 30

    Thr Val Glu Ser Cys Leu Ala Lys 45

    Asn Val Trp Lys Asp Asp Lys Thr 60

    Gly Cys Leu Trp Asn Ala Thr Gly 75 80

    Thr Gln Cys Tyr Gly Thr Trp Val 90 95

    Asn Glu Gly Gly Gly Ser Glu Gly 105 110

    Glu Gly Gly Gly Thr Lys Pro Pro 125

    Gly Tyr Thr Tyr Ile Asn Pro Leu 140

    Glu Gln Asn Pro Ala Asn Pro Asn 155 160

    Leu Asn Thr Phe Met Phe Gln Asn 170 175

    Ala Leu Thr Val Tyr Thr Gly Thr 185 190

    Lys Thr Tyr Tyr Gln Tyr Thr Pro 205

    2018241075 03 Oct 2018

    185

    Val Ser Ser Lys Ala Met Tyr Asp Ala Tyr Trp Asn 210 215 220

    Asp Cys Ala Phe His Ser Gly Phe Asn Glu Asp Pro 225 230 235

    Gly Lys Phe Arg

    Phe Val Cys Glu 240

    Tyr Gln Gly Gln Ser Ser Asp Leu Pro Gln Pro Pro 245 250

    Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Ser Glu 260 265

    Glu Gly Gly Gly Ser Gly Gly Gly Ser Gly Ser Gly 275 280

    Glu Lys Met Ala Asn Ala Asn Lys Gly Ala Met Thr 290 295 300

    Glu Asn Ala Leu Gln Ser Asp Ala Lys Gly Lys Leu 305 310 315

    Thr Asp Tyr Gly Ala Ala Ile Asp Gly Phe Ile Gly 325 330

    Leu Ala Asn Gly Asn Gly Ala Thr Gly Asp Phe Ala 340 345

    Gln Met Ala Gln Val Gly Asp Gly Asp Asn Ser Pro 355 360

    Phe Arg Gln Tyr Leu Pro Ser Leu Pro Gln Ser Val 370 375 380

    Val Asn Ala Gly 255

    Gly Gly Gly Ser 270

    Asp Phe Asp Tyr 285

    Glu Asn Ala Asp

    Asp Ser Val Ala 320

    Asp Val Ser Gly 335

    Gly Ser Asn Ser 350

    Leu Met Asn Asn 365

    Glu Cys Arg Pro

    Phe Val Phe Ser Ala Gly Lys Pro Tyr Glu Phe Ser 385 390 395

    Ile Asp Cys Asp 400

    Lys Ile Asn Leu Phe Arg Gly Val Phe Ala Phe Leu 405 410

    Leu Tyr Val Ala 415

    Thr Phe Met Tyr Val Phe Ser Thr Phe Ala Asn Ile 420 425

    Leu Arg Asn Lys 430

    Glu Ser <210> 595 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 595 cgttgatatc gctagcctat gc

    2018241075 03 Oct 2018

    186 <210> 596 <211> 30 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 596 gataggctta gctagcccgg agaacgaagg . 30 <210> 597 <211> 37 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 597 ctttcacagc ggtttcgcta gcgacccttt tgtctgc 37 <210> 598 <211> 50 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 598 ctttcacagc ggtttcgcta gcgacccttt tgtcagcgag taccagggtc 50 <210> 599 <211> 37 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide ' <400> 599 · gactgtctcg gcagcatgcg ccatacgatc atcgttg 37 <210> 600 <211> 37 <212> DNA <213> Artificial Sequence <220>

    2018241075 03 Oct 2018

    187 <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> CDS <222> (2)..(25) <400> 600 c aac gat gat cgt atg gcg cat get gccgagacag tc Asn Asp Asp Arg Met Ala His Ala

    1 5 <210> 601 <211> 8 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic peptide <400> 601

    Asn Asp Asp Arg Met Ala His Ala 1 5 <210> 602 <211> 37 <212> DNA <213> Artificial Sequence <220> · <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 602 ctttcacagc ggtttgcatg cagacccttt tgtctgc <210> 603 <211> 50 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 603 ctttcacagc ggtttgcatg cagacccttt tgteagegag taccagggtc <210> 604 <211> 7 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Illustrative peptide <400> 604

    Tyr Ala Asp Ser Val Lys Gly 1 5

    2018241075 03 Oct 2018

    188 <210> 605 <211> 21 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 605 cctcgacagc gaagtgcaca g 21 <210> 606 <211> 38 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 606 ggctgagtca agacgctctg tgcacttcgc tgtcgagg 38 <210> 607 <211> 7 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Illustrative peptide <400> 607

    Gln Ser Ala Leu Thr Gln Pro 1 5 <210> 608 <211> 22 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Primer <400> 608 cctctgtcac agtgcacaag ac

    2018241075 03 Oct 2018

    189

    <210> 609 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 609 cctctgtcac agtgcacaag acatccagat gacccagtct cc

    <210> 610 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide Sequence: Synthetic <400> 610 gggaggatgg agactgggtc gtctggatgt cttgtgcact gtgacagagg

    <210> 611 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial peptide Sequence: Illustrative <400> 611 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser 1 5 10

    <210> <211> <212> <213> 612 20 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Primer <400> 612

    gactgggtgt agtgatctag 20 <210> 613 <211> 28 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 613 ggtgtagtga tcttctagtg acaactct 28

    2018241075 03 Oct 2018

    190 <210> 614 <211> 6 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic peptide <400> 614

    Val Ser Ser Arg Asp Asn 1 5 <210> 615 <211> 15 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220> · <221> CDS <222> (1)..(15) <400> 615 tac tat tgt geg aaa

    Tyr Tyr Cys Ala Lys 1 5 <210> 616 <211> 5 ' <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic peptide <400> 616

    Tyr Tyr Cys Ala Lys

    1 5 <210> 617 <211> 36

    2018241075 03 Oct 2018

    191 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 617 ggtgccgata ggcttgcatg caccggagaa cgaagg 36 <210> 618 .

    <211> 95 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 618 cgcttcacta agtctagaga caactctaag aatactctct acttgcagat gaacagctta 60 agggctgagg acactgcagt ctactattgt acgag 95 <210> 619 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (4)..(7) <223> A, T, C, G, other or unknown <400> 619 gatnnnnatc 10 <210> 620 <211> 10 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: MALIA3-derived peptide <400> 620

    Met Lys Leu Leu Asn Val Ile Asn Phe Val 1 5 ' 10 <210> 621

    2018241075 03 Oct 2018

    192 <211> 29 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: CJRA05-derived peptide

    <400> 621 Met Ser Val Leu Val Tyr Ser Phe Ala Ser Phe Val Leu Gly 1 5 10 Leu Arg Ser Gly Ile Thr Tyr Phe Thr Arg Leu Met Glu 20 25

    <210> <211> <212> <213> 622 15 DNA Artificial Sequence <220> <223> Description of Artificial Sequence: Illustrative nucleotide sequence

    <400> 622 tttttttttt ttttt

    <210> 623 <211> 87 <212> PRT <213> Unknown Organism <220> <223> Description of Unknown Organism: MALIA3-derived peptide <400> 623

    Met 1 Ile Lys Val Glu 5 Ile Lys Pro Ser Gln 10 Ala Gln Phe Thr Thr 15 Arg Ser Gly Val Ser 20 Arg Gln Gly Lys Pro 25 Tyr Ser Leu Asn Glu 30 Gln Leu Cys Tyr Val 35 Asp Leu Gly Asn Glu 40 Tyr Pro Val Leu Val 45 Lys Ile Thr Leu Asp 50 Glu Gly Gln Pro Ala 55 Tyr Ala Pro Gly Leu 60 Tyr Thr Val His Leu Ser Ser Phe Lys Val Gly Gln Phe Gly Ser Leu Met Ile Asp Arg

    Leu Arg Leu Val Pro Ala Lys 85

    65 70 75 80

    2018241075 03 Oct 2018

    193 <210> 624 <211> 29 <212> PRT <213> Unknown Organism <220>

    <223> Description of Unknown Organism: MALIA3-derived peptide <400> 624

    Met Ser Val Leu Val Tyr Ser Phe Ala Ser Phe Val Leu Gly Trp Cys 15 10 15

    Leu Arg Ser Gly Ile Thr Tyr Phe Thr Arg Leu Met Glu 20 25 <210> 625 <211> 10 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <220>

    <221> modified_base <222> (7)..(10) <223> A, T, C, G, other or unknown <400> 625 ctcttcnnnn .

    <210> 626 <211> 87 <212> PRT <213> Artificial Sequence <220>

    <223> Description of Artificial Sequence: CJRA05-derived peptide <400> 626

    Met 1 Ile Lys Val Glu 5 Ile Lys Pro Ser Gln 10 Ala Gln Phe Thr Thr 15 Arg Ser Gly Val Ser 20 Arg Gln Gly Lys Pro 25 Tyr Ser Leu Asn Glu 30 Gln Leu Cys Tyr Val 35 Asp Leu Gly Asn Glu 40 Tyr Pro Val Leu Val 45 Lys Ile Thr Leu Asp 50 Glu Gly Gln Pro Ala 55 Tyr Ala Pro Gly Leu 60 Tyr Thr Val His Leu Ser Ser Phe Lys Val Gly Gln Phe Gly Ser Leu Met Ile Asp Arg

    2018241075 03 Oct 2018

    194

    65 70 Leu Arg Leu Val Pro Ala Lys 85 <210> 627 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial peptide <400> 627 Met Lys Leu Leu Asn Val Ile Asn 1 5 <210> 628 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 628 gacccagtct ccatcctcc <210> 629 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 629

    gactcagtct ccactctcc

    Sequence: CJRA05-derived

    Phe Val 10

    Sequence: Synthetic

    Sequence: Synthetic

    75 80 <210> 630 <211> 19 <212> DNA <213> Artificial Sequence <220>

    <223> Description of Artificial oligonucleotide <400> 630 gacgcagtct ccaggcacc

    Sequence: Synthetic

    2018241075 03 Oct 2018

    195

    <210> 631 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 631 gacgcagtct ccagccacc <210> 632 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 632 gtctcctgga cagtcgatc <210> 633 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 633

    ggccttggga cagacagtc

    Sequence: Synthetic

    Sequence: Synthetic

    Sequence: Synthetic

    <210> 634 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial oligonucleotide <400> 634 gtctcctgga cagtcagtc

    Sequence: Synthetic <210> 635 <211> 19 <212> DNA <213> Artificial Sequence

    2018241075 03 Oct 2018

    196 <220>

    <223> Description of Artificial Sequence: Synthetic oligonucleotide <400> 635 ggccccaggg cagagggtc 19