CN117203335A - Generation of cyclic polyribonucleotides in eukaryotic systems - Google Patents

Abstract

The present disclosure relates generally to methods for producing, purifying, and using circular RNAs from eukaryotic systems.

Description

Production of cyclic polyribonucleotides in eukaryotic systems

Citation of priority application

This international patent application filed under the Patent Cooperation Treaty claims the benefit of U.S. Provisional Patent Application Serial No. 63/189,619 filed on May 17, 2021 and U.S. Provisional Patent Application Serial No. 63/166,467 filed on March 26, 2021.

Sequence Listing

This application contains a sequence listing, which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created on March 23, 2022, named VL70004WO1_ST25, and is 316,185 bytes in size. Also incorporated by reference in its entirety is the sequence listing submitted in U.S. Provisional Patent Application Serial No. 63/189,610, which was created on May 17, 2021, named 51484-005001_Sequence Listing_5_17_21_ST25, and is 300,429 bytes in size. Also incorporated herein by reference in its entirety is the sequence listing filed in U.S. Provisional Patent Application Serial No. 63/166,467, created on March 25, 2021, entitled 51484-003001_Sequence_Listing_3.25.21_ST25, and having a size of 166,651 bytes.

Background Art

Cyclic polyribonucleotide is a subclass of polyribonucleotide, exists in the form of continuous ring. Endogenous cyclic polyribonucleotide is generally expressed in human tissue and cell. Most of endogenous cyclic polyribonucleotide is produced by backsplicing, and mainly plays non-coding role. It is proposed that synthetic cyclic polyribonucleotide (comprising cyclic polyribonucleotide of coding protein) is used for various therapeutic applications and engineering applications. Need to produce, purify and use the method for cyclic polyribonucleotide.

Summary of the invention

The present disclosure provides compositions and methods for producing, purifying, and using circular RNA.

In the first aspect, the characteristics of the present disclosure are eukaryotic systems for making polyribonucleotide cyclization, and the eukaryotic system comprises: (a) polyribonucleotides (e.g., linear polyribonucleotides), the polyribonucleotides have formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein: (A) comprises 5' self-cleaving ribozymes; (B) comprises 5' annealing regions; (C) comprises polyribonucleotide cargo (cargo); (D) comprises 3' annealing regions; and (E) comprises 3' self-cleaving ribozymes; and (b) comprises eukaryotic cells of RNA ligase. The linear polyribonucleotides can include, for example, other elements outside or between any one of element (A), (B), (C), (D) and (E). For example, any one of element (A), (B), (C), (D) and/or (E) can be separated by spacer sequences, as described herein.

On the other hand, present disclosure provides a kind of eukaryotic system for making polyribonucleotide cyclization, and this eukaryotic system comprises: (a) polyribonucleotide (for example, linear polyribonucleotide), this polyribonucleotide comprises (A), (B), (C), (D) and (E) operably connected with 5' to 3' direction: (A) 5' self-cleavage ribozyme; (B) 5' annealing zone; (C) polyribonucleotide load; (D) 3' annealing zone; and (E) 3' self-cleavage ribozyme; and (b) eukaryotic cell comprising RNA ligase.This linear polyribonucleotide can include, for example, outside or between any one of element (A), (B), (C), (D) and (E).For example, any one of element (A), (B), (C), (D) and/or (E) can be separated by spacer sequence, as described herein.

On the other hand, the present disclosure provides a method for producing circular RNA, the method including contacting (a) with (b) in a eukaryotic cell: (a) polyribonucleotide (e.g., linear polyribonucleotide), the polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein: (A) comprises a 5' self-cleaving ribozyme; (B) comprises a 5' annealing region; (C) comprises a polyribonucleotide cargo; (D) comprises a 3' annealing region; and (E) comprises a 3' self-cleaving ribozyme; and (b) RNA ligase. In some embodiments, the cleavage of the 5' self-cleaving ribozyme and the cleavage of the 3' self-cleaving ribozyme produce ligase-compatible linear polyribonucleotides. In some embodiments, the RNA ligase connects the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide, thereby producing circular RNA. In some embodiments, the circular RNA is separated from a eukaryotic cell. In some embodiments, the RNA ligase is endogenous to the eukaryotic cell. In some embodiments, the RNA ligase is heterologous to the eukaryotic cell.

On the other hand, the present disclosure provides a method for producing circular RNA, the method comprising contacting (a) with (b) in a eukaryotic cell: (a) polyribonucleotides (e.g., linear polyribonucleotides), the polyribonucleotides comprising (A), (B), (C), (D) and (E) operably linked in a 5' to 3' direction: (A) 5' self-cleaving ribozyme; (B) 5' annealing region; (C) polyribonucleotide cargo; (D) 3' annealing region; and (E) 3' self-cleaving ribozyme; and (b) RNA ligase. In some embodiments, the cleavage of the 5' self-cleaving ribozyme and the cleavage of the 3' self-cleaving ribozyme produce ligase-compatible linear polyribonucleotides. In some embodiments, the RNA ligase connects the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide, thereby producing circular RNA. In some embodiments, the circular RNA is separated from a eukaryotic cell. In some embodiments, the RNA ligase is endogenous to the eukaryotic cell. In some embodiments, the RNA ligase is heterologous to the eukaryotic cell.

On the other hand, the present disclosure provides a kind of eukaryotic cell, the eukaryotic cell comprises: (a) polyribonucleotide (for example, linear polyribonucleotide), the polyribonucleotide has the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein: (A) comprises 5' self-cleaving ribozyme; (B) comprises 5' annealing zone; (C) comprises polyribonucleotide cargo; (D) comprises 3' annealing zone; and (E) comprises 3' self-cleaving ribozyme; and (b) RNA ligase. In some embodiments, the cutting of the 5' self-cleaving ribozyme and the cutting of the 3' self-cleaving ribozyme produce ligase-compatible linear polyribonucleotides. In some embodiments, the RNA ligase can connect the 5' end and 3' end of the ligase-compatible linear polyribonucleotide to produce circular RNA. In some embodiments, the RNA ligase is endogenous to the eukaryotic cell. In some embodiments, the RNA ligase is heterologous to the eukaryotic cell. In some embodiments, the eukaryotic cell further comprises circular RNA.

On the other hand, the present disclosure provides a kind of eukaryotic cell, and the eukaryotic cell comprises: (a) polyribonucleotide (for example, linear polyribonucleotide), and the polyribonucleotide comprises (A), (B), (C), (D) and (E) operably connected in the 5' to 3' direction: (A) 5' self-cleaving ribozyme; (B) 5' annealing zone; (C) polyribonucleotide cargo; (D) 3' annealing zone; and (E) 3' self-cleaving ribozyme; and (b) RNA ligase. In some embodiments, the cutting of the 5' self-cleaving ribozyme and the cutting of the 3' self-cleaving ribozyme produce ligase-compatible linear polyribonucleotides. In some embodiments, the RNA ligase can connect the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide to produce circular RNA. In some embodiments, the RNA ligase is endogenous to the eukaryotic cell. In some embodiments, the RNA ligase is heterologous to the eukaryotic cell. In some embodiments, the eukaryotic cell further comprises circular RNA.

In some embodiments, the 5' self-cleaving ribozyme is capable of self-cleavage at a site within 10 ribonucleotides of the 3' end of the 5' self-cleaving ribozyme or at a site at the 3' end of the 5' self-cleaving ribozyme.

In some embodiments, the 5' self-cleaving ribozyme is a ribozyme selected from the following: Hammerhead ribozyme, Hairpin ribozyme, Hepatitis D virus ribozyme (HDV), Varkud satellite (VS) ribozyme, glmS ribozyme, Twister ribozyme, Twister sister ribozyme, Hatchet ribozyme and Pistol ribozyme. In some embodiments, the 5' self-cleaving ribozyme is a hammerhead ribozyme. In some embodiments, the 5' self-cleaving ribozyme includes a region having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the 5' self-cleaving ribozyme includes the nucleic acid sequence of SEQ ID NO: 2. In some embodiments, the 5' self-cleaving ribozyme comprises a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or its corresponding RNA equivalent, or a catalytically competent fragment thereof. In some embodiments, the 5' self-cleaving ribozyme comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof. In some embodiments, the 5' self-cleaving ribozyme comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof.

In some embodiments, the 3' self-cleaving ribozyme is capable of self-cleavage at a site within 10 ribonucleotides of the 5' end of the 3' self-cleaving ribozyme or at a site at the 5' end of the 3' self-cleaving ribozyme.

In some embodiments, the 3' self-cleaving ribozyme is a ribozyme selected from the following: hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme (HDV), Varkud satellite (VS) ribozyme, glmS ribozyme, twist ribozyme, twist sister ribozyme, axe ribozyme and pistol ribozyme. In some embodiments, the 3' self-cleaving ribozyme is a hepatitis delta virus (HDV) ribozyme. In some embodiments, the 3' self-cleaving ribozyme includes a region having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the 3' self-cleaving ribozyme includes a nucleic acid sequence of SEQ ID NO: 13. In some embodiments, the 3' self-cleaving ribozyme includes a nucleic acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with any one of SEQ ID NO: 38-585, or its corresponding RNA equivalent, or its catalytically competent fragment. In some embodiments, the 3' self-cleaving ribozyme comprises a nucleic acid sequence having at least 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof. In some embodiments, the 3' self-cleaving ribozyme comprises a nucleic acid sequence of any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof.

In some embodiments, the 5' self-cleaving ribozyme and the 3' self-cleaving ribozyme produce a ligase-compatible linear polyribonucleotide. In some embodiments, the cleavage of the 5' self-cleaving ribozyme produces a free 5'-hydroxyl group, and the cleavage of the 3' self-cleaving ribozyme produces a free 2', 3'-cyclic phosphate group.

In some embodiments, the 5' and 3' self-cleaving ribozymes share at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. In some embodiments, the 5' and 3' self-cleaving ribozymes are from the same self-cleaving ribozyme family. In some embodiments, the 5' and 3' self-cleaving ribozymes share 100% sequence identity.

In some embodiments, the 5' and 3' self-cleaving ribozymes share less than 100%, 99%, 95%, 90%, 85%, or 80% sequence identity. In some embodiments, the 5' and 3' self-cleaving ribozymes are not from the same family of self-cleaving ribozymes.

In some embodiments, the 5' annealing region has 2 to 100 ribonucleotides (e.g., 2 to 100, 2 to 80, 2 to 50, 2 to 30, 2 to 20, 5 to 100, 5 to 80, 5 to 50, 5 to 30, 5 to 20, 10 to 100, 10 to 80, 10 to 50, or 10 to 30 ribonucleotides). In some embodiments, the 3' annealing region has 2 to 100 ribonucleotides (e.g., 2 to 100, 2 to 80, 2 to 50, 2 to 30, 2 to 20, 5 to 100, 5 to 80, 5 to 50, 5 to 30, 5 to 20, 10 to 100, 10 to 80, 10 to 50, or 10 to 30 ribonucleotides).

In some embodiments, the 5' annealing region and the 3' annealing region each include a complementary region (e.g., form a pair of complementary regions). In some embodiments, the 5' annealing region includes a 5' complementary region having between 2 and 50 ribonucleotides (e.g., 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50 ribonucleotides); and the 3' annealing region includes a 3' complementary region having between 2 and 50 ribonucleotides (e.g., 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50 ribonucleotides). In some embodiments, the 5' complementary region and the 3' complementary region have between 50% and 100% sequence complementarity (eg, between 60%-100%, 70%-100%, 80%-100%, 90%-100%, or 100% sequence complementarity).

In some embodiments, the 5' complementary region and the 3' complementary region have a binding free energy of less than -5 kcal/mol (e.g., less than -10 kcal/mol, less than -20 kcal/mol, or less than -30 kcal/mol). In some embodiments, the 5' complementary region and the 3' complementary region have a binding Tm of at least 10°C, at least 15°C, at least 20°C, at least 30°C, at least 40°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, or at least 90°C. In some embodiments, the 5' complementary region and the 3' complementary region include no more than 10 mismatches, such as 10, 9, 8, 7, 6, 5, 4, 3, or 2 mismatches, or 1 mismatch. In some embodiments, the 5' complementary region and the 3' complementary region do not include any mismatches.

In some embodiments, the 5' annealing region and the 3' annealing region each include a non-complementary region. In some embodiments, the 5' annealing region further includes a 5' non-complementary region having between 2 and 50 ribonucleotides (e.g., 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50 ribonucleotides). In some embodiments, the 3' annealing region further includes a 3' non-complementary region having between 2 and 50 ribonucleotides (e.g., 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50 ribonucleotides). In some embodiments, the 5' non-complementary region is located 5' of the 5' complementary region (e.g., between the 5' self-cleaving ribozyme and the 5' complementary region). In some embodiments, the 3' non-complementary region is located 3' of the 3' complementary region (e.g., between the 3' complementary region and the 3' self-cleaving ribozyme). In some embodiments, the 5' non-complementary region and the 3' non-complementary region have a sequence complementarity between 0% and 50% (e.g., between 0%-40%, 0%-30%, 0%-20%, 0%-10%, or 0%). In some embodiments, the 5' non-complementary region and the 3' non-complementary region have a binding free energy greater than -5 kcal/mol. In some embodiments, the 5' complementary region and the 3' complementary region have a binding Tm of less than 10°C. In some embodiments, the 5' non-complementary region and the 3' non-complementary region include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches. In some embodiments, the 5' annealing region and the 3' annealing region do not include any non-complementary regions.

In embodiments, the 5' annealing region and the 3' annealing region have a high GC percentage (calculated as the number of GC nucleotides divided by the total nucleotides, multiplied by 100), i.e., wherein a relatively large number of GC pairs are involved in annealing between the 5' annealing region and the 3' annealing region, for example, wherein the GC percentage is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or even about 100%. For example, in embodiments where the 5' and 3' annealing regions are short (e.g., where each annealing region is 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in length), an increased GC percentage in the annealing region will increase the annealing strength between the two regions. In some embodiments, the 5' annealing region includes a region having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the 5' annealing region comprises the nucleic acid sequence of SEQ ID NO: 4. In some embodiments, the 3' annealing region comprises a region having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 12. In some embodiments, the 3' annealing region comprises the nucleic acid sequence of SEQ ID NO: 12.

In certain embodiments, the polyribonucleotide loading material includes coding sequence or includes non-coding sequence or includes coding sequence and non-coding sequence combination.In certain embodiments, the polyribonucleotide loading material includes two or more coding sequences (for example, 2,3,4,5,6,7,8,9 or 10 or more coding sequences), two or more non-coding sequences (for example, 2,3,4,5,6,7,8,9 or 10 or more non-coding sequences), or its combination.In the case where the polyribonucleotide loading material includes two or more coding sequences, the coding sequence can be at least one copy of each coding sequence in two or more copies or two or more different coding sequences of a single coding sequence.In the case where the polyribonucleotide loading material includes two or more non-coding sequences, the non-coding sequence can be at least one copy of each non-coding sequence in two or more copies or two or more different non-coding sequences of a single non-coding sequence.In certain embodiments, the polyribonucleotide loading material includes at least one coding sequence and at least one non-coding sequence.

In some embodiments, the polyribonucleotide payload comprises at least one non-coding RNA sequence. In some embodiments, the at least one non-coding RNA sequence comprises at least one RNA selected from the group consisting of: RNA aptamers, long non-coding RNA (lncRNA), transfer RNA derived fragments (tRF), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and Piwi interaction RNA (piRNA); or a fragment of any one of these RNAs. In some embodiments, the at least one non-coding RNA sequence comprises regulatory RNA. In some embodiments, the at least one non-coding RNA sequence trans-regulates the target sequence.

In some embodiments, the trans-regulation of the at least one non-coding RNA sequence to the target sequence is the up-regulation of the target sequence expression. In some embodiments, the trans-regulation of the at least one non-coding RNA sequence to the target sequence is the down-regulation of the target sequence expression. In some embodiments, the trans-regulation of the at least one non-coding RNA sequence to the target sequence is the inducible expression of the target sequence. For example, the at least one non-coding RNA sequence can be induced by environmental conditions (for example, light, temperature, water or nutrient availability), by circadian rhythm, by endogenous or exogenously provided inducing agents (for example, small RNA, ligands). In some embodiments, the at least one non-coding RNA sequence can be induced by the physiological state (for example, growth phase, transcriptional regulation state and intracellular metabolite concentration) of the eukaryotic system. For example, an exogenously provided ligand (for example, arabinose, rhamnose or IPTG) can be provided to use an inducible promoter (for example, PBAD, Prha and lacUV5) to induce expression.

In some embodiments, the at least one non-coding RNA sequence comprises an RNA selected from the group consisting of: small interfering RNA (siRNA) or a precursor thereof, double-stranded RNA (dsRNA) or at least partially double-stranded RNA [e.g., RNA comprising one or more stem-loops]; hairpin RNA (hpRNA), micro RNA (miRNA) or a precursor thereof [e.g., pre-miRNA or pri-miRNA]; phase small interfering RNA (phasiRNA) or a precursor thereof; heterochromatin small interfering RNA (hcsiRNA) or a precursor thereof; and natural antisense short interfering RNA (natsiRNA) or a precursor thereof.

In some embodiments, the at least one non-coding RNA sequence comprises a guide RNA (gRNA) or a precursor thereof.

In some embodiments, the target sequence comprises the nucleotide sequence of a gene of the test genome. In some embodiments, the test genome is a genome of a vertebrate, an invertebrate, a fungus, an oomycete, a plant, or a microorganism. In some embodiments, the test genome is a genome of a human, a non-human mammal, a reptile, a bird, an amphibian, or a fish. In some embodiments, the test genome is a genome of an insect, an arachnid, a nematode, or a mollusk. In some embodiments, the test genome is a genome of a monocot, a dicot, a gymnosperm, or a eukaryotic algae. In some embodiments, the test genome is a genome of a bacterium, a fungus, an oomycete, or an archaea. In some embodiments, the target sequence comprises the nucleotide sequence of a gene found in a plurality of test genomes (e.g., in the genomes of a plurality of species within a given genus).

In some embodiments, the polyribonucleotide loading material comprises a coding sequence for a polypeptide. In some embodiments, the polyribonucleotide loading material comprises an IRES operably connected to a coding sequence for a polypeptide. In some embodiments, the polyribonucleotide loading material comprises a Kozak sequence operably connected to an expression sequence for a polypeptide. In some embodiments, the polyribonucleotide loading material comprises an RNA sequence encoding a polypeptide having a biological effect on a subject. In some embodiments, the polypeptide is, for example, a therapeutic polypeptide for a human or non-human animal. In some embodiments, the polypeptide is a polypeptide having a sequence encoded in the genome of a vertebrate (e.g., non-human mammals, reptiles, birds, amphibians, or fish), an invertebrate (e.g., insects, arachnids, nematodes, or molluscs), a plant (e.g., monocots, dicots, gymnosperms, eukaryotic algae), or a microorganism (e.g., bacteria, fungi, archaea, oomycetes). In some embodiments, the polypeptide has a biological effect when contacted with a vertebrate, invertebrate, or plant, or when contacted with a vertebrate cell, an invertebrate cell, a microbial cell, or a plant cell. In some embodiments, the polypeptide is a plant modifying polypeptide. In some embodiments, the polypeptide increases the fitness of a vertebrate, an invertebrate, or plant, or increases the fitness of a vertebrate cell, an invertebrate cell, a microbial cell, or a plant cell, when contacted with the following. In some embodiments, the polypeptide decreases the fitness of a vertebrate, an invertebrate, or plant, or decreases the fitness of a vertebrate cell, an invertebrate cell, a microbial cell, or a plant cell, when contacted with the following.

In certain embodiments, the polyribonucleotide load comprises a coded polypeptide and has an RNA sequence of a nucleotide sequence for expressing in a subject or an organism through codon optimization. The codon optimized method for expressing in a particular type of organism is known in the art and is provided as a part of a commercial vector or polypeptide design service. See, for example, U.S. Patent number 6,180,774 (for expressing in monocotyledons), 7,741,118 (for expressing in dicotyledons) and 5,786,464 and 6,114,148 (both for expressing in mammals), all of which are incorporated herein by reference in their entirety. Codon optimization can be performed using any of several publicly available tools, for example, the various codon optimization tools provided at, for example, www[dot]idtdna[dot]com/pages/tools/codon-optimization-tool; www[dot]novoprolabs[dot]com/tools/codon-optimization, en[dot]vectorbuilder[dot]com/tool/codon-optimization[dot]html, where a codon usage table for the appropriate genus of the subject can be selected from a drop-down menu in the portal.

In some embodiments, the subject comprises (a) eukaryotic cells; or (b) prokaryotic cells. Embodiments of such cells include immortalized cell lines and primary cell lines. Embodiments include cells located in tissues, organs, or complete multicellular organisms. For example, in an embodiment, a cyclic polyribonucleotide as described in the present disclosure (or a eukaryotic cell containing the cyclic polyribonucleotide) is delivered in a targeted manner to one or more specific cells, tissues, or organs in a multicellular organism.

In some embodiments, the subject comprises a vertebrate, an invertebrate, a fungus, an oomycete, a plant, or a microorganism. In some embodiments, the vertebrate is selected from a human, a non-human mammal (e.g., Mus musculus), a reptile (e.g., Anolis carolinensis), a bird (e.g., Gallus domesticus), an amphibian (e.g., Xenopus tropicalis), or a fish (e.g., Danio rerio (zebrafish)). In some embodiments, the invertebrate is selected from an insect (e.g., Leptinotarsa decemlineata), an arachnid (e.g., Scorpio maurus), a nematode (e.g., Meloidogyne incognita), or a mollusk (e.g., Cornu aspersum). In some embodiments, the plant is selected from a monocot (e.g., Zea mays), a dicot (e.g., Glycine max), a gymnosperm (e.g., Pinus strobus), or a eukaryotic alga (e.g., Caulerpa sertularioides). In some embodiments, the microorganism is selected from a bacterium (e.g., Escherichia coli), a fungus (e.g., Saccharomyces cerevisiae or Pichia pastoris), an oomycete (e.g., Pythium oligandrum, Phytophthora infestans, and other Phytophthora spp.), or an archaeon (e.g., Pyrococcus furiosus).

In certain embodiments, the linear polyribonucleotide further includes a spacer region between the 5' annealing zone and the polyribonucleotide load, a length of at least 5 polyribonucleotides. In certain embodiments, the linear polyribonucleotide further includes a spacer region between the 5' annealing zone and the polyribonucleotide load, a length of 5 and 1000 polyribonucleotides. In certain embodiments, the spacer region includes a poly-A sequence. In certain embodiments, the spacer region includes a poly-A-C sequence.

In certain embodiments, the linear polyribonucleotide is at least 1kb. In certain embodiments, the linear polyribonucleotide is 1kb to 20kb. In certain embodiments, the linear polyribonucleotide is 100 to about 20,000 nucleotides. In certain embodiments, the size of the linear RNA is at least 100, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, 6,000, 7,000, 8,000, 9,000 or 10,000 nucleotides.

In some embodiments, the RNA ligase is endogenous to the eukaryotic cell (e.g., the RNA ligase is naturally present in the cell). In some embodiments, the RNA ligase is heterologous to the eukaryotic cell (e.g., the RNA ligase is not naturally present in the cell, e.g., the cell has been genetically engineered to express or overexpress the RNA ligase). In some embodiments, the RNA ligase is provided to the eukaryotic cell by transcribing an exogenous polynucleotide into an mRNA encoding the RNA ligase in the eukaryotic cell and translating the mRNA encoding the RNA ligase. In some embodiments, the RNA ligase is provided to the eukaryotic cell as an exogenous protein (e.g., the RNA ligase is expressed extracellularly and provided to the cell).

In some embodiments, the RNA ligase is a tRNA ligase. In some embodiments, the tRNA ligase is T4 ligase, RtcB ligase, TRL-1 ligase, Rnl1 ligase, Rnl2 ligase, LIG1 ligase, LIG2 ligase, PNK/PNL ligase, PF0027 ligase, thpR ligT ligase, ytlPor ligase, or a variant thereof.

In some embodiments, the RNA ligase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 586-602.

In some embodiments, the RNA ligase is selected from the group consisting of a plant RNA ligase, a plastid (e.g., chloroplast) RNA ligase, an RNA ligase from an archaea, a bacterial RNA ligase, a eukaryotic RNA ligase, a viral RNA ligase, or a mitochondrial RNA ligase, or a variant thereof.

In certain embodiments, the linear polyribonucleotide is transcribed from deoxyribonucleic acid, and the deoxyribonucleic acid includes an RNA polymerase promoter operably connected to the sequence encoding the linear polyribonucleotide as described herein. In certain embodiments, the RNA polymerase promoter is heterologous to the sequence encoding the linear polyribonucleotide. In certain embodiments, the RNA polymerase promoter is a T7 promoter, a T6 promoter, a T4 promoter, a T3 promoter, a SP3 promoter, a SP6 promoter, a CaMV 35S, an opine promoter, a plant ubiquitin promoter, a rice actin 1 promoter, an ADH-1 promoter, a GPD promoter, a CMV promoter, an EF1α promoter, a CAG promoter, a PGK promoter, a U6 nuclear promoter, a TRE promoter, an OpIE2 promoter, or an OpIE1 promoter. In some embodiments, the RNA polymerase promoter provides specificity of expression of a sequence encoding a linear polynucleotide; for example, a promoter can be selected to provide cell, tissue, or organ-specific expression, time-specific expression (e.g., specific to circadian rhythm, cell cycle, or seasonality), or development-specific expression. In some embodiments, the RNA polymerase promoter is a promoter of a plant small RNA or microRNA gene or a promoter of an animal small RNA or microRNA gene; see, for example, U.S. Patent Nos. 9,976,152 and 7,786,351; de Rie (2017) Nature Biotechnol. [Natural Biotechnology], 35: 872-878. In some embodiments of any aspect described herein, the disclosure provides a eukaryotic system for cyclizing polyribonucleotides, the eukaryotic system comprising: (a) polydeoxyribonucleotides encoding linear polyribonucleotides described herein (e.g., cDNA, circular DNA vectors, or linear DNA vectors), and (b) a eukaryotic cell comprising an RNA ligase.

In some embodiments, the eukaryotic cell is provided with an exogenous polyribonucleotide comprising the linear polynucleotide. In some embodiments, the linear polyribonucleotide is transiently transcribed from the exogenous recombinant DNA molecule provided to the eukaryotic cell. In some embodiments, the linear polyribonucleotide is transcribed from the exogenous DNA molecule provided to the eukaryotic cell in the eukaryotic cell. In some embodiments, the exogenous DNA molecule is not integrated into the genome of the eukaryotic cell. In some embodiments, the exogenous DNA molecule comprises a heterologous promoter operably connected to the DNA encoding the linear polyribonucleotide. In certain embodiments, the heterologous promoter is selected from the group consisting of: T7 promoter, T6 promoter, T4 promoter, T3 promoter, SP3 promoter, SP6 promoter, CaMV 35S, opine promoter, plant ubiquitin promoter, rice actin 1 promoter, ADH-1 promoter, GPD promoter, CMV promoter, EF1α promoter, CAG promoter, PGK promoter, U6 nuclear promoter, TRE promoter, OpIE2 promoter or OpIE1 promoter. In certain embodiments, in the eukaryotic cell, linear polyribonucleotides are transcribed from the recombinant DNA molecules incorporated into the genome of the eukaryotic cell.

In some embodiments, the eukaryotic cells are grown in culture medium. In some embodiments, the eukaryotic cells are contained in a bioreactor.

In some embodiments, the eukaryotic cell is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic cell is selected from the group consisting of a unicellular fungal cell, a unicellular animal cell, a unicellular plant cell, a unicellular algal cell, an oomycete cell, a protist cell, and a protozoan cell. In embodiments, the eukaryotic cell is a cell of a multicellular eukaryotic organism. In some embodiments, the multicellular eukaryotic organism is selected from the group consisting of a vertebrate, an invertebrate, a multicellular fungus, a multicellular oomycete, a multicellular algae, and a multicellular plant.

In another aspect, the disclosure provides a cyclic polyribonucleotide produced by a eukaryotic system described herein or any method comprising a eukaryotic system.

On the other hand, the present disclosure provides a method for improving the subject by providing a composition or formulation as described herein to the subject. In some embodiments, the composition or formulation is a nucleic acid molecule or includes a nucleic acid molecule (e.g., a DNA molecule or RNA molecule as described herein), and the nucleic acid molecule is provided to a eukaryotic subject. In some embodiments, the composition or formulation is a eukaryotic cell as described herein, or includes a eukaryotic cell as described herein.

On the other hand, the present disclosure provides a method for treating a subject's illness by providing a composition or formulation as described herein to a subject in need thereof. In some embodiments, the composition or formulation is a nucleic acid molecule or includes a nucleic acid molecule (e.g., a DNA molecule or RNA molecule as described herein), and the nucleic acid molecule is provided to a eukaryotic subject. In some embodiments, the composition or formulation is a eukaryotic cell as described herein or includes a eukaryotic cell as described herein.

In another aspect, the disclosure provides a method of providing a cyclic polyribonucleotide to a subject by providing the subject with a eukaryotic cell described herein.

In another aspect, the present disclosure provides a formulation comprising a eukaryotic system, eukaryotic cell, or polyribonucleotide as described herein. In some embodiments, the formulation is a pharmaceutical formulation, a veterinary formulation, or an agricultural formulation.

In another aspect, the disclosure provides a formulation comprising the eukaryotic cells described herein. In some embodiments, the eukaryotic cells are dried or frozen. In some embodiments, the formulation is a pharmaceutical formulation, a veterinary formulation, or an agricultural formulation.

definition

In order to facilitate understanding of the present disclosure, multiple terms are defined below. The terms defined herein have the meanings commonly understood by those of ordinary skill in the field relevant to the present disclosure. Terms such as "a, an" and "the" are not intended to refer only to a single entity, but include general categories that can be illustrated using specific examples. The term "or" is used to mean "and/or", unless explicitly stated to refer only to alternatives or alternatives are mutually exclusive, although the present disclosure supports the definition of referring only to alternatives and "and/or".

The terminology herein is used to describe particular embodiments, but their usage should not be regarded as limiting, except as recited in the claims.

As used herein, any value provided within a range of values includes the upper and lower limits, and any value contained within the upper and lower limits.

As used herein, the terms "circRNA" or "cyclic polyribonucleotide" or "circular RNA" or "cyclic polyribonucleotide molecule" are used interchangeably and refer to a polyribonucleotide molecule having a structure without free ends (i.e., without free 3' and/or 5' ends), such as a polyribonucleotide molecule that forms a ring or annular structure through covalent or non-covalent bonds.

As used herein, the term "cyclization efficiency" is a measure of the resulting circular polyribonucleotide relative to its non-cyclic starting material.

The phrase "compound, composition, product, etc. for treatment, regulation, etc." is to be understood as meaning a compound, composition, product, etc. which is suitable per se for the indicated purpose of treatment, regulation, etc. The phrase "compound, composition, product, etc. for treatment, regulation, etc." additionally discloses as a preferred embodiment that such compound, composition, product, etc. is for treatment, regulation, etc.

The words "compound, composition, product, etc. for use in..." or "use of a compound, composition, product, etc. in the manufacture of a medicament, pharmaceutical composition, veterinary composition, diagnostic composition, etc. for..." indicate that such compound, composition, product, etc. is to be used in a therapeutic method that can be practiced on the human or animal body. They are considered to be equivalent disclosures of the embodiments and claims relating to methods of treatment, etc. If an embodiment or a claim thus refers to "a compound for use in treating a human or animal suspected of having a disease", this is also considered to disclose "the use of a compound in the manufacture of a medicament for treating a human or animal suspected of having a disease" or "a method of treatment by administering a compound to a human or animal suspected of having a disease".

As used herein, the terms "disease," "disorder," and "condition" each refer to a subhealth condition, eg, a condition that is typically or would be diagnosed or treated by a medical professional.

"Heterologous" means occurring in a context different from that which occurs in nature (native). A "heterologous" polynucleotide sequence indicates that the polynucleotide sequence is used in a manner different from that found in the natural genome of the sequence. For example, a "heterologous promoter" is used to drive transcription of a sequence that is not a sequence that is naturally transcribed by the promoter; thus, a "heterologous promoter" sequence is typically included in an expression construct by recombinant nucleic acid technology. The term "heterologous" is also used to refer to a given sequence that is placed in a non-naturally occurring relationship with another sequence; for example, a heterologous coding or non-coding nucleotide sequence is typically inserted into a genome by genome transformation technology, resulting in a genetically modified genome or a recombinant genome.

As used herein, "improving the fitness of a subject" or "promoting the fitness of a subject" refers to any beneficial change in physiology or any activity performed by the subject organism resulting from the administration of the peptides or polypeptides described herein, including but not limited to any one or more of the following desired effects: (1) increasing tolerance to biotic or abiotic stress by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 10% or more; (2) increasing tolerance to biotic or abiotic stress by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 10% or more; 0% or more; (2) increase yield or biomass by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (3) adjust flowering time by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increase resistance to pests or pathogens by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more %, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) increase resistance to herbicides by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) increase the population of a subject organism (e.g., an agriculturally important insect) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. 0%, 90%, 95%, 99%, 100% or more; (6) increasing the reproductive rate of a subject organism (e.g., an insect, such as a bee or a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) increasing the migration of a subject organism (e.g., an insect, such as a bee or a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; %, 90%, 95%, 99%, 100% or more; (8) increase the body weight of a subject organism (e.g., an insect, such as a bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (9) increase the metabolic rate or activity of a subject organism (e.g., an insect, such as a bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70% or more , 80%, 90%, 95%, 99%, 100% or more; (10) increasing pollination (e.g., the number of plants pollinated in a given period of time) by a subject organism (e.g., an insect, such as a bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (11) increasing the production of a byproduct (e.g., from honey) by a subject organism (e.g., an insect, such as a bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (12) increasing the nutrient (e.g., protein, fatty acid, or amino acid) content of a subject organism (e.g., an insect) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. or (13) increasing the resistance of a subject organism to a pesticidal agent (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a thiophosphate, e.g., fenitrothion)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more, (14) increasing the health of a subject organism (e.g., a human or non-human animal) or reducing disease in a subject organism (e.g., a human or non-human animal). The increase in host fitness can be determined compared to a subject organism to which the modulator is not administered. In contrast, "reducing the fitness of a subject" refers to any adverse change in physiology or any activity performed by the subject organism resulting from the administration of a peptide or polypeptide as described herein, including but not limited to any one or more of the following desired effects: (1) reducing tolerance to biotic or abiotic stress by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (2) reducing productivity or production; (3) adjusting the flowering time by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (4) reducing the resistance to pests or pathogens by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more , 70%, 80%, 90%, 95%, 99%, 100% or more; (4) reduce resistance to herbicides by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (5) reduce the population of a test organism (e.g., an agriculturally important insect) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. (6) reduce the reproductive rate of a subject organism (e.g., an insect, such as a bee or a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (7) reduce the migration of a subject organism (e.g., an insect, such as a bee or a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. 5%, 99%, 100% or more; (8) reducing the body weight of a subject organism (e.g., an insect, such as a bee or a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (9) reducing the metabolic rate or activity of a subject organism (e.g., an insect, such as a bee or a silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more. (10) reducing pollination (e.g., the number of plants pollinated in a given period of time) by a subject organism (e.g., an insect, such as a bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (11) reducing the production of byproducts (e.g., honey from bees) by a subject organism (e.g., an insect, such as a bee or silkworm) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; (12) reducing the content of nutrients (e.g., proteins, fatty acids, or amino acids) in a subject organism (e.g., an insect) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more; or (13) reducing the resistance of the subject organism to a pesticide (e.g., a neonicotinoid (e.g., imidacloprid) or an organophosphorus insecticide (e.g., a thiophosphate, e.g., fenitrothion)) by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% or more, (14) reducing the health of the subject organism (e.g., a human or non-human animal) or reducing disease in the subject organism (e.g., a human or non-human animal). A reduction in host fitness can be determined compared to a subject organism to which the modulator is not administered. It will be apparent to one skilled in the art that certain changes in the physiology, phenotype, or activity of a subject (e.g., adjustments in the timing of flowering of a plant) can be considered to increase the fitness of the subject or decrease the fitness of the subject, depending on the context (e.g., to adapt to changes in climate or other environmental conditions). For example, a delay in flowering time (e.g., about a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 100% reduction in the number of plants in a population that flower on a given calendar date) can be a beneficial adaptation to a later or cooler spring and thus be considered to increase the fitness of a plant; conversely, the same delay in flowering time in the context of an earlier or warmer spring can be considered to decrease the fitness of a plant.

As used herein, the terms "linear RNA" or "linear polyribonucleotide" or "linear polyribonucleotide molecule" are used interchangeably and refer to polyribonucleotide molecules having 5' and 3' ends. One or both of the 5' and 3' ends can be free ends or joined to another part. Linear RNA includes RNA that has not undergone cyclization (e.g., pre-cyclization) and can be used as a starting material for cyclization.

As used herein, the term "modified ribonucleotide" means a nucleotide having at least one modification to the sugar, nucleobase, or internucleoside linkage.

The term "pharmaceutical composition" is intended to also disclose that the cyclic or linear polyribonucleotide comprised in the pharmaceutical composition can be used for treating the human or animal body by therapy.

As used herein, the term "polynucleotide" means a molecule comprising one or more nucleic acid subunits or nucleotides, and can be used interchangeably with "nucleic acid" or "oligonucleotide". A polynucleotide may include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U) or variants thereof. A nucleotide may include a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphate (PO ₃ ) groups. A nucleotide may include a nucleobase, a pentose (ribose or deoxyribose) and one or more phosphate groups. A ribonucleotide is a nucleotide in which the sugar is ribose. A polyribonucleotide or ribonucleic acid or RNA may refer to a macromolecule comprising a plurality of ribonucleotides polymerized via a phosphodiester bond. A deoxyribonucleotide is a nucleotide in which the sugar is deoxyribose.

As used herein, the term "polyribonucleotide loading material" herein includes any sequence containing at least one polyribonucleotide. In an embodiment, the polyribonucleotide loading material includes one or more coding sequences, wherein each coding sequence encoding polypeptide. In an embodiment, the polyribonucleotide loading material includes one or more non-coding sequences, such as polyribonucleotides with regulating or catalytic functions. In an embodiment, the polyribonucleotide loading material includes a combination of coding sequences and non-coding sequences. In an embodiment, the polyribonucleotide loading material includes one or more polyribonucleotide sequences as described herein, such as one or more regulatory elements, internal ribosome entry site (IRES) elements, and/or spacer sequences.

As used herein, elements of a nucleic acid construct or vector are "operably connected or operably linked" if they are located on the construct or vector such that they can perform their function (e.g., promote transcription or terminate transcription). For example, a DNA construct comprising a promoter operably linked to a DNA sequence encoding a linear precursor RNA indicates that the DNA sequence encoding the linear precursor RNA can be transcribed to form a linear precursor RNA, for example, which can then be circularized into a circular RNA using the methods provided herein.

Polydeoxyribonucleotide or deoxyribonucleic acid or DNA means a macromolecule including a plurality of deoxyribonucleotides polymerized via a phosphodiester bond. Nucleotide can be a nucleoside monophosphate or a nucleoside polyphosphate. Nucleotide means a deoxyribonucleoside polyphosphate including a detectable label (such as a luminescent label) or a marker (e.g., a fluorophore), such as, for example, a deoxyribonucleoside triphosphate (dNTP), which can be selected from deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP) dNTP. Nucleotide can include any subunit that can be incorporated into a growing nucleic acid chain. This subunit can be A, C, G, T or U, or any other subunit that is specific to one or more complementary A, C, G, T or U or complementary to purine (i.e., A or G or its variants) or pyrimidine (i.e., C, T or U or its variants). In some instances, the polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or its derivatives or variants. In some cases, just to name a few, the polynucleotide is short interfering RNA (siRNA), microRNA (miRNA), plasmid DNA (pDNA), short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre-mRNA), antisense RNA (asRNA), and covers nucleotide sequence and any structural embodiment thereof, such as single-stranded, double-stranded, triple-stranded, spiral, hairpin, etc. In some cases, the polynucleotide molecule is circular. The polynucleotide can have various lengths. Nucleic acid molecules can have at least about 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2kb, 3kb, 4kb, 5kb, 10kb, 50kb or a greater length. Polynucleotides can be isolated from cells or tissues. Embodiments of polynucleotide sequences include isolated and purified DNA/RNA molecules, synthetic DNA/RNA molecules and synthetic DNA/RNA analogs.

Embodiments of polynucleotides (e.g., polyribonucleotides or polydeoxyribonucleotides) include polynucleotides containing one or more nucleotide variants, including one or more non-standard nucleotides, one or more non-natural nucleotides, one or more nucleotide analogs and/or modified nucleotides. Examples of modified nucleotides include, but are not limited to, diaminopurine, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxy 5-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, 2,6-diaminopurine, etc. In some cases, the nucleotide includes modifications in its phosphate portion, including modifications to the triphosphate portion. Non-limiting examples of such modifications include phosphate chains of greater length (e.g., phosphate chains with 4, 5, 6, 7, 8, 9, 10 or more phosphate moieties) and modifications with thiol moieties (e.g., α-thiotriphosphates and β-thiotriphosphates). In an embodiment, the nucleic acid molecule is modified at the base moiety (e.g., at one or more atoms that are typically available for hydrogen bonding with complementary nucleotides and/or at one or more atoms that are typically unable to hydrogen bond with complementary nucleotides), the sugar moiety, or the phosphate backbone. In an embodiment, the nucleic acid molecule contains amine-modified groups such as aminoallyl 1-dUTP (aa-dUTP) and aminohexyl acrylamide-dCTP (aha-dCTP) to allow covalent attachment of amine-reactive moieties such as N-hydroxysuccinimide ester (NHS). Substitutes for standard DNA base pairs or RNA base pairs in the oligonucleotides disclosed herein can provide higher sites/cubic mm density, higher safety (resistance to accidental or purposeful synthesis of natural toxins), easier differentiation of light-programmed polymerases, or lower secondary structures. Such alternative base pairs compatible with native and mutant polymerases for de novo and/or amplification synthesis are described in Betz K, Malyshev DA, Lavergne T, Welte W, Diederichs K, Dwyer TJ, Ordoukhanian P, Romesberg FE, Marx A. Nat. Chem. Biol. 2012 Jul;8(7):612-4, which is incorporated herein by reference for all purposes.

As used herein, "polypeptide" means a polymer of amino acid residues (natural or non-natural) most often linked together by peptide bonds. As used herein, the term refers to proteins, polypeptides and peptides of any size, structure or function. Polypeptides can include gene products, naturally occurring polypeptides, synthetic polypeptides, homologues, orthologues, paralogues, fragments and other equivalents, variants and analogs of the aforementioned substances. Polypeptides can be unimolecular or multimolecular complexes, such as dimers, trimers or tetramers. They can also include single-chain or multi-chain polypeptides (such as antibodies or insulin), and can be associated or connected. The most common disulfide bonds are present in multi-chain polypeptides. The term polypeptide can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

As used herein, "precursor linear polyribonucleotides" or "precursor linear RNAs" refer to linear RNA molecules created by transcription (e.g., in vivo transcription) in a eukaryotic system (e.g., from a polydeoxyribonucleotide template provided herein). Precursor linear RNAs are linear RNAs before cleavage by one or more self-cleaving ribozymes. After cleavage by one or more self-cleaving ribozymes, linear RNAs are referred to as "ligase-compatible linear polyribonucleotides" or "ligase-compatible RNAs."

As used herein, the term "plant modifying polypeptide" refers to a polypeptide that can alter the genetic characteristics (e.g., increase gene expression, decrease gene expression, or otherwise change the nucleotide sequence of DNA or RNA), epigenetic characteristics, or biochemical or physiological characteristics of a plant in a manner that results in an increase or decrease in the fitness of the plant.

As used herein, the term "regulatory element" is to modify the expression or transcriptional part of the nucleotide sequence operably connected thereto, such as nucleotide sequence.Regulatory element includes promoter, transcription factor recognition site, terminator element, small RNA recognition site (small RNA (such as microRNA) is combined with it and cuts) and transcription stabilizing element (see, for example, the stabilizing element described in U.S. Patent Application Publication 2007/0011761). For example, in an embodiment, a regulator (such as a promoter) modifies the expression of coding or non-coding sequence in circular or linear polyribonucleotides. In another embodiment, a regulator (such as small RNA recognition and cleavage site) modifies the expression of RNA transcripts, such as by limiting its expression in specific cells, tissues or organs (see, for example, U.S. Patent number 8,334,430 and 9,139,838).

As used herein, the term "RNA equivalent" refers to an RNA sequence that is the RNA equivalent of a DNA sequence. Therefore, the RNA equivalent of a DNA sequence refers to a DNA sequence in which each thymidine (T) residue is replaced by a uridine (U) residue. For example, the disclosure provides a DNA sequence of a ribozyme identified by a bioinformatics method. The disclosure specifically contemplates that any of these DNA sequences can be converted into a corresponding RNA sequence and included in the RNA molecules described herein.

As used herein, the term "sequence identity" is determined by comparing two peptides or two nucleotide sequences using a global or local alignment algorithm. When a sequence shares at least a certain minimum percentage of sequence identity when optimally aligned (for example, when compared using default parameters by a program such as GAP or BESTFIT), these sequences are referred to as "substantially identical" or "substantially similar". GAP uses the Needleman and Wunsch global alignment algorithm to compare the entire length of two sequences, thereby maximizing the number of matches and maximizing the number of gaps. Typically, using the GAP default parameters, gaps generate penalty points = 50 (nucleotides) / 8 (proteins), and gap extension penalty points = 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is Blosum62 (Henikoff and Henikoff, 1992, PNAS [Proceedings of the National Academy of Sciences of the United States] 89, 915-919). Sequence alignments and percentage sequence identity scores are determined, for example, using computer programs such as GCG Wisconsin Software Package Version 10.3 or EmbossWin Version 2.10.0 (using program "needle") obtained from Accelrys Inc., 9685 Scranton Road, San Diego, CA, USA 92121-3752. Alternatively or additionally, percent identity is determined by searching a database using, for example, algorithms such as FASTA, BLAST, etc. Sequence identity refers to sequence identity over the entire length of the sequence.

As used herein, "structured" with respect to RNA refers to an RNA sequence predicted by the RNAFold software or similar prediction tools to form an ordered or predictable secondary or tertiary structure (e.g., a hairpin loop) with itself or other sequences in the same RNA molecule.

As used herein, "ribozyme" refers to a catalytic RNA or a catalytic region of an RNA. A "self-cleaving ribozyme" is a ribozyme that is capable of catalyzing a cleavage reaction that occurs at a nucleotide site within the ribozyme sequence itself or at the end of the ribozyme sequence itself.

As used herein, "ribozyme" refers to a catalytic RNA or a catalytic region of an RNA. A "self-cleaving ribozyme" is a ribozyme that is capable of catalyzing a cleavage reaction that occurs at a nucleotide site within the ribozyme sequence itself or at the end of the ribozyme sequence itself.

As used herein, the term "subject" refers to an organism, such as an animal, plant, or microorganism. In an embodiment, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In an embodiment, the subject is a human, including an adult and a non-adult (infant and child). In an embodiment, the subject is a non-human mammal. In an embodiment, the subject is a non-human mammal, such as a non-human primate (e.g., monkey, ape), an ungulate (e.g., bovine, including cattle, buffalo, bison, sheep, goat, and musk ox; pig; camelid, including camel, llama, and alpaca; deer, antelope; and equine, including horse and donkey), a carnivore (e.g., dog, cat), a rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or a lagomorph (e.g., rabbit, hare). In an embodiment, the subject is a bird, such as a member of the following avian taxonomic group: Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathus (e.g., ostrich, emu), Columbiformes (e.g., pigeon, pigeon), or Psittaciformes (e.g., parrot). In an embodiment, the subject is an invertebrate, such as an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, worm, or mollusk. In an embodiment, the subject is an invertebrate agricultural pest or an invertebrate parasitic on an invertebrate or vertebrate host. In an embodiment, the subject is a plant, such as angiosperm (which can be a dicot or monocot) or gymnosperm (e.g., conifer, cycad, Gnetum plant, Ginkgo), fern, horsetail plant, lycophyte, or bryophyte. In an embodiment, the subject is a eukaryotic algae (single cell or multicell). In embodiments, the subject is a plant of agricultural or horticultural importance, such as row crops, fruit-producing plants and trees, vegetables, trees, and ornamental plants (including ornamental flowers, shrubs, trees, ground covers, and turf grasses).

Plants and plant cells are plants and plant cells of any species of interest, including dicots and monocots. Plants of interest include row crops, fruit-producing plants and trees, vegetables, trees, and ornamental plants (including ornamental flowers, shrubs, trees, ground covers, and turf grasses). Examples of commercially important cultivated crops, trees, and plants include: alfalfa (Medicago sativa), almonds (Prunus dulcis), apples (Malus xdomestica), apricots (Prunus armeniaca, P. brigantine, P. mandshurica, P. mume, P. sibirica), asparagus (Asparagus officinalis), bananas (Musa spp.), barley (Hordeum vulgare), beans (Phaseolus spp.), blueberries and cranberries (Vaccinium spp.), cocoa (Theobroma spp.), cacao), canola and rapeseed or rapeseed (Brassica napus), Polish canola (Brassica rapa) and related cruciferous vegetables, including broccoli, kale, cabbage, and radishes (Brassica carinata, B. juncea, B. oleracea, B. napus, B. nigra, and B. rapa, and hybrids of these), carnation (Dianthus caryophyllus), carrot (Daucuscarota sativus), cassava (Manihot esculentum), cherry (Prunus avium), chickpea (Cicer arietinum), chicory (Cichorium intybus), red pepper and other capsicum peppers (Capsicum annuum, C. frutescens, C. chinense, C. pubescens, C. baccatum), chrysanthemum (Chrysanthemum spp.), coconut (Cocos nucifera), coffee (Coffea spp., including Coffea arabica and Coffea canephora), cotton (Gossypium hirsutum L.), cowpea (Vigna unguiculata and other Vigna spp.), fava bean (Vava beans (Vicia faba), cucumbers (Cucumis sativus), currants and gooseberries (Ribes spp.), dates (Phoenix dactylifera), duckweed (Lemnoideae), eggplant or dwarf melon (Solanum melongena), eucalyptus (Eucalyptus spp.), flax (Linumus itatissumum L.), geraniums (Pelargonium spp.), grapefruit (Citrus x paradisi), grapes (Vitus spp.), including wine grapes (Vitus vinifera and their hybrids), guava (Psidium guajava), hops (Humulus lupulus), ramie and cannabis (Cannabis sativa and Cannabis spp.), iris (Iris spp.), lemon (Citrus limon), lettuce (Lactuca sativa), lime (Citrus spp.), corn (Zea mays L.), mango (Mangifera indica), mangosteen (Garcinia mangostana), melon (Cucumis melo), millet (Setaria spp.), Echinochloa spp. spp.), Eleusine spp., Panicum spp., Pennisetum spp.), oats (Avena sativa), oil palm (Ellis quineensis), olives (Olea europaea), onions (Allium cepa) and other alliums (Allium spp.), oranges (Citrus sinensis), papayas (Carica papaya), peaches and nectarines (Prunus persica), pears (Pyrus spp.), peas (Pisa sativum), peanuts (Arachis hypogaea), peonies (Paeonia spp.), spp.), petunia (Petunia spp.), pineapple (Ananas comosus), plantains (Musa spp.), plum (Prunus domestica), poinsettia (Euphorbia pulcherrima), poplar (Populus spp.), potato (Solanum tuberosum), pumpkin and squash (Cucurbita pepo, C. maximus, C. moschata), rice (Oryza sativa L.), rose (Rosas spp.), rubber (Hevea brasiliensis), rye (Secale cereale), safflower (Carthamus tinctorius L.), sesame seed (Sesame indium), sorghum (Sorghum bicolor), soybean (Glycine max L.), strawberry (Fragaria spp., Fragaria x ananassa), sugar beet (Beta vulgaris), sugarcane (Saccharum spp.), sunflower (Helianthus annuus), sweet potato (Ipomoea batatas), tangerine (Citrus tangerina), tea (Camellia sinensis), tobacco (Nicotiana tabacum L.), tomato (Solanum lycopersicum or Lycopersicon esculentum), tulips (Tulipa spp.), walnuts (Juglans spp. L.), watermelon (Citrullus lanatus), wheat (Triticum aestivum), and yams (Discorea spp.).

Many invertebrates are considered pests because they can damage resources important to humans or cause or transmit disease in humans, non-human animals (particularly livestock), or plants. Efforts to control pest invertebrates often employ synthetic chemicals that themselves may have undesirable effects due to their toxicity (including to humans and other non-target organisms, such as beneficial invertebrates), lack of specificity, persistence in the environment, and transport through the food chain.

Invertebrate agricultural pests that damage plants (especially domesticated plants grown as crops) include, but are not limited to, arthropods (e.g., insects, arachnids, myriapods), nematodes, flatworms, and mollusks. Important agricultural invertebrate pests include representative pests of the following insects: Coleoptera (beetles), Diptera (flies), Lepidoptera (butterflies, moths), Orthoptera (grasshoppers, locusts), Thysanoptera (thrips), and Hemiptera (stink bugs), Arachnids (such as mites and ticks), various worms (such as nematodes (roundworms) and platyhelminthes (flatworms)), and mollusks (such as slugs and snails).

Examples of agricultural insect pests include aphids, aldigids, phylloxera, leaf miners, whiteflies, caterpillars (butterfly or moth larvae), mealybugs, scale insects, grasshoppers, locusts, flies, thrips, earwigs, stink bugs, flea beetles, weevils, stem borers, sharpshooters, root or stem borers, leafhoppers, leaf miners, and midges. Non-limiting specific examples of important Lepidoptera agricultural pests include, for example, the diamondback moth (Plutellaxylostella), various "borers" (e.g., Diparopsis spp., Earias spp., Pectinophora spp., and Helicoverpa spp., including corn earworm, Helicoverpa zea, and cotton bollworm (Helicoverpa armigera), European corn borer (Ostrinia nubilalis), black cutworm (Agrotis ipsilon), "armyworms" (e.g., Spodoptera frugiperda, Spodoptera exigua, Spodoptera spp.), and "barkworms" (e.g., Spodoptera spp., Spodoptera spp., Spodoptera spp., and Spodoptera spp.), including corn earworm, Helicoverpa zea, and cotton bollworm (Helicoverpa armigera), European corn borer (Ostrinia nubilalis), black cutworm (Agrotis ipsilon), "armyworms" (e.g., Spodoptera frugiperda, Spodoptera exigua, Spodoptera littoralis, Pseudaletia unipuncta, Maize stalk borer (Papaipema nebris), Bean cutworm (Striacosta albicosta), Gypsy moth (Lymatria spp.), Cabbage worm (Pieris rapae), Red bell moth (Pectinophora gossypiella), Peach moth (Synanthedon exitiosa), Squash vine moth (Melittia cucurbitae), Apple moth (Cydiapomonella), Pear borer (Grapholita molesta), Indian grain borer (Plodia interpunctella), Greater wax moth (Galleria mellonella), Tobacco hornworm (Manduca sphenanthera) sexta, Manducaquinquemaculata, Lymantria dispar, Euproctis chrysorrhoea, Trichoplusia ni, Mamestra brassicae, Anticarsiagemmatalis, Pseudoplusia includens, Epinotiaaporema, Heliothis virescens, Scripophaga incertulus, Sesamia spp., Buseola fusca, Cnaphalocrocis medinalis and Chilo suppressalis. Non-limiting specific examples of important Coleoptera (beetle) agricultural pests include, for example, the Colorado potato beetle (Leptinotarsa decemlineata) and other Leptinotarsa species (Leptinotarsa decemlineata). spp.), for example, L. juncta (false potato beetle), L. haldemani (Haldeman's green potato beetle), L. lineolata (Asteraceae shrub leaf beetle), L. behrensi, L. collinsi, L. defecta, L. heydeni, L. peninsularis, L. rubiginosa, L. texana, L. tlascalana, L. tumamoca, and L. typographica; "corn rootworms" and "cucumber beetles", including the western corn rootworm (Diabrotica virgifera virgifera), the northern corn rootworm (Diabrotica virgifera), the northern corn rootworm (Diabrotica spp.), the southern corn rootworm (Diabrotica spp.), the northern ... rootworm) (northern corn rootworm (D. barberi), southern corn rootworm (D. undecimpunctata howardi), cucurbit beetle (D. speciosa), banded cucumber beetle (D. balteata), striped cucumber beetle (Acalymma vittatum), and western striped cucumber beetle (A. trivittatum); "flea beetles", for example, Chaetocnema pulicaria, Phyllotreta spp., and Psylliodes spp.; "seed beetles", for example, Stenolophus lecontei and Clivinia impressifrons; cereal leaf beetles (Oulema pulicaria), Phyllotreta spp., and Psylliodes spp.; "corn seed beetles", for example, Stenolophus lecontei and Clivinia impressifrons; melanopus); Japanese beetle (Popillia japonica) and other "white grubs", e.g., Phyllophaga spp., Cyclocephala spp.; khaprabeetle (Trogoderma granarium); date stone beetle (Coccotrypes dactyliperda); boll weevil (Anthonomus grandis grandis); stem borer (Dectes texanus); "nematodes" and "clickers", e.g., Melanotus spp., Agriotes spp. mancus), and the beet beetle (Limonius dubitans). Non-limiting specific examples of important Hemiptera (stink bug) agricultural pests include, for example, brown stinkbug (Halyomorpha halys), green stinkbug (Chinavia hilaris); weevils, for example, Sphenophorus maidis; spittlebugs, for example, meadow spittlebug (Philaenus spumarius); leafhoppers, for example, potato leafhopper (Empoasca fabae), beet leafhopper (Circulifer tenellus), blue-green sharpshooter (Graphocephala atropunctata), glassy-winged sharpshooter (Homalodisca vitripennis), maize leafhopper (Cicadulina mbila), two-spottedleafhopper (Sophonia rufofascia), common brown leafhopper (Orosiusorientalis), rice green leafhopper (Nephotettix spp.), and white apple leafhopper (Typhlocyba pomaria); aphids (e.g., Rhopalosiphum spp., Aphis spp., Myzus spp.), grape phylloxera (Daktulosphaira vitifoliae), and psyllids, e.g., Asian citrus psyllid (Diaphorina citri), African citrus psyllid (Trioza Other examples of important agricultural pests include thrips (e.g., Frankliniella occidentalis, F. tritici, Thrips simplex, T. palmi); members of the order Diptera, including Delias spp., fruit flies (e.g., Drosophila suzukii and other Drosophila spp., Ceratitis capitata, Bactrocera spp.), leaf miners (Liriomyza spp.), and midges (e.g., Mayetiola destructor).

Other invertebrates that cause agricultural damage include plant-feeding mites, for example, two-spotted or red spider mites (Tetranychus urticae) and spruce spider mites (Oligonychus unungui); various nematodes or roundworms, for example, Meloidogyne spp., including M. incognita (southern root-knot), M. enterlobii (guava root-knot), M. javanica (Javanica root-knot), M. hapla (northern root-knot), and M. arenaria (peanut root-knot), Longidorus spp., Aphelenchoides spp., Ditylenchus spp. spp.), Globodera spp. and other Globodera spp., Nacobbus spp., Heterodera spp., Bursaphelenchus xylophilus and other Bursaphelenchus spp., Pratylenchus spp., Trichodorus spp., Xiphinema index, Xiphinema diversicaudatum and other Xiphinema spp.; and snails and slugs (e.g., Deroceras spp., Vaginulus plebius), and Veronicaleydigi).

Pest invertebrates also include those that damage man-made structures or food storage, or otherwise cause a nuisance, for example, drywood and subterranean termites, carpenter ants, weevils (e.g., Acanthoscelides spp., Callosobruchus spp., Sitophilus spp.), flour beetles (Tribolium castaneum, Tribolium confusum) and other beetles (e.g., Stegobium paniceum, Trogoderma granarium, Oryzaephilus spp.), moths (e.g., greater wax moth, which damages beehives; Plodia interpunctella, Ephestiakuehniella, Tinea spp., Tineola spp.)), bark beetles, and mites (e.g., Acarus siro, Glycophagus destructor.

Many invertebrates are considered human or veterinary pests (eg, invertebrates that bite or parasitize humans or other animals), and many are vectors of pathogenic microorganisms (eg, bacteria, viruses). Examples of these include the order Diptera, such as biting flies and midges (e.g., Phlebotomus spp., Lutzomyia spp., Tabanus spp., Chrysops spp., Haematopota spp., Simulium spp.) and blowflies (screwworm flies) (e.g., Cochliomyia macellaria, C. hominivorax, C. aldrichi and C. minima; also Chrysomya rufifacies and Chrysomya megacephala, tsetse flies (Glossina spp.), skin flies (Dermatobia hominis), hominis, Dermatobia spp.); mosquitoes (e.g., Aedes spp., Anopheles spp., Culex spp., Culiseta spp.); bed bugs (e.g., Cimex lectularius, Cimex hemipterus) and "kissing bugs" (Triatoma spp.); members of the insect orders Phthiraptera (sucking and chewing lice, e.g., Pediculus humanus, Pthirus pubis) and Siphonaptera (fleas, e.g., Tungapenetrans). Parasitic arachnids also include important disease vectors; examples include ticks (e.g., Ixodes scapularis, Ixodes pacificus, pacificus, Ixodes ricinus, Ixodes cookie, Amblyomma americanum, Amblyomma maculatum, Dermacentor variabilis, Dermacentor andersoni, Dermacentor albipictus, Rhipicephalus sanguineus, Rhipicephalus microplus, Rhipicephalus annulatus, Haemaphysalis longicornis, and Hyalomma spp.), and mites, including scabies (Sarcoptes scabiei and other Sarcoptes spp.), horse itch mites (Psoroptes spp.), and scabiei. spp.), chiggers (Trombicula alfreddugesi, fall chiggers (Trombicula alfreddugesi), demodex mites (Demodex folliculorum, Demodex brevis, Demodex canis), bee mites (e.g., Varroa destructor, Varroa jacobosoni, and other Varroa spp.), tracheal mites (Acarapis woodi), and Tropilaelaps spp. Parasites that can infect humans and/or non-human animals include ectoparasites (such as leeches (a type of annelid)) and endoparasites, collectively referred to as "helminths," which infect the digestive tract, skin, muscle, or other tissues or organs. Helminths include members of the phyla Annelida (ringed worms or arthropods), Platyhelminthes (flatworms, eg, tapeworms, flukes), Nematoda (roundworms), and Acanthocephala (spine-headed worms). Examples of parasitic nematodes include Ascaris lumbricoides, Ascaris spp., Parascaris spp., Baylisascaris spp., Brugia malayi, Brugia timori, Wuchereria bancrofti, Loa loa, Mansonella streptocerca, Mansonella ozzardi, Mansonella perstans, Onchocerca volvulus, Dirofilaria immitis and other Dirofilaria spp., Dracunculus medinensis, Ancylostoma duodenale), Ancyclostoma celanicum, and other Ancylostoma spp., Necatora americanus and other Necator spp., Angriostrongylus spp., Uncinaria stenocephala, Bunostomum phlebotomum, Enterobius vermicularis, Enterobius gregorii and other Enterobius spp., Strongyloides stercoralis, Strongyloides fuelleborni, Strongyloides papillosus, Strongyloides ransomi and other Strongyloides spp.), Thelazia californiensis, Thelazia callipaeda, Trichuris trichiura, Trichuris vulpis, Trichinella spiralis, Trichinella britovi, Trichinella nelson, Trichinella nativa, Toxocaracanis, Toxocara cati, Toxascaris leonina, Wuchereria bancrofti, and Haemonchus contortus. Examples of parasitic flatworms include Taenia saginata, Taenia solium, Taenia multiceps, Diphyllobothrium latum, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Hymenolepis nana, Hymenolepis diminuta, Spirometra erinaceieuropaei, Schistosoma haematobium, Schistosoma mansoni mansoni), Schistosoma japonicum, Schistosoma intercalatum, Schistosoma mekongi, Fasciolopis buski, Heterophyesheterophyes, Fasciola hepatica, Fasciola gigantica, Clonorchis sinensis, Clonorchis vivirrini, Dicrocoelium dendriticum, Gastrodiscoides hominis, Metagonimus yokogawai, Metorchis conjunctus, Opisthorchisviverrine, Opisthorchis felis felineus, Paragonimus westermani, Paragonimus africanus, Paragonimus spp., Echinostoma echinatum, and Trichobilharzia regenti.

Endoparasitic protozoan invertebrates include Axanthamoeba spp., Balamuthia mandrillaris, Babesia divergens, Babesia bigemina, Babesia equi, Babesia microfti, Babesia duncani, Balantidium coli, Blastocystis spp., Cryptosporidium spp., Cyclospora cayetanensis, Dientamoeba fragili, Entamoeba histolytica, Giardia lamblia, and Cyclospora cayetanensis. lamblia, Isosporabelli, Leishmania spp., Naegleria fowleri, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium knowlesi, Rhinosporidium seeberi, Sarcosystis spp., Toxoplasma gondii, Trichomonas vaginalis, Trypanosoma brucei, Trypanosoma cruzi.

As used herein, the terms "treat" or "treating" refer to the preventive or therapeutic treatment of a disease or disorder (e.g., an infectious disease, cancer, poisoning, or allergic reaction) of a subject. The effect of treatment can include reversing, alleviating, reducing the severity of the disease, curing the disease, inhibiting the progression of the disease, reducing the likelihood of recurrence of the disease, or one or more symptoms or manifestations of the disease or disorder, stabilizing (i.e., not worsening) the state of the disease or disorder, and/or preventing the spread of the disease or disorder, as compared to the state and/or condition of the disease or disorder in the absence of therapeutic treatment. Embodiments include treating plants to control diseases or adverse conditions caused by or associated with invertebrate pests or microbial (e.g., bacterial, fungal, oomycete, or viral) pathogens. Embodiments include treating plants to increase the innate defenses or immune capacity of the plant to tolerate pest or pathogen pressure.

As used herein, the term "termination element" is a moiety that terminates the translation of a coding sequence in a circular or linear polyribonucleotide, such as a nucleic acid sequence.

As used herein, the term "translation efficiency" is the rate or amount of protein or peptide production from ribonucleotide transcripts. In some embodiments, translation efficiency can be expressed as the amount of protein or peptide produced from a given amount of transcripts encoding a protein or peptide, such as within a given time period, such as in a given translation system (e.g., a eukaryotic system such as a eukaryotic cell).

As used herein, the term "translation initiation sequence" is a nucleic acid sequence that initiates translation of a coding sequence in a circular or linear polyribonucleotide.

As used herein, the term "therapeutic polypeptide" refers to a polypeptide that provides some therapeutic benefit when administered to a subject or expressed in a subject. In embodiments, the therapeutic polypeptide is used to treat or prevent a disease, disorder, or condition in a subject by administering the therapeutic polypeptide to the subject or by expressing the therapeutic polypeptide in the subject. In alternative embodiments, the therapeutic polypeptide is expressed in a cell and the cell is administered to a subject to provide a therapeutic benefit.

As used herein, "vector" means a section of DNA, which is synthesized (e.g., using PCR), or taken from a foreign DNA fragment that can or has been inserted into a virus, plasmid, or a cell of a higher organism for cloning and/or expression purposes. In certain embodiments, the vector can be stably maintained in an organism. The vector may include, for example, an origin of replication, a selectable marker or a reporter gene, such as antibiotic resistance or GFP, and/or a multiple cloning site (MCS). The term includes linear DNA fragments (e.g., PCR products, linearized plasmid fragments), plasmid vectors, viral vectors, clay mids, bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), etc. In one embodiment, the vector provided herein includes a multiple cloning site (MCS). In another embodiment, the vector provided herein does not include an MCS.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are intended to illustrate one or more features, aspects, or embodiments of the present disclosure and are not intended to be limiting.

1 is a schematic diagram depicting the design of an exemplary DNA construct to produce a ligase-compatible linear RNA and subsequent circularization by contacting the ligase-compatible linear RNA with an RNA ligase in a eukaryotic host cell.

2 is a schematic diagram depicting transcription of a DNA construct to produce a ligase-compatible linear RNA and transcription of a DNA construct to produce an RNA ligase, and subsequent circularization by contacting the ligase-compatible linear RNA with a heterologous RNA ligase in a eukaryotic host cell.

Fig. 3 shows the PCR amplification of RNA samples, which shows that circularized RNA is successfully produced in E. coli. Single bands indicate the expression of linear precursors and the correct ribozyme processing of predicted "unit length" amplicon. The ladder-like pattern indicates circularization, and higher molecular weight bands are observed, which indicates that due to the amplification twice around the circularized RNA molecule, an amplicon of twice unit length is caused. Two constructs were tested: min1 (the length after "unit length" or ribozyme processing is 275nt; twice unit length is 550nt) and min2 ("unit length" is 128nt; twice unit length is 256nt). Lane 1: min1, in vitro transcription, no ligase. Lane 2: min2, in vitro transcription, no ligase. Lane 3: min1, in vitro transcription, with RtcB ligase. Lane 4: min2, in vitro transcription, with RtcB ligase. Lane 5: min1, in vivo transcription in E. coli. Lane 6: min2, in vivo transcription in E. coli.

Fig. 4 shows RT-PCR analysis of total RNA of transformed maize and Arabidopsis cells sampled 6h and 16h after transformation. Lane 1: cells transformed with "min1" construct (unit length = 275nt; two-fold unit length = 550). Lane 2: cells transformed with Nanoluc construct. Lane 3: cells transformed with "min2" construct (unit length = 128nt; two-fold unit length = 256nt). Lanes 1 and 3 show characteristic ladder-like banding patterns, which indicate that linear RNA precursors are successfully circularized in vivo. Ladder-like banding patterns are not clearly observed in RNA from cells transformed with Nanoluc constructs; secondary structure prediction indicates that this may be due to the separation of Nanoluc sequences (823nt) and min2-ike sequences caused by endonuclease cleavage at the single-stranded region of RNA. See Examples 18, 19, 20 and 28.

FIG. 5 shows RT-PCR analysis of total RNA from transformed yeast (Saccharomyces cerevisiae) cells. Lanes 1-4: samples subjected to RT and PCR. Lanes 5-8: PCR samples not subjected to RT (negative control). Lanes 1 and 5: wild-type yeast (negative control). Lanes 2 and 6: yeast transformed with Nanoluc construct. Lanes 3 and 7: yeast transformed with "min1" construct. Lanes 4 and 8: yeast transformed with "min2" construct. Lanes 3 and 4 show a characteristic ladder-like banding pattern, which indicates that the linear RNA precursor is successfully circularized in vivo. See Examples 18, 23, and 28.

Figure 6 shows RT-PCR analysis of total RNA from transformed SF9 (Spodoptera frugiperda) insect cells. Lanes 1-5: samples subjected to RT and PCR. Lanes 6-10: PCR samples not subjected to RT (negative control). Lanes 1 and 6: non-transfected SF9 (negative control). Lanes 2 and 7: SF9 cells transformed with an empty bacmid vector (negative control). Lanes 3 and 8: SF9 cells transformed with the Nanoluc construct. Lanes 4 and 9: SF9 cells transformed with the "min1" construct. Lanes 5 and 10: SF9 cells transformed with the "min2" construct. Lanes 4 and 5 show a characteristic ladder-like banding pattern, which indicates successful circularization of the linear RNA precursor in vivo. See Examples 18, 25, 26 and 28.

FIG. 7 shows RT-PCR analysis of total RNA from transformed HeLa and HEK 293T (Homo sapiens) human cells. Leftmost lane: RNA size ladder. The gel shows samples of repeated transformation experiments for each DNA construct as indicated by the label. Negative controls are untransformed HeLa and HEK 293T cells, respectively. Lanes from HeLa or HEK 293T cells transformed with "min1" constructs or with "min2" constructs show characteristic ladder-like banding patterns, indicating successful circularization of linear RNA precursors in vivo. See Examples 18, 27, and 28.

DETAILED DESCRIPTION

In general, the present disclosure provides compositions and methods for producing, purifying, and using circular RNA from eukaryotic systems.

Polynucleotide

The present disclosure features cyclic polyribonucleotide compositions and methods of making cyclic polyribonucleotides.

In an embodiment, cyclic polyribonucleotide is produced from linear polyribonucleotide (for example, by connecting the compatible end of the ligase of linear polyribonucleotide).In an embodiment, linear polyribonucleotide is transcribed from polydeoxyribonucleotide template (for example, carrier, linearization carrier or cDNA).Therefore, the characteristics of present disclosure are polydeoxyribonucleotide, linear polyribonucleotide and cyclic polyribonucleotide compositions that can be used for producing cyclic polyribonucleotide.

Template polydeoxyribonucleotide

The present disclosure is characterized by the polydeoxyribonucleotides for preparing circular RNA. The polydeoxyribonucleotides include the following items operably connected in the 5' to 3' direction: (A) 5' self-cleaving ribozyme; (B) 5' annealing zone; (C) polyribonucleotide load; (D) 3' annealing zone; and (E) 3' self-cleaving ribozyme. In an embodiment, the polydeoxyribonucleotides include, for example, other elements outside or between any one of element (A), (B), (C), (D) and (E). In an embodiment, as described herein, any one of element (A), (B), (C), (D) and/or (E) is separated by a spacer sequence. Fig. 1 provides the design of exemplary template polydeoxyribonucleotides.

In embodiments, the polydeoxyribonucleotide is, for example, a circular DNA vector, a linearized DNA vector, or linear DNA (eg, cDNA (eg, generated from a DNA vector)).

In some embodiments, the polydeoxyribonucleotide further comprises an RNA polymerase promoter operably linked to a sequence encoding a linear RNA as described herein. In embodiments, the RNA polymerase promoter is heterologous to the sequence encoding the linear RNA. In some embodiments, the RNA polymerase promoter is a T7 promoter, a T6 promoter, a T4 promoter, a T3 promoter, an SP6 viral promoter, or an SP3 promoter.

In some embodiments, the polydeoxyribonucleotide comprises a multiple cloning site (MCS).

In some embodiments, the polydeoxyribonucleotides are used to generate circular RNAs ranging in size from about 100 to about 20,000 nucleotides. In some embodiments, the circular RNA is at least 100, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, or 5,000 nucleotides in size. In some embodiments, the circular RNA is no more than 20,000, 15,000, 10,000, 9,000, 8,000, 7,000, 6,000, 5,000, or 4,000 nucleotides in size.

Precursor linear polyribonucleotide

The characteristics of the present disclosure are also to include the following linear polyribonucleotides (for example, precursor linear polyribonucleotides) operably connected in the 5' to 3' direction: (A) 5' self-cleaving ribozyme; (B) 5' annealing zone; (C) polyribonucleotide cargo; (D) 3' annealing zone; and (E) 3' self-cleaving ribozyme. The linear polyribonucleotide may include, for example, other elements outside or between any one of element (A), (B), (C), (D) and (E). For example, any one of element (A), (B), (C), (D) and/or (E) may be separated by a spacer sequence, as described herein.

In certain embodiments, provided herein are methods for generating a precursor linear RNA by transcription in a eukaryotic system (e.g., in vivo transcription) using a polydeoxyribonucleotide provided herein (e.g., a vector, a linearized vector, or a cDNA) as a template (e.g., a vector, a linearized vector, or a cDNA in which an RNA polymerase promoter is placed upstream of a region encoding a linear RNA as provided herein).

Fig. 2 is a schematic diagram describing an exemplary process for producing circular RNA from precursor linear RNA.For example, polydeoxyribonucleotide templates can be transcribed to produce precursor linear RNA.When expressing, under suitable conditions, and not in a particular order, 5' and 3' self-cleaving ribozymes each undergo cleavage reaction, thereby producing ligase-compatible ends (for example, 5'-hydroxyl and 2', 3'-cyclic phosphoric acid) and 5' and 3' annealing zones make free ends approach.Therefore, precursor linear polyribonucleotides produce ligase-compatible polyribonucleotides, which can be connected (for example, in the presence of ligase) to produce cyclic polyribonucleotides.

Ligase-compatible linear polyribonucleotide

The present disclosure is also characterized by including the following linear polyribonucleotides (e.g., ligase-compatible linear polyribonucleotides) operably connected in the 5' to 3' direction: (B) 5' annealing zone; (C) polyribonucleotide load; and (D) 3' annealing zone. The linear polyribonucleotides may include, for example, other elements outside or between any one of element (B), (C) and (D). For example, as described herein, any element (B), (C) and/or (D) may be separated by a spacer sequence.

In certain embodiments, the ligase-compatible linear polyribonucleotide comprises a free 5'-hydroxyl group. In certain embodiments, the ligase-compatible linear polyribonucleotide comprises a free 2', 3'-cyclic phosphate.

In some embodiments, and under appropriate conditions, the 3' annealing region and the 5' annealing region promote association of the free 3' and 5' ends (eg, through partial or full complementarity, such as hybridization, that results in a thermodynamically favorable association).

In some embodiments, the proximity of a free hydroxyl group at the 5' end to a free 2',3'-cyclic phosphate at the 3' end facilitates recognition by a ligase, thereby improving the efficiency of cyclization.

Cyclic polyribonucleotide

In some embodiments, the present disclosure provides circular RNA.

In some embodiments, the circular RNA comprises a first annealing region, a polynucleotide cargo, and a second annealing region. In some embodiments, the first annealing region is connected to the second annealing region to form a circular polyribonucleotide.

In some embodiments, the circular RNA is produced by a polydeoxyribonucleotide template, a precursor linear RNA, and/or a ligase-compatible linear RNA as described herein (see, e.g., FIG. 2 ). In some embodiments, the circular RNA is produced by any of the methods described herein.

In some embodiments, the cyclic polyribonucleotide is at least about 20 nucleotides, at least about 30 nucleotides, at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides.

In certain embodiments, this cyclic polyribonucleotide has enough sizes to hold the binding site of ribosome.In certain embodiments, the size of cyclic polyribonucleotide is the length that is enough to encode useful polypeptide, for example, at least 20,000 nucleotide, at least 15,000 nucleotide, at least 10,000 nucleotide, at least 7,500 nucleotide, at least 5,000 nucleotide, at least 4,000 nucleotide, at least 3,000 nucleotide, at least 2,000 nucleotide, at least 1,000 nucleotide, at least 500 nucleotide, at least 1400 nucleotide, at least 300 nucleotide, at least 200 nucleotide or at least 100 nucleotide.

In certain embodiments, this cyclic polyribonucleotide comprises one or more elements described in other places of this paper.In certain embodiments, these elements can be separated from each other by spacer sequence.In certain embodiments, these elements can be separated from each other by 1 ribonucleotide, 2 nucleotide, about 5 nucleotide, about 10 nucleotide, about 15 nucleotide, about 20 nucleotide, about 30 nucleotide, about 40 nucleotide, about 50 nucleotide, about 60 nucleotide, about 80 nucleotide, about 100 nucleotide, about 150 nucleotide, about 200 nucleotide, about 250 nucleotide, about 300 nucleotide, about 400 nucleotide, about 500 nucleotide, about 600 nucleotide, about 700 nucleotide, about 800 nucleotide, about 900 nucleotide, about 1000 nucleotide, about 1kb at most, at least about 1000 nucleotide or the nucleotide of any amount therebetween.In certain embodiments, one or more elements are adjacent to each other, for example, lack spacer element.

In certain embodiments, cyclic polyribonucleotide can comprise one or more repeat elements described in other places of this paper.In certain embodiments, cyclic polyribonucleotide comprises one or more modifications described in other places of this paper.In one embodiment, circular RNA contains at least one nucleoside modification.In one embodiment, the nucleoside of up to 100% circular RNA is modified.In one embodiment, at least one nucleoside modification is uridine modification or adenosine modification.

As the result of its cyclization, cyclic polyribonucleotide may include some features that make it different from linear RNA.For example, as compared with linear RNA, cyclic polyribonucleotide is less likely to be degraded by exonuclease.Like this, cyclic polyribonucleotide is more stable than linear RNA, especially when hatched in the presence of exonuclease.The stability of the increase of cyclic polyribonucleotide compared with linear RNA makes cyclic polyribonucleotide more useful as the cell transformation reagent producing polypeptide, and compared with linear RNA, storage is easier and the time is longer.The stability of the cyclic polyribonucleotide treated with exonuclease can be tested using the method of this area standard, and these methods determine whether RNA degradation (for example, by gel electrophoresis) has occurred.In addition, different from linear RNA, when cyclic polyribonucleotide and phosphatase (such as calf intestine phosphatase) were hatched together, cyclic polyribonucleotide was less likely to dephosphorylate.

Ribozyme

Polynucleotide compositions as described herein may include one or more self-cleaving ribozymes, such as one or more self-cleaving ribozymes as described herein. Ribozymes are catalytic RNA or the catalytic region of RNA. Self-cleaving ribozymes are ribozymes that can catalyze the cleavage reaction that occurs at the nucleotide site in the ribozyme sequence itself or at the end of the ribozyme sequence itself.

Exemplary self-cleaving ribozymes are known in the art and/or provided herein. Exemplary self-cleaving ribozymes include hammerhead, hairpin, hepatitis delta virus ribozyme (HDV), Varkud satellite (VS), glmS ribozyme, torsion ribozyme, torsion sister ribozyme, axe ribozyme and pistol ribozyme. Additional exemplary self-cleaving ribozymes are described below. In some embodiments, the 5' self-cleaving ribozyme is a hammerhead ribozyme.

In some embodiments, the polyribonucleotides of the present disclosure include a first (e.g., 5') self-cleaving ribozyme. In some embodiments, the ribozyme is selected from any ribozyme described herein. In some embodiments, the polyribonucleotides of the present disclosure include a second (e.g., 3') self-cleaving ribozyme. In some embodiments, the ribozyme is selected from any ribozyme described herein.

In some embodiments, the 5' and 3' self-cleaving ribozymes share at least 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. In some embodiments, the 5' and 3' self-cleaving ribozymes are from the same self-cleaving ribozyme family. In some embodiments, the 5' and 3' self-cleaving ribozymes share 100% sequence identity.

In some embodiments, the 5' and 3' self-cleaving ribozymes share less than 100%, 99%, 95%, 90%, 85%, or 80% sequence identity. In some embodiments, the 5' and 3' self-cleaving ribozymes are not from the same family of self-cleaving ribozymes.

In certain embodiments, the cutting of this 5' self-cleaving ribozyme produces the free 5'-hydroxyl residue on the corresponding linear polyribonucleotide.In certain embodiments, this 5' self-cleaving ribozyme can be positioned at the site of 10 ribonucleotides in the 3' end of this 5' self-cleaving ribozyme or be positioned at the site of 3' end of this 5' self-cleaving ribozyme and carry out self-cleavage.

In certain embodiments, the cutting of this 3' self-cleaving ribozyme produces the free 3'-hydroxyl residue on the corresponding linear polyribonucleotide.In certain embodiments, this 3' self-cleaving ribozyme can be positioned at the site of 10 ribonucleotides in the 5' end of this 3' self-cleaving ribozyme or be positioned at the site of the 5' end of this 3' self-cleaving ribozyme and carry out self-cleavage.

The following are exemplary self-cleaving ribozymes contemplated by the present disclosure. This list should not be considered to limit the scope of the present disclosure.

The following self-cleaving ribozyme families were identified using RFam. RFam is a public database that contains extensive annotations to non-coding RNA elements and sequences, and is in principle an RNA analogue of the PFam database that manages protein family members. The distinctive feature of the RFam database is that in combination with primary sequence information, RNA secondary structure is the main predictor of family members. Non-coding RNA is divided into multiple families based on evolution from a common ancestor. These evolutionary relationships are determined by: establishing a common secondary structure for the putative RNA family, and then performing a specialized version of the multiple sequence alignment.

Twist: Twist ribozymes (e.g., Twist P1, P5, P3) are believed to be members of a family of small self-cleaving ribozymes that include hammerhead ribozymes, hairpin ribozymes, hepatitis delta virus (HDV) ribozymes, Varkud satellite (VS) ribozymes, and glmS ribozymes. Twist ribozymes produce 2',3'-cyclic phosphate and 5' hydroxyl products. For examples of Twist P1 ribozymes, see http://rfam.xfam.org/family/RF03160; for examples of Twist P3 ribozymes, see http://rfam.xfam.org/family/RF03154; and for examples of Twist P5 ribozymes, see http://rfam.xfam.org/family/RF02684.

Twister Sister: Twister Sister ribozymes (TS) are self-cleaving ribozymes with structural similarity to the twister ribozyme family. The catalytic products are ring 2', 3' phosphate and 5'-hydroxyl group. For examples of twister sister ribozymes, see http://rfam.xfam.org/family/RF02681.

Ax: The Ax ribozyme is a self-cleaving ribozyme discovered by bioinformatics analysis. For examples of Ax ribozymes, see http://rfam.xfam.org/family/RF02678.

HDV: Hepatitis D virus (HDV) ribozyme is a self-cleaving ribozyme in the hepatitis D virus. For examples of HDV ribozymes, see http://rfam.xfam.org/family/RF00094.

Pistol ribozymes: Pistol ribozymes are self-cleaving ribozymes. Pistol ribozymes were discovered by comparative genomic analysis. Mass spectrometry revealed that the product contains 5'-hydroxyl and 2',3'-cyclic phosphate functional groups. For examples of pistol ribozymes, see http://rfam.xfam.org/family/RF02679.

HHR type 1: Hammerhead ribozymes are self-cleaving ribozymes that catalyze reversible cleavage and ligation reactions at specific sites within RNA molecules. For examples of HHR type 1 ribozymes, see http://rfam.xfam.org/family/RF00163.

HHR type 2: Hammerhead ribozymes are self-cleaving ribozymes that catalyze reversible cleavage and ligation reactions at specific sites within RNA molecules. For examples of HHR type 2 ribozymes, see http://rfam.xfam.org/family/RF02276.

HHR type 3: Hammerhead ribozymes are self-cleaving ribozymes that catalyze reversible cleavage and ligation reactions at specific sites within RNA molecules. These RNA structural motifs are found throughout nature. For examples of HHR type 3 ribozymes, see http://rfam.xfam.org/family/RF00008.

HH9: Hammerhead ribozymes are self-cleaving ribozymes that catalyze reversible cleavage and ligation reactions at specific sites within RNA molecules. For examples of HH9 ribozymes, see http://rfam.xfam.org/family/RF02275.

HH10: Hammerhead ribozymes are self-cleaving ribozymes that catalyze reversible cleavage and ligation reactions at specific sites within RNA molecules. For examples of HH10 ribozymes, see http://rfam.xfam.org/family/RF02277.

glmS: The glucosamine-6-phosphate riboswitch ribozyme (glmS ribozyme) is an RNA structure that resides in the 5' untranslated region (UTR) of the mRNA transcript of the glmS gene. For examples of glmS ribozymes, see http://rfam.xfam.org/family/RF00234.

GIR1: Lariat capping ribozyme (formerly known as GIR1 branching ribozyme) is an approximately 180 nt ribozyme with significant similarities to group I ribozymes. For examples of GIR1 ribozymes, see http://rfam.xfam.org/family/RF01807.

CPEB3: The mammalian CPEB3 ribozyme is a self-cleaving noncoding RNA located in the second intron of the CPEB3 gene. For examples of CPEB ribozymes, see http://rfam.xfam.org/family/RF00622.

drz-Agam 1 and drz-Agam 2: The drz-Agam-1 and drz-Agam 2 ribozymes were discovered using restriction structure descriptors and are very similar to the HDV and CPEB3 ribozymes. For an example of the drz-Agam 1 ribozyme, see http://rfam.xfam.org/family/RF01787 and for an example of the drz-Agam 2 ribozyme, see http://rfam.xfam.org/family/RF01788.

Hairpin: A hairpin ribozyme is a small portion of RNA that can act as a ribozyme. Similar to the hammerhead ribozyme, it is found in RNA satellites of plant viruses. For examples of hairpin ribozymes, see http://rfam.xfam.org/family/RF00173.

RAGATH-1: RNA structural motifs discovered using bioinformatics algorithms. These RNAs contain strong similarities to known ribozymes such as, but not limited to, hammerhead and HDV ribozymes. For examples of RAGATH-1 ribozymes, see http://rfam.xfam.org/family/RF03152.

RAGATH-5: RNA structural motifs discovered using bioinformatics algorithms. These RNAs contain strong similarities to known ribozymes such as, but not limited to, hammerhead and HDV ribozymes. For examples of RAGATH-5 ribozymes, see http://rfam.xfam.org/family/RF02685.

RAGATH-6: RNA structural motifs discovered using bioinformatics algorithms. These RNAs contain strong similarities to known ribozymes such as, but not limited to, hammerhead and HDV ribozymes. For examples of RAGATH-6 ribozymes, see http://rfam.xfam.org/family/RF02686.

RAGATH-13: RNA structural motifs discovered using bioinformatics algorithms. These RNAs contain strong similarities to known ribozymes such as, but not limited to, hammerhead and HDV ribozymes. For examples of RAGATH-13 ribozymes, see http://rfam.xfam.org/family/RF02688.

In some embodiments, the self-cleaving ribozyme is a ribozyme described herein (e.g., from a class described herein), or a catalytically active fragment or portion thereof. In some embodiments, the ribozyme comprises a sequence at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 38-585, or its corresponding RNA equivalent. In some embodiments, the self-cleaving ribozyme is a ribozyme described herein (e.g., from a class described herein), or a catalytically active fragment or portion thereof. In some embodiments, the ribozyme comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 38-585, or its corresponding RNA equivalent. In some embodiments, the ribozyme comprises a sequence at least 95%, 96%, 97%, 98%, or 99% identical to any one of SEQ ID NO: 38-585, or its corresponding RNA equivalent. In some embodiments, the ribozyme comprises a sequence of any one of SEQ ID NO: 38-585. In embodiments, the self-cleaving ribozyme is a fragment of the ribozyme of any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, e.g., a fragment containing at least 20 contiguous nucleotides (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, or 60 contiguous nucleotides) of the complete ribozyme sequence and having at least 30% (e.g., at least about 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%) of the catalytic activity of the complete ribozyme. In some embodiments, the ribozyme comprises a catalytic region (e.g., a region capable of self-cleavage) of any one of SEQ ID NOs: 38-585, or a corresponding RNA equivalent thereof, wherein the region is at least 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, or 50 nucleotides in length, or the region is between 10-200 nucleotides, 10-100 nucleotides, 10-50 nucleotides, 10-30 nucleotides, 10-200 nucleotides, 20-100 nucleotides, 20-50 nucleotides, or 20-30 nucleotides.

Annealing area

The polynucleotide compositions described herein can include two or more annealing regions, such as two or more annealing regions described herein. Annealing regions or pairs of annealing regions are those containing portions with a high degree of complementarity that promote hybridization under appropriate conditions.

The annealing zone includes at least the complementary region described below. The high complementarity of the complementary region promotes the association of the annealing zone pair. In the case where the first annealing zone (e.g., 5' annealing zone) is located at or near the 5' end of the linear RNA and the second annealing zone (e.g., 3' annealing zone) is located at or near the 3' end of the linear RNA, the association of these annealing zones brings the 5' and 3' ends closer together. In some embodiments, this association facilitates the circularization of the linear RNA by the connection of the 5' and 3' ends.

In an embodiment, the annealing zone further includes a non-complementary region as described below. The non-complementary region can be added to the complementary region to allow the end of the RNA to remain flexible, unstructured, or less structured than the complementary region. The availability of free 5' and 3' ends of flexible and/or single strands supports connection, and therefore supports cyclization efficiency.

In some embodiments, each annealing region comprises 2 to 100 ribonucleotides (e.g., 5 to 80, 5 to 50, 5 to 30, 5 to 20, 10 to 100, 10 to 80, 10 to 50, or 10 to 30 ribonucleotides). In some embodiments, the 5' annealing region has 2 to 100 ribonucleotides (e.g., 2 to 100, 2 to 80, 2 to 50, 2 to 30, 2 to 20, 5 to 100, 5 to 80, 5 to 50, 5 to 30, 5 to 20, 10 to 100, 10 to 80, 10 to 50, or 10 to 30 ribonucleotides). In some embodiments, the 3' annealing region has 2 to 100 ribonucleotides (e.g., 2 to 100, 2 to 80, 2 to 50, 2 to 30, 2 to 20, 5 to 100, 5 to 80, 5 to 50, 5 to 30, 5 to 20, 10 to 100, 10 to 80, 10 to 50, or 10 to 30 ribonucleotides).

Complementary region

A complementary region is a region that, under appropriate conditions, is favorable for associating with a corresponding complementary region. For example, a pair of complementary regions may share a high degree of sequence complementarity (e.g., a first complementary region is at least in part the reverse complement of a second complementary region). When two complementary regions associate (e.g., hybridize), they may form a highly structured secondary structure, such as a stem or stem-loop.

In some embodiments, the polyribonucleotide comprises a 5' complementary region and a 3' complementary region. In some embodiments, the 5' annealing region comprises a 5' complementary region having between 2 and 50 ribonucleotides (e.g., 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50 ribonucleotides); and the 3' annealing region comprises a 3' complementary region having between 2 and 50 ribonucleotides (e.g., 2-40, 2-30, 2-20, 2-10, 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20, or 20-50).

In some embodiments, the 5' complementary region and the 3' complementary region have between 50% and 100% sequence complementarity (eg, between 60%-100%, 70%-100%, 80%-100%, 90%-100%, or 100% sequence complementarity).

In some embodiments, the 5' complementary region and the 3' complementary region have a binding free energy of less than -5 kcal/mol (eg, less than -10 kcal/mol, less than -20 kcal/mol, or less than -30 kcal/mol).

In some embodiments, the 5' complementary region and the 3' complementary region have a combined Tm of at least 10°C, at least 15°C, at least 20°C, at least 30°C, at least 40°C, at least 50°C, at least 60°C, at least 70°C, at least 80°C, or at least 90°C.

In some embodiments, the 5' complementary region and the 3' complementary region include at least one but not more than 10 mismatches, such as 10, 9, 8, 7, 6, 5, 4, 3, or 2 mismatches, or 1 mismatch (i.e., when the 5' complementary region and the 3' complementary region hybridize to each other). For example, a mismatch can be a nucleotide in the 5' complementary region and a nucleotide in the 3' complementary region that are opposite to each other (i.e., when the 5' complementary region and the 3' complementary region hybridize) but do not form a Watson-Crick base pair. For example, a mismatch can be an unpaired nucleotide that forms a kink or bulge in the 5' complementary region or the 3' complementary region. In some embodiments, the 5' complementary region and the 3' complementary region do not include any mismatches.

Non-complementary region

A non-complementary region is a region that is not favorable for associating with a corresponding non-complementary region under appropriate conditions. For example, a pair of non-complementary regions may share a low degree of sequence complementarity (e.g., the first non-complementary region is not the reverse complement of the second non-complementary region). When two non-complementary regions are in proximity, they do not form a highly structured secondary structure, such as a stem or stem loop.

In certain embodiments, the polyribonucleotide comprises a 5' non-complementary region and a 3' non-complementary region. In certain embodiments, the 5' non-complementary region has ribonucleotides between 5 and 50 (e.g., 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20 or 20-50 ribonucleotides). In certain embodiments, the 3' non-complementary region has ribonucleotides between 5 and 50 (e.g., 5-40, 5-30, 5-20, 5-10, 10-50, 10-40, 10-30, 10-20 or 20-50 ribonucleotides).

In some embodiments, the 5' non-complementary region is located 5' to the 5' complementary region (e.g., between the 5' self-cleaving ribozyme and the 5' complementary region). In some embodiments, the 3' non-complementary region is located 3' to the 3' complementary region (e.g., between the 3' complementary region and the 3' self-cleaving ribozyme).

In some embodiments, the 5' non-complementary region and the 3' non-complementary region have between 0% and 50% sequence complementarity (eg, between 0%-40%, 0%-30%, 0%-20%, 0%-10%, or 0% sequence complementarity).

In some embodiments, the 5' non-complementary region and the 3' non-complementary region have a binding free energy greater than -5 kcal/mol.

In some embodiments, the 5' complementary region and the 3' complementary region have a binding Tm of less than 10°C.

In some embodiments, the 5' non-complementary region and the 3' non-complementary region comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mismatches.

Polyribonucleotide loading

The polyribonucleotide cargo described herein includes any sequence containing at least one polyribonucleotide.

For example; The polyribonucleotide cargo can comprise at least about 40 nucleotides, at least about 50 nucleotides, at least about 75 nucleotides, at least about 100 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 1,000 nucleotides, at least about 2,000 nucleotides, at least about 5,000 nucleotides, at least about 6,000 nucleotides, at least about 7,000 nucleotides, at least about 8,000 nucleotides, at least about 9,000 nucleotides, at least about 10,000 nucleotides, at least about 12,000 nucleotides, at least about 14,000 nucleotides, at least about 15,000 nucleotides, at least about 16,000 nucleotides, at least about 17,000 nucleotides, at least about 18,000 nucleotides, at least about 19,000 nucleotides, or at least about 20,000 nucleotides. In some embodiments, the polyribonucleotide cargo comprises 1-20,000 nucleotides, 1-10,000 nucleotides, 1-5,000 nucleotides, 100-20,000 nucleotides, 100-10,000 nucleotides, 100-5,000 nucleotides, 500-20,000 nucleotides, 500-10,000 nucleotides, 500-5,000 nucleotides, 1,000-20,000 nucleotides, 1,000-10,000 nucleotides, or 1,000-5,000 nucleotides.

In an embodiment, this polyribonucleotide loading thing comprises one or more coding (or expression) sequences, wherein each coding sequence encoding polypeptide. In an embodiment, this polyribonucleotide loading thing comprises one or more non-coding sequences. In an embodiment, this polyribonucleotide loading thing is completely composed of one or more non-coding sequences. In an embodiment, this polyribonucleotide loading thing comprises a combination of coding (or expression) sequence and non-coding sequence.

In an embodiment, the polyribonucleotide load includes multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10) of a single coding sequence. For example, the polyribonucleotide may include multiple copies of a sequence encoding a single protein. In other embodiments, the polyribonucleotide load includes at least one copy (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10 copies) of each coding sequence in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) different coding sequences. For example, the polynucleotide load may include two copies of a first coding sequence and three copies of a second coding sequence.

In an embodiment, the polyribonucleotide cargo comprises one or more copies of at least one non-coding sequence. In an embodiment, at least one non-coding RNA sequence comprises at least one RNA selected from the group consisting of: RNA aptamers, long non-coding RNA (lncRNA), transfer RNA derived fragments (tRF), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and Piwi interacting RNA (piRNA); or a fragment of any one of these RNAs. In an embodiment, at least one non-coding RNA sequence comprises at least one regulatory RNA, such as at least one RNA selected from the group consisting of: microRNA (miRNA) or miRNA precursor (see, e.g., U.S. Pat. Nos. 8,395,023, 8,946,511, 8,410,334 or 10,570,414), microRNA recognition site (see, e.g., U.S. Pat. Nos. 8,334,430 or 10,876,126), small interfering RNA (siRNA) or siRNA precursor (such as but not limited to RNA sequence forming RNA hairpin or RNA stem loop or RNA stem) (see, e.g., U.S. Pat. Nos. 8,404,927 or 10,378,012), small RNA ... point (see, e.g., U.S. Pat. No. 9,139,838), trans-acting siRNA (ta-siRNA) or ta-siRNA precursor (see, e.g., U.S. Pat. No. 8,030,473), phase sRNA or phase RNA precursor (see, e.g., U.S. Pat. No. 8,404,928), phase sRNA recognition site (see, e.g., U.S. Pat. No. 9,309,512), miRNA decoy (see, e.g., U.S. Pat. Nos. 8,946,511 or 10,435,686), miRNA cleavage blocker (see, e.g., U.S. Pat. No. 9,040,774), cis-acting riboswitch, trans-acting riboswitch, and ribozyme; all of these cited U.S. patents are incorporated herein in their entirety. In an embodiment, at least one non-coding RNA sequence includes an RNA sequence that is complementary or antisense to a target sequence (e.g., a target sequence encoded by a messenger RNA or encoded by DNA of the subject's genome); such an RNA sequence can be used to recognize and bind to a target sequence, e.g., by Watson-Crick base pairing. In an embodiment, the polyribonucleotide load includes multiple copies (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10) of a single non-coding sequence. For example, the polyribonucleotide may include multiple copies of a sequence encoding a single microRNA precursor or multiple copies of a guide RNA sequence. In other embodiments, the polyribonucleotide load includes at least one copy (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or even more than 10 copies) of each non-coding sequence in two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) different non-coding sequences. In an example, the polyribonucleotide load includes two copies of the first non-coding sequence and three copies of the second non-coding sequence. In another example, the polyribonucleotide load includes at least one copy of each miRNA precursor in two or more different miRNA precursors. In another example, the polyribonucleotide cargo includes (a) an RNA sequence complementary to or antisense to a target sequence, and (b) a ribozyme or an aptamer.

In certain embodiments, the cyclic polyribonucleotide prepared as described herein is used as effector in therapy and/or agriculture.Polyribonucleotide may include the RNA sequence of the polypeptide encoding the biological effect on the subject.In certain embodiments, the polyribonucleotide load comprises the RNA sequence of the nucleotide sequence encoded by the polypeptide and has codon optimized for expression in the subject.For example, the cyclic polyribonucleotide (for example, in medicine, veterinary or agricultural composition) prepared by the method described herein (for example, eukaryotic method as described herein) can be used to the subject.In another example, the cyclic polyribonucleotide prepared by the method described herein (for example, eukaryotic method as described herein) can be delivered to the cell.

In some embodiments, the cyclic polyribonucleotide includes any feature or any combination of features as disclosed in International Patent Publication No. WO 2019/118919 (hereby incorporated herein by reference in its entirety).

Peptide coding sequence

In certain embodiments, cyclic polyribonucleotide as herein described (for example, the polyribonucleotide loading thing of cyclic polyribonucleotide) comprises one or more encoding sequences, wherein each encoding sequence encoding polypeptide.In certain embodiments, this cyclic polyribonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more encoding sequences.

Each encoded polypeptide can be linear or branched. The length of the polypeptide can be from about 5 to about 40,000 amino acids, about 15 to about 35,000 amino acids, about 20 to about 30,000 amino acids, about 25 to about 25,000 amino acids, about 50 to about 20,000 amino acids, about 100 to about 15,000 amino acids, about 200 to about 10,000 amino acids, about 500 to about 5,000 amino acids, about 1,000 to about 2,500 amino acids, or any range therebetween. In some embodiments, it may be useful for polypeptides to have a length of less than about 40,000 amino acids, less than about 35,000 amino acids, less than about 30,000 amino acids, less than about 25,000 amino acids, less than about 20,000 amino acids, less than about 15,000 amino acids, less than about 10,000 amino acids, less than about 9,000 amino acids, less than about 8,000 amino acids, less than about 7,000 amino acids, less than about 6,000 amino acids. , less than about 5,000 amino acids, less than about 4,000 amino acids, less than about 3,000 amino acids, less than about 2,500 amino acids, less than about 2,000 amino acids, less than about 1,500 amino acids, less than about 1,000 amino acids, less than about 900 amino acids, less than about 800 amino acids, less than about 700 amino acids, less than about 600 amino acids, less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less.

The polypeptide included herein may include naturally occurring polypeptides or non-naturally occurring polypeptides. In some cases, the polypeptide may be a functional fragment or variant of a reference polypeptide (e.g., an enzymatically active fragment or variant of an enzyme). For example, the polypeptide may be a functionally active variant of any polypeptide described herein, such as having at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of a polypeptide described herein or a naturally occurring polypeptide in a specified region or throughout the sequence. In some cases, a polypeptide can be at least 50% (e.g., at least 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or more) identical to a protein of interest.

Some examples of polypeptides include, but are not limited to, fluorescent tags or markers, antigens, therapeutic polypeptides, or polypeptides for agricultural applications.

The therapeutic polypeptide can be a hormone, a neurotransmitter, a growth factor, an enzyme (e.g., an oxidoreductase, a metabolic enzyme, a mitochondrial enzyme, an oxygenase, a dehydrogenase, an ATP-independent enzyme, a lysosomal enzyme, a desaturase), a cytokine, an antigen-binding polypeptide (e.g., an antigen-binding antibody or antibody-like fragment, such as a single-chain antibody, a nanobody or other polypeptide containing an Ig heavy chain and/or light chain), an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an interferon, an interleukin, and a thrombolytic agent.

In some cases, the circular polyribonucleotide expresses a non-human protein.

Polypeptides for agricultural applications can be bacteriocins, lysins, antimicrobial polypeptides, antifungal polypeptides, node C-rich peptides, bacterial cell regulatory peptides, peptide toxins, pesticidal polypeptides (e.g., insecticidal polypeptides and/or nematicidal polypeptides), antigen-binding polypeptides (e.g., antigen-binding antibodies or antibody-like fragments, such as single-chain antibodies, nanobodies or other polypeptides containing Ig heavy chains and/or light chains), enzymes (e.g., nucleases, amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones, and transcription factors.

In certain embodiments, cyclic polyribonucleotides express antibodies, such as antibody fragments or a part thereof. In certain embodiments, the antibodies expressed by cyclic polyribonucleotides can be of any isotype, such as IgA, IgD, IgE, IgG, IgM. In certain embodiments, cyclic polyribonucleotides express a part of antibodies, such as light chain, heavy chain, Fc fragment, CDR (complementarity determining region), Fv fragment or Fab fragment, its other part. In certain embodiments, cyclic polyribonucleotides express one or more parts of antibodies. For example, cyclic polyribonucleotides can include more than one encoding sequence, wherein each expresses a part of antibodies, and its sum can constitute antibody. In some cases, cyclic polyribonucleotides include an encoding sequence encoding antibody heavy chain and another encoding sequence encoding antibody light chain. In some cases, when cyclic polyribonucleotides are expressed in cell (for example, eukaryotic cell) environment, light chain and heavy chain can be subjected to suitable modification, folding or other post-translational modifications to form functional antibodies.

In an embodiment, the polypeptide includes a plurality of polypeptides, for example, multiple copies of a polypeptide sequence, or a plurality of different polypeptide sequences. In an embodiment, the plurality of polypeptides are connected by a linker amino acid or a spacer amino acid.

In an embodiment, the polynucleotide payload includes a sequence encoding a signal peptide. Many signal peptide sequences have been described, for example, the Tat (twin arginine translocation) signal sequence is typically an N-terminal peptide sequence containing a consensus SRRxFLK "twin arginine" motif, which is used to translocate a folded protein containing such a Tat signal peptide across a lipid bilayer. See also, for example, the Signal Peptide Database publicly available at www[dot]signalpeptide[dot]de. Signal peptides can also be used to direct proteins to specific organelles; see, for example, the experimentally determined and computationally predicted signal peptides disclosed in the Spdb signal peptide database, which is publicly available at proline[dot]bic[dot]nus[dot]edu[dot]sg/spdb.

In an embodiment, the polynucleotide cargo includes a sequence encoding a cell penetrating peptide (CPP). Hundreds of CPP sequences have been described; see, for example, the database of cell penetrating peptides CPPsite, which is publicly available at crdd[dot]osdd[dot]net/raghava/cppsite/. An example of a commonly used CPP sequence is a polyarginine sequence, such as octa-arginine or nona-arginine, which can be fused to the C-terminus of a CGI peptide.

In an embodiment, the polynucleotide cargo comprises a sequence encoding a self-assembling peptide; see, e.g., Miki et al. (2021) Nature Communications, 21:3412, DOI: 10.1038/s41467-021-23794-6.

Therapeutic peptides

In certain embodiments, cyclic polyribonucleotides as herein described (for example, the polyribonucleotide load of cyclic polyribonucleotides) include at least one coding sequence encoding therapeutic polypeptides.Therapeutic polypeptides are polypeptides that provide some therapeutic benefits when used to a subject or expressed in a subject.Therapeutic polypeptides can be used to treat or prevent disease, disorder or illness or its symptom to a subject or to express in a subject.In certain embodiments, cyclic polyribonucleotides encode two, three, four, five, six, seven, eight, nine, ten or more therapeutic polypeptides.

In certain embodiments, the cyclic polyribonucleotide comprises a coding sequence encoding a therapeutic protein. The protein can treat a disease in a subject in need. In certain embodiments, the therapeutic protein can compensate for a mutated, under-expressed, or missing protein in a subject in need. In certain embodiments, the therapeutic protein can target a cell, tissue, or virus in a subject in need, interact with or combine with a cell, tissue, or virus in a subject in need.

The therapeutic polypeptide may be a polypeptide that can be secreted from a cell or localized in the cytoplasm, nucleus, or membrane compartment of a cell.

The therapeutic polypeptide can be a hormone, a neurotransmitter, a growth factor, an enzyme (e.g., an oxidoreductase, a metabolic enzyme, a mitochondrial enzyme, an oxygenase, a dehydrogenase, an ATP-independent enzyme, a lysosomal enzyme, a desaturase), a cytokine, a transcription factor, an antigen-binding polypeptide (e.g., an antigen-binding antibody or antibody-like fragment, such as a single-chain antibody, a nanobody or other polypeptide containing an Ig heavy chain and/or light chain), an Fc fusion protein, an anticoagulant, a blood factor, a bone morphogenetic protein, an interferon, an interleukin, a thrombolytic agent, an antigen (e.g., a tumor, a virus, or a bacterial antigen ), nucleases (e.g., nucleases, such as Cas proteins, e.g., Cas9), membrane proteins (e.g., chimeric antigen receptors (CARs), transmembrane receptors, G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), antigen receptors, ion channels, or membrane transporters), secreted proteins, gene editing proteins (e.g., CRISPR-Cas, TALENs, or zinc fingers), or gene writing proteins (see, e.g., International Patent Application Publication No. WO/2020/047124, which is incorporated herein by reference in its entirety).

In certain embodiments, therapeutic polypeptide is an antibody, for example, a full-length antibody, an antibody fragment, or a part thereof. In certain embodiments, the antibody expressed by the cyclic polyribonucleotide can be of any isotype, such as IgA, IgD, IgE, IgG, IgM. In certain embodiments, the cyclic polyribonucleotide expresses a part of an antibody, such as a light chain, a heavy chain, an Fc fragment, a CDR (complementarity determining region), an Fv fragment, or a Fab fragment, its other part. In certain embodiments, the cyclic polyribonucleotide expresses one or more parts of an antibody. For example, the cyclic polyribonucleotide can include more than one coding sequence, wherein each of which expresses a part of an antibody, and its sum can constitute an antibody. In some cases, the cyclic polyribonucleotide includes a coding sequence encoding an antibody heavy chain and another coding sequence encoding an antibody light chain. When the cyclic polyribonucleotide is expressed in a cell, the light chain and the heavy chain can be subjected to appropriate modification, folding or other post-translational modifications to form a functional antibody.

In certain embodiments, the cyclic polyribonucleotide prepared as described herein is used as effector in therapy and/or agriculture.For example, the cyclic polyribonucleotide prepared by the method described herein (for example, acellular method as described herein) (for example, in medicine, veterinary or agricultural composition) can be applied to the subject. In an embodiment, the subject is a vertebrate (for example, a mammal, a bird, a fish, a reptile or an amphibian). In an embodiment, the subject is a person. In an embodiment, the method subject is a non-human mammal. In an embodiment, the subject is a non-human mammal, such as a non-human primate (for example, a monkey, an ape), an ungulate (for example, a cattle, a buffalo, a sheep, a goat, a pig, a camel, a llama, an alpaca, a deer, a horse, a donkey), a carnivore (for example, a dog, a cat), a rodent (for example, a rat, a mouse) or a lagomorph (for example, a rabbit). In an embodiment, the subject is a bird, such as a member of the following avian taxonomic group: Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathus (e.g., ostrich, emu), Columbiformes (e.g., pigeon, pigeon), or Psittaciformes (e.g., parrot). In an embodiment, the subject is an invertebrate, such as an arthropod (e.g., insect, arachnid, crustacean), nematode, annelid, worm, or mollusk. In an embodiment, the subject is an invertebrate agricultural pest or an invertebrate parasitic on an invertebrate or vertebrate host. In an embodiment, the subject is a plant, such as angiosperm (which can be a dicot or monocot) or gymnosperm (e.g., conifer, cycad, Gnetum plant, Ginkgo), fern, horsetail plant, lycophyte, or bryophyte. In an embodiment, the subject is a eukaryotic algae (single cell or multicell). In embodiments, the subject is a plant of agricultural or horticultural importance, such as row crops, fruit-producing plants and trees, vegetables, trees, and ornamental plants (including ornamental flowers, shrubs, trees, ground covers, and turf grasses).

Plant Modified Peptides

In certain embodiments, cyclic polyribonucleotides as herein described (for example, the polyribonucleotide cargo of cyclic polyribonucleotides) include at least one coding sequence encoding plant modification polypeptides.Plant modification polypeptides refer to polypeptides that can change the genetic characteristics (for example, increase gene expression, reduce gene expression or otherwise change the nucleotide sequence of DNA or RNA) of plants in a manner that causes plant fitness to improve or reduce, epigenetic characteristics, or physiological or biochemical characteristics.In certain embodiments, cyclic polyribonucleotides encode two, three, four, five, six, seven, eight, nine, ten or more different plant modification polypeptides, or multiple copies of one or more plant modification polypeptides.Plant modification polypeptides can increase the fitness of various plants, or can be the plant modification polypeptides of targeting one or more specific plants (for example, specific species or genus of plant).

Examples of polypeptides useful herein may include enzymes (e.g., metabolic recombinases, helicases, integrases, RNases, DNases, or ubiquitinated proteins), pore-forming proteins, signaling ligands, cell-penetrating peptides, transcription factors, receptors, antibodies, nanobodies, gene editing proteins (e.g., CRISPR-Cas endonucleases, TALENs, or zinc fingers), gene writing proteins (see, e.g., International Patent Application Publication No. WO/2020/047124, which is incorporated herein by reference in its entirety), riboproteins, protein aptamers, or chaperone proteins.

Agricultural peptides

In certain embodiments, cyclic polyribonucleotides as described herein (e.g., polyribonucleotide cargoes of cyclic polyribonucleotides) include at least one coding sequence encoding an agricultural polypeptide. An agricultural polypeptide is a polypeptide suitable for agricultural use. In an embodiment, an agricultural polypeptide is applied to a plant or seed (e.g., by foliar spraying, dusting, injection, or seed coating) or applied to the environment of a plant (e.g., by soil immersion or granular soil application), causing the fitness of the plant to change. Embodiments of agricultural polypeptides include polypeptides that change the level, activity, or metabolism of one or more microorganisms that reside in a plant or non-human animal host or reside thereon, and the change causes the fitness of the host to improve. In certain embodiments, the agricultural polypeptide is a plant polypeptide. In certain embodiments, the agricultural polypeptide is an insect polypeptide. In certain embodiments, when contacted with a non-human vertebrate, an invertebrate, a microorganism, or a plant cell, the agricultural polypeptide has a biological effect.

In some embodiments, the circular polyribonucleotide encodes two, three, four, five, six, seven, eight, nine, ten or more agricultural polypeptides, or multiple copies of one or more agricultural polypeptides.

The embodiment of the polypeptide that can be used for agricultural application includes for example bacteriocin, lysin, antimicrobial peptide, the peptide rich in nodule C and bacterial cell regulatory peptide.Such polypeptide can be used for changing the level, activity or metabolism of target microorganism to improve the fitness of insect (such as, honeybee and silkworm).The embodiment of agriculturally useful polypeptide includes peptide toxin, such as those naturally produced by insect pathogenic bacteria (for example, Bacillus thuringiensis (Bacillus thuringiensis), Photorhabdus luminescens (Photorhabdus luminescens), Serratia entomophila (Serratia entomophila) or Xenorhabdus nematophila (Xenorhabdus nematophila)), as known in the art.The embodiment of agriculturally useful polypeptide includes polypeptide (including small peptides, such as cyclic dipeptides or diketopiperazine) for controlling important harmful organisms or pathogens in agriculture, such as antimicrobial polypeptides or antifungal polypeptides for controlling plant diseases, or pest-killing polypeptides (such as insects or nematodes) for controlling invertebrate pests (such as insects or nematodes), such as insect-killing polypeptides and/or nematode-killing polypeptides. Embodiments of agriculturally useful polypeptides include antibodies, nanobodies, and fragments thereof, e.g., antibodies or nanobody fragments that retain at least some (e.g., at least 10%) specific binding activity of an intact antibody or nanobody. Embodiments of agriculturally useful polypeptides include transcription factors, e.g., plant transcription factors; see, e.g., the "AtTFDB" database listing transcription factor families identified in the model plant Arabidopsis thaliana, which is publicly available at agris-knowledgebase[dot]org/AtTFDB/. Embodiments of agriculturally useful polypeptides include nucleases, e.g., exonucleases or endonucleases (e.g., Cas nucleases, such as Cas9 or Cas12a). Examples of agriculturally useful polypeptides further include cell penetrating peptides, enzymes (e.g., amylases, cellulases, peptidases, lipases, chitinases), peptide pheromones (e.g., yeast mating pheromones, invertebrate reproduction and larval signaling pheromones, see, e.g., Altstein (2004) Peptides, 25: 1373-1376).

Examples of agriculturally useful polypeptides confer beneficial agronomic traits such as herbicide tolerance, insect control, improved yield, increased fungal or oomycete resistance, increased viral resistance, increased nematode resistance, increased bacterial disease resistance, plant growth and development, improved starch yield, improved oil yield, high oil yield, improved fatty acid content, high protein yield, fruit ripening, increased animal and human nutrition, production of biopolymers, environmental stress resistance, medicinal peptides and secretable peptides, improved processing traits, improved digestibility (e.g., reduced levels of toxins or reduced levels of compounds with "anti-nutritional" properties (such as lignin, lectins, and phytates)), enzyme production, flavor, nitrogen fixation, hybrid seed yield, fiber yield, and biofuel production. Non-limiting examples of agriculturally useful polypeptides include polypeptides that confer: herbicide resistance (U.S. Pat. Nos. 6,803,501; 6,448,476; 6,248,876; 6,225,114; 6,107,549; 5,866,775; 5,804,425; 5,633,435; and 5,463,175), increased yield (U.S. Pat. Nos. RE38,446; 6,716,474; 6,663,906; 6,476,295; 6,441,277; 6,423,828; 6,399,330; 6,372,211; 6,235 ,971; 6,222,098; and 5,716,837), insect control (U.S. Pat. Nos. 6,809,078; 6,713,063; 6,686,452; 6,657,046; 6,645,497; 6,642,030; 6,639,054; 6,620,988; 6,593,293; 6,555,655; 6,538,109; 6,537,756; 6,521,442; 6,501,009; 6,468,523; 6,326,351; 6,313,378; 6,284,949; 6,281,0 16; 6,248,536; 6,242,241; 6,221,649; 6,177,615; 6,156,573; 6,153,814; 6,110,464; 6,093,695; 6,063,756; 6,063,597; 6,023,013; 5,959,09 1; 5,942,664; 5,942,658, 5,880,275; 5,763,245; 5,763,241; 10,017,549; 10,233,217; 10,487,123; 10,494,408; 10,494, 409; 10,611,806; 10,612,037; 10,669,317; 10,827,755; 11,254,950; 11,267,849; 11,130,965; 11,136,593; and 11,180,774), fungal disease resistance (U.S. Pat. Nos. 6,653,280; 6,573,361; 6,506,962; 6,316,407; 6,215,048; 5,516,671; 5,773,696; 6,121,436; 6,316,407; and 6,506,962), virus resistance ( Nos. 6,617,496; 6,608,241; 6,015,940; 6,013,864; 5,850,023; and 5,304,730), nematode resistance (U.S. Pat. No. 6,228,992), bacterial disease resistance (U.S. Pat. No. 5,516,671), plant growth and development (U.S. Pat. Nos. 6,723,897 and 6,518,488), starch yield (U.S. Pat. Nos. 6,538,181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), improved oil yield (U.S. Pat. No. 6,444,897), and improved plant growth and development (U.S. Pat. Nos. 6,723,897 and 6,518,488). ,876; 6,426,447; and 6,380,462), high oil yield (U.S. Pat. Nos. 6,495,739; 5,608,149; 6,483,008; and 6,476,295), improved fatty acid content (U.S. Pat. Nos. 6,828,475; 6,822,141; 6,770,465; 6,706,950; 6,660,849; 6,596,538; 6,589,767; 6,537,750; 6,489,461; and 6,459,018), high protein yield (U.S. Pat. No. 6,380,466), fruit ripening (U.S. Pat. No. 6,495,739; 5,608,149; 6,483,008; and 6,476,295). No. 5,512,466), increased animal and human nutrition (U.S. Pat. Nos. 6,723,837; 6,653,530; 6,5412,59; 5,985,605; and 6,171,640), biopolymers (U.S. Pat. Nos. RE37,543; 6,228,623; and U.S. Pat. Nos. 5,958,745 and 6,946,588), environmental stress resistance (U.S. Pat. No. 6,072,103), pharmaceutical peptides and secretable peptides (U.S. Pat. Nos. 6,812,379; 6,774,283; 6,140,075; and 6,080,560), improved processability shape (U.S. Patent No. 6,476,295), improved digestibility (U.S. Patent No. 6,531,648), low raffinose (U.S. Patent No. 6,166,292), industrial enzyme production (U.S. Patent No. 5,543,576), improved flavor (U.S. Patent No. 6,011,199), nitrogen fixation (U.S. Patent No. 5,229,114), hybrid seed yield (U.S. Patent No. 5,689,041), fiber yield (U.S. Patent Nos. 6,576,818; 6,271,443; 5,981,834; and 5,869,720), and biofuel production (U.S. Patent No. 5,998,700).

Exemplary Secreted Polypeptide Effectors

Exemplary secreted proteins that can be expressed are described herein, for example, in the table below.

Cytokines and Cytokine Receptors:

In some embodiments, the effectors described herein comprise a cytokine of Table 1 or a functional variant or fragment thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a protein sequence disclosed in Table 1 by reference to its UniProt ID. In some embodiments, the functional variant binds to the corresponding cytokine receptor with a Kd that is no more than 10%, 20%, 30%, 40%, or 50% higher or lower than the Kd of the corresponding wild-type cytokine for the same receptor under the same conditions. In some embodiments, the effector comprises a fusion protein comprising a first region (e.g., a cytokine polypeptide of Table 1 or a functional variant or fragment thereof) and a second heterologous region. In some embodiments, the first region is a first cytokine polypeptide of Table 1. In some embodiments, the second region is a second cytokine polypeptide of Table 1, wherein the first and second cytokine polypeptides form cytokine heterodimers with each other in wild-type cells. In some embodiments, the polypeptide of Table 1 or a functional variant thereof comprises a signal sequence, e.g., a signal sequence endogenous to the effector, or a heterologous signal sequence.

In some embodiments, the effectors described herein comprise an antibody or variant thereof that binds a cytokine from Table 1. In some embodiments, the antibody molecule comprises a signal sequence.

Table 1. Exemplary cytokines and cytokine receptors

¹ The sequence is available at the NCBI database on the World Wide Web at “ncbi.nlm.nih.gov/gene”, Maglott D et al. Gene: a gene-centered information resource at NCBI. Nucleic Acids Res. 2014. pii:gku1055.

² The sequence is available on the Uniprot database on the World Wide Web at “uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49: D1 (2021).

Peptide hormones and receptors

In some embodiments, the effectors described herein comprise a hormone of Table 2 or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a protein sequence disclosed in Table 2 by reference to its UniProt ID. In some embodiments, the functional variant binds to the corresponding receptor with a Kd that is no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type hormone for the same receptor under the same conditions. In some embodiments, the polypeptide of Table 2 or a functional variant thereof comprises a signal sequence, e.g., a signal sequence endogenous to the effector, or a heterologous signal sequence.

In some embodiments, the effectors described herein comprise an antibody molecule (e.g., scFv) that binds to a hormone from Table 2. In some embodiments, the effectors described herein comprise an antibody molecule (e.g., scFv) that binds to a hormone receptor from Table 2. In some embodiments, the antibody molecule comprises a signal sequence.

Table 2. Exemplary polypeptide hormones and receptors

¹ The sequence is available at the NCBI database on the World Wide Web at “ncbi.nlm.nih.gov/gene”, Maglott D et al. Gene: a gene-centered information resource at NCBI. Nucleic Acids Res. 2014. pii:gku1055.

² The sequence is available on the Uniprot database on the World Wide Web at “uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49: D1 (2021).

Growth Factors:

In some embodiments, the effectors described herein comprise a growth factor of Table 3 or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a protein sequence disclosed in Table 3 by reference to its UniProt ID. In some embodiments, the functional variant binds to the corresponding receptor with a Kd that is no more than 10%, 20%, 30%, 40%, or 50% higher than the Kd of the corresponding wild-type growth factor for the same receptor under the same conditions. In some embodiments, the polypeptide of Table 3 or a functional variant thereof comprises a signal sequence, e.g., a signal sequence endogenous to the effector, or a heterologous signal sequence.

In some embodiments, the effectors described herein comprise an antibody or variant thereof that binds to a growth factor of Table 3. In some embodiments, the effectors described herein comprise an antibody molecule (e.g., scFv) that binds to a growth factor receptor of Table 3. In some embodiments, the antibody molecule comprises a signal sequence.

Table 3. Exemplary Growth Factors

¹ The sequence is available at the NCBI database on the World Wide Web at “ncbi.nlm.nih.gov/gene”, Maglott D et al. Gene: a gene-centered information resource at NCBI. Nucleic Acids Res. 2014. pii:gku1055.

² The sequence is available on the Uniprot database on the World Wide Web at “uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49: D1 (2021).

Coagulation factors:

In some embodiments, the effector described herein comprises a polypeptide of Table 4 or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a protein sequence disclosed in Table 4 by reference to its UniProt ID. In some embodiments, the functional variant catalyzes the same reaction as the corresponding wild-type protein, e.g., the catalytic rate is no less than 10%, 20%, 30%, 40%, or 50% lower or higher than the wild-type protein. In some embodiments, the polypeptide of Table 4 or a functional variant thereof comprises a signal sequence, e.g., a signal sequence endogenous to the effector, or a heterologous signal sequence.

Table 4. Factors related to coagulation

¹ The sequence is available at the NCBI database on the World Wide Web at “ncbi.nlm.nih.gov/gene”, Maglott D et al. Gene: a gene-centered information resource at NCBI. Nucleic Acids Res. 2014. pii:gku1055.

² The sequence is available on the Uniprot database on the World Wide Web at “uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49: D1 (2021).

Enzyme replacement therapy:

In some embodiments, the effectors described herein comprise an enzyme of Table 5 or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to a protein sequence disclosed by reference to its UniProt ID in Table 5. In some embodiments, the functional variant catalyzes the same reaction as the corresponding wild-type protein, e.g., the catalytic rate is not less than or not more than 10%, 20%, 30%, 40%, or 50% slower than the wild-type protein.

Table 5. Exemplary enzyme effectors for enzyme deficiencies

¹ The sequence is available at the NCBI database on the World Wide Web at “ncbi.nlm.nih.gov/gene”, Maglott D et al. Gene: a gene-centered information resource at NCBI. Nucleic Acids Res. 2014. pii:gku1055.

² The sequence is available on the Uniprot database on the World Wide Web at “uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49: D1 (2021).

Other non-enzymatic effectors:

In some embodiments, the therapeutic polypeptide described herein comprises a polypeptide of Table 6 or a functional variant thereof, e.g., a protein having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the protein sequence disclosed in Table 6 by reference to its UniProt ID.

Table 6. Exemplary non-enzymatic effectors and corresponding indications

¹ The sequence is available at the NCBI database on the World Wide Web at “ncbi.nlm.nih.gov/gene”, Maglott D et al. Gene: a gene-centered information resource at NCBI. Nucleic Acids Res. 2014. pii:gku1055.

² The sequence is available on the Uniprot database on the World Wide Web at “uniprot.org/uniprot/”; UniProt: the universal protein knowledgebase in 2021. Nucleic Acids Res. 49: D1 (2021).

Regeneration, repair and fibrosis factors

The therapeutic polypeptides described herein also include, for example, growth factors or functional variants thereof as disclosed in Table 7, for example, proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the protein sequences disclosed in Table 7 by reference to their NCBI protein accession numbers. Antibodies or fragments thereof directed against such growth factors, or miRNAs that promote regeneration and repair are also included.

Table 7

¹ The sequence is available on the World Wide Web at "ncbi.nlm.nih.gov/gene" (Maglott D et al. Gene: agene-centered information resource at NCBI. Nucleic Acids Res. 2014. Pii: gku1055.)

² The sequence is available on the World Wide Web at “ncbi.nlm.nih.gov/protein/”

Conversion Factor:

The therapeutic polypeptides described herein also include conversion factors, such as protein factors that convert fibroblasts into differentiated cells, such as the factors disclosed in Table 8 or functional variants thereof, for example, proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the protein sequences disclosed in Table 8 by reference to their NCBI protein accession numbers.

Table 8: Indicative polypeptides for organ repair by transformation of fibroblasts

Target Gene accession number ¹ Protein accession number ² MESP1 Gene ID: 55897 EAX02066 ETS2 Gene ID: 2114 NP_005230 HAND2 Gene ID: 9464 NP_068808 Myocardin Gene ID: 93649 NP_001139784 ESRRA Gene ID: 2101 AAH92470 miR1 MI0000651 n/a miR133 MI000450 n/a TGF Gene ID: 7040 NP_000651.3 WNT Gene ID: 7471 NP_005421 JAK Gene ID: 3716 NP_001308784 NOTCH Gene ID: 4851 XP_011517019

¹ The sequence is available on the World Wide Web at "ncbi.nlm.nih.gov/gene" (Maglott D et al. Gene: agene-centered information resource at NCBI. Nucleic Acids Res. 2014. Pii: gku1055.)

² The sequence is available on the World Wide Web at “ncbi.nlm.nih.gov/protein/”

Proteins that stimulate cell regeneration:

The therapeutic polypeptides described herein also include proteins that stimulate cell regeneration, such as the proteins disclosed in Table 9 or functional variants thereof, for example, proteins having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the protein sequences disclosed in Table 9 by reference to their NCBI protein accession numbers.

Table 9

Target Gene accession number ¹ Protein accession number ² MST1 NG_016454 NP_066278 STK30 Gene ID: 26448 NP_036103 MST2 Gene ID: 6788 NP_006272 SAV1 Gene ID: 60485 NP_068590 LATS1 Gene ID: 9113 NP_004681 LATS2 Gene ID: 26524 NP_055387 YAP1 NG_029530 NP_001123617 CDKN2b NG_023297 NP_004927 CDKN2a NG_007485 NP_478102

¹ The sequence is available on the World Wide Web at "ncbi.nlm.nih.gov/gene" (Maglott D et al. Gene: agene-centered information resource at NCBI. Nucleic Acids Res. 2014. Pii: gku1055.)

² The sequence is available on the World Wide Web at “ncbi.nlm.nih.gov/protein/”

In certain embodiments, the cyclic polyribonucleotide comprises one or more expressed sequences (coding sequence), and is configured to be continuously expressed in cells in a subject. In certain embodiments, the cyclic polyribonucleotide is configured to make the expression of one or more expressed sequences in cells at a later time point equal to or higher than the expression at an earlier time point. In such embodiments, the expression of one or more expressed sequences can maintain a relatively stable level or can increase over time. The expression of expressed sequences can be relatively stable in the time period of extension. For example, in some cases, the expression of one or more expressed sequences in cells at least 7,8,9,10,12,14,16,18,20,22,23 or more days in the time period will not reduce 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5%. In some cases, in some cases, expression of one or more expressed sequences in the cell is maintained at a level that does not vary by more than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, or 5% for at least 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 23, or more days.

Internal ribosome entry site (IRES)

In certain embodiments, cyclic polyribonucleotides as described herein (e.g., polyribonucleotide cargo of cyclic polyribonucleotides) include one or more internal ribosome entry sites (IRES) elements. In certain embodiments, IRES is operably connected to one or more coding sequences (e.g., each IRES is operably connected to one or more coding sequences). In an embodiment, IRES is located between the 5' end of a heterologous promoter and a coding sequence.

Suitable IRES elements included in the circular polyribonucleotide include RNA sequences that can engage eukaryotic ribosomes. In certain embodiments, the IRES element is at least about 5nt, at least about 8nt, at least about 9nt, at least about 10nt, at least about 15nt, at least about 20nt, at least about 25nt, at least about 30nt, at least about 40nt, at least about 50nt, at least about 100nt, at least about 200nt, at least about 250nt, at least about 350nt or at least about 500nt.

In some embodiments, the IRES element is derived from the DNA of an organism, including but not limited to viruses, mammals, and fruit flies. Such viral DNA can be derived from, but not limited to, picornavirus complementary DNA (cDNA), encephalomyocarditis virus (EMCV) cDNA, and poliovirus cDNA. In one embodiment, the fruit fly DNA from which the IRES element is derived includes but is not limited to the antennapedia gene from Drosophila melanogaster.

In some embodiments, if present, the IRES sequence is an IRES sequence of the following viruses: Taura syndrome virus, Triatoma virus, Theiler's encephalomyelitis virus, Simian virus 40, Solenopsis invicta virus 1, Rhopalosiphum padi virus, Reticuloendotheliosis virus, Fumanpoliovirus 1, Plautia stall intestine virus, Kashmir honey bee virus, Human rhinovirus 2, Homalodisca coagulata virus-1, Human immunodeficiency virus type 1, Homalodisca coagulata virus-1, Himetobi P virus, Hepatitis C virus, Hepatitis A virus, GB hepatitis virus, Foot-and-mouth disease virus, Human enterovirus 71, Equine rhinitis virus, Ectropis obliqua) small RNA-like virus, encephalomyocarditis virus (EMCV), Drosophila C virus, crucifer tobacco virus, cricket paralysis virus, bovine viral diarrhea virus 1, black queen cell virus, aphid lethal paralysis virus, avian encephalomyelitis virus, acute bee paralysis virus, Hibiscus chlorotic ringspot virus, classical swine fever virus, human FGF2, human SFTPA1, human AML1/RUNX1, Drosophila antennapedia, human AQP4, human AT1R, human BAG-1, human BCL2, human BiP, human c-IAPl, human c-myc, human eIF4G, mouse NDST4L, human LEF1, mouse HIF1α, human n.myc, mouse Gtx, human p27kipl, human PDGF2/c-sis, human p53, human Pim-1, mouse Rbm3, Drosophila In another embodiment, the IRES is an IRES sequence of Coxsackievirus B3 (CVB3). In another embodiment, the IRES is an IRES sequence of encephalomyocarditis virus.

In some embodiments, the cyclic polyribonucleotide includes at least one IRES flanking at least one (e.g., 2, 3, 4, 5 or more) coding sequence. In some embodiments, the IRES flanks at least one (e.g., 2, 3, 4, 5 or more) coding sequence on both sides. In some embodiments, the cyclic polyribonucleotide includes one or more IRES sequences on one or both sides of each coding sequence, resulting in the separation of one or more peptides and/or one or more polypeptides.

Adjustment element

In certain embodiments, cyclic polyribonucleotide as herein described (for example, the polyribonucleotide cargo of cyclic polyribonucleotide) comprises one or more regulating elements.In certain embodiments, cyclic polyribonucleotide comprises regulating element, for example modifies the sequence of the expression of encoding sequence in the cyclic polyribonucleotide.

An adjusting element can include a sequence that is located adjacent to the coding sequence of the encoded expression product. An adjusting element can be operably connected to an adjacent sequence. As compared with the amount of the product expressed when the adjusting element is not present, the adjusting element can increase the amount of the product expressed. In addition, an adjusting element can increase the amount of the product expressed by a plurality of coding sequences connected in series. Therefore, an adjusting element can enhance the expression of one or more coding sequences. A plurality of adjusting elements are well known to those of ordinary skill in the art.

In certain embodiments, regulating element is a translation regulator. The translation regulator can regulate the translation of the coding sequence in the cyclic polyribonucleotide. The translation regulator can be a translation enhancer or a translation inhibitor. In certain embodiments, the cyclic polyribonucleotide comprises at least one translation regulator adjacent to at least one coding sequence. In certain embodiments, the cyclic polyribonucleotide comprises a translation regulator adjacent to each coding sequence. In certain embodiments, the translation regulator is present in one or both sides of each coding sequence, causing for example the separation of the coded product of one or more peptides and/or one or more polypeptides.

In certain embodiments, the polyribonucleotide payload includes at least one non-coding RNA sequence including regulatory RNA. In certain embodiments, the non-coding RNA sequence trans-regulates the target sequence. In certain embodiments, the target sequence includes the nucleotide sequence of the gene of the tested genome, wherein the tested genome is the genome of vertebrates, invertebrates, fungi, oomycetes, plants or microorganisms. In an embodiment, the subject genome is the genome of people, non-human mammals, reptiles, birds, amphibians or fish. In an embodiment, the subject genome is the genome of insects, arachnids, nematodes or molluscs. In an embodiment, the subject genome is the genome of monocots, dicots, gymnosperms or eukaryotic algae. In an embodiment, the tested genome is the genome of bacteria, fungi, oomycetes or archaebacteria. In an embodiment, the target sequence is included in the nucleotide sequence of the gene found in multiple subject genomes (for example, in the genome of multiple species in a given genus).

In some embodiments, the trans-regulation of at least one non-coding RNA sequence to the target sequence is the up-regulation of target sequence expression. In some embodiments, the trans-regulation of at least one non-coding RNA sequence to the target sequence is the down-regulation of target sequence expression. In some embodiments, the trans-regulation of at least one non-coding RNA sequence to the target sequence is the inducible expression of target sequence expression. For example, inducible expression can be induced by environmental conditions (for example, light, temperature, water, or nutrient availability), by circadian rhythm, by endogenous or exogenously provided inducing agents (for example, small RNA, ligands). In some embodiments, the at least one non-coding RNA sequence can be induced by the physiological state (for example, growth phase, transcriptional regulation state and intracellular metabolite concentration) of the eukaryotic system. For example, an exogenously provided ligand (for example, arabinose, rhamnose, or IPTG) can be provided to use an inducible promoter (for example, PBAD, Prha, and lacUV5) to induce expression.

In some embodiments, at least one non-coding RNA sequence includes a regulatory RNA selected from the group consisting of: small interfering RNA (siRNA) or a precursor thereof, double-stranded RNA (dsRNA) or at least partially double-stranded RNA (e.g., RNA comprising one or more stem loops); hairpin RNA (hpRNA), microRNA (miRNA) or a precursor thereof (e.g., pre-miRNA or pri-miRNA); phase small interfering RNA (phasiRNA) or a precursor thereof; heterochromatin small interfering RNA (hcsiRNA) or a precursor thereof; and natural antisense short interfering RNA (natsiRNA) or a precursor thereof. In some embodiments, at least one non-coding RNA sequence includes a guide RNA (gRNA) or a precursor thereof, or a heterologous RNA sequence that can be recognized and bound by a guide RNA. In some embodiments, the regulatory element is a microRNA (miRNA) or a miRNA binding site, or a siRNA or a siRNA binding site.

In some embodiments, the cyclic polyribonucleotides described herein (e.g., the polyribonucleotide cargo of cyclic polyribonucleotides) include at least one agriculturally useful non-coding RNA sequence, which, when provided to a specific plant tissue, cell, or cell type, imparts desired characteristics, such as desired characteristics associated with plant morphology, physiology, growth, development, yield, product, nutritional properties, disease or pest resistance, and/or environmental or chemical tolerance. In an embodiment, agriculturally useful non-coding RNA sequences cause targeted regulation of gene expression of endogenous genes, such as via antisense (see, e.g., U.S. Patent No. 5,107,065); inhibitory RNA ("RNAi", including regulation of gene expression via miRNA, siRNA, trans-acting siRNA, and phase sRNA-mediated mechanisms, such as, as disclosed in US2006/0200878 and US2008/0066206 and in U.S. Patent Application Serial No. 11/974,469); or co-suppression-mediated mechanisms. In an embodiment, the agriculturally useful non-coding RNA sequence is a catalytic RNA molecule (e.g., a ribozyme or a riboswitch; see, e.g., US2006/0200878) that is engineered to cleave a desired endogenous mRNA product. Agriculturally useful non-coding RNA sequences are known in the art, e.g., antisense directional RNAs that regulate gene expression in plant cells are disclosed in U.S. Patent Nos. 5,107,065 and 5,759,829, and sense directional RNAs that regulate gene expression in plants are disclosed in U.S. Patent Nos. 5,283,184 and 5,231,020. Providing agriculturally useful non-coding RNAs to plant cells can also be used to regulate gene expression in organisms associated with plants, such as invertebrate pests of plants or microbial pathogens that infect plants (e.g., bacteria, fungi, oomycetes, or viruses), or microorganisms associated with invertebrate pests of plants (e.g., symbiotic).

Translation start sequence

In certain embodiments, cyclic polyribonucleotide as herein described (for example, the polyribonucleotide cargo of cyclic polyribonucleotide) comprises at least one translation initiation sequence.In certain embodiments, cyclic polyribonucleotide comprises the translation initiation sequence operably connected with encoding sequence.

In certain embodiments, the cyclic polyribonucleotide encoding polypeptide can include a translation initiation sequence, such as a start codon. In certain embodiments, the translation initiation sequence includes a Kozak or Shine-Dalgarno sequence. In certain embodiments, the cyclic polyribonucleotide includes a translation initiation sequence adjacent to the coding sequence, such as a Kozak sequence. In certain embodiments, the translation initiation sequence is a non-coding start codon. In certain embodiments, the translation initiation sequence (for example, a Kozak sequence) is present on one side or both sides of each coding sequence, causing the separation of the coded product. In certain embodiments, the cyclic polyribonucleotide includes at least one translation initiation sequence adjacent to the coding sequence. In certain embodiments, the translation initiation sequence provides conformational flexibility for the cyclic polyribonucleotide. In certain embodiments, the translation initiation sequence is substantially in the single-stranded region of the cyclic polyribonucleotide.

Circular polyribonucleotide can comprise more than 1 start codon, for example but not limited to at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 25, at least 30, at least 35, at least 40, at least 50, at least 60 or more than 60 start codons.Translation can start on first start codon or can start in the downstream of first start codon.

In certain embodiments, the cyclic polyribonucleotide can start from a codon that is not the first start codon, such as AUG.The translation of cyclic polyribonucleotide can start from an alternative translation initiation sequence, such as but not limited to ACG, AGG, AAG, CTG/CUG, GTG/GUG, ATA/AUA, ATT/AUU, TTG/UUG.In certain embodiments, translation starts at an alternative translation initiation sequence under the selectivity conditions of, for example, stress-induced conditions.As non-limiting examples, the translation of cyclic polyribonucleotide can start at an alternative translation initiation sequence, such as ACG.As another non-limiting examples, the translation of cyclic polyribonucleotide can start at an alternative translation initiation sequence CTG/CUG.As another non-limiting examples again, the translation of cyclic polyribonucleotide can start at an alternative translation initiation sequence GTG/GUG. As yet another non-limiting example, the cyclic polyribonucleotide can start translation at a repeat-associated non-AUG (RAN) sequence, such as an alternative translation start sequence comprising a short segment of repeat RNA (e.g., CGG, GGGGCC, CAG, CTG).

Termination element

In certain embodiments, cyclic polyribonucleotide as herein described (for example, the polyribonucleotide loading thing of cyclic polyribonucleotide) comprises at least one termination element.In certain embodiments, cyclic polyribonucleotide comprises the termination element operably connected with encoding sequence.

In certain embodiments, the cyclic polyribonucleotide comprises one or more encoding sequences, and each encoding sequence may or may not have a termination element. In certain embodiments, the cyclic polyribonucleotide comprises one or more encoding sequences, and the encoding sequence lacks a termination element so that the cyclic polyribonucleotide is translated continuously. The exclusion of the termination element can cause the continuous expression of rolling circle translation or coded product.

Non-coding sequences

In certain embodiments, cyclic polyribonucleotide as herein described (for example, the polyribonucleotide loading material of cyclic polyribonucleotide) comprises one or more non-coding sequences, for example, the sequence of the expression of non-encoded polypeptide.In certain embodiments, this cyclic polyribonucleotide comprises two, three, four, five, six, seven, eight, nine, ten or more non-coding sequences.In certain embodiments, cyclic polyribonucleotide does not encode polypeptide encoding sequence.

The non-coding sequence can be a natural or synthetic sequence. In some embodiments, the non-coding sequence can change cell behavior, such as, for example, lymphocyte behavior. In some embodiments, the non-coding sequence is antisense to the cell RNA sequence.

In certain embodiments, cyclic polyribonucleotide comprises regulating nucleic acid, and this regulating nucleic acid is RNA or RNA sample structure, typically between about 5-500 base pairs (bp) (depending on specific RNA structure, for example miRNA 5-30bp, lncRNA 200-500bp) and can have the core base sequence identical (complementary) or almost identical (substantially complementary) with the coding sequence in the target gene of intracellular expression.In an embodiment, cyclic polyribonucleotide comprises the regulating nucleic acid of coding RNA precursor, and this RNA precursor can be processed into less RNA, and for example miRNA precursor (it can be from about 50 to about 1000bp) can be processed into less miRNA intermediate or mature miRNA.

Long noncoding RNA (lncRNA) is defined as a non-protein coding transcript longer than 100 nucleotides. Many lncRNAs are characterized as tissue specific. Reverse lncRNAs transcribed in the opposite direction to nearby protein coding genes account for a large proportion (e.g., about 20% of total lncRNAs in mammalian genomes) and may regulate the transcription of nearby genes. In one embodiment, the cyclic polyribonucleotides provided herein include the sense strand of lncRNA. In one embodiment, the cyclic polyribonucleotides provided herein include the antisense strand of lncRNA.

In an embodiment, the cyclic polyribonucleotide encoding and endogenous gene or gene product (for example, mRNA) all or with its at least one fragment substantially complementary or fully complementary regulatory nucleic acid.In an embodiment, the boundary between nucleic acid and intron and exon, inside between exon or with exon adjacent sequence complementarity, thus prevent the newly generated nuclear RNA transcript of specific gene from maturing into mRNA for transcription.The regulatory nucleic acid complementary to specific gene can hybridize with the mRNA of this gene and prevent its translation.Antisense regulatory nucleic acid can be DNA, RNA or its derivatives or hybrid.In certain embodiments, regulatory nucleic acid includes the protein binding site that can be combined with the protein that participates in the expression regulation of endogenous gene or exogenous gene.

In an embodiment, at least one regulating RNA of circular polyribonucleotide encoding and target transcript hybridization, wherein the length of this regulating RNA is about 5 and 30 nucleotides, about 10 and 30 nucleotides, or about 11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 or more than 30 nucleotides.In an embodiment, regulating nucleic acid and the sequence identity degree of the targeting transcript is at least 75%, at least 80%, at least 85%, at least 90% or at least 95%.

In an embodiment, the cyclic polyribonucleotide encodes a microRNA (miRNA) molecule identical to about 5 to about 25 continuous nucleotides of a target gene, or encodes a precursor of the miRNA. In certain embodiments, the miRNA has a sequence that allows miRNA to identify a specific target mRNA and to be combined with it. In an embodiment, the miRNA sequence begins with dinucleotide AA, including a content of about 30%-70% (about 30%-60%, about 40%-60% or about 45%-55%) GC, and does not have a high identity percentage with any nucleotide sequence except the target in the genome of a subject (e.g., mammal) to be introduced therein, for example, as determined by standard BLAST retrieval.

In certain embodiments, the cyclic polyribonucleotide comprises at least a miRNA (or miRNA precursor), for example 2 kinds, 3 kinds, 4 kinds, 5 kinds, 6 kinds or more kinds of miRNA or miRNA precursor.In certain embodiments, the cyclic polyribonucleotide comprises the sequence of coding miRNA (or its precursor), and this miRNA (or its precursor) has at least about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide complementarity with target sequence.

siRNA and shRNA are similar to intermediates in the processing pathway of endogenous microRNA (miRNA) genes. In certain embodiments, siRNA can act as miRNA, and vice versa. Like siRNA, microRNA uses RISC to downregulate target genes, but unlike siRNA, most animal miRNAs do not cut mRNA. On the contrary, miRNA reduces protein output by translation inhibition or poly A removal and mRNA degradation. Known miRNA binding sites are located in mRNA 3'UTR; miRNA seems to target sites that are almost completely complementary to the 2nd-8th nucleotides from the 5' end of miRNA. This region is called the seed region. Because mature siRNA and miRNA are interchangeable, exogenous siRNA downregulates mRNA with seed complementarity to siRNA.

The list of known miRNA sequences can be found in the database maintained by research organizations, such as Wellcome Trust Sanger Institute, Penn Center for Bioinformatics, Memorial Sloan Kettering Cancer Center, and European Molecular Biology Laboratory. Known effective siRNA sequences and homologous binding sites are also well presented in the relevant literature. By technology known in the art, RNAi molecules are easy to design and produce. In addition, there are computational tools that increase the chance of finding effective and specific sequence motifs.

Plant miRNAs, their precursors, and their target genes are known in the art; see, e.g., U.S. Pat. Nos. 8,697,949, 8,946,511, and 9,040,774, and also see the publicly available microRNA database "miRbase" available at miRbase[dot]org. Naturally occurring miRNA or miRNA precursor sequences can be engineered or their sequences modified so that the resulting mature miRNA recognizes and binds to a selected target sequence; examples of both engineered plant and animal miRNAs and miRNA precursors have been well documented; see, e.g., U.S. Pat. Nos. 8,410,334, 8,536,405, and 9,708,620. All cited patents and the miRNA and miRNA precursor sequences disclosed therein are incorporated herein by reference.

Spacer sequence

In certain embodiments, cyclic polyribonucleotides as described herein include one or more spacer sequences. Spacer refers to any adjacent (for example, one or more nucleotide) nucleotide sequence providing the distance between two adjacent polynucleotide districts and/or flexibility. Spacer can be present between any nucleic acid element as described herein. Spacer also can be present in nucleic acid element as described herein.

For example, wherein the nucleic acid includes any two or more of the following elements: (A) 5' self-cleaving ribozyme; (B) 5' annealing zone; (C) polyribonucleotide cargo; (D) 3' annealing zone; and/or (E) 3' self-cleaving ribozyme; the spacer region may be present between any one or more elements. As described herein, any one of the elements (A), (B), (C), (D) and/or (E) may be separated by a spacer sequence. For example, the spacer may be between (A) and (B), between (B) and (C), between (C) and (D), and/or between (D) and (E).

Spacers may also be present in nucleic acid regions described herein. For example, a polynucleotide cargo region may include one or more spacers. Spacers may separate regions within a polynucleotide cargo.

In certain embodiments, the length of the spacer sequence can be, for example, at least 5 nucleotides, at least 10 nucleotides, at least 15 nucleotides, or at least 30 nucleotides. In certain embodiments, the length of the spacer sequence is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, or 30 nucleotides. In certain embodiments, the length of the spacer sequence is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35, or 30 nucleotides. In certain embodiments, the length of the spacer sequence is between 20 and 50 nucleotides. In certain embodiments, the length of the spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides.

In certain embodiments, between 5' annealing zone and the polyribonucleotide load, the length in the spacer district can be between 5 and 1000, 5 and 900, 5 and 800, 5 and 700, 5 and 600, 5 and 500, 5 and 400, 5 and 300, 5 and 200, 5 and 100, 100 and 200, 100 and 300, 100 and 400, 100 and 500, 100 and 600, 100 and 700, 100 and 800, 100 and 900 or 100 and 1000 polyribonucleotides. The spacer sequence can be a poly-A sequence, a poly-A-C sequence, a poly-C sequence or a poly-U sequence.

The spacer sequence can be used to separate the IRES from adjacent structural elements to maintain the structure and function of the IRES or adjacent elements. The spacer specificity can be engineered according to the IRES. In certain embodiments, RNA folding computer software (such as RNAFold) can be used to guide the design of various elements (including spacers) of the vector.

In certain embodiments, polyribonucleotides include 5' spacer sequences (for example, between 5' annealing zone and polyribonucleotide load). In certain embodiments, the length of 5' spacer sequences is at least 10 nucleotides. In another embodiment, the length of 5' spacer sequences is at least 15 nucleotides. In other embodiments, the length of 5' spacer sequences is at least 30 nucleotides. In certain embodiments, the length of 5' spacer sequences is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides. In certain embodiments, the length of 5' spacer sequences is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. In certain embodiments, the length of 5' spacer sequences is between 20 and 50 nucleotides. In certain embodiments, the length of the 5' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In one embodiment, the 5' spacer sequence is a poly A sequence. In another embodiment, the 5' spacer sequence is a poly A-C sequence.

In certain embodiments, polyribonucleotides include 3' spacer sequences (for example, between 3' annealing zone and polyribonucleotide load). In certain embodiments, the length of 3' spacer sequences is at least 10 nucleotides. In another embodiment, the length of 3' spacer sequences is at least 15 nucleotides. In other embodiments, the length of 3' spacer sequences is at least 30 nucleotides. In certain embodiments, the length of 3' spacer sequences is at least 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 nucleotides. In certain embodiments, the length of 3' spacer sequences is no more than 100, 90, 80, 70, 60, 50, 45, 40, 35 or 30 nucleotides. In certain embodiments, the length of 3' spacer sequences is between 20 and 50 nucleotides. In certain embodiments, the length of the 3' spacer sequence is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In one embodiment, the 3' spacer sequence is a poly A sequence. In another embodiment, the 5' spacer sequence is a poly A-C sequence.

In one embodiment, the polyribonucleotide includes a 5' spacer sequence, but does not include a 3' spacer sequence. In another embodiment, the polyribonucleotide includes a 3' spacer sequence, but does not include a 5' spacer sequence. In another embodiment, the polyribonucleotide neither includes a 5' spacer sequence nor a 3' spacer sequence. In another embodiment, the polyribonucleotide does not include an IRES sequence. In a further embodiment, the polyribonucleotide does not include an IRES sequence, a 5' spacer sequence or a 3' spacer sequence.

In some embodiments, the spacer sequence comprises at least 3 ribonucleotides, at least 4 ribonucleotides, at least 5 ribonucleotides, at least about 8 ribonucleotides, at least about 10 ribonucleotides, at least about 12 ribonucleotides, at least about 15 ribonucleotides, at least about 20 ribonucleotides, at least about 25 ribonucleotides, at least about 30 ribonucleotides, at least about 40 ribonucleotides, at least about 50 ribonucleotides, at least about 60 ribonucleotides, at least about 70 ribonucleotides, at least about 80 ribonucleotides, nucleotides, at least about 90 ribonucleotides, at least about 100 ribonucleotides, at least about 120 ribonucleotides, at least about 150 ribonucleotides, at least about 200 ribonucleotides, at least about 250 ribonucleotides, at least about 300 ribonucleotides, at least about 400 ribonucleotides, at least about 500 ribonucleotides, at least about 600 ribonucleotides, at least about 700 ribonucleotides, at least about 800 ribonucleotides, at least about 900 ribonucleotides, or at least about 100 ribonucleotides.

Ligase

RNA ligases are a class of enzymes that use ATP to catalyze the formation of phosphodiester bonds between the ends of RNA molecules. Endogenous RNA ligases repair nucleotide breaks in single-stranded duplex RNA in plants, animals, humans, bacteria, archaea, oomycetes, and fungal cells, as well as viruses.

The present disclosure provides a method for producing circular RNA in a eukaryotic system by contacting a linear RNA (e.g., a ligase-compatible linear RNA as described herein) with an RNA ligase.

In some embodiments, the RNA ligase is endogenous to the eukaryotic cell. In some embodiments, the RNA ligase is heterologous to the eukaryotic cell. In some embodiments, the RNA ligase is provided to the eukaryotic cell by transcribing an exogenous polynucleotide into an mRNA encoding the RNA ligase in the eukaryotic cell and translating the mRNA encoding the RNA ligase. In some embodiments, the RNA ligase is provided to the eukaryotic cell by transcribing an endogenous polynucleotide into an mRNA encoding the RNA ligase in the eukaryotic cell and translating the mRNA encoding the RNA ligase; for example, a vector encoding an RNA ligase endogenous to the eukaryotic cell can be provided to the eukaryotic cell for overexpression in the eukaryotic cell. In some embodiments, the eukaryotic cell is provided with an RNA ligase as an exogenous protein.

In some embodiments, the RNA ligase is a tRNA ligase or a variant thereof. In some embodiments, the tRNA ligase is a T4 ligase, a RtcB ligase, a TRL-1 ligase, and a Rnl1 ligase, a Rnl2 ligase, a LIG1 ligase, a LIG2 ligase, a PNK/PNL ligase, a PF0027 ligase, a thpR ligT ligase, a ytlPor ligase, or a variant thereof (e.g., a mutant variant that retains ligase function).

In some embodiments, the RNA ligase is a plant RNA ligase or a variant thereof. In some embodiments, the RNA ligase is a chloroplast RNA ligase or a variant thereof. In embodiments, the RNA ligase is a eukaryotic algae RNA ligase or a variant thereof. In some embodiments, the RNA ligase is an RNA ligase from an archaea or a variant thereof. In some embodiments, the RNA ligase is a bacterial RNA ligase or a variant thereof. In some embodiments, the RNA ligase is a eukaryotic RNA ligase or a variant thereof. In some embodiments, the RNA ligase is a viral RNA ligase or a variant thereof. In some embodiments, the RNA ligase is a mitochondrial RNA ligase or a variant thereof.

In some embodiments, the RNA ligase is a ligase described in Table 10 or a variant thereof. In some embodiments, the RNA ligase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 586-602.

Table 10: Exemplary tRNA ligases

Generation method

The present disclosure also provides a method for producing circular RNA in a eukaryotic system. Fig. 2 is a schematic diagram depicting an exemplary process for producing circular RNA from a precursor linear RNA. In certain embodiments, exogenous polyribonucleotides (e.g., linear polyribonucleotides as described herein or DNA molecules encoding for transcribing linear polyribonucleotides as described herein) are provided to eukaryotic cells. The linear polyribonucleotides can be transcribed from the exogenous DNA molecules provided to the eukaryotic cells in the eukaryotic cells. The linear polyribonucleotides can be transcribed from the exogenous recombinant DNA molecules provided to the eukaryotic cells in ...

In certain embodiments, this DNA molecule comprises a heterologous promoter operably connected to the DNA encoding this linear polyribonucleotide. This heterologous promoter can be a T7 promoter, a T6 promoter, a T4 promoter, a T3 promoter, an SP3 promoter or an SP6 promoter. In certain embodiments, this heterologous promoter can be a constitutive promoter. In certain embodiments, this heterologous promoter can be an inducible promoter. For example, the heterologous promoter can be a cauliflower mosaic virus (CaMV) 35S promoter, an opine promoter, a plant ubiquitin (Ubi) promoter, a rice actin 1 promoter, an alcohol dehydrogenase (ADH-1) promoter (e.g., corn ADH-1 and yeast ADH-1), a glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter (e.g., Saccharomyces cerevisiae GPD promoter), a cytomegalovirus (CMV) promoter (e.g., a human cytomegalovirus promoter), an elongation factor 1 alpha (EF1α) promoter (e.g., human EF1α), a chicken β-actin gene (CAG) promoter, a phosphoglycerate kinase gene (PGK) promoter, a U6 nuclear promoter (e.g., a human U6 nuclear promoter), a tetracycline response element (TRE) promoter, an OPIE2 promoter (e.g., a baculovirus OpIE2 promoter), an OpIE1 promoter (e.g., a baculovirus OpIE1 promoter). Other useful promoters for eukaryotic systems include those disclosed in the Eukaryotic Protein Database, publicly available online at https://[dot]edp[dot]epfl[dot]ch.

When expressing in cell, 5' and 3' self-cleaving ribozyme experience cleavage reaction separately, thereby produce ligase compatible end (for example, 5'-hydroxyl and 2', 3'-cyclic phosphate) and 5' and 3' annealing zone make free end approach.Therefore, precursor linear polyribonucleotide produces ligase compatible polyribonucleotide, it can be connected (for example, in the presence of ligase) to produce cyclic polyribonucleotide.

Linear RNA is transcribed (for example, transcribed in vivo) from DNA template in eukaryotic system, precursor linear RNA self-cutting to form ligase-compatible linear RNA and ligase-compatible linear RNA connection to produce circular RNA in eukaryotic cell.In certain embodiments, the transcription (for example, transcribed in vivo) of this linear polyribonucleotide in eukaryotic system is carried out in eukaryotic cell with endogenous ligase.In certain embodiments, this endogenous ligase is overexpressed.In certain embodiments, the transcription (for example, transcribed in vivo) of this linear polyribonucleotide in eukaryotic system is carried out in eukaryotic cell with heterologous ligase.

In some embodiments, the eukaryotic cell includes an RNA ligase, such as an RNA ligase described herein. In some embodiments, the RNA ligase is endogenous to the eukaryotic cell. In some embodiments, the RNA ligase is heterologous to the eukaryotic cell. When the RNA ligase is heterologous to the cell, the RNA ligase can be provided to the cell as an exogenous RNA ligase, or can be encoded by a polynucleotide provided to the cell. When the RNA ligase is endogenous to the cell, the RNA ligase can be overexpressed in the cell by providing the cell with polyribonucleotides encoding the expression of the RNA ligase.

In an embodiment, the eukaryotic cell comprising the polyribonucleotides described herein is a unicellular eukaryotic cell. In some embodiments, the unicellular eukaryotic cell is a unicellular fungal cell, such as a yeast cell (e.g., Saccharomyces cerevisiae and other Saccharomyces species (Saccharomyces spp.), Brettanomyces species (Brettanomyces spp.), Schizosaccharomyces species (Schizosaccharomyces spp.), Torulaspora species (Torulaspora spp.), and Pichia species (Pichia spp.)). In some embodiments, the unicellular eukaryotic cell is a unicellular animal cell. The unicellular animal cell can be a cell or a daughter cell thereof separated from a multicellular animal and grown in culture. In some embodiments, the unicellular animal cell can be dedifferentiated. In some embodiments, the unicellular eukaryotic cell is a unicellular plant cell. The unicellular plant cell can be a cell or a daughter cell thereof separated from a multicellular plant and grown in culture. In some embodiments, the unicellular plant cell can be dedifferentiated. In some embodiments, the unicellular plant cell is from a plant callus. In an embodiment, the unicellular cell is a plant cell protoplast. In some embodiments, the unicellular eukaryotic cell is a unicellular eukaryotic algae cell, such as a unicellular green algae, a diatom, an euglena, or a dinoflagellate. Non-limiting examples of target unicellular eukaryotic algae include Dunaliella salina, Chlorella vulgaris, Chlorella zofingiensis, Haematococcus pluvialis, Neochlorisoleoabundans and other Neochloris spp., Protosiphonbotryoides, Botryococcus braunii, Cryptococcus spp., Chlamydomonas reinhardtii and other Chlamydomonas spp. In some embodiments, the eukaryotic cell is an oomycete cell. In some embodiments, the unicellular eukaryotic cell is a protist cell. In some embodiments, the unicellular eukaryotic cell is a protozoan cell.

In some embodiments, the eukaryotic cell is a cell of a multicellular eukaryotic organism. For example, the multicellular eukaryotic organism can be selected from the group consisting of a vertebrate, an invertebrate, a multicellular fungus, a multicellular oomycete, a multicellular algae, and a multicellular plant. In some embodiments, the eukaryotic organism is a human. In some embodiments, the eukaryotic organism is a non-human vertebrate. In some embodiments, the eukaryotic organism is an invertebrate. In some embodiments, the eukaryotic organism is a multicellular fungus or a multicellular oomycete. In some embodiments, the eukaryotic organism is a multicellular plant. In embodiments, the eukaryotic cell is a human cell or a cell of a non-human mammal, such as a non-human primate (e.g., monkey, ape), an ungulate (e.g., bovine, including cattle, buffalo, bison, sheep, goats, and musk oxen; pigs; camelids, including camels, llamas, and alpacas; deer, antelopes; and equids, including horses and donkeys), a carnivore (e.g., dog, cat), a rodent (e.g., rat, mouse, guinea pig, hamster, squirrel), or a lagomorph (e.g., rabbit, hare). In embodiments, the eukaryotic cell is a cell of a bird, such as a member of the following avian taxa: Galliformes (e.g., chicken, turkey, pheasant, quail), Anseriformes (e.g., duck, goose), Paleognathus (e.g., ostrich, emu), Columbiformes (e.g., pigeon, pigeon), or Psittaciformes (e.g., parrot). In an embodiment, the eukaryotic cell is a cell of an arthropod (e.g., an insect, arachnid, crustacean), a nematode, an annelid, a worm, or a mollusk. In an embodiment, the eukaryotic cell is a cell of a multicellular plant, such as angiosperms (which can be dicots or monocots) or gymnosperms (e.g., conifers, cycads, gnetum, ginkgo), ferns, horsetail plants, lycophytes, or bryophytes. In an embodiment, the eukaryotic cell is a cell of a eukaryotic multicellular algae. In an embodiment, the eukaryotic cell is a cell of a plant with agricultural or horticultural importance, such as row crops, plants and trees producing fruits, vegetables, trees, and ornamental plants (including ornamental flowers, shrubs, trees, ground covers, and lawn grasses); see, for example, a non-limiting list of commercially important cultivated plant species listed in the paragraph describing "subjects" above.

The eukaryotic cells can be grown in a culture medium. The eukaryotic cells can be contained in a bioreactor.

Purification method

The present disclosure provides the method for purifying cyclic polyribonucleotides from eukaryotic cells.For example, the purification of laboratory scale research can be carried out by the following: add phenol, chloroform and isoamyl alcohol (Sigma (Sigma): P3803), and vortex to destroy eukaryotic cells and RNA (for example, the cyclized RNA molecule formed from linear precursor RNA) is extracted into aqueous phase.Aqueous phase is washed with chloroform to remove residual phenol, and RNA is precipitated from aqueous phase by adding ethanol.The precipitate containing RNA can be air-dried and resuspended in water or aqueous buffer such as nuclease-free.

Bioreactor

Eukaryotic cell as described herein can be included in a bioreactor.In certain embodiments, any method for producing cyclic polyribonucleotide as described herein can be carried out in a bioreactor.Bioreactor refers to any container for carrying out a chemical process involving an organism or a biochemically active substance derived from such an organism therein.Especially, a bioreactor can be compatible with the method for producing circular RNA using a eukaryotic system as described herein.The container for a bioreactor can include a culture bottle, a dish or a bag, which can be single-use (disposable), autoclavable or sterilizable.A bioreactor can be made of glass, or it can also be based on polymer, or it can also be made of other materials.

Examples of bioreactors include, but are not limited to, stirred tank (e.g., well mixed) bioreactors and tubular (e.g., plug flow) bioreactors, airlift bioreactors, membrane stirred tanks, rotary filter stirred tanks, vibrating mixers, fluidized bed reactors, and membrane bioreactors. The mode of operating a bioreactor can be an intermittent or continuous process. When reagents and product streams continuously enter and exit the system, the bioreactor is continuous. An intermittent bioreactor can have a continuous recirculation flow, but there is no continuous reagent feed or product harvest. An intermittent bioreactor can have a continuous recirculation flow, but there is no continuous nutrient feed or product harvest. For intermittent harvesting and batch feeding (fed-batch or batchfed) cultivation, cells are inoculated in a culture medium similar to the composition of the batch culture medium at a lower viable cell density. Allow cells to grow exponentially without substantially external manipulation until nutrients are somewhat exhausted and the cells approach a stable growth phase. At this point, for intermittent harvesting batch feeding processes, part of the cells and products can be harvested, and the removed culture medium is supplemented with fresh culture medium. The process can be repeated several times. For the production of recombinant proteins, a batch feed process can be used. When cells grow exponentially, but nutrients are gradually exhausted, concentrated feed medium (e.g., 10-15 times concentrated basal medium) is added continuously or intermittently to supply additional nutrients, thereby allowing further increase in cell concentration and conversion period. Fresh culture medium can be added in a manner proportional to cell concentration without removing the culture medium (broth). In order to accommodate the added culture medium, the batch feed culture starts with a volume far below the full capacity of the bioreactor (e.g., about 40% to 50% of the maximum volume).

Some methods of present disclosure are for the large-scale production of cyclic polyribonucleotide.For large-scale production method, the method can be carried out in the volume of 1 liter (L) to 50L or larger (for example, 5L, 10L, 15L, 20L, 25L, 30L, 35L, 40L, 45L, 50L or larger). In some embodiments, the method can be carried out in a volume of 5L to 10L, 5L to 15L, 5L to 20L, 5L to 25L, 5L to 30L, 5L to 35L, 5L to 40L, 5L to 45L, 10L to 15L, 10L to 20L, 10L to 25L, 20L to 30L, 10L to 35L, 10L to 40L, 10L to 45L, 10L to 50L, 15L to 20L, 15L to 25L, 15L to 30L, 15L to 35L, 15L to 40L, 15L to 45L, or 15 to 50L.

In some embodiments, the bioreactor can produce at least 1g of circular RNA. In some embodiments, the bioreactor can produce 1-200g of circular RNA (e.g., 1-10g, 1-20g, 1-50g, 10-50g, 10-100g, 50-100g, or 50-200g of circular RNA). In some embodiments, the amount produced is measured per liter (e.g., 1-200g/liter), per batch or reaction (e.g., 1-200g/batch or reaction), or per unit time (e.g., 1-200g/hour or/day).

In some embodiments, more than one bioreactor can be used in series to increase production capacity (eg, one, two, three, four, five, six, seven, eight, or nine bioreactors can be used in series).

How to use

In some embodiments, the compositions or formulations described herein are used as effectors in therapy and/or agriculture.

In some embodiments, the present disclosure provides a method for improving the subject by providing a composition or preparation as described herein to the subject. In some embodiments, the composition or preparation is a nucleic acid molecule or includes a nucleic acid molecule (e.g., a DNA molecule or RNA molecule as described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is a eukaryotic cell as described herein or includes a eukaryotic cell as described herein.

In some embodiments, the present disclosure provides a method for treating a subject's disease by providing a composition or preparation as described herein to a subject in need thereof. In some embodiments, the composition or preparation is a nucleic acid molecule or includes a nucleic acid molecule (e.g., a DNA molecule or RNA molecule as described herein), and the polynucleotide is provided to a eukaryotic subject. In some embodiments, the composition or preparation is a eukaryotic cell as described herein or includes a eukaryotic cell as described herein.

In some embodiments, the disclosure provides a method of providing cyclic polyribonucleotides to a subject by providing the subject with a eukaryotic cell described herein.

In some embodiments, the subject includes eukaryotic cells. In some embodiments, the subject includes vertebrates, invertebrates, fungi, oomycetes, plants, or microorganisms. In some embodiments, the subject is a vertebrate (e.g., a mammal, a bird, a fish, a reptile, or an amphibian). In some embodiments, the subject is a human. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a non-human mammal, such as a non-human primate, an ungulate, a carnivore, a rodent, or a lagomorph. In some embodiments, the subject is a bird, a reptile, or an amphibian. In some embodiments, the subject is an invertebrate (e.g., an insect, an arachnid, a nematode, or a mollusk). In some embodiments, the subject is a plant or eukaryotic algae. In some embodiments, the subject is a plant, such as an angiosperm (which can be a dicot or a monocot) or a gymnosperm (e.g., a conifer, a cycad, a gnetophyte, a ginkgo), a fern, a horsetail plant, a lycophyte, or a bryophyte. In embodiments, the subject is a plant of agricultural or horticultural importance, such as a row crop, fruit, vegetable, tree, or ornamental plant. In some embodiments, the microorganism is selected from bacteria, fungi, oomycetes, or archaea.

Preparation or composition

In some embodiments of the present disclosure, cyclic polyribonucleotides as described herein (e.g., cyclic polyribonucleotides prepared using a eukaryotic system by the methods described herein) can be provided as preparations or compositions, for example, for delivery to a cell, plant, invertebrate, non-human vertebrate, or human subject's composition, such as agriculture, veterinary or pharmaceutical composition. In certain embodiments, the present disclosure provides a kind of eukaryotic cell (e.g., eukaryotic cell prepared using a eukaryotic system by the methods described herein), which can be formulated as, for example, for delivery to a cell, plant, invertebrate, non-human vertebrate, or human subject's composition, such as agriculture, veterinary or pharmaceutical composition. In certain embodiments, a eukaryotic system as described herein in an appropriate composition (e.g., in agriculture, veterinary or pharmaceutical formulation) is provided to a subject.

Therefore, in some embodiments, the present disclosure also relates to compositions, these compositions include cyclic polyribonucleotides (for example, cyclic polyribonucleotides prepared by eukaryotic method as described herein) or eukaryotic cells and pharmaceutically acceptable carriers comprising the cyclic polyribonucleotides. In one aspect, the present disclosure provides pharmaceutical compositions or veterinary compositions, these pharmaceutical compositions or veterinary compositions include effective dose of polyribonucleotides as described herein (or eukaryotic cells comprising the polyribonucleotides) and pharmaceutically acceptable excipients. The pharmaceutical compositions or veterinary compositions of the present disclosure can include polyribonucleotides as described herein (or eukaryotic cells comprising the polyribonucleotides) and one or more pharmaceutically or physiologically acceptable carriers, excipients or diluents.

In certain embodiments, pharmaceutically acceptable carriers can be components that are nontoxic to a subject except active ingredients in a pharmaceutical composition or veterinary composition. Pharmaceutically acceptable carriers can include, but are not limited to, buffers, excipients, stabilizers, or preservatives. Examples of pharmaceutically acceptable carriers are physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents, and absorption delay agents, such as salts, buffers, sugars, antioxidants, aqueous or non-aqueous carriers, preservatives, wetting agents, surfactants, or emulsifiers, or combinations thereof. The amount of one or more pharmaceutically acceptable carriers in a pharmaceutical composition or veterinary composition can be determined experimentally based on the activity of one or more carriers and the characteristics of the desired formulation (such as stability and/or minimum oxidation).

In some embodiments, such compositions may include buffers such as acetic acid, citric acid, histidine, boric acid, formic acid, succinic acid, phosphoric acid, carbonic acid, malic acid, aspartic acid, Tris buffer, HEPPSO, HEPES, neutral buffered saline, phosphate buffered saline, etc.; carbohydrates such as glucose, sucrose, mannose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); antibacterial and antifungal agents; and preservatives.

In certain embodiments, the compositions of the present disclosure can be formulated for a variety of parenteral or non-parenteral administration modes. In one embodiment, these compositions can be formulated for infusion or intravenous administration. The compositions disclosed herein can be provided as sterile liquid preparations such as isotonic aqueous solutions, emulsions, suspensions, dispersions or viscous compositions that can be buffered to a desired pH. Formulations suitable for oral use can include liquid solutions, capsules, medicine bags, tablets, lozenges and lozenges, powder liquid suspensions and emulsions in appropriate liquids.

The pharmaceutical composition or veterinary composition of the present disclosure can be administered in a manner suitable for the disease or condition to be treated or prevented. The amount and frequency of administration will be determined by factors such as the condition of the subject, and the type and severity of the subject's disease or condition, although appropriate dosages can be determined by clinical trials.

In an embodiment, the cyclic polyribonucleotide described in this disclosure is provided with the formulation suitable for agricultural application, for example as liquid solution or emulsion or suspension, concentrate (liquid, emulsion, suspension, gel or solid), powder, particle, paste, gel, bait or seed coating or seed treatment agent.The embodiment of this type of agricultural formulation is applied to the environment of plant or plant, for example as foliar spray, dusting application, particle application, root or soil soaking, processing in ditch, particle soil processing, bait, hydroponic solution or implantable or injectable formulation.Some embodiments of this type of agricultural formulation include extra components, such as excipient, diluent, surfactant, spreading agent, adhesive, safener, stabilizer, buffer, drift control agent, retainer, oil concentrate, defoamer, foam marker, fragrance, carrier or encapsulating agent. Useful adjuvants for agricultural formulations include those disclosed in the Compendium of Herbicide Adjuvants, 13th Edition (2016), publicly available online at www[dot]herbicide-adjuvants[dot]com. In embodiments, an agricultural formulation containing a cyclic polyribonucleotide as described in the present disclosure (or a eukaryotic cell containing the cyclic polyribonucleotide) further contains one or more components selected from the group consisting of: a carrier, a surfactant, a wetting agent, a spreading agent, a cationic lipid, a silicone, a silicone surfactant, an antioxidant, a polynucleotide herbicidal molecule, a non-polynucleotide herbicidal molecule, a non-polynucleotide pesticidal molecule, a safener, an insect pheromone, an insect attractant, and an insect growth regulator.

Example

Various embodiments of the eukaryotic systems, eukaryotic cells, formulations, methods, and other compositions described herein are set forth in the following numbered sets of embodiments.

1. A eukaryotic system for cyclizing polyribonucleotides, the eukaryotic system comprising:

(a) a linear polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein: (A) comprises a 5' self-cleaving ribozyme; (B) comprises a 5' annealing region; (C) comprises a polyribonucleotide cargo; (D) comprises a 3' annealing region; and (E) comprises a 3' self-cleaving ribozyme; and

(b) Eukaryotic cell containing RNA ligase.

2. The eukaryotic system of embodiment 1, wherein the 5' self-cleaving ribozyme is capable of self-cleavage at a site within 10 ribonucleotides of the 3' end of the 5' self-cleaving ribozyme or at a site at the 3' end of the 5' self-cleaving ribozyme.

3. The eukaryotic system of embodiment 1 or 2, wherein the 5' self-cleaving ribozyme is a ribozyme selected from the group consisting of a hammerhead ribozyme, a hairpin ribozyme, a hepatitis delta virus ribozyme (HDV), a Varkud satellite (VS) ribozyme, a glmS ribozyme, a twister ribozyme, a twister sister ribozyme, an axe ribozyme, and a pistol ribozyme.

4. The eukaryotic system of embodiment 3, wherein the 5' self-cleaving ribozyme is a hammerhead ribozyme.

5. The eukaryotic system of any one of embodiments 1-4, wherein the 5' self-cleaving ribozyme comprises a region having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO: 2.

6. The eukaryotic system of embodiment 5, wherein the 5' self-cleaving ribozyme comprises the nucleic acid sequence of SEQ ID NO: 2.

7. The eukaryotic system of embodiment 1 or 2, wherein the 5' self-cleaving ribozyme comprises a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof.

8. The eukaryotic system of embodiment 7, wherein the 5' self-cleaving ribozyme comprises the nucleic acid sequence of any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof.

9. The eukaryotic system of any one of embodiments 1-8, wherein the 3' self-cleaving ribozyme is capable of self-cleavage at a site within 10 ribonucleotides of the 5' end of the 3' self-cleaving ribozyme or at a site at the 5' end of the 3' self-cleaving ribozyme.

10. The eukaryotic system of any one of embodiments 1-9, wherein the 3' self-cleaving ribozyme is a ribozyme selected from the group consisting of a hammerhead ribozyme, a hairpin ribozyme, a hepatitis delta virus ribozyme (HDV), a Varkud satellite (VS) ribozyme, a glmS ribozyme, a twister ribozyme, a twister sister ribozyme, an axe ribozyme, and a pistol ribozyme.

11. The eukaryotic system of embodiment 10, wherein the 3' self-cleaving ribozyme is a hepatitis delta virus (HDV) ribozyme.

12. The eukaryotic system of any one of embodiments 1-10, wherein the 3' self-cleaving ribozyme comprises a region having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:13.

13. The eukaryotic system of embodiment 12, wherein the 3' self-cleaving ribozyme comprises the nucleic acid sequence of SEQ ID NO:13.

14. The eukaryotic system of any one of embodiments 1-9, wherein the 3' self-cleaving ribozyme comprises a nucleic acid sequence having at least 95% sequence identity to any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof.

15. The eukaryotic system of embodiment 14, wherein the 3' self-cleaving ribozyme comprises the nucleic acid sequence of any one of SEQ ID NOs: 38-585, or its corresponding RNA equivalent, or a catalytically competent fragment thereof.

16. The eukaryotic system of any one of embodiments 1-15, wherein cleavage by the 5' self-cleaving ribozyme and cleavage by the 3' self-cleaving ribozyme produce a ligase-compatible linear polyribonucleotide.

17. The eukaryotic system of any one of embodiments 1-16, wherein cleavage by the 5' self-cleaving ribozyme produces a free 5'-hydroxyl group, and cleavage by the 3' self-cleaving ribozyme produces a free 2',3'-cyclic phosphate group.

18. The eukaryotic system of any one of embodiments 1-17, wherein the 5' annealing region has 2 to 100 ribonucleotides.

19. The eukaryotic system of any one of embodiments 1-18, wherein the 3' annealing region has 2 to 100 ribonucleotides.

20. The eukaryotic system of one of embodiments 1-19, wherein the 5' annealing region comprises a 5' complementary region having between 2 and 50 ribonucleotides; and the 3' annealing region comprises a 3' complementary region having between 2 and 50 ribonucleotides; and wherein the 5' complementary region and the 3' complementary region have a sequence complementarity between 50% and 100%; or wherein the 5' complementary region and the 3' complementary region have a binding free energy lower than -5 kcal/mol; or wherein the 5' complementary region and the 3' complementary region have a binding Tm of at least 10°C.

21. The eukaryotic system of embodiment 20, wherein the 5' annealing zone further comprises a 5' non-complementary region having between 2 and 50 ribonucleotides and located 5' to the 5' complementary region; and the 3' annealing zone further comprises a 3' non-complementary region having between 2 and 50 ribonucleotides and located 3' to the 3' complementary region; and wherein the 5' non-complementary region and the 3' non-complementary region have a sequence complementarity between 0% and 50%; or wherein the 5' non-complementary region and the 3' non-complementary region have a binding free energy greater than -5 kcal/mol; or wherein the 5' non-complementary region and the 3' non-complementary region have a binding Tm less than 10°C.

22. The eukaryotic system of any one of embodiments 1-21, wherein the 5' annealing region comprises a region having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

23. The eukaryotic system of embodiment 22, wherein the 5' annealing region comprises the nucleic acid sequence of SEQ ID NO: 4.

24. The eukaryotic system of any one of embodiments 1-23, wherein the 3' annealing region comprises a region having at least 85% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

25. The eukaryotic system of embodiment 24, wherein the 3' annealing region comprises the nucleic acid sequence of SEQ ID NO:12.

26. The eukaryotic system of any one of embodiments 1-25, wherein the polyribonucleotide cargo comprises a coding sequence, or comprises a non-coding sequence, or comprises a combination of a coding sequence and a non-coding sequence.

27. The eukaryotic system of embodiment 26, wherein the polyribonucleotide cargo comprises at least one non-coding RNA sequence.

28. The eukaryotic system of embodiment 26 or 27, wherein the at least one non-coding RNA sequence comprises at least one RNA selected from the group consisting of: an RNA aptamer, a long non-coding RNA (lncRNA), a transfer RNA-derived fragment (tRF), a transfer RNA (tRNA), a ribosomal RNA (rRNA), a small nuclear RNA (snRNA), a small nucleolar RNA (snoRNA), and a Piwi-interacting RNA (piRNA); or a fragment of any of these RNAs.

29. The eukaryotic system of embodiment 26 or 27, wherein the at least one non-coding RNA sequence comprises a regulatory RNA.

30. The eukaryotic system of embodiment 29, wherein the at least one non-coding RNA sequence trans-regulates the target sequence.

31. The eukaryotic system of embodiment 30, wherein the target sequence comprises a nucleotide sequence of a gene of the subject genome.

32. The eukaryotic system of embodiment 30 or 31, wherein trans-regulation of the target sequence by the at least one non-coding RNA sequence is upregulation of expression of the target sequence.

33. The eukaryotic system of embodiment 30 or 31, wherein trans-regulation of the target sequence by the at least one non-coding RNA sequence is down-regulation of expression of the target sequence.

34. The eukaryotic system of embodiment 30 or 31, wherein trans-regulation of the target sequence by the at least one non-coding RNA sequence is inducible expression of the target sequence.

35. The eukaryotic system of embodiment 27, wherein the at least one non-coding RNA sequence comprises an RNA selected from the group consisting of: small interfering RNA (siRNA) or a precursor thereof, double-stranded RNA (dsRNA) or at least partially double-stranded RNA; hairpin RNA (hpRNA), micro RNA (miRNA), or a precursor thereof; phase small interfering RNA (phasiRNA) or a precursor thereof; heterochromatin small interfering RNA (hcsiRNA) or a precursor thereof; and natural antisense short interfering RNA (natsiRNA) or a precursor thereof.

36. The eukaryotic system of embodiment 27, wherein the at least one non-coding RNA sequence comprises a guide RNA (gRNA) or a precursor thereof.

37. The eukaryotic system of any one of embodiments 26-36, wherein the polyribonucleotide cargo comprises a coding sequence encoding a polypeptide.

38. The eukaryotic system of any one of embodiments 26-37, wherein the polyribonucleotide cargo comprises an IRES operably linked to a coding sequence encoding a polypeptide.

39. The eukaryotic system of any one of embodiments 26-37, wherein the polyribonucleotide cargo comprises a Kozak sequence operably linked to an expression sequence encoding a polypeptide.

40. The eukaryotic system of any one of embodiments 26-39, wherein the polyribonucleotide cargo comprises an RNA sequence encoding a polypeptide having a biological effect on a subject.

41. The eukaryotic system of embodiment 39 or 40, wherein the polyribonucleotide cargo comprises an RNA sequence encoding a polypeptide and having a nucleotide sequence codon-optimized for expression in the subject.

42. The eukaryotic system of any one of embodiments 39-41, wherein the subject comprises (a) a eukaryotic cell; or (b) a prokaryotic cell.

43. The eukaryotic system of any one of embodiments 39-42, wherein the subject comprises a vertebrate, an invertebrate, a fungus, an oomycete, a plant, or a microorganism.

44. The eukaryotic system of embodiment 43, wherein the vertebrate is selected from a human, a non-human mammal, a reptile, a bird, an amphibian, or a fish.

45. The eukaryotic system of embodiment 43, wherein the invertebrate is selected from insects, arachnids, nematodes, or molluscs.

46. The eukaryotic system of embodiment 43, wherein the plant is selected from a monocot, a dicot, a gymnosperm, or a eukaryotic alga.

47. The eukaryotic system of embodiment 43, wherein the microorganism is selected from bacteria, fungi, oomycetes, or archaea.

48. The eukaryotic system of any one of embodiments 1-47, wherein the linear polyribonucleotide further comprises a spacer region between the 5' annealing region and the polyribonucleotide cargo, the length of which is at least 5 polyribonucleotides.

49. The eukaryotic system of any one of embodiments 1-48, wherein the linear polyribonucleotide further comprises a spacer region between the 5' annealing region and the polyribonucleotide cargo, the length of which is between 5 and 1000 polyribonucleotides.

50. The eukaryotic system of embodiment 48 or 49, wherein the spacer region comprises a poly A sequence.

51. The eukaryotic system of embodiment 48 or 49, wherein the spacer region comprises a poly A-C sequence.

52. The eukaryotic system of any one of embodiments 1-51, wherein the linear polyribonucleotide is at least 1 kb.

53. The eukaryotic system of any one of embodiments 1-52, wherein the linear polyribonucleotide is 1 kb to 20 kb.

54. The eukaryotic system of any one of embodiments 1-53, wherein the RNA ligase is endogenous to the eukaryotic cell.

55. The eukaryotic system of any one of embodiments 1-53, wherein the RNA ligase is heterologous to the eukaryotic cell.

56. The eukaryotic system of embodiments 1-55, wherein the RNA ligase is provided to the eukaryotic cell by transcribing an exogenous polynucleotide into an mRNA encoding the RNA ligase in the eukaryotic cell and translating the mRNA encoding the RNA ligase.

57. The eukaryotic system of embodiment 55, wherein RNA ligase is provided to the eukaryotic cell as an exogenous protein.

58. The eukaryotic system of any one of embodiments 1-57, wherein the RNA ligase is a tRNA ligase.

59. The eukaryotic system of embodiment 58, wherein the tRNA ligase is T4 ligase, RtcB ligase, TRL-1 ligase, Rnl1 ligase, Rnl2 ligase, LIG1 ligase, LIG2 ligase, PNK/PNL ligase, PF0027 ligase, thpR ligT ligase, ytlPor ligase, or a variant thereof.

60. The eukaryotic system of embodiment 59, wherein the RNA ligase comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 586-602.

61. The eukaryotic system of any one of embodiments 1-57, wherein the RNA ligase is selected from the group consisting of a plant RNA ligase, a chloroplast RNA ligase, an RNA ligase from an archaea, a bacterial RNA ligase, a eukaryotic RNA ligase, a viral RNA ligase, or a mitochondrial RNA ligase, or a variant thereof.

62. The eukaryotic system of any one of embodiments 1-61, wherein the eukaryotic cell is provided with exogenous polyribonucleotides comprising the linear polynucleotide.

63. The eukaryotic system of any one of embodiments 1-62, wherein the linear polyribonucleotide is transiently transcribed in the eukaryotic cell from an exogenous recombinant DNA molecule provided to the eukaryotic cell.

64. The eukaryotic system of any one of embodiments 1-62, wherein the linear polyribonucleotide is transcribed in the eukaryotic cell from an exogenous DNA molecule provided to the eukaryotic cell.

65. The eukaryotic system of embodiment 63 or 64, wherein the exogenous DNA molecule is not integrated into the genome of the eukaryotic cell.

66. The eukaryotic system of any one of embodiments 63-65, wherein the exogenous DNA molecule comprises a heterologous promoter operably linked to the DNA encoding the linear polyribonucleotides.

67. The eukaryotic system of embodiment 66, wherein the heterologous promoter is selected from the group consisting of: T7 promoter, T6 promoter, T4 promoter, T3 promoter, SP3 promoter, SP6 promoter, CaMV 35S, opine promoter, plant ubiquitin, rice actin 1, ADH-1 promoter, GPD promoter, CMV promoter, EF1α promoter, CAG promoter, PGK promoter, U6 nuclear promoter, TRE promoter, OpIE2 promoter, or OpIE1 promoter.

68. A eukaryotic system as described in embodiment 63 or 64, wherein the linear polyribonucleotide is transcribed in the eukaryotic cell from a recombinant DNA molecule incorporated into the genome of the eukaryotic cell.

69. The eukaryotic system of any one of embodiments 1-68, wherein the eukaryotic cell is grown in culture medium.

70. The eukaryotic system of embodiment 69, wherein the eukaryotic cells are contained in a bioreactor.

71. The eukaryotic system of any one of embodiments 1-69, wherein the eukaryotic cell is a unicellular eukaryotic cell.

72. The eukaryotic system of embodiment 71, wherein the unicellular eukaryotic cell is selected from the group consisting of a unicellular fungal cell, an oomycete cell, a unicellular animal cell, a unicellular plant cell, a unicellular algae cell, a protist cell, and a protozoan cell.

73. The eukaryotic system of any one of embodiments 1-72, wherein the eukaryotic cell is a multicellular eukaryote.

74. The eukaryotic cell of embodiment 73, wherein the multicellular eukaryote is selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular oomycetes, multicellular algae, and multicellular plants.

75. A formulation comprising the eukaryotic system of any one of embodiments 1-74.

76. The formulation of embodiment 75, wherein the formulation is a pharmaceutical formulation, a veterinary formulation, or an agricultural formulation.

77. A method for producing circular RNA, the method comprising contacting (a) with (b) in a eukaryotic cell:

(a) a linear polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein: (A) comprises a 5' self-cleaving ribozyme; (B) comprises a 5' annealing region; (C) comprises a polyribonucleotide cargo; (D) comprises a 3' annealing region; and (E) comprises a 3' self-cleaving ribozyme; and

(b) RNA ligase; wherein cleavage by the 5' self-cleaving ribozyme and cleavage by the 3' self-cleaving ribozyme produce a ligase-compatible linear polyribonucleotide, and wherein the RNA ligase ligates the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide, thereby producing a circular RNA.

78. The method of embodiment 77, wherein the circular RNA is isolated from the eukaryotic cell.

79. The method of embodiment 77 or 78, wherein the RNA ligase is endogenous to the eukaryotic cell.

80. The method of embodiment 77 or 78, wherein the RNA ligase is heterologous to the eukaryotic cell.

81. A circular RNA produced by the method of any one of embodiments 77-80.

82. A formulation comprising the circular RNA of embodiment 81.

83. The formulation of embodiment 82, wherein the formulation is a pharmaceutical formulation, a veterinary formulation, or an agricultural formulation.

84. A method of treating a disorder in a subject in need thereof, the method comprising providing the subject with the formulation of embodiment 82 or 83.

85. A eukaryotic cell, comprising:

(a) a linear polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein: (A) comprises a 5' self-cleaving ribozyme; (B) comprises a 5' annealing region; (C) comprises a polyribonucleotide cargo; (D) comprises a 3' annealing region; and (E) comprises a 3' self-cleaving ribozyme; and

(b) RNA ligase; wherein cleavage by the 5' self-cleaving ribozyme and cleavage by the 3' self-cleaving ribozyme produce a ligase-compatible linear polyribonucleotide, and wherein the RNA ligase is capable of ligating the 5' end and the 3' end of the ligase-compatible linear polyribonucleotide to produce a circular RNA.

86. The eukaryotic cell of embodiment 85, wherein the RNA ligase is endogenous to the eukaryotic cell.

87. The eukaryotic cell of embodiment 85, wherein the RNA ligase is heterologous to the eukaryotic cell.

88. The eukaryotic cell of embodiment 85, further comprising the circular RNA.

89. A method for providing circular RNA to a subject, the method comprising providing the eukaryotic cell of embodiment 88 to the subject.

90. A method of treating a condition in a subject in need thereof, the method comprising providing the subject with the eukaryotic cell of embodiment 88.

91. A formulation comprising the eukaryotic cells of embodiment 88.

92. The formulation of embodiment 91, wherein the eukaryotic cells are dried or frozen.

93. The formulation of embodiment 91 or 92, wherein the formulation is a pharmaceutical formulation, a veterinary formulation, or an agricultural formulation.

94. A method of treating a disorder in a subject in need thereof, the method comprising providing to the subject the formulation of any one of embodiments 91-93.

Examples

The following examples are presented to provide one of ordinary skill in the art with a description of how the compositions and methods described herein may be used, prepared and evaluated, and are intended to be purely exemplary of the present disclosure and are not intended to limit the scope of what the inventors regard as their invention. Table 11 summarizes the examples provided.

Table 11: Summary of examples of generating functional circular RNA in eukaryotic cells

Example 1: Production of circular RNA in maize protoplast plant cells

This example describes the design, production, and purification of circular RNA from a eukaryotic system including the plant cell maize protoplasts. A schematic diagram depicting the design of an exemplary DNA construct for producing circular RNA in maize protoplast cells is provided in FIG. 1 . The DNA construct was designed using the HBT plasmid and encodes from 5' to 3': a constitutive promoter and enhancer, such as the 35S promoter with an enhancer (SEQ ID NO: 1); a 5' self-cleaving ribozyme that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 2); a 5' annealing region (SEQ ID NO: 4); a polyribonucleotide cargo, including, for example, an aptamer (such as Pepper (SEQ ID NO: 6)), an IRES (such as EMCV IRES (SEQ ID NO: 8)), and a coding sequence (such as nanoluciferase (SEQ ID NO: 10)); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme that cuts at its 5' end, such as the hepatitis delta virus ribozyme (SEQ ID NO: 13); and a transcription terminator sequence, such as the NOS terminator (SEQ ID NO: 15).

The DNA construct is transcribed to produce a linear RNA, which includes from 5' to 3': a 5' self-cleaving ribozyme (SEQ ID NO: 3) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 5); an internal ribosome entry site (IRES) (SEQ ID NO: 9); a coding region encoding a polypeptide (SEQ ID NO: 11); a 3' annealing region (SEQ ID NO: 12); and a 3' self-cleaving ribozyme (SEQ ID NO: 14) that cuts at its 5' end. After expression, the linear RNA self-cleaves to produce a ligase-compatible linear RNA with a free 5' hydroxyl and a free 3' monophosphate. The ligase-compatible linear RNA is cyclized by adding RNA ligase. A schematic diagram depicting the process of cyclization in corn protoplast cells is provided in FIG. 2.

The DNA construct encoding RNA ligase is designed to maintain RNA ligase expression in corn protoplast plant cells. The DNA construct is constructed based on the HBT plasmid and includes from 5' to 3': a promoter for constitutive expression of RNA ligase, such as the 35S promoter (SEQ ID NO: 17); a coding sequence encoding RNA ligase, such as AtRNL-monocotyledonous RNA ligase, which is a codon-optimized Arabidopsis thaliana RNA ligase (Uniprot reference AT1G07910) (SEQ ID NO: 18); and a transcription terminator sequence, such as the NOS terminator sequence (SEQ ID NO: 16). The DNA construct is transcribed to produce an RNA sequence of RNA ligase, which is then translated to produce RNA ligase in corn protoplast plant cells.

The DNA construct designed as described in the coding nanoluciferase and RNA ligase was transformed into corn B73 protoplasts. According to the improved leaf flesh protoplast preparation protocol as described in molbio.[dot]mgh.[dot]harvard.[dot]edu/sheenweb/protocols_reg.[dot]html, corn B73 protoplasts were isolated for 8-10 day old seedlings. This protocol is commonly used for monocots, such as maize and rice (Oryza sativa).

Prepare an enzyme solution containing 0.6M mannitol, 10mM MES (pH 5.7), 1.5% Cellulase RIO, and 0.3% Metase RIO. Heat the enzyme solution at 50°C-55°C for 10 minutes to inactivate the protease and accelerate enzyme dissolution. Then cool the solution to room temperature, then add 1mM _CaCl2 , 5mM mercaptoethanol, and 0.1% bovine serum albumin. Pass the enzyme solution through a 0.45μm filter, and prepare a wash solution containing 0.6M mannitol, 4mM MES (pH 5.7), and 20mM KCl.

Additionally, leaves of the plant were obtained and the middle 6-8 cm were cut out. Ten leaf segments were stacked and cut into 0.5 mm wide strips without damaging the leaves. The leaf strips were completely immersed in the enzyme solution in a petri dish, covered with aluminum foil, and vacuum was applied for 30 minutes to penetrate the leaf tissue. The petri dish was transferred to a platform shaker and incubated for an additional 2.5 hours of digestion with gentle shaking at 40 rpm. After digestion, the enzyme solution containing the protoplasts was carefully transferred to a round-bottom tube through a 35 μm nylon mesh using a serological pipette. The petri dish was then rinsed with 5 mL of washing solution and also filtered through a mesh. The protoplast suspension was centrifuged at 1200 rpm for 2 minutes in a swinging bucket centrifuge. As much supernatant as possible was aspirated without touching the precipitate; the precipitate was gently washed once with 20 mL of washing buffer and the supernatant was carefully removed. The precipitate was then resuspended by gently vortexing in a small amount of washing solution and then resuspended in 10-20 mL of washing buffer. Pipe is placed upright on ice and continues 30 minutes to 4 hours, but is no more than 4 hours.After leaving standstill on ice, remove supernatant by suction, and make sediment resuspended with the lavage buffer solution between 2mL and 5mL.Use hemocytometer to measure the concentration of protoplasts, and with lavage buffer solution, concentration is adjusted to 2x 10 ⁵ individual protoplasts/mL.

Protoplasts were then PEG-transfected as described by Niu and Sheen (2011). Briefly, 10 μL of DNA vector (10 μg per vector), 100 μL of protoplasts in wash solution, and 110 μL of PEG solution (40% (w/v) PEG 4000 (Sigma-Aldrich), 0.2 M mannitol, and 0.1 M CaCl ₂ ) were incubated at room temperature for 5-10 minutes. 440 μL of wash solution was added and gently mixed by inversion to stop transfection. The protoplasts were then precipitated by spinning at 110 x g for 1 minute, and the supernatant was removed. In each well of a 12-well tissue culture plate, the protoplasts were gently resuspended with 500 μL of an incubation solution comprising 0.6 M mannitol, 4 mM MES (pH 5.7), and 4 mM KCl, and incubated for 12, 24, and 48 hours.

The production of RNA in protoplast cells was monitored by harvesting cells from 100 μL protoplast cells and measuring aptamer fluorescence. In order to measure the production of RNA using aptamer fluorescence, protoplast cells were supplemented with 500 nM HBC525, which emits fluorescence when combined with the Pepper aptamer in the RNA load, see Chen et al. (2019) Nature Biotechnol. [Natural Biotechnology], 37: 1287-1293, doi: 10.1038 / s41587-019-0249-1. The amount of RNA produced from the DNA construct was quantified by measuring the fluorescence at 525 nm using a spectrophotometer.

The RNA produced by the protoplast cells was then extracted from the cells. RNA extraction was performed by centrifuging 1 mL of protoplast cells, resuspending the cell pellet in TRIzol (Thermo Fisher Scientific), and adding the resuspended pellet to Direct-zol RNA microprep (Zymo Research). The extracted RNA was eluted in 15 μL of nuclease-free water.

Gel shift and/or poly A polymerase methods were used to confirm that the linear RNA was circularized in eukaryotic systems including maize protoplast cells.

Example 2: Production of circular RNA in Nicotiana benthamiana plant cells

This example describes the design, production, and purification of circular RNA from eukaryotic systems including plant cells from Nicotiana benthamiana. A schematic diagram depicting the design of an exemplary DNA construct for producing circular RNA in Nicotiana benthamiana plant cells is provided in FIG. 1 . The DNA construct was designed using the pCAMBIA-1302 plasmid (Abcam) and encodes from 5' to 3': a constitutive promoter, such as the 35S promoter (SEQ ID NO: 19); a 5' self-cleaving ribozyme that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 2); a 5' annealing region (SEQ ID NO: 4); a polyribonucleotide cargo, including, for example, an aptamer (such as Pepper (SEQ ID NO: 6)), an IRES (such as EMCV IRES (SEQ ID NO: 8)), and a coding sequence (such as nanoluciferase (SEQ ID NO: 10)); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme that cuts at its 5' end, such as the hepatitis delta virus ribozyme (SEQ ID NO: 13).

The DNA construct is transcribed to produce a linear RNA, which includes from 5' to 3': a 5' self-cleaving ribozyme (SEQ ID NO: 3) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 5); an internal ribosome entry site (IRES) (SEQ ID NO: 9); a coding region encoding a polypeptide (SEQ ID NO: 11); a 3' annealing region (SEQ ID NO: 12); and a 3' self-cleaving ribozyme (SEQ ID NO: 14) that cuts at its 5' end. After expression, the linear RNA self-cleaves to produce a ligase-compatible linear RNA with a free 5' hydroxyl and a free 3' monophosphate. The ligase-compatible linear RNA is cyclized by adding an RNA ligase. A schematic diagram depicting the process of cyclization in a plant cell is provided in FIG. 2.

Design the DNA construct encoding RNA ligase to maintain the RNA ligase expression in the Nicotiana benthamiana plant cell. The DNA construct is constructed based on the pCAMBIA-1302 plasmid (Abcam) and includes from 5' to 3': a promoter for constitutive expression of RNA ligase, such as 35S promoter (SEQ ID NO: 17); a coding sequence encoding RNA ligase, such as AtRNL, Arabidopsis RNA ligase (Uniprot reference AT1G07910) (SEQ ID NO: 20); and a transcription terminator sequence, such as NOS terminator sequence (SEQ ID NO: 16). The DNA construct is transcribed to produce the RNA sequence of RNA ligase, which is then translated to produce RNA ligase in plant cells.

The DNA construct was transformed into the Agrobacterium GV3101 strain (Lifeasible). The Agrobacterium infiltration of Nicotiana benthamiana was performed according to the method of Norkunas et al., 2018. In brief, a single colony of the recombinant bacteria was inoculated into a liquid LB medium containing kanamycin (50 mg/L) and rifampicin (25 mg/L). The culture was then incubated overnight at 28 ° C with shaking. The bacteria were precipitated and resuspended in MMA basic medium (including 10mM MES (pH 5.6), 10mM MgCl2, and 200μM acetosyringone) to an OD600 of 1.0. The culture was then incubated at room temperature with gentle shaking for 2-4 hours. The culture of the recombinant bacteria carrying the plasmid with the RNA payload sequence and the recombinant bacteria carrying the plasmid with the RNA ligase were mixed at 1:1 and then delivered to the back of the leaves of the 1-2 month-old plantlets using a blunt plastic syringe and applying gentle pressure.

The production of RNA in N. benthamiana cells was monitored by measuring aptamer fluorescence. In order to measure the production of RNA using aptamer fluorescence, 500nM HBC525 (which fluoresces when bound to the Pepper aptamer in the RNA load, see Chen et al. (2019) Nature Biotechnol. [Natural Biotechnology], 37: 1287-1293, doi: 10.1038/s41587-019-0249-1) was delivered to the back of leaves transformed with Agrobacterium. The amount of RNA produced from the DNA construct was quantified by measuring the fluorescence at 525nm using a spectrophotometer.

The RNA produced by the N. benthamiana cells was then extracted from the cells. RNA extraction was performed by harvesting the infiltrated leaves and grinding the samples in TRIzol (Thermo Fisher Scientific) and adding the resuspended pellet to Direct-zolRNA microprep (Zymo Research). The extracted RNA was eluted in 15 μL of nuclease-free water.

Gel shift methods and/or poly A polymerase methods were used to confirm that the linear RNA that was circularized was circularized in eukaryotic systems including Nicotiana benthamiana cells.

Example 3: Production of circular RNA in Saccharomyces cerevisiae cells

This example describes the design, production, and purification of circular RNA from eukaryotic systems including Saccharomyces cerevisiae cells. A schematic diagram depicting the design of an exemplary DNA construct for producing circular RNA in Saccharomyces cerevisiae cells is provided in FIG. 1 . The DNA construct was designed using the pYES2 plasmid (Thermo Fisher Scientific) and encodes from 5' to 3': a promoter for inducing RNA expression, such as the pGAL promoter (SEQ ID NO:21); a 5' self-cleaving ribozyme that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO:2); a 5' annealing region (SEQ ID NO:4); a polyribonucleotide cargo, including, for example, an aptamer (such as Pepper (SEQ ID NO:6)), an IRES (such as EMCV IRES (SEQ ID NO:8)), and a coding sequence (such as nanoluciferase (SEQ ID NO:10)); a 3' annealing region (SEQ ID NO:12); a 3' self-cleaving ribozyme that cuts at its 5' end, such as the hepatitis delta virus ribozyme (SEQ ID NO:13); and a transcription terminator sequence, such as the CYC1 terminator (SEQ ID NO:23).

The DNA construct is transcribed to produce a linear RNA, which includes from 5' to 3': a 5' self-cleaving ribozyme (SEQ ID NO: 3) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 5); an internal ribosome entry site (IRES) (SEQ ID NO: 9); a coding region encoding a polypeptide (SEQ ID NO: 11); a 3' annealing region (SEQ ID NO: 12); and a 3' self-cleaving ribozyme (SEQ ID NO: 14) that cuts at its 5' end. After expression, the linear RNA self-cleaves to produce a ligase-compatible linear RNA with a free 5' hydroxyl and a free 3' monophosphate. The ligase-compatible linear RNA is cyclized by adding RNA ligase. A schematic diagram depicting the process of cyclization in a Saccharomyces cerevisiae cell is provided in FIG. 2.

The DNA construct encoding RNA ligase is designed to maintain RNA ligase expression in fungal cells. The DNA construct is constructed based on the pYES2 plasmid and includes from 5' to 3': a promoter for inducible expression of RNA ligase, such as pGAL (SEQ ID NO: 22); a coding sequence encoding RNA ligase, such as Kluyveromyces lactis tRNA ligase (GenBank: CAG98435.1); and a transcription terminator sequence, such as the CYC1 terminator sequence (SEQ ID NO: 24). The DNA construct is transcribed to produce an RNA sequence of RNA ligase, which is then translated to produce RNA ligase in Saccharomyces cerevisiae fungal cells.

According to the pYES2 plasmid manual (Thermo Fisher Scientific), the DNA construct encoding the polyribonucleotide load and the DNA construct encoding the RNA ligase are both transformed into competent INVSc1 cells. SC-U selection plates are used to select transformants. Cells are maintained in the SC-U medium.

The production of RNA in fungal cells was monitored by harvesting cells from 1 mL of yeast and measuring aptamer fluorescence. In order to measure the production of RNA using aptamer fluorescence, protoplast cells were supplemented with 500 nM HBC525, which fluoresces when combined with the Pepper aptamer in the RNA load, see Chen et al. (2019) Nature Biotechnol. [Natural Biotechnology], 37: 1287-1293, doi: 10.1038 / s41587-019-0249-1. The amount of RNA produced from the DNA construct was quantified by measuring the fluorescence at 525 nm using a spectrophotometer.

RNA produced by the fungal cells was then extracted from the yeast cells. RNA extraction was performed by centrifuging 1 mL of yeast cells, resuspending the cell pellet in TRIzol (Thermo Fisher Scientific), and adding the resuspended pellet to Direct-zol RNA microprep (Zymo Research). The extracted RNA was eluted in 15 μL of nuclease-free water.

The linear RNA that is circularized is confirmed to be circularized in eukaryotic systems including Saccharomyces cerevisiae cells using gel shift methods and/or poly A polymerase methods.

Example 4: Characterization of circular RNA

This example describes how to characterize the extracted circular RNA produced by the methods described in Examples 1, 2, and 3.

In order to characterize the circular RNA generated by the methods described in Examples 1, 2, and 3, 1 μg of extracted RNA was boiled in 50% formamide and loaded onto a 6% PAGE urea gel for denaturing electrophoresis. After separating the nucleotides, the gel was stained with ethidium bromide and imaged. The circularity of the RNA was confirmed by observing the gel shift of the circular RNA compared to the linear RNA species. Additionally or alternatively, in order to characterize the circular RNA, 1 μg of extracted RNA was treated with poly A polymerase (New England Biolabs) according to the manufacturer's instructions.

Alternatively, circular RNA is characterized by the following: according to the manufacturer's instructions, 1 μg of extracted RNA is processed with poly A tail polymerase (New England Biolabs, Inc.). In 1 hour reaction at 37°C, a poly A tail of about 100, 200, or 300 nucleotides is enzymatically added to the linear polyribonucleotide. The poly A tail is not added to the circular polyribonucleotide because they do not have free 3' ends. After processing with the poly A tail, the product undergoes gel electrophoresis on 6% PAGE urea gel. The gel obtained compares the untreated sample with the RNA extract processed through poly A polymerase to identify the change in the molecular weight of the linear RNA compared with the molecular weight of the circular RNA observed without change.

Example 5: Expression of functional proteins from circular RNA

This example describes how to confirm that functional protein is expressed from circular RNA generated by the methods described in Examples 1, 2, and 3.

The production of functional Nanoluciferase proteins encoded by the DNA constructs described in Examples 1, 2 and 3 was measured using the wheat germ extract (WGE) in vitro translation system (Promega Corporation) and the insect cell extract (ICE) in vitro translation system (Promega Corporation).

According to the manufacturer's instructions, wheat germ extract (WGE) in vitro translation system (Puluo Mag company) is used to measure the expression of Nanoluc RNA reporter. In brief, the RNA extracted by 1 μg as described in Example 1, 2 and 3 is heated to 75 ℃ for 5 minutes, then cooled on the bench at room temperature for 20 minutes. RNA is transferred to 1x wheat germ extract and incubated at 30 ℃ for 1 hour. The mixture is placed on ice and diluted 4 times with water. The product of in vitro translation reaction is then analyzed in Nano-Glo luciferase assay (Puluo Mag company). The wheat germ extract product of 10 μl is mixed with the Nano-Glo assay buffer (Puluo Mag company) of 10 μl, and luminescence is measured in a spectrophotometer.

Alternatively, according to the manufacturer's instructions, the expression of Nanoluc RNA reporter is measured using insect cell extract (ICE) in vitro translation system (Puluo Mag company). In brief, the RNA extracted by 1 μg as described in Example 1, 2 and 3 is heated to 75 ° C for 5 minutes, then cooled on the bench at room temperature for 20 minutes. RNA is transferred to 1x insect cell extract and incubated at 30 ° C for 1 hour. The mixture is placed on ice and diluted 4 times with water. The product of in vitro translation reaction is then analyzed in Nano-Glo luciferase assay (Puluo Mag company). The insect cell extraction product of 10 μl is mixed with the Nano-Glo assay buffer (Puluo Mag company) of 10 μl, and luminescence is measured in a spectrophotometer.

Example 6: Design of RNA constructs for circularization in insect cells

This example describes the design, production, and purification of circular RNA from eukaryotic systems including insect cells. A schematic diagram depicting the design of an exemplary DNA construct for producing circular RNA in insect cells is provided in FIG. The DNA construct encodes from 5' to 3': a promoter for inducing RNA expression, such as a codon-optimized OpIE1 promoter (SEQ ID NO: 25); a 5' self-cleaving ribozyme (SEQ ID NO: 4) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 4); a polyribonucleotide payload, including, for example, an aptamer, such as Pepper (SEQ ID NO: 6); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme (SEQ ID NO: 13) that cuts at its 5' end; and a transcription terminator sequence (SEQ ID NO: 27).

The DNA construct is transcribed to produce a linear RNA, which includes from 5' to 3': a 5' self-cleaving ribozyme (SEQ ID NO: 3) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 5); a coding region encoding a polypeptide, such as an aptamer Pepper (SEQ ID NO: 7); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme (SEQ ID NO: 14) that cuts at its 5' end. After expression, the linear RNA self-cleaves to produce a ligase-compatible linear RNA with a free 5' hydroxyl and a free 3' monophosphate. The ligase-compatible linear RNA is cyclized by adding RNA ligase. A schematic diagram depicting the process of cyclization in insect cells is provided in FIG. 2.

Another DNA construct encoding RNA ligase is designed to maintain RNA ligase expression in insect cells. The DNA construct is constructed and includes from 5' to 3': a promoter for inducing RNA ligase expression in a coding sequence encoding RNA ligase (such as RNA 2', 3'-cyclic phosphate and 5'-OH (RtCB) ligase (GenBank: CAG33456.1)), such as the T7lac polymerase promoter (SEQ ID NO: 29) for driving expression in a 3' to 5' direction; and a transcription terminator sequence (SEQ ID NO: 30).

Example 7: Generation of RNA constructs that enter insect cells

This example describes the transfection of RNA constructs into insect cells and the subsequent production of circular RNA.

The linear RNA constructs described in Example 6 were cloned into the pFastBac donor plasmid for expression in Spodoptera frugiperda cells as described previously (ThermoFisher, USA). These constructs were then transformed into competent DH10Bac E. coli cells and Lac7- E. coli cells so that they contained the recombinant bacmid containing the constructs described in Example 6. SF9 or SF21 cells were co-transfected with CELLFECTIN reagent (ThermoFisher, USA) and the bacmid containing the constructs described in Example 6. Circularization of the constructs was performed by induction with IPTG. SF9 or SF21 cells were cultured as a monolayer or in suspension and RNA was then collected.

Example 8: Purification of circular RNA from insect cells

This example describes the purification of circular RNA from insect cells.

Then the cell culture described in Example 7 was ultracentrifuged at 80,000 x g for 75 minutes to remove the remaining virus and supernatant from the cell pellet. Once the supernatant is removed, the cell pellet is washed with phosphate buffered saline and centrifuged at 1,000 x g for 1 minute. The cells are then resuspended in Tri reagent (Sigma Millipore, USA). The cells are then subjected to freeze-thaw cycles from -80 ° C or from liquid nitrogen to lyse the cells in preparation for RNA extraction. The cells are then centrifuged at 12,000 x g for 1 minute at 4 ° C to precipitate cell debris, and the supernatant is transferred to a new tube in preparation for RNA purification. RNA purification is performed using RNA cleaning and concentrator columns as previously described (Zymo, USA). In order to confirm that the RNA produced from insect cells is a circular species, the purified RNA is then treated with exonuclease. Then in the case of compared with single-stranded RNA, the remaining RNA is run on PAGE gel to confirm the enrichment of circular RNA molecules.

Example 9: Production of circular RNA in insect cells

This example describes the design, production, and purification of circular RNA from eukaryotic systems including insect cells. A schematic diagram depicting the design of an exemplary DNA construct for producing circular RNA in insect cells is provided in FIG. The DNA construct encodes from 5' to 3': a promoter for inducing RNA expression, such as the OpIE1 promoter (SEQ ID NO: 25); a 5' self-cleaving ribozyme that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 2); a 5' annealing region (SEQ ID NO: 4); a polyribonucleotide cargo, including, for example, an aptamer, such as Pepper (SEQ ID NO: 6); an IRES, such as the EMCV IRES (SEQ ID NO: 8); and an expressed protein, such as the 3X-Flag protein (SEQ ID NO: 36); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme that cuts at its 5' end (SEQ ID NO: 13); and a transcription terminator sequence, such as the IE1 terminator sequence (SEQ ID NO: 28).

The DNA construct is transcribed to produce a linear RNA, which includes from 5' to 3': a 5' self-cleaving ribozyme (SEQ ID NO: 3) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 5); a coding region encoding a polypeptide, such as an aptamer Pepper (SEQ ID NO: 7); an expression sequence, such as a 3X-FLAG protein (SEQ ID NO: 37); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme (SEQ ID NO: 14) that cuts at its 5' end. After expression, the linear RNA self-cleaves to produce a ligase-compatible linear RNA with a free 5' hydroxyl and a free 3' monophosphate. The ligase-compatible linear RNA is cyclized by adding RNA ligase. A schematic diagram depicting the process of cyclization in insect cells is provided in FIG. 2.

Another DNA construct encoding RNA ligase is designed to maintain RNA ligase expression in insect cells. The DNA construct is constructed and includes from 5' to 3': a promoter for inducing RNA ligase expression in a coding sequence encoding RNA ligase (such as RNA 2', 3'-cyclic phosphate and 5'-OH (RtCB) ligase (GenBank: CAG33456.1)), such as the T7lac polymerase promoter (SEQ ID NO: 29) for driving expression in a 3' to 5' direction; and a transcription terminator sequence (SEQ ID NO: 30).

Circular RNA is produced in Spodoptera frugiperda SF9 or SF21 cells. Circular RNA is purified and incubated in wheat germ extract for 4 to 8 hours for efficient protein translation. In order to confirm the expression of 3X-FLAG peptides from circular RNA, circular RNA is incubated in anti-FLAG coated plates and then detected by ELISA assay according to the manufacturer's protocol (Sigma Millipore, USA). Protease treated and untreated proteins are compared to confirm efficient protein expression.

Example 10: Design of RNA constructs for circularization and expression in mammalian cells

This example describes the design of a DNA vector for expressing RNA and RNA ligase in mammalian cells. A schematic diagram depicting the design of an exemplary DNA construct for producing circular RNA in mammalian cells is provided in FIG. 1 . The DNA construct using the pcDNA3.1 plasmid backbone was modified at the multiple cloning site to include, from 5' to 3': a constitutive promoter for inducing RNA expression, such as a codon-optimized CMV promoter (SEQ ID NO:31); a 5' self-cleaving ribozyme that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO:2); a 5' annealing region (SEQ ID NO:4); a polyribonucleotide cargo, including, for example, an aptamer, such as Pepper (SEQ ID NO:6); an IRES, such as an EMCV IRES (SEQ ID NO:8); and an expressed protein, such as the reporter protein NanoLuc (SEQ ID NO:10); a 3' annealing region (SEQ ID NO:12); a 3' self-cleaving ribozyme that cuts at its 5' end (SEQ ID NO:13); and a transcription terminator sequence, such as SV40 (SEQ ID NO:33).

The DNA construct is transcribed to produce a linear RNA, which includes from 5' to 3': a 5' self-cleaving ribozyme (SEQ ID NO: 3) that cuts at its 3' end; a 5' annealing region (SEQ ID NO: 5); a coding region encoding a polypeptide, such as an aptamer Pepper (SEQ ID NO: 7); a 3' annealing region (SEQ ID NO: 12); a 3' self-cleaving ribozyme (SEQ ID NO: 14) that cuts at its 5' end. After expression, the linear RNA self-cleaves to produce a ligase-compatible linear RNA with a free 5' hydroxyl and a free 3' monophosphate. The ligase-compatible linear RNA is cyclized by adding RNA ligase. A schematic diagram depicting the process of cyclization in mammalian cells is provided in FIG. 2.

Another DNA construct encoding RNA ligase is designed to maintain RNA ligase expression in insect cells. The DNA construct is constructed and includes from 5' to 3': a promoter for inducing RNA ligase expression in a coding sequence encoding RNA ligase (such as RNA 2', 3'-cyclic phosphate and 5'-OH (RtCB) ligase (GenBank: CAG33456.1)), such as the TREG3G promoter (SEQ ID NO: 35) for driving expression in a 3' to 5' direction; and a transcription terminator sequence (SEQ ID NO: 30).

Example 11: Transfection of mammalian cells

This example describes transfection of DNA constructs into mammalian cells. The DNA constructs described in Examples 9 and 10 were transformed into HEK293 Tet-On 3G cells (Takara Bio). Cells were maintained in 1× Dulbecco's Modified Eagle Medium (DMEM) (Life Technologies) with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ml streptomycin under standard tissue culture conditions. Cells were plated for transfection using FuGENE HD (Promega) according to the manufacturer's instructions using OptiMEM ^™ I Low Serum Medium (Thermo Fisher Scientific).

Example 12: Monitoring RNA production in mammalian cells

This example describes the use of fluorescent aptamer Pepper to monitor RNA production in mammalian cells. The production of RNA in mammalian cells is monitored by harvesting cells from 1 mL and measuring aptamer fluorescence. In order to measure the production of RNA using aptamer fluorescence, mammalian cells are supplemented with 500nM HBC525, which emits fluorescence when combined with the Pepper aptamer in the RNA load, see Chen et al. (2019) Nature Biotechnol. [Natural Biotechnology], 37: 1287-1293, doi: 10.1038 / s41587-019-0249-1. The amount of RNA produced from the DNA construct is quantified by measuring the fluorescence at 525nm using a spectrophotometer.

Example 13: Extraction of RNA from mammalian cells

This example describes the extraction of RNA from mammalian cells. RNA produced by the mammalian cells described in Example 12 was then extracted. RNA extraction was performed by removing the culture medium and stripping the cells with 1× phosphate buffered saline (Thermo Fisher Scientific) and resuspending the cells in TRIzol™ LS reagent (Invitrogen) and purifying RNA according to the manufacturer's instructions. Total RNA concentration was measured and normalized using a micro-spectrophotometer (e.g., NanoDrop 2000 (Thermo Fisher Scientific)).

Example 14: Confirmation of circular RNA produced in mammalian cells

This example describes the use of a gel shift method to isolate and identify circular RNAs produced in mammalian cells from total RNA.

The linear RNA that circularizes in mammalian cells was confirmed to be circularized using the gel shift method. To characterize circular RNA, 1 μg of extracted RNA was boiled in 50% formamide and loaded onto a 6% PAGE urea gel for denaturing electrophoresis. After separation of the nucleotides, the gel was stained with ethidium bromide and imaged. The circularity of the RNA was confirmed by observing the gel shift of the circular RNA compared to the linear RNA species.

Example 15: Confirmation of circular RNA produced in mammalian cells

This example describes the use of poly A polymerase method to separate and confirm circular RNA from total RNA. Circular RNA is characterized by the following: according to the manufacturer's instructions, 1 μg of extracted RNA is processed with poly A tail polymerase (New England Biolabs). In the 1 hour reaction at 37 ° C, a poly A tail of about 100, 200 or 300 nucleotides is enzymatically added to the linear polyribonucleotide. The poly A tail is not added to the circular polyribonucleotide because they do not have a free 3 ' end. After being treated with the poly A tail, the product undergoes gel electrophoresis on a 6% PAGE urea gel. The resulting gel compares the untreated sample with the RNA extract treated with poly A polymerase to identify the change in the molecular weight of the linear RNA compared to the molecular weight of the observed circular RNA without change.

Example 16: Measurement of circularization efficiency of circular RNA in mammalian cells

This example describes the efficiency of measuring circular RNA production. The RNA production efficiency in mammalian cells is calculated as (the quality of the circular RNA produced)/(the quality of the total RNA). The amount of circular RNA produced by mammalian cells is measured by using aptamer fluorescence. Aptamer fluorescence is measured by: staining a 6% PAGE urea gel containing the isolated target RNA and a standard curve of homologous RNA produced by in vitro transcription (IVT) with 500nM HBC525, which emits fluorescence when combined with the Pepper aptamer in the RNA load; and using ImageJ software to analyze the relative brightness of the fluorescence. The quality is then calculated using the standard curve and divided by the total RNA quality measured in Example 15.

Example 17: Characterization of proteins produced by circular RNA produced in mammalian cells

This example shows that the circular RNA produced in mammalian cells is functional and can express reporter proteins. The production of functional nano-luciferase proteins encoded by the above-mentioned DNA constructs is measured using a rabbit reticulocyte lysate translation system. According to the manufacturer's instructions, the expression of Nanoluc RNA reporters is measured using an in vitro translation system (Promega) treated with rabbit reticulocyte lysate (RRL) nuclease. In brief, 1 μg of RNA extracted as described in Example 14 is heated to 75 ° C for 5 minutes, then cooled at room temperature on the bench for 20 minutes. RNA is transferred to 70% RRL and incubated at 30 ° C for 1 hour. The mixture is placed on ice and diluted 4 times with water. The product of the in vitro translation reaction is then analyzed in Nano-Glo luciferase assay (Promega). 10 μl of RRL product is mixed with 10 μl of Nano-Glo assay buffer (Promega), and luminescence is measured in a spectrophotometer.

Example 18: Detection of Cyclization of Linear Polyribonucleotides in Cells

This example describes the general method of using RT-PCR to confirm the circular conformation of polyribonucleotides in cells. This method is suitable for analyzing RNA samples from any cell, prokaryotic or eukaryotic cells.

The method is described here with the analysis of RNA from prokaryotic cells.Total RNA preparation from Escherichia coli bacterial cells is used as the template of reverse transcriptase (RT) reaction.Random hexamer initiation reaction is used.Linear polyribonucleotides have produced complementary DNA (cDNA), and this complementary DNA (cDNA) has a length shorter than "unit length" (i.e., 5' and 3' distance between ribozyme cleavage sites).Due to rolling circle amplification, cyclic polyribonucleotides have produced cDNA of shorter (shorter than unit length) and longer (longer than unit length) length.Use the oligonucleotide primers in the polyribonucleotide sequence, the cDNA product from RT reaction is used as the template in the PCR reaction.The PCR amplification of unit length cDNA has produced the amplicon of unit length.The PCR amplification that is longer than the cDNA of unit length has produced the amplicon of unit length and the amplicon that is longer than unit length (typically integer multiple unit length, modal is twice unit length), and this has generated characteristic ladder pattern on gel. In the absence of RNA ligase, the linear polyribonucleotide generated in vitro is used as the negative control of cyclic polyribonucleotide RT-PCR signal; These PCRs have generated the amplicon of the unit length lacking ladder pattern. The cyclic polyribonucleotide generated by contacting the linear polyribonucleotide generated in vitro with RNA ligase is used as the positive control of cyclic polyribonucleotide RT-PCR signal; These PCRs have generated the amplicon longer than unit length in ladder pattern. In this way, the RT-PCR carried out by the total RNA from bacterial cells (these bacterial cells contain the linear polyribonucleotide precursors predetermined for cyclization by RNA ligase) shows that the amplicon longer than unit length has characteristic ladder pattern, which confirms the cyclization of linear precursors, and the total RNA separated from the bacterial cells lacking polyribonucleotide or lacking RNA ligase does not show this pattern. Fig. 3 shows the example of the cyclization of linear polyribonucleotides in bacterial cells and the RT-PCR detection of cyclization RNA product. Two kinds of constructs have been tested, which encode linear polyribonucleotide precursors " min1 " (SEQ ID NO:603) (it has the unprocessed length of 392nt and the processed length of 275nt after ribozyme cutting) and " min2 " (SEQ ID NO:604) (it has the unprocessed length of 245nt and the processed length of 128nt after ribozyme cutting). The cyclization of min1 is indicated by a ladder pattern, and this ladder pattern is formed by the band from unit length amplicon (275nt) and twice unit length amplicon (550nt). The cyclization of min 2 is indicated by a ladder pattern, and this ladder pattern is formed by the band from unit length amplicon (128nt) and twice unit length amplicon (256nt).

The alternative method of the cyclization of verification linear RNA precursor uses digoxigenin labeling and Northern blot analysis.In brief, the UTP of DIG labeling is used to replace UTP, and the PCR amplicon of the DNA construct of the linear polyribonucleotide precursor of coding is used as template, and the RNA molecule of digoxigenin labeling is used to transcribe in vitro according to the explanation of manufacturers in SP6 Mega IVT test kit.Sample to be analyzed is extracted as the total RNA from the bacterial cell of transfection, separated by gel electrophoresis, then transferred to nitrocellulose filter.According to the scheme of manufacturers (DIG Northern Starter test kit, Roche (Roche), 12039672910) preparation is designed to have the digoxigenin labeling probe with the complementary sequence of linear polyribonucleotide precursor, it is purified (for example, using Monarch50ug RNA purification column), and is used for making RNA visualization on nitrocellulose filter.

Example 19: Production of circularized RNA in maize cells

This example describes the recombinant DNA vector that is used to provide linear polyribonucleotide precursors and heterologous ligase to plant cells for transcription and cyclization of these linear polyribonucleotides. More specifically, this example describes the successful production of circular RNA in corn cells.

Synthetic DNA vector is to express linear polyribonucleotide precursor in vegetable cell.In example, carrier is built on HBT plasmid, and this plasmid can be from Arabidopsis Biological Resource Center, Ohio State University, Columbus, Ohio (Arabidopsis Biological Resource Center, Ohio State University, Columbus OH), 43210 obtains (stock number HBT-sGFP (S65T)/CD3-911). The linear polyribonucleotide precursor includes from 5' to 3': (a) a 35S promoter (SEQ ID NO:605) with an enhancer for constitutive RNA expression; (b) a self-cleaving RNA that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO:606); (c) a 5' annealing region (SEQ ID NO:607); (d) a polyribonucleotide cargo, including a Pepper adapter (SEQ ID NO:608), an EMCV IRES (SEQ ID NO:609), and a NanoLuc (SEQ ID NO:610); (e) a 3' annealing region (SEQ ID NO:611); (f) a self-cleaving RNA that cuts at its 5' end, such as a hepatitis delta virus ribozyme (SEQ ID NO:612); and (g) a transcription terminator sequence, the NOS terminator (SEQ ID NO:613).

A second DNA vector for heterologous expression of RNA ligase in monocot cells was synthesized. This vector was also constructed on the HBT plasmid and included from 5' to 3': (a) a 35S promoter with an enhancer (SEQ ID NO: 605) for constitutive expression in plants; (b) an RNA ligase identified from Arabidopsis thaliana and codon-optimized for expression in monocots (SEQ ID NO: 615); and (c) a transcription terminator sequence, the NOS terminator (SEQ ID NO: 613).

Next is the general procedure for preparing monocot protoplasts. Maize (Zea mays) B73 protoplasts were isolated from 8-10 day old seedlings according to the mesophyll protoplast preparation protocol (modified from the protocol publicly available at molbio[dot]mgh[dot]Harvard[dot]edu/sheenweb/protocols_reg[dot]html). This protocol is generally suitable for monocots, such as maize (Zea mays) and rice (Oryza sativa). An enzyme solution containing 0.6 mol mannitol, 10 mmol MES (pH 5.7), 1.5% Cellulase RIO, and 0.3% Metase RIO was prepared. The enzyme solution was heated at 50-55 degrees Celsius for 10 minutes to inactivate the protease and accelerate enzyme dissolution and cooled to room temperature, then 1 mmol CaCl2, 5 mmol mercaptoethanol, and 0.1% bovine serum albumin were added. The enzyme solution was passed through a 0.45 micron filter. A wash solution containing 0.6 M mannitol, 4 mM MES (pH 5.7), and 20 mM KCl was prepared.

A second batch of leaves from the plant was obtained and the middle 6-8 cm was cut out. Ten leaf segments were stacked and cut into 0.5 mm wide strips without damaging the leaves. The leaf strips were completely immersed in the enzyme solution in the culture dish, covered with aluminum foil, and vacuum was applied for 30 minutes to penetrate the leaf tissue. The culture dish was transferred to a platform shaker and incubated for an additional 2.5 hours of digestion under gentle shaking (40 rpm). After digestion, the enzyme solution containing the protoplasts was carefully transferred to a round-bottom tube through a 35 micron nylon mesh using a serological pipette; the culture dish was rinsed with 5 ml of washing solution and also filtered through a mesh. The protoplast suspension was centrifuged at 1200 rpm for 2 minutes in a swing bucket centrifuge. As much supernatant as possible was aspirated without touching the precipitate; the precipitate was gently washed once with 20 ml of washing buffer and the supernatant was carefully removed. The precipitate was resuspended by gently vortexing in a small amount of washing solution and then resuspended in 10-20 ml of washing buffer. The pipe is placed upright on ice for 30 minutes-4 hours (not longer than 4 hours). After standing on ice, the supernatant is removed by suction, and the sediment is resuspended with 2-5 ml of washing buffer. The concentration of protoplasts is measured using a hemocytometer, and the concentration is adjusted to 2x 10^5 individual protoplasts/ml with washing buffer.

The general procedure for producing circular RNA in plant cells was followed. Protoplasts were transfected with polyethylene glycol (PEG) as described by Niu and Sheen (2011). Briefly, 10 μl of DNA vector (10 μg per vector), 100 μl of protoplasts in wash solution, and 110 μl of PEG solution (40% (w/v) PEG 4000 (Sigma-Aldrich), 0.2 M mannitol, and 0.1 M CaCl2) were incubated at room temperature for 5-10 min. Transfection was stopped by adding 440 μl of wash solution and gently mixing by inversion. The protoplasts were then pelleted by spinning at 110 x g for 1 min, and the supernatant was removed. The protoplasts were gently resuspended in 500 μl of incubation solution (0.6 M mannitol, 4 mM MES (pH 5.7), and 4 mM KCl) per well of a 12-well tissue culture plate and incubated for 12, 24, and 48 h.

RNA production was monitored by harvesting an aliquot of the cells and measuring aptamer fluorescence. Aptamer fluorescence was measured by supplementing with 500 nM HBC525, which fluoresces upon binding to the Pepper aptamer in the RNA cargo.

RNA extraction was performed by centrifuging 1 ml of protoplast cells, resuspending the cell pellet in TRIzol (Thermo Fisher Scientific, catalog number 15596026) and adding to Direct-zol RNA microprep (Zymo Research, catalog number R2060). Total RNA was eluted in 15 μl of nuclease-free water.

RNA can be characterized by a suitable method.For gel shift analysis, 1 microgram of extracted RNA is boiled in 50% formamide and loaded onto 6% PAGE urea gel for denaturing gel electrophoresis.After separating nucleotides, the gel is stained with ethidium bromide and imaged.The observation of the gel shift of circular RNA species compared to linear RNA species confirms the cyclization in plant cells.For poly A polymerase analysis, according to the manufacturer's instructions, 1 microgram of extracted RNA is processed with poly A tail polymerase (catalog number M0276S, New England Biolabs, Ipswich, Massachusetts (MA)).In 1 hour reaction at 37 degrees Celsius, the poly A tail of about 100nt, about 200nt or about 300nt is enzymatically added to linear nucleotides.Cyclic nucleotides do not have free 3' ends, so they cannot add poly A tails.As mentioned above, the product of the poly A tail reaction is run on 6% PAGE urea gel. Comparison of untreated and poly A polymerase treated RNA extracts revealed an increase in the molecular weight of linear species and no change in the molecular weight of circular species.

RNA production efficiency is calculated as (mass of desired RNA produced)/(mass of total RNA). One measure of quality can be obtained by aptamer fluorescence from circular RNA that includes a fluorescent RNA aptamer, such as a Pepper aptamer, in the cargo sequence; fluorescence is measured by staining a 6% PAGE urea gel containing isolated RNA from in vivo transcribed samples and a standard curve of in vitro transcribed homologous RNA with 500 nM HBC525 and analyzing relative brightness using ImageJ software. The mass of a given RNA of interest is then calculated using the standard curve and divided by the total RNA mass.

In an example, circular RNA is produced in cells of the monocot maize (Zea mays; "corn"). The DNA vector constructed on the HBT plasmid contains from 5' to 3': (a) a cauliflower mosaic virus (CaMV) 35S promoter with an enhancer (SEQ ID NO:605) for constitutive RNA expression; (b) a self-cleaving RNA that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO:606); (c) a 5' annealing region (SEQ ID NO:607); (d) a polyribonucleotide cargo, including a Pepper aptamer (SEQ ID NO:608), an EMCV IRES (SEQ ID NO:609), and NanoLuc (SEQ ID NO:610); (e) a 3' annealing region (SEQ ID NO:611); (f) a self-cleaving RNA that cuts at its 5' end, such as a hepatitis D virus ribozyme (SEQ ID NO:612); and (g) a transcription terminator sequence, the NOS terminator (SEQ ID NO:613).

Maize (B73) protoplasts were prepared at a concentration of 4 x 10^5 protoplasts/ml according to the general procedure described above. Protoplasts were transfected using a DNA vector driven by the CaMV 35s promoter encoding a linear polyribonucleotide precursor and a DNA vector encoding an Arabidopsis RNA ligase codon-optimized for expression in monocots according to the general procedure described above and incubated for 6h and 16h.

RNA extraction was performed using the Quick-RNA Plant Miniprep Kit from Zymo Research (Irvine, CA) according to the manufacturer's protocol. Briefly, 1 ml of transfected protoplasts were harvested and resuspended in 800 μl of RNA lysis buffer. After centrifugation, 400 μl of supernatant was collected and passed through a series of Zymo columns, and RNA was then eluted in 30 μl of nuclease-free water.

The RNA was analyzed using the RT-PCR method described above in Example 18. Figure 4 demonstrates the presence of amplicons longer than unit length, confirming successful production of circularized RNA in maize cells.

Example 20: Production of circularized RNA in Arabidopsis cells

This example has been described and is used to provide the recombinant DNA vector of linear polyribonucleotide precursor and heterologous ligase to plant cell, is used for transcribing and the cyclization of this linear polyribonucleotide.More specifically, this example has been described and produces circular RNA in dicotyledonous plant cell.

In the example, circular RNA is produced in cells of the dicotyledonous plant Arabidopsis thaliana. The DNA vector constructed on the HBT plasmid contains from 5' to 3': (a) a cauliflower mosaic virus (CaMV) 35S promoter (SEQ ID NO: 605) with an enhancer for constitutive RNA expression; (b) a self-cleaving RNA that cuts at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 606); (c) a 5' annealing region (SEQ ID NO: 607); (d) a polyribonucleotide cargo, which includes a Pepper adapter (SEQ ID NO: 608), an EMCV IRES (SEQ ID NO: 609), and a NanoLuc (SEQ ID NO: 610); (e) a 3' annealing region (SEQ ID NO: 611); (f) a self-cleaving RNA that cuts at its 5' end, such as a hepatitis D virus ribozyme (SEQ ID NO: 612); and (g) a transcription terminator sequence, the NOS terminator (SEQ ID NO: 613).

A second DNA vector for heterologous expression of RNA ligase in dicotyledonous plant cells was synthesized. This vector was also constructed on the HBT plasmid and included from 5' to 3': (a) a 35S promoter with an enhancer (SEQ ID NO: 605) for constitutive expression in plants; (b) an RNA ligase identified from Arabidopsis thaliana (see AT1G07910, DOI: 10.1261/rna.043752.113); and (c) a transcription terminator sequence, the NOS terminator (SEQ ID NO: 613).

According to the general procedure of preparing dicotyledonous plant protoplasts, Arabidopsis protoplasts are prepared. The preparation contains an enzyme solution of 0.4 mol mannitol, 20 mmoles MES (pH 5.7), 20 mmoles KCl, 1.5% cellulase R10, and 0.4% analyte R10. The enzyme solution is heated at 50-55 degrees Celsius for 10 minutes to dissolve and cool to room temperature with inactivation of protease and accelerated enzyme, and then 10 mmoles CaCl2, 1 mmoles mercaptoethanol, and 0.1% bovine serum albumin are added. The enzyme solution is passed through a 0.45 micron filter. The preparation contains a W5 solution of 154 mmoles NaCl, 125 mmoles CaCl2, 2 mmoles MES (pH 5.7), and 5 mmoles KCl. The preparation contains a WI solution of 0.5 mol mannitol, 4 mmoles MES (pH 5.7), and 20 mmoles KCl. MMg solution containing 0.4 mM mannitol, 15 mM MgCl2, and 4 mM MES (pH 5.7).

Obtain the fully expanded leaves of the plant, cut the middle part into 0.5 to 1 mm strips without crushing the edges. Immediately transfer these leaf strips and fully immerse them in the enzyme solution in the culture dish covered with aluminum foil. The culture dish is transferred to a platform oscillator and incubated for an additional 2.5 to 3 hours of digestion under gentle shaking (40rpm). After digestion, an equal volume of W5 solution is added to the enzyme solution containing protoplasts, and the resulting solution is carefully transferred to a round-bottomed tube through a 35-micron nylon mesh using a serological pipette; the culture dish is rinsed with 5 milliliters of W5 solution, and also filtered through a mesh. The protoplast suspension is centrifuged for 2 minutes at 100x g in a swing bucket centrifuge. Supernatant is aspirated as much as possible without contacting the precipitate; the precipitate is gently resuspended in 0.5 milliliters of W5 solution. The concentration of protoplasts is measured using a hemocytometer, and the concentration is adjusted to 4x 10^5 protoplasts/ml with MMg solution.

Next is the general procedure for producing circular RNA in dicotyledonous plant cells. Protoplasts were isolated from fully expanded leaves of three-week-old Arabidopsis thaliana grown on half-strength MS medium according to the general protoplast procedure described above. Protoplasts were transfected with a DNA vector driven by the CaMV 35s promoter encoding a linear polyribonucleotide precursor and a DNA vector encoding Arabidopsis thaliana RNA ligase. Protoplasts were transfected with PEG as described by Niu and Sheen (2011). Briefly, 10 μl of DNA vector (10 μg per vector), 100 μl of protoplasts in wash solution, and 110 μl of PEG solution (40% (w/v) PEG 4000 (Sigma-Aldrich), 0.2 M mannitol, and 0.1 M CaCl2) were incubated at room temperature for 5-10 min. Transfection was stopped by adding 440 μl of wash solution and gently mixing by inversion. The protoplasts were then pelleted by spinning at 110 x g for 2 min, and the supernatant was removed. The protoplasts were gently resuspended in 500 μl of incubation solution (0.6 M mannitol, 4 mM MES (pH 5.7), and 4 mM KCl) per well of a 12-well tissue culture plate. The transfected Arabidopsis cells were incubated for 6 h and 16 h.

RNA extraction was performed using the Quick-RNA Plant Miniprep Kit from Zymo Research (Irvine, CA) according to the manufacturer's protocol. Briefly, 1 ml of transfected protoplasts were harvested and resuspended in 800 μl of RNA lysis buffer. After centrifugation, 400 μl of supernatant was collected and passed through a series of Zymo columns, and RNA was then eluted in 30 μl of nuclease-free water.

The RNA was analyzed using the RT-PCR method described above in Example 18. Figure 4 shows the presence of amplicons longer than unit length, confirming successful production of circularized RNA in Arabidopsis cells.

Example 21: Production of circularized RNA in tobacco plants

This example has been described and is used to provide the recombinant DNA vector of linear polyribonucleotide precursor and heterologous ligase to plant, is used for transcribing and the cyclization of this linear polyribonucleotide.More specifically, this example has been described and produces circular RNA in tobacco plant.

In an example, circular RNAs are produced in the leaves of the dicot plant tobacco (Nicotiana benthamiana). The DNA vector constructed on the pCAMBIA-1302 plasmid (Cat. No. ab275760, Abcam, Cambridge, UK) contains from 5' to 3': (a) the cauliflower mosaic virus (CaMV) 35S promoter with an enhancer (SEQ ID NO: 605) for constitutive RNA expression; (b) a self-cleaving RNA that cleaves at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 606); (c) a 5' annealing region (SEQ ID NO: 607); (d) a polyribonucleotide cargo, which includes a Pepper aptamer (SEQ ID NO: 608), an EMCV IRES (SEQ ID NO: 609), and a NanoLuc (SEQ ID NO: 610); (e) a 3' annealing region (SEQ ID NO: 611); (f) a self-cleaving RNA that cleaves at its 5' end, such as a hepatitis D virus ribozyme (SEQ ID NO: 612). NO:612); and (g) a transcription terminator sequence, NOS terminator (SEQ ID NO:613).

A second DNA vector for heterologous expression of RNA ligase in dicotyledonous plant cells was synthesized. This vector was also constructed on the pCAMBIA-1302 plasmid and included from 5' to 3': (a) a 35S promoter (SEQ ID NO: 605) with an enhancer for constitutive expression in plants; (b) an RNA ligase identified from Arabidopsis thaliana; and (c) a transcription terminator sequence, the NOS terminator (SEQ ID NO: 613).

The DNA vector was transiently transformed into Agrobacterium tumefaciens GV3101 strain (Catalog No. ACC-100, Lifeasible, Shirley, New York). Agrobacterium was infiltrated ("Agroinfiltration") into leaves of Nicotiana benthamiana according to the method from Norkunas et al. (2018) (DOI: 10.1186/s13007-018-0343-2). In brief, a single colony of recombinant Agrobacterium bacteria was inoculated into liquid LB medium containing kanamycin (50 mg/L) and rifampicin (25 mg/L). The culture was incubated overnight at 28°C with shaking. The bacteria were precipitated and resuspended in MMA (10 mM MES (pH 5.6), 10 mM MgCl2, 200 micromolar acetosyringone) to OD600 = 1.0. The culture was incubated at room temperature with gentle shaking for 2-4 hours. The culture from the recombinant bacteria carrying the plasmid encoding the linear RNA precursor with the RNA cargo sequence and the recombinant bacteria carrying the plasmid with the RNA ligase were mixed 1:1 and then delivered to the dorsal side of the leaves of 1-2 month old plantlets using a blunt-tipped plastic syringe and applying gentle pressure.

RNA production was monitored by measuring aptamer fluorescence. Aptamer fluorescence was measured by delivering 500 nM HBC525 to the underside of Agrobacterium-infiltrated leaves. HBC525 fluoresces when bound to the Pepper aptamer in the RNA cargo.

RNA extraction was performed by harvesting infiltrated leaves and grinding the samples in TRIzol (Thermo Fisher Scientific, catalog number 15596026) and adding to Direct-zol RNA microprep (Zymo Research, catalog number R2060). Total RNA was eluted in nuclease-free water as described in Example 19 and can be characterized by gel shift assay or by poly A polymerase assay.

RNA was analyzed using the RT-PCR method described above in Example 18. The presence of amplicons longer than unit length confirmed successful production of circularized RNA in transiently transfected tobacco leaves.

Example 22: Production of circular RNA in unicellular green algae

This example describes a recombinant DNA vector for providing linear polyribonucleotide precursors and heterologous ligases to algae for transcription and cyclization of the linear polyribonucleotides. More specifically, this example describes the production of circular RNA in the unicellular green alga Chlorella vulgaris.

In the examples, circular RNAs were produced in the single-cell green alga Chlorella vulgaris grown in suspension culture. The DNA vector constructed on the pCAMBIA-1302 plasmid (Cat. No. ab275760, Abcam, Cambridge, UK) contains from 5' to 3': (a) the cauliflower mosaic virus (CaMV) 35S promoter with an enhancer (SEQ ID NO: 605) for constitutive RNA expression; (b) a self-cleaving RNA that cleaves at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 606); (c) a 5' annealing region (SEQ ID NO: 607); (d) a polyribonucleotide cargo, including Pepper aptamers (SEQ ID NO: 608), EMCVIRES (SEQ ID NO: 609), and NanoLuc (SEQ ID NO: 610); (e) a 3' annealing region (SEQ ID NO: 611); (f) a self-cleaving RNA that cleaves at its 5' end, such as a hepatitis D virus ribozyme (SEQ ID NO: 612). NO:612); and (g) a transcription terminator sequence, NOS terminator (SEQ ID NO:613).

A second DNA vector for heterologous expression of RNA ligase in dicotyledonous plant cells was synthesized. This vector was also constructed on the pCAMBIA-1302 plasmid and included from 5' to 3': (a) a 35S promoter (SEQ ID NO: 605) with an enhancer for constitutive expression in plants; (b) an RNA ligase identified from Arabidopsis thaliana; and (c) a transcription terminator sequence, the NOS terminator (SEQ ID NO: 613).

These DNA vectors were transformed into common Chlorella according to the method described in Kumar et al. (2017) (DOI: 10.1007/s10811-018-1396-3). Briefly, protoplasts were prepared from cultured Chlorella cells by enzymatic cell wall digestion for up to 15 h with gentle rotation at 50 rpm in the dark. The two DNA vectors were transformed into Chlorella protoplast cells by electroporation with Bio-Rad's Gene Pulser Xcell electroporation system (Bio-Rad, Hercules, California). After electroporation, the cells were then transferred to a 12-well plate containing BG11 medium (1.5 g/L NaNO3, 0.04 g/L K2HPO4, 0.075 g/L MgSO4.7H2O, 0.036 g/L CaCl2.2H2O, 0.006 g/L citric acid, 0.006 g/L ammonium ferric citrate, 0.001 g/L EDTA, 0.02 g/L Na2CO3, 1 ml/L trace metal mixture A5; Stanier et al. (1971) DOI: 10.1128/br.35.2.171-205.1971). The cells were cultured in the dark at 25°C for 24 h. The cells were harvested and plated onto BG11 agar plates containing 70 μg/ml hygromycin and incubated at 25 °C with 60 μmol photons m-1s-1 under continuous fluorescent light.

RNA production was monitored by harvesting an aliquot of Chlorella cells and measuring aptamer fluorescence. Aptamer fluorescence was measured by supplementing with 500 nM HBC525, which fluoresces upon binding to the Pepper aptamer in the RNA cargo.

RNA extraction was performed by centrifuging 1 ml of cultured cells, resuspending the cell pellet in TRIzol (Thermo Fisher Scientific, catalog number 15596026) and adding to Direct-zol RNA microprep (Zymo Research, catalog number R2060). Total RNA was eluted in nuclease-free water as described in Example 19 and can be characterized by gel shift assay or by poly A polymerase assay.

Example 23: Production of circular RNA in yeast

This example has been described and is used to provide the recombinant DNA vector of linear polyribonucleotide precursor and heterologous ligase to yeast cell, is used for transcribing and the cyclization of this linear polyribonucleotide.More specifically, this example has been described and in yeast Saccharomyces cerevisiae, produces circular RNA.

In an example, circular RNA is produced in yeast Saccharomyces cerevisiae. The DNA vector constructed on the pYES2 yeast expression plasmid (Cat. No. V82520, Thermo Fisher Scientific, Waltham, Massachusetts) contains from 5' to 3': (a) GAL1 promoter (SEQ ID NO: 614) for inducible RNA expression; (b) self-cleaving RNA that cuts at its 3' end, such as hammerhead ribozyme (SEQ ID NO: 606); (c) 5' annealing region (SEQ ID NO: 607); (d) polyribonucleotide cargo, which includes Pepper adapter (SEQ ID NO: 608), EMCV IRES (SEQ ID NO: 609), and NanoLuc (SEQ ID NO: 610); (e) 3' annealing region (SEQ ID NO: 611); (f) self-cleaving RNA that cuts at its 5' end, such as hepatitis D virus ribozyme (SEQ ID NO: 612). NO:612); and (g) a transcription terminator sequence, CYC1 terminator (SEQ ID NO:616). (Alternative DNA vectors for yeast include PSF-TEFI-URA3 plasmid (Catalog No. OGS534, Sigma-Aldrich, St. Louis, MO); alternative promoters include constitutive promoters, such as the TEF1 promoter for constitutive RNA expression.)

A second DNA vector for heterologous expression of RNA ligase in dicotyledonous plant cells was synthesized. This vector was also constructed on the pYES2 plasmid and included from 5' to 3': (a) GAL1 promoter (SEQ ID NO: 614) for inducible expression; (b) KlaTrl1, a tRNA ligase identified from Kluyveromyces lactis (GenBank: CAG98435.1, DOI: 10.1261/rna.043752.113, SEQ ID NO: 617); and (c) transcription terminator sequence, CYC1 terminator (SEQ ID NO: 616).

The two DNA constructs were transformed into competent INVSc1 Saccharomyces cerevisiae cells according to the pYES2 plasmid manual. Transformants were selected on SC-U selection plates and the cells were maintained in SC-U medium.

RNA production was monitored by harvesting aliquots of transformed yeast cells and measuring aptamer fluorescence. Aptamer fluorescence was measured by supplementing 500 nM HBC525, which fluoresces upon binding to the Pepper aptamer in the RNA cargo.

RNA extraction was performed by centrifuging 1 ml of cultured cells, resuspending the cell pellet in TRIzol (Thermo Fisher Scientific, catalog number 15596026) and adding to Direct-zol RNA microprep (Zymo Research, catalog number R2060). Total RNA was eluted in nuclease-free water and characterized by gel shift assay or by poly A polymerase assay as described in Example 19.

RNA was analyzed using the RT-PCR method described above in Example 18. The characteristic ladder-like banding pattern on the gel caused by amplicons longer than a unit length (most commonly two unit lengths) confirmed the successful production of circularized RNA in the transformed S. cerevisiae cells as shown in FIG5 .

Example 24: Functionality of circularized RNA cargo comprising coding sequences.

The functionality of the cyclized RNA product can be tested (for example, for the circular RNA produced in the experiment described in Example 19-23) to determine whether the Nanoluc luciferase coding sequence as a part of the load of the cyclized RNA can be translated and play a role.According to the manufacturer's instructions, the expression of the Nanoluc RNA reporter is measured using wheat germ extract (WGE) in vitro translation system (catalog number L4380, Plomec Corporation, Madison City (Madison), Wisconsin (WI)). In brief, 1 microgram of extracted RNA is heated to 75°C for 5 minutes and then cooled on the bench for 20 minutes. RNA is transferred to 1x wheat germ extract and incubated at 30°C for 1 hour. The mixture is placed on ice and diluted 4 times with water. The resulting translation reaction product is analyzed using Nano-Glo luciferase assay (catalog number N1110, Plomec Corporation, Madison City, Wisconsin), and Nanoluc luciferase is measured in a spectrophotometer. Luminescence above background indicates functional luciferase translation from the circular RNA.

Also according to the manufacturer's instructions, the expression of Nanoluc RNA reporter is measured using insect cell extract (ICE) in vitro translation system (catalog number L1101, Plomec Corporation, Madison, Wisconsin). In brief, 1 microgram of extracted RNA is heated to 75 ° C for 5 minutes, then cooled on the bench for 20 minutes. RNA is transferred to 1x insect cell extract and incubated at 30 ° C for 1 hour. The mixture is placed on ice and diluted 4 times with water. The resulting translation reaction product is analyzed using Nano-Glo luciferase assay (catalog number N1110, Plomec Corporation, Madison, Wisconsin), and Nanoluc luciferase luminescence is measured in a spectrophotometer. Luminescence higher than background indicates that functional luciferase is translated from circular RNA.

Example 25: Production of circular RNA in insect cells

This example describes the recombinant DNA vector for providing linear polyribonucleotide precursors and heterologous ligase to insect cells, for transcription and cyclization of these linear polyribonucleotides. More specifically, this example describes producing circular RNA in Fall Armyworm (spodoptera, Lepidoptera) cells.

Examples of DNA constructs encoding linear polyribonucleotide precursors for producing circular RNA in insect cells include the following. In a non-limiting example, the DNA construct includes from 5' to 3': (a) OpIE1 promoter (SEQ ID NO:618) or inducible T7lac polymerase promoter (SEQ ID NO:619); (b) self-cleaving RNA that cuts at its 3' end, such as hammerhead ribozyme (SEQ ID NO:606); (c) 5' annealing region (SEQ ID NO:607); (d) polyribonucleotide cargo, which includes Pepper adapter (SEQ ID NO:608), EMCV IRES (SEQ ID NO:609), and NanoLuc (SEQ ID NO:610); (e) 3' annealing region (SEQ ID NO:611); (f) self-cleaving RNA that cuts at its 5' end, such as hepatitis delta virus ribozyme (SEQ ID NO:612); and (g) transcription terminator sequence (SEQ ID NO:620). In another example, the DNA construct includes from 5' to 3': (a) the left arm sequence of bacterial transposon Tn7 for generating recombinant bacmid DNA (SEQ ID NO: 621); (b) a polyhedrin promoter (SEQ ID NO: 622) for driving ribonucleotide transcription; (c) a self-cleaving RNA that cleaves at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 606); (d) a 5' annealing region (SEQ ID NO: 607); (e) a polyribonucleotide cargo, which includes a Pepper aptamer (SEQ ID NO: 608), an EMCV IRES (SEQ ID NO: 609), and a NanoLuc (SEQ ID NO: 610); (f) a 3' annealing region (SEQ ID NO: 611); (g) a self-cleaving RNA that cleaves at its 5' end, such as a hepatitis D virus ribozyme (SEQ ID NO: 612); (h) an SV40 poly (A) signal sequence (SEQ ID NO:623); and (i) the bacterial transposon Tn7 right arm sequence (SEQ ID NO:624) used to generate recombinant bacmid DNA.

An example of a second DNA construct for providing RNA ligase to insect cells includes, from 5' to 3', an inducible T7 lac polymerase promoter (SEQ ID NO:619) operably linked to a DNA sequence encoding a heterologous RtCB ligase (SEQ ID NO:625), followed by a transcription terminator sequence (SEQ ID NO:620).

DNA constructs encoding linear polyribonucleotide precursors for producing circular RNA in insect cells and encoding heterologous RtCB ligases were cloned into pFastBac donor plasmids for expression in Spodoptera frugiperda SF9 or SF21 cells (available from Thermo Fisher Scientific, Waltham, MA). They were then transformed into competent DH10Bac E. coli cells and Lac7 E. coli cells to generate recombinant bacmids. Spodoptera frugiperda SF9 or SF21 cells were co-transfected with CELLFECTIN reagent (Thermo Fisher Scientific, Waltham, MA) and recombinant bacmids containing linear polyribonucleotide precursor DNA constructs and heterologous RtCB ligases. Circularization of the linear polyribonucleotide precursors was achieved by inducing the heterologous RtCB ligase with IPTG. SF9 or SF21 cells were cultured as a monolayer or in suspension, and RNA was then collected.

In another example, a DNA construct encoding a linear polyribonucleotide precursor for producing circular RNA in insect cells and encoding a heterologous RtCB ligase was cloned into a pFastBac1 donor plasmid between BamHI and NotI in the MCS region and transformed into competent DH10Bac E. coli cells using the Bac to Bac Baculovirus Expression System (Cat. No. 10359016, Thermo Fisher Scientific, Waltham, MA) to generate a recombinant bacmid. The recombinant bacmid DNA was quantified by NanodropOne (Thermo Fisher Scientific, Waltham, MA). Spodoptera frugiperda SF9 or SF21 cells were co-transfected with CELLFECTIN reagent (Thermo Fisher Scientific, Waltham, MA) and the recombinant bacmid containing the linear polyribonucleotide precursor DNA construct and the heterologous RtCB ligase. Circularization of the linear polyribonucleotide precursor was achieved by inducing the heterologous RtCB ligase with IPTG. SF9 cells were cultured in monolayer at 27°C in the dark. 72 hours after transfection, cells were collected for RNA extraction. These RNA samples were subjected to RT-PCR as described in Example 18. The presence of amplicons longer than the unit length with a characteristic ladder pattern confirmed the circularization of the linear precursor (Figure 6). This indicates that circular RNA was successfully produced in insect cells.

Optionally, the cell culture is then ultracentrifuged at 80,000 x g for 75 minutes to remove remaining viruses and supernatant from the cell pellet. Once the supernatant is removed, the cell pellet is washed with phosphate buffered saline and centrifuged at 1,000 x g for 1 minute. The cells are then resuspended in Tri reagent (Sigma Millipore, USA). The cells are then subjected to freeze-thaw cycles from -80 ° C or from liquid nitrogen to lyse cells in preparation for RNA extraction. The cells are then centrifuged at 4 ° C for 1 minute at 12,000 x g to precipitate cell debris, and the supernatant is transferred to a new tube in preparation for RNA purification. RNA cleaning and concentrator columns (Zymo, USA) are used to purify RNA. In order to confirm that the RNA produced from insect cells is a circular species, the purified RNA is then processed with an exonuclease mixture (New England Biolabs, Inc.) containing RNase R and exonuclease T to degrade single-stranded RNA molecules. The remaining RNA was then run on a PAGE gel and compared to the single-stranded RNA to confirm the enrichment of circular RNA molecules.

Example 26: Production of circular RNA in insect cells and characterization of cargo-encoded polypeptides

This example describes the recombinant DNA vector for providing linear polyribonucleotide precursors and heterologous ligase to insect cells, for transcribing and cyclization of these linear polyribonucleotides. More specifically, this example describes the characterization of the cyclic RNA and the polypeptide of encoding that produce the coding sequence payload in the greedy fall armyworm cell.

In this example, the DNA construct encoding the linear polyribonucleotide precursor includes from 5' to 3': (a) OpIE1 promoter (SEQ ID NO:618); (b) self-cleaving RNA that cuts at its 3' end, such as hammerhead ribozyme (SEQ ID NO:606); (c) 5' annealing region (SEQ ID NO:607); (d) 5' EMCV IRES (SEQ ID NO:609); (e) 3X-Flag peptide coding sequence (SEQ ID NO:628); (f) 3' annealing region (SEQ ID NO:611); (g) self-cleaving RNA that cuts at its 5' end, such as hepatitis delta virus ribozyme (SEQ ID NO:612); and (h) transcription terminator sequence (SEQ ID NO:620).

The DNA construct encoding RNA ligase included, from 5' to 3', an inducible T7lac polymerase promoter (SEQ ID NO: 619) operably linked to a DNA sequence encoding a heterologous RtCB ligase (SEQ ID NO: 625), followed by a transcription terminator sequence (SEQ ID NO: 620).

Circularized RNA was produced in SF9 and SF21 cells according to the procedure as in Example 25. Circular RNA was purified and incubated in wheat germ extract for 4 to 8 hours for efficient protein translation. To confirm the expression of 3X-FLAG peptide, proteins from in vitro translation reactions were incubated in anti-FLAG coated plates (Catalog No. P2983, Millipore-Sigma) and detected by ELISA. Proteinase treated and untreated proteins were compared to confirm efficient protein expression.

Example 27: Production of circular RNA in mammalian cells

This example has been described and is used to provide the recombinant DNA vector of linear polyribonucleotide precursor and heterologous ligase to mammalian cell, is used for transcribing and the cyclization of this linear polyribonucleotide.More particularly, this example has been described and in mammalian cell line (particularly human embryonic kidney (HEK 293) cell and human cervical epithelium (HeLa) cell), produces the circular RNA that carries the encoding sequence payload.

In this example, a DNA construct encoding a linear polyribonucleotide precursor was constructed by modifying the multiple cloning site of the pcDNA3.1 plasmid to include (1) the following items for expressing the linear RNA precursor in a 5' to 3' direction: (a) a CMV promoter (SEQ ID NO: 626); (b) a self-cleaving RNA that cleaves at its 3' end, such as a hammerhead ribozyme (SEQ ID NO: 606); (c) a 5' annealing region (SEQ ID NO: 607); (d) a polyribonucleotide cargo, including a Pepper aptamer (SEQ ID NO: 608), an EMCV IRES (SEQ ID NO: 609), and a NanoLuc (SEQ ID NO: 610); (e) a 3' annealing region (SEQ ID NO: 611); (f) a self-cleaving RNA that cleaves at its 5' end, such as a hepatitis delta virus ribozyme (SEQ ID NO: 612); and (g) an SV40 transcription terminator sequence (SEQ ID NO: 613). NO:627); (2) the following for RNA ligase expression in a 5' to 3' direction: (a) a codon-optimized, inducible TRE3G promoter (SEQ ID NO:629) operably linked to DNA encoding a heterologous RtcB ligase (SEQ ID NO:625) followed by an SV40 transcription terminator sequence (SEQ ID NO:627).

The vector was transformed into human embryonic kidney HEK 293 Tet-On 3G cells (Catalog No. CRL-3216, American Type Culture Collection, Manassas, VA) or immortalized human cervical epithelial HeLa cells (Catalog No. CCL-2, American Type Culture Collection, Manassas, VA). Cells were maintained in 1×DMEM (Life Technologies 11995-065) containing 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin under standard tissue culture conditions. Cells were plated for transfection using OptiMEM ^™ I low serum medium (Thermo Fisher 31985) according to the manufacturer's instructions using FuGENE HD (Promega 2311) or Lipofectamine ^™ 3000 reagent (Thermo Fisher L3000001).

RNA production was monitored by harvesting cells from 1 ml culture samples and measuring aptamer fluorescence. Aptamer fluorescence was measured by supplementing 500 nM HBC525, which fluoresces when bound to the Pepper aptamer in the RNA load (providing a reference). Fluorescence was measured at 525 nm.

RNA was harvested from cells by removing the culture medium and stripping the cells with 1x phosphate buffered saline (PBS) (Thermo Fisher Scientific 10010031). The cell suspension was mixed with TRIzol ^™ LS reagent (Invitrogen 10296010) and RNA was purified according to the manufacturer's instructions. Total RNA concentration values were normalized using NanoDrop 2000 (Thermo Fisher Scientific) and can be characterized by gel shift assay or by poly A polymerase assay, as described in Example 19.

According to the manufacturer's instructions, the expression of Nanoluc reporters was measured using a rabbit reticulocyte lysate, nuclease-treated (RRL) in vitro translation system (Catalog No. L4960, Promega, Madison, Wisconsin). Briefly, 1 microgram of extracted RNA was heated to 75°C for 5 minutes and then cooled on the bench for 20 minutes. The RNA was transferred to 70% RRL and incubated at 30°C for 1 hour. The mixture was placed on ice and diluted 4 times with water. The products of the in vitro translation reaction were analyzed using the Nano-Glo luciferase assay (Catalog No. N1110, Promega, Madison, Wisconsin); 10 microliters of the RRL product was mixed with 10 microliters of Nano-Glo assay buffer (Promega) and luminescence was measured in a spectrophotometer.

Example 28: Confirmation of Circularization of RNA Produced in Vivo in Various Eukaryotic Cells

This example describes the use of RT-PCR to verify the circular conformation of polyribonucleotides produced as linear precursors transcribed in vivo in various eukaryotic cells and to confirm that these linear precursors are successfully circularized in vivo.

The RT-PCR analysis scheme described in Example 18 was used to evaluate the in vivo transcription and cyclization of RNA from eukaryotic cells, including monocots (corn), dicots (Arthropoda), yeast, insects, and mammals (human). As described in Examples 18-27, yeast cells, insect SF9 cells, corn protoplast cells, Arabidopsis protoplast cells, and human HEK293 and HeLa cells were transformed with appropriate DNA vectors encoding the respective linear polyribonucleotide precursors "min1" (SEQ ID NO: 603) (which has an unprocessed length of 392nt and a processed length of 275nt after ribozyme cleavage), or "min2" (SEQ ID NO: 604) (which has an unprocessed length of 245nt and a processed length of 128nt after ribozyme cleavage). Total RNA prepared from transformed eukaryotic cells was used as a template for reverse transcriptase (RT) reactions. In the PCR reaction using oligonucleotide primers AAGGATGTGTTCCCTAGGAGGGTGG (SEQ ID NO:630) and GAAAGGGGATAGTACCTGGGAGGGGG (SEQ ID NO:631), the cDNA products of these RT reactions are used as templates. In the absence of RNA ligase, the linear polyribonucleotide generated in vitro is used as the negative control of the cyclic polyribonucleotide RT-PCR signal; These PCRs have generated the amplicon of the unit length lacking the ladder pattern. The cyclic polyribonucleotide generated by contacting the linear polyribonucleotide generated in vitro with RNA ligase is used as the positive control of the cyclic polyribonucleotide RT-PCR signal; These PCRs have generated the amplicon longer than the unit length (typically being an integral multiple of the unit length), and these amplicons have generated characteristic ladder-like band patterns on gel. The cyclization of min1 is indicated by ladder pattern, and this ladder pattern is formed by the band from unit length amplicon (275nt) and twice unit length amplicon (550nt), and also observes faint three times unit length band occasionally.The cyclization of min2 is indicated by ladder pattern, and this ladder pattern is formed by the band from unit length amplicon (128nt) and twice unit length amplicon (256nt), and also observes faint three times unit length occasionally.The RT-PCR analysis of the total RNA that obtains from yeast cell, insect SF9 cell, corn protoplast cell, Arabidopsis protoplast cell and people HEK293 and HeLa cell with the DNA construct transformed of coding linear polyribonucleotide precursor all shows that the amplicon that is longer than unit length has the characteristic ladder pattern of indicating this linear precursor cyclization, and does not show this pattern (Fig. 4, Fig. 5, Fig. 6 and Fig. 7) from the total RNA that yeast, insect, plant or mammalian cell separation that lack this polyribonucleotide are not shown. These results confirm the successful generation of circular RNA by in vivo transcription of linear RNA precursors and circularization of linear RNA precursors in eukaryotic cells.

All the patents and patent disclosures of quotations mentioned in this application are incorporated herein by reference in their entirety. All materials and methods disclosed and claimed herein can be prepared and used as indicated by the above disclosure and by example, without the need to carry out too much experimentation. Although the materials and methods related to the present invention have been described in terms of embodiments and illustrative examples, it will be apparent to those skilled in the art that, without departing from the concept, spirit and scope of the present invention, substitutions and changes can be made to the materials and methods described herein. Therefore, the breadth and scope of the present invention should not be limited by any of the above-mentioned examples, but should only be defined according to the aforementioned embodiments, the following claims and their equivalents.

Sequence Listing

<110> FLAGSHIP PIONEERING INNOVATIONS VII, LLC

<120> Production of cyclic polyribonucleotides in eukaryotic systems

<130> P13752WO00

<150> 63/189619

<151> 2021-05-17

<150> 63/166467

<151> 2021-03-26

<160> 631

<170> PatentIn Version 3.5

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cccgccgcat ttaagtgcta ccataacttc cacagggccg aggacattag tccagataac 1380

ctcttctaca agctcgtggt ccatgtgcat tccgattctg ggtttcgtcg gtatcacaag 1440

gagatgagac acatgccatc tctttggccg ctgtaccgcg ggttctttgt agacatcaac 1500

ctgttcaagt cgaacaaggg cagagatctc atggcgctca aaagcattga taatgccagc 1560

gagaatgatg gaaggggcga aaaggacggg ctggcagatg atgacgccaa cctcatgatc 1620

aaaatgaagt tcttgacata caagctaagg acattcctca ttagaaatgg cttgtctatc 1680

cttttcaagg atggtgcggc tgcttataaa acttactatc tccgccaaat gaagatatgg 1740

ggcacgtcag acggcaaaca gaaggagctt tgtaagatgc tcgacgaatg ggcggcttac 1800

atcaggagga aatgcggaaa tgaccagcta tcatcctcaa cgtatctgtc cgaggctgaa 1860

ccattcctag agcagtacgc caaaagatcc ccaaaaaatc atattttaat tggctctgcc 1920

gggaacttag ttagaactga agatttcctt gccatagttg atggggactt ggatgaggag 1980

ggagacttag tgaagaagca gggtgtaacc ccagcaaccc cggaacctgc tgttaaggag 2040

gccgtacaaa aggatgaagg cttgatcgtc ttttttcctg gcatccccgg aagtgccaag 2100

agcgccctgt gcaaagagct gctcaacgcc cctggcggct ttggtgacga ccgtcccgtt 2160

cacactttga tgggtgacct cgtcaagggt aagtactggc ctaaggtggc ggatgagcgg 2220

agaaagaagc cacaatcaat catgctcgcc gataaaaatg cgcccaacga agacgtctgg 2280

cgtcaaatag aggatatgtg tagacgtacg agggcgtccg ccgtccctat tgtggctgat 2340

tcagagggaa ccgacacaaa cccttacagc ctggatgctc tcgcagtttt tatgttccgc 2400

gtcctgcagc gggtgaacca tccaggtaaa cttgacaagg agagttccaa cgctggatat 2460

gttctcctga tgttttacca cctctatgag ggcaagaaca gaaatgaatt tgaatcagaa 2520

ctgattgaac gatttggatc attgataaag atgccgctgc ttaaatcaga taggaccccc 2580

cttccggatc ctgtcaaatc tgttcttgaa gaaggcattg atctgtttaa tcttcattct 2640

agacgtcacg gccggcttga gtcaacaaag gggacgtacg cggcggagtg gacaaagtgg 2700

gagaagcaac tgcgggatac cctagtagcc aattctgagt atttaagctc catccaagtg 2760

ccgttcgaga gcatggttca tcaggtgaga gaagaattga agaccatcgc gaaaggagat 2820

tataaacccc cctccagcga aaagcgcaag catggcagca tagtcttcgc ggcaataaat 2880

ttacctgcta cacaggttca ctcgcttttg gaaaaactag cagccgccaa cccgaccatg 2940

aggtctttcc ttgaaggcaa gaaaaagagt atacaggaaa agttggagcg gtcacatgta 3000

accctggctc ataagaggtc gcatggtgtc gcgacggttg cgtcctatag ccagcacttg 3060

aatagggagg tgccagtgga gctcacggag cttatctaca atgataagat ggccgcgtta 3120

acggcgcacg ttgggtcagt cgacggcgag accgtcgtca gtaaaaacga gtggcctcat 3180

gtaacactgt ggacggcaga gggcgtgact gctaaggaag cgaacactct gccacaactg 3240

tacttagagg ggaaagcatc gcgcctcgtg atcgacccgc cggtgtcgat atccggcccg 3300

ttggaatttttttga 3315

<210> 21

<211> 451

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 21

60

ccucgucuuc accggucgcg uuccugaaac gcagaugugc cucgcgccgc acugcuccga 120

acaauaaaga uucuacaaua cuagcuuuua uggguuaugaa gaggaaaaau uggcaguaac 180

cuggccccac aaaccuucaa augaacgaau caaauuaaca accauaggau gauaaugcga 240

uuaguuuuuu agccuuauuu cugggguaau uaaucagcga agcgaugauu uuugaucuau 300

uaacagauau auaaaugcaa aaacugcaua accacuuuaa cuaauacuuu caacauuuuc 360

gguuuguauu acuucuuauu caaauguaau aaaaguauca acaaaaaauu guuaauauac 420

cucuauacuu uaacgucaag gagaaaaaac c 451

<210> 22

<211> 451

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 22

acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60

cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120

acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180

ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240

ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300

taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc 360

ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac 420

ctctatactt taacgtcaag gagaaaaaac c 451

<210> 23

<211> 249

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 23

aucauguaau uaguuauguc acgcuuacau ucacgcccuc cccccacauc cgcucuaacc 60

gaaaaggaag gaguuagaca accugaaguc uaggucccua uuuauuuuuu uauaguuaug 120

uuaguauuaa gaacguuauu uauauuucaa auuuuucuuu uuuuucugua cagacgcgug 180

uacgcaugua acauuauacu gaaaaccuug cuugagaagg uuuugggacg cucgaaggcu 240

uuaauuugc 249

<210> 24

<211> 249

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 24

atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 60

gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 120

ttagtattaa gaacgttattatatttcaa atttttcttt tttttctgta cagacgcgtg 180

tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 240

ttaatttgc 249

<210> 25

<211> 351

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 25

uuggucaugc gaaacacgca cggcgcgcgc acgcagcuua gcacaaacgc gucguugcac 60

gcgcccaccg cuaaccgcag gccaaucggu cggccggccu cauauccgcu caccagccgc 120

guccuaucgg gcgcggcuuc cgcgcccauu uugaauaaau aaacgauaac gccguuggug 180

gcgugaggca uguaaaaggg uuacaucauu aucuuguucg ccauccgguu gguauaaaua 240

gacguucaug uugguuuuug uuucaguugc aaguuggcug cggcgcgcgc agcaccuuug 300

ccgggaucug ccgggcugca gcacguguug acaauuaauc aucggcauag u 351

<210> 26

<211> 351

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 26

ttggtcatgc gaaacacgca cggcgcgcgc acgcagctta gcacaaacgc gtcgttgcac 60

gcgcccaccg ctaaccgcag gccaatcggt cggccggcct catatccgct caccagccgc 120

gtcctatcgg gcgcggcttc cgcgcccatt ttgaataaat aaacgataac gccgttggtg 180

gcgtgaggca tgtaaaaggg ttacatcatt atcttgttcg ccatccggtt ggtataaata 240

gacgttcatg ttggtttttg tttcagttgc aagttggctg cggcgcgcgc agcacctttg 300

ccgggatctg ccgggctgca gcacgtgttg acaattaatc atcggcatag t 351

<210> 27

<211> 307

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 27

60

auuaucgagg ggccguuguu gguguggggu uuugcauaga aauaacaaug ggaguuggcg 120

acguugcugc gccaacacca ccucccuucc cuccuuucau cauguaucug uagauaaaau 180

aaaauauuaa accuaaaaac aagaccgcgc cuaucaacaa aaugauaggc auuaacuugc 240

cgcugacgcu gucacuaacg uuggacgauu ugccgacuaa accuucaucg cccaguaacc 300

aaucuag 307

<210> 28

<211> 307

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 28

ttatacatat attttgaatt taattaatta tacatatatt ttatattatt tttgtctttt 60

attatcgagg ggccgttgtt ggtgtggggt tttgcataga aataacaatg ggagttggcg 120

acgttgctgc gccaacacca cctcccttcc ctcctttcat catgtatctg tagataaaat 180

aaaatattaa acctaaaaac aagaccgcgc ctatcaacaa aatgataggc attaacttgc 240

cgctgacgct gtcactaacg ttggacgatt tgccgactaa accttcatcg cccagtaacc 300

aatctag 307

<210> 29

<211> 43

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 29

ggaattgtta tccgctcaca attccctata gtgagtcgta tta 43

<210> 30

<211> 540

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 30

gcacaaggac aaaaaagcag gccagaaacg aaacgaccac gaaaagcccc ggaaaaaggg 60

accaaaggcg gccggagagg ccgagcgcgc agcaacaaaa cggcgaagag ccgacaaaca 120

agcggaagcc gagacaccgg gaaaggccac caggagcgga cccgaccaaa ggggcgaacc 180

ggcggcgggg gcgggaaggc gggaagaacg cgcgcgaccg cgaacggcgg aagaccgccg 240

aaaaccggca gagcgcgcag gcgagaaacg gccggccgca cgggcgacca cggccggaaa 300

cggaaaaggg ccgggaaaac caccgaacgc gagcaaaggg cgagcgaagc cggacaggga 360

acgcaaaaaa ccccgccgaa accaaaacaa aacaccggga acgcgggaac cggaaccaca 420

gaaacgccga gagcggacca gggggaagcg cacccggcac gcggaaggaa cgccacggga 480

cacacgacgg cacaaaaaga gagcaggaaa cgcgagacgg ccgcgcggac ggcgacagga 540

<210> 31

<211> 499

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 31

cguuacauaa cuuacgguaa auggcccgcc uggcugaccg cccaacgacc cccgcccauu 60

120

auggguggag uauuuacggu aaacugccca cuuggcagua caucaagugu aucauaugcc 180

aaguacgccc ccuauugacg ucaaugacgg uaaauggccc gccuggcauu augcccagua 240

caugaccuua ugggacuuuc cuacuuggca guacaucuac guauuaguca ucgcuauuac 300

cauggugaug cgguuuuggc aguacaucaa ugggcgugga uagcgguuug acucacgggg 360

auuuccaagu cuccacccca uugacgucaa ugggaguuug uuuuggcacc aaaaucaacg 420

ggacuuucca aaaugucgua acaacuccgc cccauugacg caaaugggcg guaggcgugu 480

acggugggaggucuauaua 499

<210> 32

<211> 508

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 32

cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60

gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 120

atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 180

aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 240

catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 300

catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 360

atttccaagt ctccacccca ttgacgtcaa tggggagtttg ttttggcacc aaaatcaacg 420

ggactttcca aaatgtcgta acaactccgcccattgacg caaatgggcg gtaggcgtgt 480

acggtggggag gtctatataa gcagagct 508

<210> 33

<211> 192

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 33

cagacaugau aagauacauu gaugaguuug gacaaaccac aacuagaaug cagugaaaaa 60

120

auaaacaagu uaacaacaac aauugcauuc auuuuauguu ucagguucag ggggaggugu 180

gggagguuuu uu 192

<210> 34

<211> 192

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 34

cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg cagtgaaaaa 60

aatgctttat ttgtgaaatt tgtgatgcta ttgctttatttgtaaccatt ataagctgca 120

ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt 180

gggaggtttt tt 192

<210> 35

<211> 379

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 35

gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag 60

agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttatccc 120

tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta 180

cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag 240

agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg 300

aaccgtcaga tcgcctggag caattccaca acacttttgt ctttaccaa ctttccgtac 360

cacttcctac cctcgtaaa 379

<210> 36

<211> 66

<212> RNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 36

gacuacaaag accaugacgg ugauuauaaa gaucaugaca ucgauuacaa ggaugacgau 60

gacaag 66

<210> 37

<211> 66

<212> DNA

<213> Artificial sequence

<220>

<223> Synthetic constructs

<400> 37

gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat 60

gacaag 66

<210> 38

<211> 61

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D66A6B_12908

<400> 38

caugcucagc ggucccaagu ccgcaucaaa gccugagggc ugcaguaaag guacugagcu 60

g 61

<210> 39

<211> 76

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3 type torsional ribozyme, URS0000D6AAF0_12908

<400> 39

uuauuuagcc gucuaaaguc ggcaaugaau ugagauagca cccuguaaau uuucagggug 60

uaaacaaacu aaauga 76

<210> 40

<211> 72

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D6663E_12908

<400> 40

uuaauugccg guugccaguc cguuaaauug ugagcagucc ggccauug ccggauuaaa 60

caaaccaauu aa 72

<210> 41

<211> 72

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D6C266_12908

<400> 41

uuaguuaacg guugcacguc cgauaaauug ugagcagucc cggagcaauc cgggauuaaa 60

caaacuaacu aa 72

<210> 42

<211> 74

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D6AF2A_12908

<400> 42

ugauuuaggc guuccaaacc gccgcaaauu gugaggacug cucgccaaaa gcgggcagua 60

aacaaguuaa auca 74

<210> 43

<211> 80

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D6A2C3_12908

<400> 43

aauucuugcg guucaaaguc cgcguaaaau ccagaugaca cauucccgua auaaacggga 60

guguguaaug aacaagaauu 80

<210> 44

<211> 73

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D6726E_12908

<400> 44

acaccccaccu guuacaaguc aggacagaag cagaguaacg guugcuuacg caaccgguaa 60

ugcuacugggugu 73

<210> 45

<211> 74

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P3-type torsades de pointes, URS0000D66C2E_12908

<400> 45

caauaaagcg guuacaagcc cgcaaaaaua gcagaguaau gucgcgauag cgcggcauua 60

augcagcuuu aug 74

<210> 46

<211> 72

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D659B0_12908

<400> 46

uguuuaaugc agccaugagu auuuaauacu augaagguga uaagcuccuu guaaaguaau 60

gcagaaucga ca 72

<210> 47

<211> 57

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6D1CA_12908

<400> 47

gccguaaagc cacuaugacc ggguugcaag ucccggcugc gauaggcuga gcacggu 57

<210> 48

<211> 119

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D67E2B_12908

<400> 48

guucuaaugc agccagcacg acuuugucau agauaaaaua ucauuaauac acuauuuaca 60

cagauguaug cgauuacuag ugcugggagu ccuaagccuc cauaaaugca gaagggaac 119

<210> 49

<211> 93

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D68054_12908

<400> 49

ucuguaaccc caccaccgug gacauccugg cagggauaau ggccaggaug aucauggugg 60

agguccaaag uccucaaaag aggggauggc aga 93

<210> 50

<211> 84

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6D330_12908

<400> 50

acaauaaugc ggccucgcua ccaauacgca uuuauuagua uugguaacgu gacaguccca 60

agccuguaaa acgcagaggg uugu 84

<210> 51

<211> 57

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D6A800_12908

<400> 51

gucguaaugc agccguugcc acgugccaag ucguggauua gaaaugcaga ggcggaa 57

<210> 52

<211> 61

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D68297_12908

<400> 52

gguguaaugc gacucgcuca cagagcgaca gguucacagu ccuacaaacg cagaugacac 60

c 61

<210> 53

<211> 97

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D68DD8_12908

<400> 53

agcuuaauac agguagauaa gcaagcaagg ugcggcuauc uacacggucc caacuccgua 60

aagguuagag ugacaacuaa ucgaaguaga gggagcu 97

<210> 54

<211> 52

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D66D37_12908

<400> 54

uaaauaaugu cgccaaugga gguaucaagc ccucauaaag acagagauaa aa 52

<210> 55

<211> 73

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D68577_12908

<400> 55

acguuaaugu ggcuguaugu gugggugcac acacauacac uagucccaag ccuagguaaa 60

cacagaggga uug 73

<210> 56

<211> 62

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D68F79_12908

<400> 56

aaaguaaugc aacuacaaga aauuguaucg gugacaaguc cgagauaaau gcagagucau 60

uu 62

<210> 57

<211> 56

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D68EE0_12908

<400> 57

ucuguaauga ugccgauggc gguugcaagc ccgcaggaag aaacucagag cacaga 56

<210> 58

<211> 55

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D67CC2_12908

<400> 58

acuauaaucu ugccaucguc aguuccaagc cugagugaga aaaagagagg auagu 55

<210> 59

<211> 58

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D65864_12908

<400> 59

acucuaaccc agcggcaauc uuuugcccgu guccgaagcc acuaaugggg acgggagu 58

<210> 60

<211> 55

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D68DB5_12908

<400> 60

ccgcuaacca ugccguggcc agucccaagc cuggauguga aaaugggagg ggcgg 55

<210> 61

<211> 60

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6B540_12908

<400> 61

uuuuuaauga agccacagug aucacuguga ggguccuaag ccccuaauuc agaagggaaa 60

<210> 62

<211> 68

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D6A03C_12908

<400> 62

uguguaaugc uacuaugaua gcacauugcg aaucauacgg guugcaaguc ccucaagcag 60

agcacacg 68

<210> 63

<211> 51

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6C02F_12908

<400> 63

uuuuuaaccc agccagagac ggucacaagc ccgugaaaug gggaguggaa a 51

<210> 64

<211> 58

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D6AF09_12908

<400> 64

gcucuaaugu ggccacccga cagggugugu guuucaagcc accaacacag agaagagc 58

<210> 65

<211> 83

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D67A5B_12908

<400> 65

gguguaacac ggcuauaguc aggcauuaca agauuaaguc cugcuauaaa ggucuaaagc 60

ccuuguaaac aguggauagc acu 83

<210> 66

<211> 57

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D66DD2_12908

<400> 66

uguguaaugc gagcauugua uggucacaac uccauaauua aaaacgcaga gugcaca 57

<210> 67

<211> 56

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D667E4_12908

<400> 67

gcuuuaacac agccaaagaa gguuccaagc ccuuuaguga aauuguggag gaaagc 56

<210> 68

<211> 67

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6A251_12908

<400> 68

aaaguaaugc agccgcccgc cgcgcgcggg gacgucggua gcaagcccgu guaaugcaga 60

guuuucu 67

<210> 69

<211> 61

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6A995_12908

<400> 69

gcuuuaaugc ggcccguuuu gauacggcag guuacaagcc cugguaaacg cagaguagag 60

c 61

<210> 70

<211> 69

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D6A5FC_12908

<400> 70

uucguaaugc ggccgugcug guaacguucc agcgcgacgg ucccaagccc gaaaaacgca 60

gagggagaa 69

<210> 71

<211> 80

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D67156_12908

<400> 71

ccgguaaugc ggcacgcgug gucacaagcc caccgcccuu cguugagcgg aaacguucac 60

guugggacgc agagugacgg 80

<210> 72

<211> 53

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6CC8F_12908

<400> 72

ggcuuaacuc agccaacggc gguccaaagc ccgcguguaa ugaggaugga gcc 53

<210> 73

<211> 66

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D65A05_12908

<400> 73

agcguaaugu agccuagucc gacuuuggac uagaggguuc acagccccuu uaauacagau 60

gacgca 66

<210> 74

<211> 81

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsades de pointes, URS0000D6967F_12908

<400> 74

gguguaaagc uacuaaacag gcaauacaaa aauaaguccu guuuaaaggu ucaaaguccu 60

uguaaaaaag cugaugacac g 81

<210> 75

<211> 62

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6755D_12908

<400> 75

ccucuaaugc ggcccggcau ggugccggac ggugguaagc ccgugcaaac gcagaaccua 60

gg 62

<210> 76

<211> 52

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D68D61_12908

<400> 76

cucguaaugc ggcgaaccgg uggcaagccc ggugguggac gcagagccag ag 52

<210> 77

<211> 53

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D67BA2_12908

<400> 77

uccucaaugc ggcaagccgg ugacaagccc ggcgguagac gcagagucaa gga 53

<210> 78

<211> 62

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6B09E_12908

<400> 78

uuuguaaugu ggccuaaauu uuuauuuaga acguuccaag ccguuaaaac acagaggaca 60

aa 62

<210> 79

<211> 55

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D65D7A_12908

<400> 79

cucuuaaagu ugccuaagaa cguugcaagc cguuuuacga aaaacugagc aagaa 55

<210> 80

<211> 71

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D694CE_12908

<400> 80

auuguaaugc agcauauaga uguauuaaca ccuauauaga guucaaagcc ucuacaaaug 60

cagaugacaa u 71

<210> 81

<211> 72

<212> RNA

<213> Pea aphid (Acyrthosiphon pisum)

<220>

<223> Acyrthosiphon pisum (Pea aphid) P1-type torsades de pointes, URS0000D68632_7029

<400> 81

uuuuuaauca uaccaguagu cuaauuuuua gauuacugac aguccuaagu cuguaaaaaa 60

ugagaaggga aa 72

<210> 82

<211> 106

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D67356_12908

<400> 82

aacucagcua gggagaguau aacaucaug uugacgagac cuagacgaaa cacagaggaa 60

aauuauuaau cacuggauag uauuaguaau gacucugugu ccauga 106

<210> 83

<211> 77

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D6976A_12908

<400> 83

uugucagcua aggagacaga aaaauuaucu acugaugaga cuuagccgaa accaccucuu 60

uuaggggugg ucuagau 77

<210> 84

<211> 85

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D6B94F_12908

<400> 84

60

guuguacguu auuagguauc uauga 85

<210> 85

<211> 119

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D698D3_12908

<400> 85

aacucagcua gggagaguag cgagcauuac guaauacuac guauuacucc aauaacauug 60

ucacugauga gaccuagacg aaacuacggu aaacauuugc aucauacugu agucugaua 119

<210> 86

<211> 76

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D68882_12908

<400> 86

aagacagccu aggagucuau aaaauaugug cugacgagac uaggacgaaa cuauccucag 60

uugaggauag uccacu 76

<210> 87

<211> 73

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D6A535_12908

<400> 87

aagacagucu aggagucuau aaaauuguua cugaagagac uagaacgaaa cuucuuuaau 60

uagaagucua aca 73

<210> 88

<211> 63

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D6B98C_12908

<400> 88

aacucaacca ggagaguaua aaauguuuac ugaugagacu ggacgaaacc aauagguuua 60

aac 63

<210> 89

<211> 74

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence RAGATH-1 hammerhead ribozyme, URS0000D68B88_12908

<400> 89

aagacaucca ggagucuaua aaauagucac ugaugagacu ggacgaaacc ucugcuauau 60

guagaggucu gauu 74

<210> 90

<211> 163

<212> DNA

<213> Unknown

<220>

<223> Veillonella sp. CAG:933 genome scaffold, scf58, HF986131.1

<400> 90

attgcctgtg aaggtagtgc atatttttat tattagatca tcagaagatg acaagcatgt 60

ggggcgtaag tagtattttt atgcgggaga agaagaatgg caattgttct aattagtact 120

gataattgca aatactatga tcgtgcggac gttaaaatca tgc 163

<210> 91

<211> 176

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-X_000328, BAAZ01000328.1

<400> 91

ttataatgtt agcataaata caataaagtt aatgcagtag aaatactgcg ctctttaagg 60

tgagaatcct tgacaagcat gtggggctta tatctattca tacagagcaa gtacgtacgg 120

gaaagcttaa aatactcatc tgtaaaatag tattagtgca gactttaaaa tcatgc 176

<210> 92

<211> 128

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F1-T_010313, BAAV01010313.1

<400> 92

acagaaaaag aagctaaaga agcaagaaag tattactgtg agaatcagta ataagcatgt 60

ggggctttatg tctttatcaaa agggtggcca acttttagat agcattagtg cggacgttaa 120

aatcatgc 128

<210> 93

<211> 157

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 1091142135580, whole genome shotgun sequence, AACY021400709.1

<400> 93

acattttgtg gttttaaggg ttaatcctta aggttgataa accttgacaa gcctatgggg 60

ctactatagt attctcttat tacgggtaag agtatcaagc ataagcgaaa ttccgtgctt 120

atgtaatgct aagttagtgc agacttaaaa attaggc 157

<210> 94

<211> 170

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: In-A_012728, BABB01012728.1

<400> 94

cttgttcgtg agaataggtg caattgccta aatgaatgtc ttcagaagat gacaaacctg 60

tggggcgtaa gtaataaaga gtctgaaaga ttgcagataa gagtatgcac ttatggcaa 120

tatgcatacc agaataattt attatgatcg tgcggacgtt aaaatcaggt 170

<210> 95

<211> 159

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-X_000328, BAAZ01000328.1

<400> 95

tcagtctgtg aagatagagt atacgtcctc agaagatgac aaacctgtgg ggcgtaagta 60

aatgcatatc gtatattatt cccttgaata cggcaatagc gggtaatatc cgagatactc 120

gtatttgtgt ttataatcgt gcagacgtta aaatcaggt 159

<210> 96

<211> 78

<212> DNA

<213> Damara Mole (Fukomys damarensis)

<220>

<223> Damara mole (Fukomys damarensis) contig 106618, whole genome shotgun sequence, AYUG01106618.1

<400> 96

ggaggataac agggggccac agcacaagcg ttcacgtcgc agcccctgtc ggattctgag 60

gaatctgcga attctgca 78

<210> 97

<211> 78

<212> DNA

<213> Eurasian wild boar (Sus scrofa)

<220>

<223> Eurasian wild boar (Sus scrofa) TJ Tabasco isolate Duroc breed chromosome 14, whole genome shotgun sequence, CM000825.5

<400> 97

agaggataac tggcagccac agtagaagca ttcacattgt ggtccatgtc agattctggt 60

gaatttgcaa attctgct 78

<210> 98

<211> 78

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_60, whole genome shotgun sequence, AKHW03000178.1

<400> 98

tttatgtcac tggggggccat agcggaagtg ttcatatcat ggccccaatc ggattccaac 60

aaatctgaga attctgct 78

<210> 99

<211> 77

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 145668, whole genome shotgun sequence, AFYH01145668.1

<400> 99

gggtactatt gggggacccgt agcaggagcg ttcacatcgc ggtccctgtc agactattac 60

agtctgcgaa tcctgct 77

<210> 100

<211> 83

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_55, whole genome shotgun sequence, AKHW03006769.1

<400> 100

attgcagctt aggggggccat agcagaagca ttcatgttgc agcccctgtc aggtaatagc 60

tggtaatacc tgctaattctgat 83

<210> 101

<211> 79

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 100904, whole genome shotgun sequence, AFYH01100904.1

<400> 101

attgtttatt ttgggggcca tagcagaagt gttcatgtcg cggcccctgt cagattctta 60

tgaatctgcaaattctgct 79

<210> 102

<211> 78

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 227694, whole genome shotgun sequence, AFYH01227694.1

<400> 102

ttacccacaa ctggggccat agcagaagcg ttcatgtcgc ggcccctgtc atattcttac 60

aaacctgtga attctgct 78

<210> 103

<211> 75

<212> DNA

<213> Florida lancelet (Branchiostoma floridae)

<220>

<223> Florida lancelet (Branchiostoma floridae) genome scaffold BRAFLscaffold_190, whole genome shotgun sequence, GG666606.1

<400> 103

cgccactaca tgggggccac agaaggagcg ttcacgtcgc ggtccctgtc aggtgttcta 60

cctgcggatc cttct 75

<210> 104

<211> 83

<212> DNA

<213> Chinese alligator (Alligator sinensis)

<220>

<223> Unplaced genome scaffold scaffold150_1 of the Chinese alligator (Alligator sinensis), whole genome shotgun sequence, KE695878.1

<400> 104

agcagttggc taggggtcat agtagaagtg ttcatgccac aacccctgtc aggtaatacc 60

tagtaatacc tgcaaattct gct 83

<210> 105

<211> 81

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_1.1, whole genome shotgun sequence, AKHW03001485.1

<400> 105

agaggtcaca agtccgaggc cgcggcagaa gtgctcacgg cacgggccct gtcagattcc 60

agcgaatctg caaattctgc t 81

<210> 106

<211> 77

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_58, whole genome shotgun sequence, AKHW03000416.1

<400> 106

caggggttgc atgaggccat agcaaaagca ctcacagtgc tgccctgtca gattccaaca 60

aatctgcaaa ttctgct 77

<210> 107

<211> 83

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_121, whole genome shotgun sequence, AKHW03004037.1

<400> 107

aatgctttga tgggggtcat agcagaagca ttaatgttgt gacccctgtc aggtaatacc 60

tgataatacc tgtgaattct gct 83

<210> 108

<211> 78

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 110885, whole genome shotgun sequence, AFYH01110885.1

<400> 108

tgcacatcta tgggggcctt agcagaagca ttcacgtcgc agcccctgtc ggattcttaa 60

gaatttgcgaattctgct 78

<210> 109

<211> 78

<212> DNA

<213> Chinese alligator (Alligator sinensis)

<220>

<223> Unplaced genome scaffold scaffold277_1 of the Chinese alligator (Alligator sinensis), whole genome shotgun sequence, KE695937.1

<400> 109

caattaagat gcagggccac agcagacatg tttatgttgt ggtccctgtc ggattctaat 60

gaatctgaga attctgct 78

<210> 110

<211> 75

<212> DNA

<213> Purple sea urchin (Strongylocentrotus purpuratus)

<220>

<223> Purple sea urchin (Strongylocentrotus purpuratus) contig 100549_fixed, whole genome shotgun sequence, AAGJ05100549.1

<400> 110

acagtaaaaa agtggggcca ttgaaggagc gttcacgtcg tggtccctgt cagatgaaaa 60

tctgcgaatccttca 75

<210> 111

<211> 76

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_244, whole genome shotgun sequence, AKHW03003332.1

<400> 111

agttgctata acggccacaa cagaaatgtt cacatcgtgg ccccggtcag attccagcaa 60

atctgcaaat tctgct 76

<210> 112

<211> 81

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_72, whole genome shotgun sequence, AKHW03000533.1

<400> 112

agaggttaca agtgcaaggc cagagcagaa gtgttcacag catagccctt gtcagatacc 60

aatgaatctg tgaattctgc t 81

<210> 113

<211> 78

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 070068, whole genome shotgun sequence, AFYH01070068.1

<400> 113

agcttgcgaa tggggggccat agcagaagag ttcacgtcgc ggcccctgtc agagttctac 60

gaatttgcgaattctgct 78

<210> 114

<211> 78

<212> DNA

<213> Nine-banded Armadillo (Dasypus novemcinctus)

<220>

<223> Nine-banded armadillo (Dasypus novemcinctus) cont2.425401, whole genome shotgun sequence, AAGV020425402.1

<400> 114

atagaagata atggggccac agcagaagca ttcatgttgc agcccttgtg agattcaagt 60

gaatctgtga attctgct 78

<210> 115

<211> 104

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.106 genome scaffold, whole genome shotgun sequence, CH477291.1

<400> 115

tacccagcaa atcctatccc tacctcctta aggtactggc tgaagtacga gtaactttag 60

gaaagatcgg gtaaccaacc ccggtccaat tctgactgag aagg 104

<210> 116

<211> 96

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.16 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663677.1

<400> 116

cactggcaaa atccgatccc tgcctccacg tggcgctgct ggatgtcggt tttggtgagg 60

cttatcacct cagccaagac ctaaccaaag ggacgg 96

<210> 117

<211> 116

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 96443, whole genome shotgun sequence, JXUM01096443.1

<400> 117

ttcccaacaa ctcctatccc tacctcctcg tgacactcac tggaccgcca gctactttag 60

acaagatcgg ataacccacc ctgacggata atttggccgt tggctgacag ggcagg 116

<210> 118

<211> 133

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.33 genome scaffold, whole genome shotgun sequence, CH477218.1

<400> 118

tgctcagcaa ctcctatccc tacctcctcg tggtactggt acgagtatgg gtggtaccgg 60

tacgagtaac cttggggaag atcgggtaac caatcccggg gggggaactt tggtcgtatg 120

cagacaggga agg 133

<210> 119

<211> 123

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.2284 genome scaffold, whole genome shotgun sequence, CH479147.1

<400> 119

tgtccagtaa ctcctatccc tacctccccg tggtgccgcc tggggtacga gtaatcgtag 60

gcaacattgg gtaaccaacc ctgacaggga aggctcctct cttctgtatg ctgacaggga 120

agg 123

<210> 120

<211> 109

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 57437, whole genome shotgun sequence, JXUM01057437.1

<400> 120

tgccccagcaa ctcttatccc tacttcctcg tggtaccagc cggaaactac gagaaaccta 60

agggaagatc gggtaaccac aagtgtggcg ggggcgcaga gggggggag 109

<210> 121

<211> 92

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 160006, whole genome shotgun sequence, JXUM01160006.1

<400> 121

tgcccagcac ctcctatccc tgcctccacg cggtagggaa gatcgggtaa ccaaccccgg 60

tgagaagttt ggtcgtaggc tgacagggaa gg 92

<210> 122

<211> 106

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.267 genome scaffold, whole genome shotgun sequence, CH477452.1

<400> 122

tgcacagcaa ctcctatccc tatctcctcg cggtactgac cgaggtacga gcaaccttag 60

ggaagatcgg gttctgcaaa cctagagcgt ctgtacatgg agtagg 106

<210> 123

<211> 110

<212> DNA

<213> Anopheles sinensis

<220>

<223> Anopheles sinensis unplaced genome scaffold AS2_scf7180000690996, whole genome shotgun sequence.

<400> 123

tattcttgaa ctccgatccc aacctcctcg tggtgctagc tgaagtatga tcttggaact 60

tattaagttc ttcagcacat tgtgcaacga tcgtatacca atagggacgg 110

<210> 124

<211> 135

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.263 genome scaffold, whole genome shotgun sequence, KE524294.1

<400> 124

tgcctagcaa ctcctatccc tacctcttca tggtactgcc cggggtactg gccggagtat 60

tagcaactca agcaattaga gaagatcggg taactaaccc cggtctcaac tttgatcgta 120

tgctgatatg gaagg 135

<210> 125

<211> 99

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 149242, whole genome shotgun sequence, JXUM01149242.1

<400> 125

tgcccagcaa ctccaatccc tacatccgcg aggtaccggt tgtagactac gagcaccgag 60

caaccggtgg taactttggt cgtattctga cagggaagg 99

<210> 126

<211> 76

<212> DNA

<213> Longbearded sandfly (Lutzomyia longipalpis)

<220>

<223> Lutzomyia longipalpis contig 2844, whole genome shotgun sequence, AJWK01002842.1

<400> 126

ggtaatccaa ctcctacttc aacctccacg tggtgacacc tgggcaccca atttattggg 60

tggctaactg aagagg 76

<210> 127

<211> 113

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.353 genome scaffold, whole genome shotgun sequence, CH477538.1

<400> 127

tgctcagcag ctcctatctc tacctcgtcg cattactggc cggggtccga gcaaccttat 60

ggaaaatcgc cccaaccccg agggaaactt tggtcgtatg ctgacaggga agg 113

<210> 128

<211> 64

<212> DNA

<213> Trichomalopsis sarcophagae

<220>

<223> Trichomalopsis sarcophagae Alberta strain scaffold25490, whole genome shotgun sequence, NNAY01025263.1

<400> 128

ggcgtacaaa atcctatcgt gcaacctccc cgtggtgtat gccgggttat gctaatgcgg 60

aagg 64

<210> 129

<211> 76

<212> DNA

<213> Pea aphid (Acyrthosiphon pisum)

<220>

<223> Acyrthosiphon pisum LSR1 strain contig 29506, whole genome shotgun sequence, ABLF02028779.1

<400> 129

ggtcggtgaa gtcctacccc caccaccacg tggtgccgac tggaaacgga actccggttc 60

cagccaacgggggagg 76

<210> 130

<211> 99

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 110469, whole genome shotgun sequence, JXUM01110469.1

<400> 130

tgccccagcaa ctcctatccc tacctcctcg cggtaccggc cggaaactat aaggcaatct 60

agcgctcatc acccttctct ctcaagcaaa cacagaaga 99

<210> 131

<211> 95

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.12 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663633.1

<400> 131

cattggcaaa atcctattcc tacctcctcg tggtgctggt ggatgagggc atgctgagtc 60

tcactagctc agtatgtctt aactaaaagg gaagg 95

<210> 132

<211> 95

<212> DNA

<213> Eye-spotted gar (Lepisosteus oculatus)

<220>

<223> Eyed gar (Lepisosteus oculatus), linkage group LG14, whole genome shotgun sequence, CM001417.1

<400> 132

ggctggcaaa atcctatcac cacctcctcg cggtgccagg tggatacggc tggatacaac 60

tggatacaac gactcgttgg aactaacggt gaagg 95

<210> 133

<211> 96

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.686 genome scaffold, whole genome shotgun sequence, CH477871.1

<400> 133

tggccagcac ctcccatccc cacctccttg tggtactggc cagggtacga gcaaccaatc 60

ccggtggaca ctcttgtcgt atgctgacag ggaaag 96

<210> 134

<211> 126

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.594 genome scaffold, whole genome shotgun sequence, CH477779.1

<400> 134

tgcccagcaa ctcttatccc tacctcctct tacttcctcg tggtaatggc cagggtacga 60

gcaaccttag ggaagatcgg ataaccaacc ctggtgagag ctctcgtcgt atgctggcag 120

ggaagg 126

<210> 135

<211> 98

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 77081, whole genome shotgun sequence, JXUM01077081.1

<400> 135

taaccaggaa ctcctatccc tacctccccg cggtactgac cgggatacga tcagtcccaa 60

tcaccgtggg aactttggtc gtatgctgac agggaagg 98

<210> 136

<211> 88

<212> DNA

<213> Anopheles farauti

<220>

<223> Unplaced genome scaffold supercont2.12 of Anopheles farauti FAR1 strain, whole genome shotgun sequence, KI915051.1

<400> 136

ggccggcaaa gcccgacccc cacctcctcg tggtgccggc tggatgcata agaccctacc 60

cgtcgtgggt tgcagccaac gggggcgg 88

<210> 137

<211> 89

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.1120 genome scaffold, whole genome shotgun sequence, CH478303.1

<400> 137

tgccaagaaa ctcctcccca acctcctcgt ggtactggcc gggctacgag taaccttgga 60

gaactttagt cgtatgatga caagaaagg 89

<210> 138

<211> 97

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 8119, whole genome shotgun sequence, JXUM01008119.1

<400> 138

tgccccagcaa ctcttatccc tacctccacg tggtaccgca cagaaaaaaa aatattcatg 60

taaaattcag cgacaaatca tgcacataaa gggaatg 97

<210> 139

<211> 130

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.1096 genome scaffold, whole genome shotgun sequence, CH478279.1

<400> 139

tgctcagtaa ctcctatccc tgacctcccc gaggtgccgg ctggggtgcg aacaacccaa 60

aggttgaaag gcgaattcac gtagcctaat gagctcaaag cgaactcagg tcgcatgctg 120

acagggaagg 130

<210> 140

<211> 80

<212> DNA

<213> Rhodnius prolixus

<220>

<223> Rhodnius prolixus Rhodnius_prolixus-3.0.3-200.47, whole genome shotgun sequence, ACPB03013890.1

<400> 140

tgctcggtaa aatctgatct ctacctcctt gtggtcctac caggaccttt tacctactaa 60

gaataggccaacagagacgg 80

<210> 141

<211> 102

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 176146, whole genome shotgun sequence, JXUM01176146.1

<400> 141

tgcccagcaa caccaaccc tacctccgcg gggcaccagc cggactgcat gcggctgtat 60

gcggactaca tgggaccttt ggtcgtaggc tgacagggaa gg 102

<210> 142

<211> 109

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 103962, whole genome shotgun sequence, JXUM01103962.1

<400> 142

tgccccagcaa ctcctatccc tacctcctcg tggtaccggc cggaaactat gattagcatc 60

acggggatca tcaagaataa tttcggaccg cacaagctaa atgggtgag 109

<210> 143

<211> 97

<212> DNA

<213> Anopheles albimanus

<220>

<223> Anopheles albimanus ALBI9_A strain cont1.2834, whole genome shotgun sequence, APCK01002835.1

<400> 143

cgtctcggaa cacctatctc tacctccacg tggtgcctgc tggattatgg tgcatgcgac 60

ggtacagctc acatgaacca tataccgaca gagaagg 97

<210> 144

<211> 81

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 45626, whole genome shotgun sequence, JXUM01045626.1

<400> 144

tgcccagcaa ctcctatccc tacctcctcg tggtactggt tggaaactac gctggaatca 60

acgtccgagt tccagggaag g 81

<210> 145

<211> 82

<212> DNA

<213> Anopheles albimanus

<220>

<223> Anopheles albimanus ALBI9_A strain chromosome 3L, whole genome shotgun sequence, CM008154.1

<400> 145

aactcggaac tcctatcctc acctccacgt ggtgccggct ggaatatgat tgtattagtc 60

tatcatatac agacgaggaa gg 82

<210> 146

<211> 108

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.1004 genome scaffold, whole genome shotgun sequence, CH478188.1

<400> 146

tgcccagcaa ctcctatccc tacctcctcg tggtactggc cggggtacga gttgttgatc 60

taagcaaccg gaagtccatg tccatgatca aagcacccat agaggaag 108

<210> 147

<211> 94

<212> DNA

<213> Anopheles stephensi

<220>

<223> Anopheles stephensi SDA-500 strain unplaced genome scaffold supercont1.615, whole genome shotgun sequence, KB664972.1

<400> 147

tgctttagaa ctccgatctc aaacctcctc gtggtgctgg ctggaggata attgttgcac 60

attttacaca acaattattc actgattgag acgg 94

<210> 148

<211> 94

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.438 genome scaffold, whole genome shotgun sequence, CH477623.1

<400> 148

agcccagcaa ctcctatccc tacctcctcg tggtactggc cggctgcgaa aggcctggaa 60

aagtttcaga aaatggagtc gctaaaaccg aagg 94

<210> 149

<211> 120

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.461 genome scaffold, whole genome shotgun sequence, CH477646.1

<400> 149

tgcccaataa ttcctatccc tatctcccca cgatgccgcc cagagtacga gtaatcatct 60

ttccgatctt ttccagtaat caaccccggt gagaccttgg tcgtatgctg acaagaaagg 120

<210> 150

<211> 134

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.161 genome scaffold, whole genome shotgun sequence, CH477346.1

<400> 150

tgcccagcaa ctcctatccc tacctcctcg tggtactggc cggggtacga gtaaccttgg 60

ggaagtagta ggaagtagta ggaaggagta accaaccccc ggtgggaact ttggtcgtat 120

gctgacagga aagg 134

<210> 151

<211> 80

<212> DNA

<213> Red castaneum

<220>

<223> Tribolium castaneum, Georgia GA2, linkage group LGX, whole genome shotgun sequence, CM000276.3

<400> 151

tcctggcaaa aatgctctaa acctccacgt ggttcttgct ggacaaatta gttattagct 60

aatttgacca attagagcaa 80

<210> 152

<211> 89

<212> DNA

<213> Anopheles farauti

<220>

<223> Unplaced genome scaffold supercont2.15 of Anopheles farauti FAR1 strain, whole genome shotgun sequence, KI915054.1

<400> 152

gcctttggaa ctccgttttc taacctccac gtggtgctgg ctggaatatg gtctttcctt 60

tatggtcgat catatacaaa tagaaacgg 89

<210> 153

<211> 105

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.281 genome scaffold, whole genome shotgun sequence, CH477466.1

<400> 153

tgttcatcaa ctcctatccc tacctcctcg cggtactgtc cggggtacga gcaaccttag 60

agaagatccc gcaacggctt cgtggcgcga gccgagatgt gcagg 105

<210> 154

<211> 104

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 134552, whole genome shotgun sequence, JXUM01134552.1

<400> 154

aacccagtaa ctccgatccc ttccttcacg cggcgccggc cggggtgcga ccatccgaaa 60

ggtagattaa gcttgaagct taggtcgtat gttgacaggg aagg 104

<210> 155

<211> 119

<212> DNA

<213> Unknown

<220>

<223> Culex pipiens quinquefasciatus supercont3.1335 genome scaffold, whole genome shotgun sequence, DS233147.1

<400> 155

tgttcagtaa ctccgatacc ctggcctccc cgcggcgctg gccgggatac tagtaaccat 60

tggagagatc gggtaaccaa ccccggtggg aactatggta gtatgctgac agggtaagg 119

<210> 156

<211> 83

<212> DNA

<213> Anopheles epiroticus

<220>

<223> Unplaced genome scaffold supercont1.133 of Anopheles epiroticus epiroticus2 strain, whole genome shotgun sequence, KB669981.1

<400> 156

ggccgacaaa accctctccc aacctccacg tggtgtcggc tggaaagtgc ctcatgtaat 60

gttgcattta ccaactggga agg 83

<210> 157

<211> 104

<212> DNA

<213> Anopheles albimanus

<220>

<223> Anopheles albimanus ALBI9_A strain chromosome 3R, whole genome shotgun sequence, CM008155.1

<400> 157

aatctcggaa ctcctatccc cacctcctcg tggtgccggc tggaatatgg tagatgtgca 60

tggtatccga ccaatatcat cttaccatat acagacgggg aagg 104

<210> 158

<211> 92

<212> DNA

<213> Aedes aegypti

<220>

<223> Aedes aegypti Liverpool strain supercont1.294 genome scaffold, whole genome shotgun sequence, CH477479.1

<400> 158

tgccaagcat ctcctatccc taccatctcg tggtactggc cgtggtacga gcctcccaga 60

tgggaacgat ggtcgtatgg tgacagcgaa gg 92

<210> 159

<211> 55

<212> DNA

<213> White Talisman Jump (Folsomia candida)

<220>

<223> Folsomia candida VU strain Fcan01_Sc032 population, whole genome shotgun sequence, LNIX01000032.1

<400> 159

aacaaatata cgggtgcccc cgtactgatg aggccatggc aggccgaaat ttgtg 55

<210> 160

<211> 55

<212> DNA

<213> Paenibacillus wynnii

<220>

<223> Paenibacillus wynnii strain DSM 18334 unitig_3_1r, whole genome shotgun sequence, JQCR01000003.1

<400> 160

cttgctttatg gactcagttc actgacgagc tcgtgagatt cgagcgaaaa gtatc 55

<210> 161

<211> 60

<212> DNA

<213> Agaricus bisporus

<220>

<223> Agaricus bisporus var. burnettii JB137-S8 unplaced genome scaffold AGABI1scaffold_33, whole genome shotgun sequence, JH971417.1

<400> 161

gtcggattag ggcagcggtt aagccctctg atgagcccct tcgcaagggc gaaatccgca 60

<210> 162

<211> 58

<212> DNA

<213> Eucalyptus grandis

<220>

<223> Eucalyptus grandis BRASUZ1 cultivated species unplaced genome scaffold scaffold_11, whole genome shotgun sequence, KK198763.1

<400> 162

aattagttgg gagttgatgc tgctctcctg atgaggccat agcaggccga aaccagtt 58

<210> 163

<211> 58

<212> DNA

<213> Eucalyptus grandis

<220>

<223> Eucalyptus grandis BRASUZ1 cultivated species unplaced genome scaffold scaffold_2, whole genome shotgun sequence, KK198754.1

<400> 163

aattggttgg gagctaatgc tattctcctg acgaggccat ggcaggctga aactattt 58

<210> 164

<211> 75

<212> DNA

<213> laboriosa (Habropoda laboriosa)

<220>

<223> Habropoda laboriosa contig 20310, whole genome shotgun sequence, LHQN01020310.1

<400> 164

gtggcgtctg gggcatggac cggctacatc agcctcactg atgagtctgt ggtcggtctc 60

gagacgaaac gcttc 75

<210> 165

<211> 57

<212> DNA

<213> Unknown

<220>

<223> Desulfobulbus sp. Tol-SR contig_572, whole genome shotgun sequence, JROS01000118.1

<400> 165

gtgatgtctg cggctgaatc tgccgcactg acgagcccat ccagggcgaa acatcca 57

<210> 166

<211> 58

<212> DNA

<213> Orchesella cincta

<220>

<223> Orchesella cincta Ocin01_Sc3888, whole genome shotgun sequence, LJIJ01003888.1

<400> 166

gacgcgtcta gaagtgaagc ccttctactg atgaggttat ggcagaccga aacgcaaa 58

<210> 167

<211> 58

<212> DNA

<213> Sunflower (Helianthus annuus)

<220>

<223> Sunflower (Helianthus annuus) linkage group 3, whole genome shotgun sequence, CM007892.1

<400> 167

cactagttga gagttgtcgc tggtttcctg atgagtccaa ggcaagacaa aaccagta 58

<210> 168

<211> 56

<212> DNA

<213> Unknown

<220>

<223> Citreicella sp. 357 C357_106, whole genome shotgun sequence, AJKJ01000094.1

<400> 168

cccaggtacc cggatgtgtt ttccgggctg atgagtccgt gaggacgaaa cctggg 56

<210> 169

<211> 67

<212> DNA

<213> Green monkey (Chlorocebus sabaeus)

<220>

<223> Green monkey (Chlorocebus sabaeus) 1994-021 isolate chromosome 4, whole genome shotgun sequence, CM001944.2

<400> 169

attcagtcag gagttttttc tgctgatgag ttcctggtct tgctaacttc aaagaacgaa 60

gctgcag 67

<210> 170

<211> 60

<212> DNA

<213> Fomitopsis pinicola

<220>

<223> Fomitopsis pinicola FP-58527 SS1 unplaced genome scaffold FOMPIscaffold_81, whole genome shotgun sequence, KE504202.1

<400> 170

ggacggtcgg ggcagcgggt aagccccctg acgaggactt tcgcaggtcc gaaaccgctg 60

<210> 171

<211> 75

<212> DNA

<213> Trichomalopsis sarcophagae

<220>

<223> Trichomalopsis sarcophagae Alberta strain scaffold10693, whole genome shotgun sequence, NNAY01010628.1

<400> 171

tgcgcgtctg aggcagggtt accatcggat gccttatactga cgagtccacg atggtaacct 60

gggacgaaac gcaac 75

<210> 172

<211> 60

<212> DNA

<213> Unknown

<220>

<223> Grape (Vitis vinifera), whole genome shotgun sequence of line PN40024, untargeted chromosome 13, chr13, FN597036.1

<400> 172

aactggtcaa gagctggagt cattcccctg atgaatccat gaatcaggat gaaaccagtt 60

<210> 173

<211> 57

<212> DNA

<213> Unknown

<220>

<223> Candidatus Uhrbacteria bacterium RIFOXYB2_FULL_45_11 rifoxyb2_full_scaffold_3973, whole genome shotgun sequence, MGFD01000034.1

<400> 173

tttttgtctt tagatacagt atctaaactg atgagtcctg taaggacgaa acaaaag 57

<210> 174

<211> 61

<212> DNA

<213> Unknown

<220>

<223> Asian citrus Psyllid, Diaphorina citri - Florida strain, whole genome shotgun sequence, AWGM01152003.1

<400> 174

aacgcgtctt aggctgctct caggtgctag ctgatgagtt ccaacaagaa cgaaacgcgt 60

c 61

<210> 175

<211> 79

<212> DNA

<213> Dufourea novaeangliae

<220>

<223> Dufourea novaeangliae contig 3158, whole genome shotgun sequence, LGHO01003158.1

<400> 175

caggcgtctg gggttggggt cgtctaccgt cagtcccact gacgaatctt ggttgacgat 60

tctcgagacg aaacgccat 79

<210> 176

<211> 58

<212> DNA

<213> Eucalyptus grandis

<220>

<223> Eucalyptus grandis cultivar BRASUZ1 unplaced genome scaffold_1, whole genome shotgun sequence, KK198753.1

<400> 176

aactggtcag gagcttatgc taccatccta atgaggccat ggtaggccga aaccagtt 58

<210> 177

<211> 58

<212> DNA

<213> Clementine (Citrus clementina)

<220>

<223> Unplaced genome scaffold scaffold_5 of Citrus clementina Clemenules cultivar, whole genome shotgun sequence, KI536799.1

<400> 177

cactggttgg gaactgaagc cgttctcctg acgagcccac ggtagggcga aaccagtc 58

<210> 178

<211> 56

<212> DNA

<213> Echinostoma caproni

<220>

<223> Genome assembly of Echinostoma caproni Egyptian strain, scaffold: ECPE_scaffold0022838, LL256423.1

<400> 178

ctggagtgat atttgctgat atttactgat gagctccaat aagagcgaaa ctcgag 56

<210> 179

<211> 58

<212> DNA

<213> Unknown

<220>

<223> Grape (Vitis vinifera), whole genome shotgun sequence of line PN40024, chromosome 6, chr6, FN597024.1

<400> 179

aactagttgg gagctagagc cattcccctt atgagtccat ggcaagacga aaccagtc 58

<210> 180

<211> 68

<212> DNA

<213> Unknown

<220>

<223> Candidatus Uhrbacteria bacterium RIFOXYC2_FULL_47_19 rifoxyc2_full_scaffold_469, whole genome shotgun sequence, MGFG01000021.1

<400> 180

accacttctg ccgttgagta cggcactgat gagtccattc gattgtaaac agcaggacga 60

aaagtaaa 68

<210> 181

<211> 55

<212> DNA

<213> Unknown

<220>

<223> Candidatus Taylorbacteria bacterium RIFCSPLOWO2_02_FULL_46_40 rifcsplowo2_02_scaffold_68864, whole genome shotgun sequence, MHSH01000051.1

<400> 181

cgttgctctc ggaatgtgta ttccgactga tgagtccaaa aggacgaaag cagaa 55

<210> 182

<211> 56

<212> DNA

<213> Unknown

<220>

<223> Omnitrophica bacteria tentative species CG1_02_46_14 cg1_0.2_scaffold_5404_c, whole genome shotgun sequence, MNVS01000076.1

<400> 182

cggctgtttc ccgatgtgtt atcgggactg atgagtccga aaggacgaaa cagcgt 56

<210> 183

<211> 58

<212> DNA

<213> Rhizobium phage

<220>

<223> Rhizobium phage RHEph01, complete genome, JX483873.1

<400> 183

aataggtacg gggctgatgc tgccccgctg atgaggccaa gctatggccg aaaccatc 58

<210> 184

<211> 58

<212> DNA

<213> Eucalyptus grandis

<220>

<223> Eucalyptus grandis BRASUZ1 cultivated species unplaced genome scaffold scaffold_11, whole genome shotgun sequence, KK198763.1

<400> 184

aactggtcga gagttgatgt cgctctcttg acgaggccat ggcaggtcga aaccaatt 58

<210> 185

<211> 58

<212> DNA

<213> Octopus bimaculoides

<220>

<223> Octopus bimaculoides Scaffold62703_contig_4, whole genome shotgun sequence, LGKD01404090.1

<400> 185

aatgagtcaa gtgacgcgaa catctctgat gagaccctca aaaaggtcga aattcgat 58

<210> 186

<211> 57

<212> DNA

<213> Perkinsus marinus

<220>

<223> Perkinsus marinus ATCC 50983 gcontig_1104296167808, whole genome shotgun sequence, AAXJ01016906.1

<400> 186

ggtgtgtctg gcgccgttag ccactgatga gtccctgtgg tgaggacgaa acactac 57

<210> 187

<211> 56

<212> DNA

<213> Cornetzi leafcutter ant (Trachymyrmex cornetzi)

<220>

<223> Trachymyrmex cornetzi contig 48241, whole genome shotgun sequence, LKEY01048241.1

<400> 187

tatatgtcag tttgcgtttg ctctgaggag ggctcaggaa tgagccgaaa catgta 56

<210> 188

<211> 55

<212> DNA

<213> Unknown

<220>

<223> Parcubacteria (Giovannonibacteria) bacteria GW2011_GWF2_42_19 UV11_C0020, whole genome shotgun sequence, LCDF01000020.1

<400> 188

ccactgtcct agagtgtgta ctctagctga tgagtcggaa acgacgaaac agaaa 55

<210> 189

<211> 53

<212> DNA

<213> Octopus bimaculoides

<220>

<223> Octopus bimaculoides Scaffold54493_contig_334, whole genome shotgun sequence, LGKD01378372.1.

<400> 189

ccgaagtcga gctgtcttaa ttgatgaggc gaaggaaaat gccgaaacta cgc 53

<210> 190

<211> 74

<212> DNA

<213> Dufourea novaeangliae

<220>

<223> Dufourea novaeangliae contig 944, whole genome shotgun sequence, LGHO01000944.1

<400> 190

cccgcgtcta aggcagggtc tgctagaaaa gccttatactga cgagtccact agcatgccca 60

ggacgaaacgctcc 74

<210> 191

<211> 60

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0008277, LL965256.1

<400> 191

tggatgtata ttcatgatat aggattgctg atgagtccca aagataggac gaaacaaccg 60

<210> 192

<211> 55

<212> DNA

<213> Pleurotus ostreatus

<220>

<223> Pleurotus ostreatus PC15 unplaced genome scaffold_10, whole genome shotgun sequence, KL198013.1

<400> 192

tttgtgttgg gaggtgtgtg cctctcctga tgaatccaaa aggacgaaac acatt 55

<210> 193

<211> 99

<212> DNA

<213> Four-striped stingless bee (Melipona quadrifasciata)

<220>

<223> Melipona quadrifasciata 111107301 isolate, unplaced genome scaffold95, whole genome shotgun sequence, KQ435798.1

<400> 193

agggcgtctg gggtaggagt cactgccatc aaaacaccccc cctcccccccc ccccccccca 60

ctgatgagtc taggcagcga ctccgagacg aaacgcatc 99

<210> 194

<211> 58

<212> DNA

<213> Morning glory (Ipomoea nil)

<220>

<223> Morning glory (Ipomoea nil) DNA, scaffold: scaffold1407, cultivar: Tokyo-kokei standard, BDFN01001407.1

<400> 194

aactagtcgg gagctattga cgttcccctg atgagcccat gacgggacaa aaccagtt 58

<210> 195

<211> 52

<212> DNA

<213> Punctularia strigosozonata

<220>

<223> Punctularia strigosozonata HHB-11173 SS5 unplaced genome scaffold PUNSTscaffold_19, whole genome shotgun sequence, JH687556.1

<400> 195

gctcggtcat ctcgggcaga accctgatga gcctataaag gcgaaacagg gc 52

<210> 196

<211> 62

<212> DNA

<213> Four-striped stingless bee (Melipona quadrifasciata)

<220>

<223> Melipona quadrifasciata 111107301 isolate, unplaced genome scaffold98, whole genome shotgun sequence, KQ435803.1

<400> 196

caagcgtttt ggggccagcc ccactgatga gtctaggcag cgactccaag acgaaacgca 60

tc 62

<210> 197

<211> 55

<212> DNA

<213> Mycobacterium obuense

<220>

<223> Mycobacterium obuense UC1 strain Mobu_contig000008, whole genome shotgun sequence, LAUZ02000008.1

<400> 197

ctgctctcca gggtcaccct gctgacgagc ccgtgaaagt cgggcgaaag agccc 55

<210> 198

<211> 74

<212> DNA

<213> Unknown

<220>

<223> Candidatus Giovannonibacteria RIFCSPLOWO2_01_FULL_46_13 rifcsplowo2_01_scaffold_439, whole genome shotgun sequence, MFIE01000019.1

<400> 198

gaacgctcgc gagatgtgtg tctcgcctga tgagcccgcc aaaggcgggc aagtccaaaa 60

ggacgaaagc gtgt 74

<210> 199

<211> 54

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0102769, LL113166.1

<400> 199

aatgcatcca gtacatccac tggctgacga gtccgagata agacgaaatg catg 54

<210> 200

<211> 59

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0002697, LL959675.1

<400> 200

gacatgtctg ggatgcaggt acatccaact gacgagtccc aaatacgacg aaacatgca 59

<210> 201

<211> 69

<212> DNA

<213> Aedes albopictus

<220>

<223> Aedes albopictus Foshan isolate contig 106395, whole genome shotgun sequence, JXUM01106395.1

<400> 201

tcaaagtctt gacgaaaggc caacgggcca aaacgtcaac tgatgagtcc ttgatggacg 60

aaactttgt 69

<210> 202

<211> 55

<212> DNA

<213> Unknown

<220>

<223> Candidatus Lloydbacteria bacterium RIFCSPHIGHO2_02_FULL_54_17 rifcsphigho2_02_scaffold_4023, whole genome shotgun sequence, MHLO01000032.1

<400> 202

ttgctgtaga gaagtgcatg cttctcctga cgagtcggaa acgacgaaac agcac 55

<210> 203

<211> 110

<212> DNA

<213> Unknown

<220>

<223> Bacteria SM23_31 WORSMTZ_22961, whole genome shotgun sequence, LJUD01000105.1

<400> 203

agcagagacc gggaagggat tctcttatta tgaaaatatt gaaaatagca tgaaacacta 60

aaccccgggg atcctcccgg taatgcagcc gtagccggtc acaagcccgg 110

<210> 204

<211> 58

<212> DNA

<213> Clostridium thermocellum

<220>

<223> Clostridium thermocellum ATCC 27405, complete genome, CP000568.1

<400> 204

tccagagtga cggaacgact cttcctccgg taatgcggtg gcccggtcac aagtccgg 58

<210> 205

<211> 60

<212> DNA

<213> Unknown

<220>

<223> Candidate classification NC10 bacteria CSP1-5 XU15_C0011, whole genome shotgun sequence, LDXR01000011.1

<400> 205

cgcagagagg ggctaggcca taggcttagc tctaatgcgg cataccggtc tcaagcccgg 60

<210> 206

<211> 51

<212> DNA

<213> Unknown

<220>

<223> Black crowned crane (Balearica pavonina gibbericeps), contig 83242, whole genome shotgun sequence, JJRR01083242.1

<400> 206

tgcagatgga ataatttaat gcaactgtag ttactcaggt tccaagtcct g 51

<210> 207

<211> 63

<212> DNA

<213> Spirochaetes bacteria

<220>

<223> Borrelia GWB1_66_5 gwb1_scaffold_16834, whole genome shotgun sequence, MIAS01000104.1

<400> 207

tgcagagggg gccgggacgc gcgaagcgac tcggcctaat gcacaggccg gtcccaagtc 60

cgg 63

<210> 208

<211> 57

<212> DNA

<213> Clostridium asparagiforme

<220>

<223> Clostridium asparagiforme DSM 15981 genome scaffold Scfld9, whole genome shotgun sequence, GG657595.1

<400> 208

cgcagagcaa cggggcagca atgccccggt aatgcggggg aacggttgca accccgt 57

<210> 209

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Clostridium hungatei DSM 14427 strain CLHUN_contig000001, whole genome shotgun sequence, MZGX01000001.1

<400> 209

tgcagatggg cggccttatg gccgttaatg cgctccccgga taccgggaac ccgtccaaag 60

cccgg 65

<210> 210

<211> 60

<212> DNA

<213> Purple sea urchin (Strongylocentrotus purpuratus)

<220>

<223> Purple sea urchin (Strongylocentrotus purpuratus) contig 102072_fixed, whole genome shotgun sequence, AAGJ05102072.1

<400> 210

agggaggggg gggtattgga accaaacctc ttaaccaacc gtcgcccgtc ccaagtcggg 60

<210> 211

<211> 56

<212> DNA

<213> Unknown

<220>

<223> Desulfosporosinus sp. I2 contig 00035, whole genome shotgun sequence, JYNH01000035.1

<400> 211

cgcagagtga ccgcccatcg cgggcgggta atgcggctag ccggtcacaa gcccgg 56

<210> 212

<211> 70

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. W14A NODE_41, whole genome shotgun sequence, MBSV01000063.1

<400> 212

cgcagagcag cggagaaact gacttcgtta atgcggcctg acgtttttcg tctgacggtt 60

gcaagcccgc 70

<210> 213

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Bioreactor metagenome PBDCA2_GBB5CE401D1Q9V_left, whole genome shotgun sequence, AGTN01047810.1

<400> 213

tgcagatggg cgccttcggg cgttaatgcg ctgaaaccaa aggttccacc aggtccaaag 60

tcctg 65

<210> 214

<211> 59

<212> DNA

<213> Unknown

<220>

<223> Stromatolite metagenome 35133330, whole genome shotgun sequence, ABMG01007509.1

<400> 214

ggtgagcggc cccgcccgta aggacgggga ctaaaccaca agtccggtcg caagtccgg 59

<210> 215

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Uncultured Ruminococcaceae bacteria TS29_contig142355, whole genome shotgun sequence, ADJT01008886.1

<400> 215

tgcagagtga gaaagctcat taccgtttgg tgatgggctt ttgtaatgca gagcgccggt 60

cacaatcccg g 71

<210> 216

<211> 56

<212> DNA

<213> Unknown

<220>

<223> Freshwater sediment metagenome lwFormaldehyde_BCIB5337_x1, whole genome shotgun sequence, ABSN01019877.1

<400> 216

cgcagatgac ggtgccacca cggcaccgta atgcgacaag caggttccaa tccctg 56

<210> 217

<211> 72

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome ctg_1101668267133, whole genome shotgun sequence, ABSN01019877.1

<400> 217

tgatgagggg cggggggcca gagacccccc gttaaatcgc catgtcaacc gacatgctgg 60

tcccaagcccag 72

<210> 218

<211> 61

<212> DNA

<213> Unknown

<220>

<223> Compost metagenome contig 24470, whole genome shotgun sequence, ADGO01024387.1

<400> 218

agtgagggga tcgatctaaa ctactggctt gtttcgtgca agtcaccggt cccaagtccg 60

g 61

<210> 219

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Compost metagenome FHNL2OP04YM6SP, whole genome shotgun sequence, ADGO01161384.1

<400> 219

cgcagagcac gccctacggg gcgtaatgcg gcctcaccac tggggtgagc cagttgcaag 60

cctgg 65

<210> 220

<211> 63

<212> DNA

<213> Unknown

<220>

<223> Compost metagenome FHNL2OP04YQ5F0, whole genome shotgun sequence, ADGO01160766.1

<400> 220

cgcagagggc agcccttcgg ggctgtaatg cactccccac ctggggagcg gtcccaagtc 60

cgc 63

<210> 221

<211> 64

<212> DNA

<213> Unknown

<220>

<223> Bioreactor metagenome PBDCA2_FISUTAU01BA9VK, whole genome shotgun sequence, AGTN01403367.1

<400> 221

cgcagagtga cgggagggtt tatcggccct cccggtaatg cggcagcccg gttcgcaagc 60

ccgg 64

<210> 222

<211> 55

<212> DNA

<213> Unknown

<220>

<223> Bioreactor metagenome PBDCA2_contig37489, whole genome shotgun sequence, AGTN01271243.1

<400> 222

cgcagagtga gccgggaaac cggcttaatg cgggcagagg cggtcacaac cccgc 55

<210> 223

<211> 219

<212> DNA

<213> Unknown

<220>

<223> Naegleria sp. NG872 SSU rRNA gene group I intron, NG872 strain, AJ001399.1

<400> 223

ctgttatattgg aatttgatag ttgtgcgatg gggttcatac cttaactgcc aaaacgggac 60

cccttttggg gtataaatct tgtaaaagga ttatattccg tactaaggat atttgataat 120

atccggaatg tctagagact acacggcaag ccaattggtg gtatgaatgg atagtcccta 180

gtttttttta ccatctaggt atcccataca aaatggtaa 219

<210> 224

<211> 196

<212> DNA

<213> Didymium iridis

<220>

<223> Partial IGS, 18S rRNA gene, I-DirI gene and partial ITS1 of Didymium iridis, Pan2-16 isolate, AJ938153.1

<400> 224

ttttggttgg gttgggaagt atcatggcta atcaccatga tgcaatcggg ttgaacactt 60

aattgggtta aaacggtggg ggacgatccc gtaacatccg tcctaacggc gacagactgc 120

acggccctgc ctcttaggtg tgttcaatga acagtcgttc cgaaaggaag catccggtat 180

cccaagacaa tcaaat 196

<210> 225

<211> 200

<212> DNA

<213> Unknown

<220>

<223> Naegleria sp. NG458 group I-like ribozyme GIR1, NG458 strain, AM497931.1

<400> 225

ctgttattga aggacgttct agagtgcgat ggggttcata cctttatctg ccaaaacggg 60

acctctgttg aggtatatat tgaatattcc gtactaagga tttaatccgg aacgtctaga 120

gactacacgg cagaccattg ttggtggtat gaatggatag tccctagtga accatctagg 180

catcccatac aaaatggtta 200

<210> 226

<211> 209

<212> DNA

<213> Unknown

<220>

<223> Heterolobosea sp. BA 16S small subunit ribosomal RNA gene, partial sequence; and His-Cys box homing endonuclease gene, complete cds, DQ388519.1

<400> 226

cagctgtttt gatacatgct cgactttctt tttctcttgt gcaatggggt ttatgagtta 60

attagccaaa acgggacctt aaaaaggtgt aagtaaccgt actaagttcg taagaacgga 120

atgtctagag actacacggc tgagcgattt agctctcata aatggatagt cctcagtata 180

ccatctgagc atcccataca aaatggtta 209

<210> 227

<211> 76

<212> DNA

<213> Eubacterium ruminantium

<220>

<223> Eubacterium ruminantium ATCC 17233 genome assembly, contigs: EI46DRAFT_scaffold00014.14, FUXA01000016.1

<400> 227

agtcgtcaga gcgactataa ataggcttta ggctctgagc gtgccgaccg tcaataaaag 60

gcggtcagcg gtagca 76

<210> 228

<211> 71

<212> DNA

<213> Anopheles gambiae

<220>

<223> Anopheles gambiae strain PEST whole genome shotgun sequencing project, whole genome shotgun sequence, AAAB01006002.1

<400> 228

actcgactaa gcgagtataa aaaggtttca agcttagagc gttgataggg ataaaaacct 60

atcaggtaac a 71

<210> 229

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Tsukamurella phage TIN3, complete genome, KR011063.1

<400> 229

cctcgtcagg gcgaggttaa atagccgcat aggccctgag cgtccccgcc ccacaagggc 60

gggggacgg g 71

<210> 230

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Paenibacillus sp. TCA20 DNA, contig: PspTCA2nb10, BBIW01000010.1

<400> 230

agtcggcttg gcgactataa ataggctttt ggccaagcgc gggctcccaa ctcggggagta 60

tagca 65

<210> 231

<211> 69

<212> DNA

<213> Paenibacillus naphthalenovorans

<220>

<223> Paenibacillus naphthalenovorans strain 32O-Y, complete genome, CP013652.1

<400> 231

actcgtgcca gcgagtttaa atagaccaat aggctggcag cgttccactc ataaagagtg 60

gaggaggta 69

<210> 232

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Ruminococcus sp. SR1 5 draft genome, FP929053.1

<400> 232

agtggtcaca gccactataa acagggcttt aagctgtgag cgttgaccgt cacaacggcg 60

gtcaggtagt c 71

<210> 233

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. ASF502 genome scaffold acMal-supercont1.1, whole genome shotgun sequence, KB822441.1

<400> 233

agtagtcatg gctactataa atagagactt aagccatgag cgttcccatc tttgtgatgg 60

gaggtgtct 69

<210> 234

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Gordonia phage GTE7, complete genome, JN035618.1

<400> 234

cgtcgtctga gcgacgttaa atagccgtta ggctcagagc ggtacacctc ccctattctc 60

gggttggg 69

<210> 235

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Cellulophaga phage phi19:3, complete genome, KC821608.1

<400> 235

agccgttgca gcggcataaa ataggttatt aggctgcaag cgttcgccct taattgggcg 60

gtgtta 66

<210> 236

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Sphingobacterium sp. ML3W, complete genome, CP009278.1

<400> 236

agtcgtttga gcgacttaaa ataggtttta agctcaaagc gccccgataa taatcggggag 60

taaca 65

<210> 237

<211> 70

<212> DNA

<213> Unknown

<220>

<223> Blautia sp. YL58, complete genome, CP015405.2

<400> 237

agaggttgca acctctataa atagggcttt aagttgcaag cgttcccgct ggaaacagtg 60

ggagatagcc 70

<210> 238

<211> 66

<212> DNA

<213> Lachnospiraceae

<220>

<223> Lachnospiraceae A2 genome scaffold acPFL-supercont1.1, whole genome shotgun sequence, KE159636.1

<400> 238

agccgtccca acggctctaa aaagtccatt aagttgggag cgtccggcag aaatgccggg 60

gttgga 66

<210> 239

<211> 75

<212> DNA

<213> Clostridiales bacteria

<220>

<223> Clostridiales bacteria 42_27 Ley3_66761_scaffold_13135, whole genome shotgun sequence, MNRF01000152.1

<400> 239

gctcgtctgg gcgagggtaa atagtaatta ggcccaggc gtcttggctg gcagatctgc 60

cggtcggggg tttag 75

<210> 240

<211> 64

<212> DNA

<213> Unknown

<220>

<223> Brevibacillus phage Jenst, complete genome, KT151955.1

<400> 240

tagtgttgcg gcacttacaa gcccattaag ccgcaagcgt tagcccttcc ggggctaggt 60

tggg 64

<210> 241

<211> 74

<212> DNA

<213> Unknown

<220>

<223> Gordonia phage Orchid, complete genome, KU998253.1

<400> 241

acacgactgg acgtgtataa ataggcgtta ggtccagtgc gggtgatggt attgagtatt 60

ttggaatcgg tgcc 74

<210> 242

<211> 64

<212> DNA

<213> Soybean fermentation Bacillus glycinifermentans

<220>

<223> Bacillus glycinifermentans GO-13 strain contig_36, whole genome shotgun sequence, LECW02000030.1

<400> 242

agtcgtggcg gcaacattaa acaggcatta agccgccagc attcccctta ttggggaggt 60

tgca 64

<210> 243

<211> 69

<212> DNA

<213> Soybean fermentation Bacillus glycinifermentans

<220>

<223> Bacillus glycinifermentans GO-13 strain contig_9, whole genome shotgun sequence, LECW02000082.1

<400> 243

ggacgtgacg gcggctcaaa aaagtgcatt aagccgcaag agtttccccg tttttggggg 60

aaggtttca 69

<210> 244

<211> 80

<212> DNA

<213> Ethanoligenens harbinense

<220>

<223> Ethanoligenens harbinense YUAN-3, complete genome, CP002400.1

<400> 244

caccgtggcg gcggtgtaaa acaaacatta agccgccagc gtcccggaac aaggcatttt 60

ccgattctcc gggggttgca 80

<210> 245

<211> 70

<212> DNA

<213> Clostridiales bacteria

<220>

<223> Clostridiales bacteria 44_9 Ley3_66761_scaffold_7759, whole genome shotgun sequence, MNRG01000094.1

<400> 245

gctcgtctgg gcgaggataa acagctatta agcccaggc gttctgagtc tttaagattc 60

ggaggtttag 70

<210> 246

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Bacillus phage B4, complete genome, JN790865.1

<400> 246

agtcgtgtga gcgactataa acaggcttta ggctcacagc gtcgcggggt ttatcccccg 60

tgggtagca 69

<210> 247

<211> 70

<212> DNA

<213> Unknown

<220>

<223> Sphingobacterium sp. ML3W, complete genome, CP009278.1

<400> 247

agtggattgc gccactttaa aaaggtttta agcgtaaagc gttgcaaggt tttgagcctt 60

gcaggtaaca 70

<210> 248

<211> 64

<212> DNA

<213> Soybean fermentation Bacillus glycinifermentans

<220>

<223> Bacillus glycinifermentans GO-13 strain contig_3, whole genome shotgun sequence, LECW02000023.1

<400> 248

actcgtcaca gcgagtataa agaggcatta ggctgtgagc gttccccgtc atggggaggt 60

tgca 64

<210> 249

<211> 67

<212> DNA

<213> Unknown

<220>

<223> Draft genome of Clostridiales sp. SS3 4, FP929062.1

<400> 249

acacgttgcg ccgtgtataa atagccagtt agggcgcaag cgtcccggca ttttgccggg 60

ggtctgg 67

<210> 250

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Genome assembly of Alistipes sp. isolate CHKCI003, CHKC3, FCNT01000042.1

<400> 250

agccgttcgg gtggctataa atagacctta ggcccgaagc gtggcggcac ctgccgccgg 60

tggta 65

<210> 251

<211> 78

<212> DNA

<213> Streptococcus sobrinus

<220>

<223> Streptococcus sobrinus TCI-98 contig 00583, whole genome shotgun sequence, AGGO01000583.1

<400> 251

agtcgttgtg gcgactataa ccaagctctt taagccacaa gcgttgctga tgaggtttca 60

taacatcagc aggtagag 78

<210> 252

<211> 67

<212> DNA

<213> Paenibacillus elgii

<220>

<223> Paenibacillus elgii B69 contig 93, whole genome shotgun sequence, AFHW01000093.1

<400> 252

actggttcga gccagtaaaa aaaggccgat aagctcgaag cgttccactc ttagagtgga 60

ggaggca 67

<210> 253

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 35801239, whole genome shotgun sequence, ABLZ01250225.1

<400> 253

agtcgttagg gcgactataa acagacatta agccctaagc gtcccctact agctaggggg 60

gttgta 66

<210> 254

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome ctg_1101668203871, whole genome shotgun sequence, AACY023396520.1

<400> 254

agtcggtaga gcgactttaa aaaggcatta ggctctacgc gttccaggag gaaactcctg 60

gaggttgtt 69

<210> 255

<211> 64

<212> DNA

<213> Bacteria of the family Erysipelotrichaceae

<220>

<223> Erysipelotrichaceae bacteria 2_2_44A cont1.7, whole genome shotgun sequence, ADCZ01000007.1

<400> 255

attcgactag acgagtataa ataggtgtca ggtctagtgc ggcagggttc ttccctgcat 60

cata 64

<210> 256

<211> 69

<212> DNA

<213> Bacteria of the family Erysipelotrichaceae

<220>

<223> Erysipelotrichaceae bacteria 2_2_44A cont1.7, whole genome shotgun sequence, ADCZ01000007.1

<400> 256

aatcgactag gcgattttaa ataggtgtta agcctagtgc ggtaagaggt ataaccctct 60

tgcgtcacg 69

<210> 257

<211> 70

<212> DNA

<213> Unknown

<220>

<223> Microbial mat metagenome hsmat10_BHWZ5893_b1, whole genome shotgun sequence, ABPY01006745.1

<400> 257

gctggtcacg gccagtataa acagacatta agccgtgagc gtctcctgtt ctgtgaacgg 60

gagggttgta 70

<210> 258

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Activated sludge metagenome contig01440, whole genome shotgun sequence, AERA01001428.1

<400> 258

actcgttagg gcgagtataa atagccatta ggccctaagc gtcaatgata agctcattgg 60

gttgga 66

<210> 259

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 1096626606346, whole genome shotgun sequence, AACY020454254.1

<400> 259

agtcgtttgg gcgactataa acagacgaat aagcccaaag cgtttcctcg taagaggaag 60

gacgga 66

<210> 260

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Coral metagenome 39763165, whole genome shotgun sequence, ABNK01016853.1

<400> 260

agtcgtctga gcgactataa acagagtttt aggctcagag cgcctcccct tcgggggagg 60

gtacta 66

<210> 261

<211> 79

<212> DNA

<213> Colombian leafcutter ant (Atta colombica)

<220>

<223> Colombian leafcutter ant (Atta colombica) fungus garden Top 2030450980, whole genome shotgun sequence, AGFS01138167.1

<400> 261

actcgactag acgagtataa actacattaa gcctagtgcg ttatagccgt aaataagaag 60

taaacggcta taggttgta 79

<210> 262

<211> 70

<212> DNA

<213> Paenibacillus polymyxa

<220>

<223> Paenibacillus polymyxa E681, complete genome, CP000154.1

<400> 262

gttcgtctga gcgaacgcaa acaggccatt aagctcagag cgttcactgg attcgtccag 60

tgagattggc 70

<210> 263

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome ctg_1101667068628, whole genome shotgun sequence, AACY022661277.1

<400> 263

actggactac gccagtataa ataggcatta agcgtagtgc gttccaatgt tgtgaaacat 60

cggaggttgt t 71

<210> 264

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 1096626660187, whole genome shotgun sequence, AACY020496190.1

<400> 264

agtcgtctaa gcgactctaa aaaggcttta agcttagagc gttcgcccat attgggcgag 60

gttgta 66

<210> 265

<211> 73

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 1096626606768, whole genome shotgun sequence, AACY020454584.1

<400> 265

actggttgcg gccagtataa atagtcttta agccgcaagc gtgtcctgga gttaatcttc 60

cagggcggta gca 73

<210> 266

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome ctg_1101667160699, whole genome shotgun sequence, AACY022753348.1

<400> 266

agtcgactaa gcgactctaa acagcattta ggcttagtgc gttcccctgc tcacgcgggg 60

gaggtatgg 69

<210> 267

<211> 78

<212> DNA

<213> Lysinibacillus sphaericus

<220>

<223> Lysinibacillus sphaericus C3-41, complete genome, CP000817.1

<400> 267

actcgactaa gcgagtataa acaggcatta ggcttagagc gttctcacgt tatctgaatg 60

atgatgtgag aggttgca 78

<210> 268

<211> 55

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 32650920, whole genome shotgun sequence, ABLX01143204.1

<400> 268

actcgacagg gcgaggctaa atagcattta ggccctgagc ggctcccttc gggag 55

<210> 269

<211> 58

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 2065701, whole genome shotgun sequence, AACY021048934.1

<400> 269

gctcggtgcg gcgagcctaa atagtgcctt aggccgcacg cgttatgcat aggtggca 58

<210> 270

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Microbial mat metagenome hsmat10_BHWZ5893_b1, whole genome shotgun sequence, ABPY01006745.1

<400> 270

acaggtttgc gcctgtataa atagacatta agcgcaaagc gtcccgcaat tgttgcgggg 60

gttgta 66

<210> 271

<211> 67

<212> DNA

<213> Tolumonas auensis

<220>

<223> Tolumonas auensis DSM 9187, complete genome, CP001616.1

<400> 271

aagcgaaaca ggccccggag ggcctgtctg ccggaggtgg tgctccggta ctgatgagca 60

gcctagc 67

<210> 272

<211> 69

<212> DNA

<213> Mycobacterium vanbaalenii

<220>

<223> Mycobacterium vanbaalenii PYR-1, complete genome, CP000511.1

<400> 272

tgccgaaacg ccgactcggg tcggcgtccc tgggaggtgg cattctcagg ctgatgatgg 60

ctgccgcag 69

<210> 273

<211> 77

<212> DNA

<213> Unknown

<220>

<223> Shewanella oneidensis MR-1, complete genome, AE014299.2

<400> 273

aagcgaaaca agcaaggcgc ttaggtgcct tgcctgtctg ctcggcgtgg ttgccgagca 60

ctgatgagca gccaaag 77

<210> 274

<211> 87

<212> DNA

<213> Desulfobacteraceae bacteria

<220>

<223> Desulfobacteraceae bacteria 4572_35.1 ex4572_35.1_scaffold_634, whole genome shotgun sequence, NBLX01000010.1

<400> 274

atgcgaaacc gcgatcattt tgccgccatt ggcaaggtga tcgcggtcat cagggtgcgg 60

cgatcctgat ctgatgagca gccaaga 87

<210> 275

<211> 70

<212> DNA

<213> Desulfovibrio alkalitolerans

<220>

<223> Desulfovibrio alkalitolerans DSM 16529 ctg12, whole genome shotgun sequence, ATHI01000003.1

<400> 275

aagcgaaacc gccctgagtg ggcggtcgtt ccggagagac ggcgaccggg gcctgatgag 60

ccagccgaat 70

<210> 276

<211> 77

<212> DNA

<213> Unknown

<220>

<223> Streptomyces phage R4, complete genome, JX182370.1

<400> 276

atgcgaaaca tctcgccggc tggaccggtg aggtgtcggc ccagggcggt tcctgggtcc 60

tgacgatgca accggga 77

<210> 277

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Thermoplasmatales archaeon SG8-52-4 WOR_8-12_1532, whole genome shotgun sequence, LSSF01000016.1

<400> 277

agccgaaaca ggggtctgtg cgcccctgtc caccatgggt ggtgccatgg tgccgatgat 60

ggtagccacaa 71

<210> 278

<211> 70

<212> DNA

<213> Unknown

<220>

<223> Paenibacillus sp. MSt1 Contig_22, whole genome shotgun sequence, JNVM01000022.1

<400> 278

agccgaaacg cctcgcgata ggaggcgtcg cggggatatg gcctaccccg cctgatgatg 60

gcaggccgga 70

<210> 279

<211> 74

<212> DNA

<213> Neptunomonas antarctica

<220>

<223> Genome assembly of Neptunomonas antarctica strain DSM 22306, contig: Ga0111702_106, FTOE01000006.1

<400> 279

ccgcgaaacg cccacacctt aacgggacgg gcgtctatcc agcgtggcaa ctgggtactg 60

atgagcagcc acta 74

<210> 280

<211> 67

<212> DNA

<213> Ethanoligenens harbinense

<220>

<223> Ethanoligenens harbinense YUAN-3, complete genome, CP002400.1

<400> 280

agccgaaacg gggtgaaagc cctgtccgct ggggatggcc tcctcgcgct gatgatggca 60

ggccaac 67

<210> 281

<211> 84

<212> DNA

<213> Unknown

<220>

<223> Rhodobacterales bacteria 65-51 scnpilot_p_inoc_scaffold_125, whole genome shotgun sequence, MKWD01000005.1

<400> 281

atgcgaaacc gcatccgggg cggcgtgtgc cccgggtgcc ggtcggccgg gcgtggtggc 60

ccggtcctga tgatgcagcc ggag 84

<210> 282

<211> 68

<212> DNA

<213> Unknown

<220>

<223> Pirellula sp. SH-Sr6A, complete genome, CP011272.1

<400> 282

agccgaaacg cggtagcgat ccgcgtcgcc gatcggtggt tcgatcggcc tgacgatggc 60

agccaacc 68

<210> 283

<211> 74

<212> DNA

<213> Unknown

<220>

<223> Devosia sp. 66-22 SCNpilot_expt_1000_bf_scaffold_212, whole genome shotgun sequence, MKUZ01000009.1

<400> 283

ttgcgaaacg cctcccggct ccggctgggg gcgtcgtcca cgggtcgcgc cgtgggcctg 60

atgagcagcg acac 74

<210> 284

<211> 77

<212> DNA

<213> Unknown

<220>

<223> Genome assembly of Verrucomicrobiaceae bacterium GAS474, LT629781.1

<400> 284

tgccgaaacg gcttcctcgt gccccgaggt gccgtcctgc cgggctgagc tcccagcagc 60

tgatgaggca gctccct 77

<210> 285

<211> 68

<212> DNA

<213> Unknown

<220>

<223> Saccharothrix sp. ALI-22-I Contig71, whole genome shotgun sequence, MTQP01000067.1

<400> 285

cggcgaaacc gcctccccgg aggcggtcca cgggattggc attcccgtgc tgaggatgcc 60

tgccgagc 68

<210> 286

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Marinomonas sp. S3726 contig0030, whole genome shotgun sequence, JXYC01000030.1

<400> 286

tagcgaagcg cggctaggta tagccgcgtc aatctcgtgt agtggctaga tactgatgag 60

cagctaaaa 69

<210> 287

<211> 75

<212> DNA

<213> Unknown

<220>

<223> Ruegeria sp. ANG-R contig_12, whole genome shotgun sequence, JWLJ01000012.1

<400> 287

atgcgaaacc gtcccggtgt tcacgccggg atggtcatcg gggcgtggtg accccggtct 60

gatgagcagc cagaa 75

<210> 288

<211> 67

<212> DNA

<213> Unknown

<220>

<223> Beggiatoa sp. IS2 Ga0073106_1108, whole genome shotgun sequence, MTEL01000108.1

<400> 288

aaccgaaact cccctcacgg ggagtccgac cgggattaat cacccggcgc tgatgaggca 60

gattcct 67

<210> 289

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Streptomyces phage R4, complete genome, JX182370.1

<400> 289

tgccgaaaca cccttcgggg tgtcggggtg gggtggcgct cacctcctga cgatggcagc 60

cacga 65

<210> 290

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Lachnoclostridium sp. An76 An76_contig_9, whole genome shotgun sequence, NFHL01000009.1

<400> 290

agccgaaacg gtcagtaatg accgtcagcc gggaagtgac tgccccggct ctgatgatgg 60

caggtcatg 69

<210> 291

<211> 66

<212> DNA

<213> Herbaspirillum seropedicae

<220>

<223> Herbaspirillum seropedicae SmR1, complete genome, CP002039.1

<400> 291

agccgaaaca tcctcaaagg gtgtctctca gaggtggcct cctgagactg atgatggctg 60

gctgtg 66

<210> 292

<211> 71

<212> DNA

<213> Moritella viscosa

<220>

<223> Moritella viscosa genome assembly, LN554852.1

<400> 292

aagcgaaaca cgtcttagtg ataagtcgtg tctactcagc gttgtggttg agtactgatg 60

agcagcaact t 71

<210> 293

<211> 67

<212> DNA

<213> Fervidicella metallireducens

<220>

<223> Fervidicella metallireducens AeB contig 00024, whole genome shotgun sequence, LN554852.1

<400> 293

aaccgaaaca agggtatgtc ccttgtctgc tgaggataac ctctcagcac tgatgaggta 60

ggttaaa 67

<210> 294

<211> 67

<212> DNA

<213> Unknown

<220>

<223> Saccharothrix sp. ALI-22-I Contig71, whole genome shotgun sequence, MTQP01000067.1

<400> 294

cggcgaaacc gtccggtgtg gacggtcccg agggctggca tccctcggct gatgatgcct 60

gccaaga 67

<210> 295

<211> 61

<212> DNA

<213> Unknown

<220>

<223> Streptomyces phage R4, complete genome, JX182370.1

<400> 295

aggcgaaacg ccgtgaggcg tccggccggg tggtacccgg tcgctgatga gccagcctgc 60

t 61

<210> 296

<211> 67

<212> DNA

<213> Ethanoligenens harbinense

<220>

<223> Ethanoligenens harbinense YUAN-3, complete genome, CP002400.1

<400> 296

agccgaaacg ggactttggt cctgtctgcc gggaatggcc gcccggcact gaggatggca 60

ggctgct 67

<210> 297

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Oscillibacter sp. KLE 1745 genome scaffold 306, whole genome shotgun sequence, KI271721.1

<400> 297

agccgaaacg ccctccgggg cgtcatcggg gggagccctc ccccggtctg aagatggcag 60

ggcacg 66

<210> 298

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Subdoligranulum sp. 4_3_54A2FAA genome scaffold supercont1.5, whole genome shotgun sequence, JH414702.1

<400> 298

agccgaaaca gccctgcggg gctgtcgtgc gggggctgac cgccccgtgc ctgatgatgg 60

caggtcaag 69

<210> 299

<211> 69

<212> DNA

<213> Acinetobacter baumannii

<220>

<223> Acinetobacter baumannii strain SDF, complete genome, CU468230.2

<400> 299

aagcgaaaca caggcattcg tgcctgtgtc tactggatgt cgtgatccag tactgatgag 60

cagcgatag 69

<210> 300

<211> 66

<212> DNA

<213> Streptomyces hygroscopicus

<220>

<223> Streptomyces hygroscopicus subsp.jinggangensis 5008, complete genome, CP003275.1

<400> 300

tgccgaaacc ccttggtgag gggtcgttcc ggggtggtgc ccggagcctg acgacggcag 60

ccgccc 66

<210> 301

<211> 70

<212> DNA

<213> Ruminococcus callidus

<220>

<223> Ruminococcus callidus ATCC 27760 genome scaffold 724, whole genome shotgun sequence, KI260480.1

<400> 301

agccgaaaca gcggcagaga gccgctgtct gccggaactg gtctaccggc actgatgatg 60

gcagaccgga 70

<210> 302

<211> 65

<212> DNA

<213> Unknown

<220>

<223> Saccharothrix sp. ALI-22-I Contig71, whole genome shotgun sequence, MTQP01000067.1

<400> 302

aggcgaaacc cggctggcac cgggtccgta gggctggcat ccctgcgctg atgagcctgc 60

caacg 65

<210> 303

<211> 67

<212> DNA

<213> Unknown

<220>

<223> Blautia sp. An249 An249_contig_12, whole genome shotgun sequence, NFJL01000012.1

<400> 303

agccgaaacg gggaacttac cccgtccgct gcgggatcgc ctcccggcgc tgatgaggca 60

ggcgaga 67

<210> 304

<211> 78

<212> DNA

<213> Unknown

<220>

<223> Rhodovulum sp. P5, complete genome, CP015039.1

<400> 304

ccgcgaaacc ccgccaggcc catcggtctg gcggcggtcg gccgggcgtg gtggcccgac 60

cctgatgagc agccggag 78

<210> 305

<211> 81

<212> DNA

<213> Unknown

<220>

<223> Geobacteraceae bacteria GWC2_58_44 gwc2_scaffold_235, whole genome shotgun sequence, MGZL01000059.1

<400> 305

atgcgaaacg atcattttgc cggcgtcgac aaaatgatcg tcatcccggc gtggcggccg 60

gggtctgatg agcagccgcg g 81

<210> 306

<211> 68

<212> DNA

<213> Streptomyces yokosukanensis

<220>

<223> Streptomyces yokosukanensis DSM 40224 strain genome scaffold PRJNA299221_s003, whole genome shotgun sequence, KQ948208.1

<400> 306

cggcgaaacc cgctggtgag gcgggtcgcg aagcggtggt gcgcttcgcc tgatgatgcc 60

agccagca 68

<210> 307

<211> 84

<212> DNA

<213> Unknown

<220>

<223> Endozoicomonas sp. (Bugula neritina AB1) isolate AB1-5 ACH42_contig000207, whole genome shotgun sequence, MDLD01000207.1

<400> 307

ttgcgaaaca ctcccgccgt acctgtcccc acaggtggga gtgtcagtcc agtgtggtga 60

ctgggctctg atgagcagcc aaag 84

<210> 308

<211> 68

<212> DNA

<213> Chloroflexi bacteria

<220>

<223> Chloroflexi bacteria RBG_13_60_13 RBG_13_scaffold_3543, whole genome shotgun sequence, MGNC01000101.1

<400> 308

agccgaaacg ggggcatcgg cccccgtcgt cccgggcagt ccactgggac ctgacgaggc 60

aaagcgcg 68

<210> 309

<211> 72

<212> DNA

<213> Unknown

<220>

<223> Shewanella oneidensis MR-1, complete genome, AE014299.2

<400> 309

aagcgaaacc cgccccattc atggggcgcg gtctgtctaa tgtagtgatt aggcactgat 60

gagcagctaacc 72

<210> 310

<211> 66

<212> DNA

<213> Unknown

<220>

<223> Streptomyces phage R4, complete genome, JX182370.1

<400> 310

aggcgaaacc acccgagagg gtggtcggac cgggcggttc ccggttcctg acgatgccaa 60

ccactg 66

<210> 311

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Desulfofustis glycolicus DSM 9705 genome assembly, contigs: EJ46DRAFT_scaffold00001.1, FQXS01000001.1

<400> 311

aggcgaaacg ccggggtgac ccggcgtcgt cggagggtga tgcctccggc ctgacgatgc 60

cagttacag 69

<210> 312

<211> 68

<212> DNA

<213> Unknown

<220>

<223> Desulfovibrio sp. TomC contig00038, whole genome shotgun sequence, JSEH01000038.1

<400> 312

aggcgaaacc gttctcctcg gagcggtcgg ccgggtgtgg tggcccggcc ctgatgagcc 60

agccgctc 68

<210> 313

<211> 76

<212> DNA

<213> Desulfuromonas soudanensis

<220>

<223> Desulfuromonas soudanensis WTL strain chromosome, complete genome, CP010802.1

<400> 313

aagcgaaacg accaccccccc caggggggta gtcgtcgctc gggggtggtg ccccgggcct 60

gatgatgcag ccaagt 76

<210> 314

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Marinomonas sp. S3726 contig0020, whole genome shotgun sequence, JXYC01000020.1

<400> 314

aagcgaaaca tggctcgttg tagccgtgtc tattcagcgt agtggctggg tactgatgag 60

cagctaaaa 69

<210> 315

<211> 57

<212> DNA

<213> Faecalibacterium prausnitzii

<220>

<223> Faecalibacterium prausnitzii L2 6 draft genome, FP929045.1

<400> 315

gcggacactt tcaagggctg caccgctgcc gcaaaagcaa ccctatgcca ccgcccc 57

<210> 316

<211> 54

<212> DNA

<213> Faecalibacterium prausnitzii

<220>

<223> Faecalibacterium prausnitzii L2 6 draft genome, FP929045.1

<400> 316

acggatgcct tgacgggccg caccgaacga aaagcgaccc gatcccacac cccg 54

<210> 317

<211> 53

<212> DNA

<213> Faecalibacterium prausnitzii

<220>

<223> Faecalibacterium prausnitzii L2 6 draft genome, FP929045.1

<400> 317

acggatactc tagccgggtt gcaccgttca aagcagccca gccccagccg caa 53

<210> 318

<211> 54

<212> DNA

<213> Faecalibacterium prausnitzii

<220>

<223> Faecalibacterium prausnitzii SL3 3 draft genome, FP929046.1

<400> 318

tgggataccc tagcagggcc gcaccccaga aaagcggccc cgccccacac ccgg 54

<210> 319

<211> 53

<212> DNA

<213> Unknown

<220>

<223> Uncultured Faecalibacterium sp. TS29_contig14193, whole genome shotgun sequence, ADJT01006171.1

<400> 319

ccggatattt tggcagggct gcaccgggca aagcaacccc gccccactac ccc 53

<210> 320

<211> 53

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: In-D_005494., BABD01005494.1

<400> 320

gcggacacct cagcagggcc gcaccggaca aagcggcccc gccccaccgc cca 53

<210> 321

<211> 57

<212> DNA

<213> Unknown

<220>

<223> Uncultured Faecalibacterium sp. TS29_contig122416, whole genome shotgun sequence, ADJT01006524.1

<400> 321

gcggatgccc tggcagggtc gcaccgctca aacaaagcgg ccccgcccca taacccc 57

<210> 322

<211> 52

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F1-S_028045, BAAU01028045.1

<400> 322

tcggacactc tggcagggca agcaccgtat agcagccccg accaactacc cc 52

<210> 323

<211> 54

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: In-R_005008, BABG01005008.1

<400> 323

ccggaagccc tggcagggtg cgcaccggat aaagcggccc tacctcaccg gcac 54

<210> 324

<211> 247

<212> DNA

<213> Unknown

<220>

<223> Parasitella parasitica CBS 412.66 genome assembly, contig: contig_63, CCXP01000063.1

<400> 324

aaaagcacct cttaaatagt gatccgtaaa atgaggttca tataaaattt ttcactatat 60

gctggaaaat cttaaagctt taagtacctc aatggtaaca atcttaaaga tattacaata 120

gacaatcagc aggaaaccaa cataattcta ttatttttag taggatcctc agagactaca 180

cgtgaaacac cgtattttta ttaagaatac gctgaagata tagtccgccc cacttcgaaa 240

gatgtgg 247

<210> 325

<211> 262

<212> DNA

<213> Unknown

<220>

<223> Candidatus Taylorbacteria bacterium RIFCSPLOWO2_12_FULL_43_20 rifcsplowo2_12_scaffold_4872, whole genome shotgun sequence, MHSK01000028.1

<400> 325

ctgttatagt tctgttaatg caataaaatg taaaaacatt ttgataaact aaataaacaa 60

taatcagtac actattcaaa gctgcctcgc tctgtagtaa tacaaagcag gtccgcacca 120

tggctatatg cggggaagtc tgtaatttgc agatcatccg cagggaagtt ctaaaatttt 180

ttttagaacc cctcagagac cacacgccat gctccagctg gtttgtacca gctggatgaa 240

gatatggtcc ttcgttaaag ag 262

<210> 326

<211> 428

<212> DNA

<213> Unknown

<220>

<223> Yanofskybacteria, tentative species RIFCSPHIGHO2_02_FULL_43_15c rifcsphigho2_02_scaffold_6549, whole genome shotgun sequence, MGJT01000029.1

<400> 326

caaggcggct tgttatacttgc cgcaggggcc attgagaagc aattctcaat agcaaattcg 60

actatatgct ggaaactccg ccagtatctc taggtactat gatattatga tatatcatag 120

tgaaaatcct aagagtgatg cggacaatca gcaggcaacc ccgctcaaat ttttggaaat 180

aaaaaagtgg atttctggga ttcaggatcc ggcctcgatt agaggttgcc atcctttcgg 240

aaggatgccc tgaaatgctt cccacgcaaa atccactttc atgctagtat atcaaataaa 300

taatgcactt gtcaagtgtt tgtttctaaa aatttgagcg ggagagtcct caacgactga 360

aagtcgaacc agttatattt caaaaaaatg actggatgat acagtctgaa cttataggcg 420

actataag 428

<210> 327

<211> 501

<212> DNA

<213> Trichoplax adhaerens

<220>

<223> Mitochondria of Trichoplax adhaerens Grell Red Sea isolate, complete genome, DQ112541.1

<400> 327

aagattaaat aatataagtttttgactttt acctccggca ctttttttac tattaggttc 60

ctctttagta gaacaaggag cgggtacagg gtggacggtt gaaaggccgc ccgagttagt 120

gatgacttgg tgaaaatttt gctcaatgcg agaacatcct caaaaaaaag gtgctttggc 180

tcattgatta accctaaaaa ggtacctttt gatggcccca tgcaaaaatc cttttttacg 240

ccgaaggcgc cgtggacaac tcgccggggg cccaagccta tgggcccctc agagactaaa 300

tgcagaatat cttctatttt ttgataggcg ccgggcccct taacgggcgc cgaaggcgcc 360

caatgggagc caacgaccga tggcgccata ggcgccgaag gcgccgatag aaataaaggg 420

cccgaagcga ccgattcacc aatcggtcgc ttcggccgat ggaagataaa ggaatagtcc 480

gatccgactc taaagggtcg g 501

<210> 328

<211> 307

<212> DNA

<213> Galerina marginata

<220>

<223> Galerina marginata CBS 339.88 GALMAscaffold_102_Cont1090, whole genome shotgun sequence, AYUM01001090.1

<400> 328

ttgcctgggt tttcttaatt gaattcccga atttaaatgc tagtccaagt taaaacttgg 60

gcaagacctt caaactgacg gggaactcct aaagcttcag acaccaagcc ttatccgaa 120

agggtagggt ggccaggtta atagcctcgg gtatggtaaa agatctgaag atattacaat 180

ggacaatccg cagccaaggc cctaacgaag tgtttcactt ctatgggaca ggttcagaga 240

ctagatggag gtcggtctca tgtaaatgag gcttaaggta tagtccggct tcaagtgaaa 300

acttgtt 307

<210> 329

<211> 215

<212> DNA

<213> Sclerotinia sclerotiorum

<220>

<223> Sclerotinia sclerotiorum 1980 UF-70 mitochondria, complete genome, KT283062.1

<400> 329

ttatattttt attactaaaa aaaaaaggga aaaaacagca aataaaaaaa cttcttctta 60

ctaattgctg gaaactcctg tttaatagga caatcagcag gagcctgctg tatatgttta 120

tacagtaggg ttcttcagag actacacgta agatatccta gaatcattaa ataaatagga 180

taaagatata gtccgctctt aatagaaata ttaag 215

<210> 330

<211> 564

<212> DNA

<213> Gossypium arboreum

<220>

<223> Gossypium arboreum cultivar AKA8401 contig_3227_1, whole genome shotgun sequence, JRRC01306379.1

<400> 330

ctacggactt aattggattg agccttggta tggaaaccta ctaagtgata actttcaaat 60

tcagagaaac cctggaatga aaaatgggca atcctgagcc aaatcctatt attttattat 120

tttacgaaaa taaacatgaa caaaggttca gcaagcgaga ataagaaaaa aaggaaagga 180

taggtgcaga gactcaatgg aagctattct aacaaatggg gttgactgtt ggtaaaggaa 240

tccttatatc gaatatcgaa actctagaaa ggatgcaaga tatacctatt ttttttatag 300

gtatactaat gaaaaactat ctcaaaaaag acgtaccgaa cccgtatttttttttttatt 360

tctattatt gcaatatcaa tttatattta tatgaaaata tgaaaaataa aaagaattgt 420

tgtgaatcga ttccaagttg aagaaagaat cgaatagaat agtcattaat caaatcattc 480

actccatagt ctgataaatc ttttgaaaaa ctgattaatc ggacgagaat aaagatagag 540

tcccgttcta catgtcaata tcaa 564

<210> 331

<211> 227

<212> DNA

<213> Unknown

<220>

<223> Acanthamoeba polyphaga mummi virus, complete genome, JX962719.1

<400> 331

attccttatt ggttcctaag tatatatcga aaggtatata tggtaatagt taatcactat 60

tagaggaaaa atatcaataa ggtcatagtc aatccgcagc aaagctccta aacccgttat 120

gctagggcat ggagaatgtt caacgactaa acggatgtgg gcatgaagga attagcactt 180

cctaatgatt gcttaagata tagtctaaac ccaccagtga tggtgtt 227

<210> 332

<211> 370

<212> DNA

<213> Unknown

<220>

<223> Bacillus koreensis DSM 16467 strain scaffold4, whole genome shotgun sequence, LILC01000037.1

<400> 332

ttcgtgacgt agattatgct ttagctgcgt aagcagtaac aagcacagtc gtcctagctg 60

gtaacggcta gagatcataa ttgggtgaat tgctggaaac cccttagagc tttcttcccc 120

acagcggagt tggaaacgac agacgcgatg ggtttaagaa gaagagagat tgggcaatca 180

gcagccgagc tcctgttccg aaaggatgga gaaggttcaa cgactaggat agaccatcta 240

aaagctaaag ctcaagatga tgaaatccat aggtgaagca gtagatcatc actactgtga 300

atccgaagtg cccaacccct accgaatacg gagggtgaag atatagtcta gtcatttatg 360

aaagtaaatg 370

<210> 333

<211> 282

<212> DNA

<213> Corchorus olitorius

<220>

<223> Corchorus olitorius cv. O-4 contig 18264, whole genome shotgun sequence, AWUE01018231.1

<400> 333

ccacggactt aattaaattg gattgagctt tggcatggta acctactaag tgataacttt 60

caaattcaga gaaatcctgg aatgaaaaat gggcaatcct gagtcaaatc ctattatttc 120

acgaaaataa acaaaggttc agcaggggag acatctttaa cagctgccaa tgaatctcca 180

atatatttgg taatttccta cttatagtag ttaaagaagc tgaataacaa gcattttaag 240

gtagaagagt gctgacctgt gaggttagtg gaggtcgtgt gg 282

<210> 334

<211> 214

<212> DNA

<213> Unknown

<220>

<223> Omnitrophica WOR_2 bacteria SM23_29 WORSMTZ_35813, whole genome shotgun sequence, LJUB01000113.1

<400> 334

agcgggctgt gctcaagcgc agcttccaca ggaaactgtg gttgacaaag caggagaatt 60

gctggaagcc cctctggggt aatcagcagc cgagcccgtc attattttgg cgggaaggtt 120

cagagactat gtacctgcct cccgaaacgc aatgtccgcc tatggcggaa agtcgtggga 180

gaagatatag tccaagtccg atagtaatat cggg 214

<210> 335

<211> 243

<212> DNA

<213> Rhizopus oryzae

<220>

<223> Rhizopus oryzae RA 99-880 supercont3.83 mitochondrial scaffold, whole genome shotgun sequence, GG669565.1

<400> 335

gttcggagat ttgtggagtt caccacgggt aggtaataag ccccctcatt attagatggg 60

gataatctca ctatatgccc gaaactccta aagcccaatt tacggaaacc gtgataataa 120

ttgggataat acaatggaca atgggcagga aacagaaaat ttattctggc tcctcagaga 180

ctacatgtga aacattcatt ttaatgaatg aagatatagt cccatccatg acgagattca 240

tgg 243

<210> 336

<211> 251

<212> DNA

<213> Urartu wheat (Triticum urartu)

<220>

<223> Triticum urartu cv. G1812 contig 97470, whole genome shotgun sequence, AOTI010097470.1

<400> 336

atttgaatac aatagtattg agcccaagta aaactggatg aattgcaggg aaaactaaaa 60

atgaatttag ttaatctgca gcgaagctat tatcggcacc ttattaactt atcagttaat 120

ttgtagataa tagaacgttc aacgactaat cggtgagctg tgctagcaat aatccggaca 180

cgagcgtcca gcagaaaata attaatattt attttctgat gaaatagtct gaacaatggt 240

gtgaatcatt g 251

<210> 337

<211> 825

<212> DNA

<213> Unknown

<220>

<223> Microbotryum lychnidis-dioicae p1A1Lamole unplaced genome scaffold supercont1.89, whole genome shotgun sequence, GL541731.1

<400> 337

attttaggat tcattgtttg gtcttggttg ggactccctc acagtgatgt gggggtaaga 60

aatttcgcta tttgctgaaa cagttttacg ttgttaggta tcaagatata gtaaaatcct 120

agcagcaata ctcaatcagc agggagccgt cactttatta atgcgggttc ttcagagact 180

atacgcgaga catcttggca taactttaca gccttctata cccttttaat ggttatatca 240

acactatgag tcagcttttt tctagttagc cttttttttt ggcctgtcgt ttttgctctc 300

ttttttagaa aaacagcccg ctcattattt ttgcttttat ttttcttttt ctttttactc 360

tctttttcta ccggaggagt aaaaacagca gcgcttattt tcactcggta gaaaataaag 420

gccaaataag cgcccttttt ctgtttttat aaagcgcctc gcccagccga gctgggcgct 480

tcattcgccc agccgagctg cttttcatca aagatgaagc gcccagctcg gctgggcgag 540

gcgcttagtttttatttttg ctttttttgt cctgcttttt gattaatcaa aaaagacaaa 600

aacagcaaaa agcacaaaaa aggaaaaggc agctcggctg ggcgaggaaa aagacaaaaa 660

aataaaaaaa aagggaaaga gcatttaaca ccagacggat tacagcagat tcgatccctt 720

tacaggcaaa ttaatgtctg taactaagaa tcagctaaga cagatcatct cggccataaa 780

gcgacaagat gaagatatag tccgaacttt actcgcgaga gtaag 825

<210> 338

<211> 269

<212> DNA

<213> Unknown

<220>

<223> Candidatus Liberibacter solanacearum CLso-ZC1, complete genome, CP002371.1

<400> 338

atatgtggtt tatgtttgta aacttcataa taggtaatga aaaaaaattg tggatgtggc 60

ggaataggta gacgcagcag acttaatgtt attgggtgcc catagagaaa tcgatggagt 120

agaactgctc aaattcgggg aaagcttttg caaagctaat cccgagccaa atcttgttat 180

tcaagagagg tgtagagact ggacgggcag cacctaaggc atttaaaacg ttatggtgaa 240

gggacagtcc agaccacaaa cactgcaaa 269

<210> 339

<211> 324

<212> DNA

<213> Tortispora caseinolytica

<220>

<223> Tortispora caseinolytica NRRL Y-17796 unplaced genome scaffold CANCAscaffold_5, whole genome shotgun sequence, KV453845.1

<400> 339

agcgggtcgt tttctgaaag gaaagcggcg ttgctgaaag ctaggttcta aaacgttggg 60

ccagtcgcgc tgaaaggcgc ggctagtcgt gcatatgcac ggcgacactg tcaaattgcg 120

gcgacaccct gagagcttca agtaccaagc tagcgtcgaa agacagctag tggccgagtt 180

agtaaccctg ggtacggtaa aaaccttgaa gattgggcga gcacgcagcc aagtcctacg 240

gcgcaagcta cggatgcagt tcacagacta aatggcagtg ggcgaaagct taagatatag 300

tcgggcctct ggcgaaagcc aggt 324

<210> 340

<211> 847

<212> DNA

<213> Verticillium longisporum

<220>

<223> Genome assembly of Verticillium longisporum VL1 isolate, contig: scaffold_246, CVQH01016224.1

<220>

<221> Features not yet classified

<222> (333)..(345)

<223> n is a, c, g, or t

<220>

<221> Features not yet classified

<222> (510)..(517)

<223> n is a, c, g, or t

<220>

<221> Features not yet classified

<222> (625)..(633)

<223> n is a, c, g, or t

<400> 340

aaatcggcgt catttgagac gaggactttc gggcccgaaa gggtgtccac caacgaggac 60

cgtagcacgg cttgtgtacc gtagtctcct cggaggcgac accctcaaat tgcgggaaac 120

tcctaaagct cacgctccaa agccgtctgt gaaagcagtt cggtggccag gttaattgcc 180

tcgggtattg gaacaacgcg tgagatgcaa caatggacaa tccgcagcca agcctctaag 240

tctcttgtga ctctgggtga acgtgcttca cccagtttgc tcaaggcggg aggactcaca 300

gatcgaaacc ggagtcacga cctctggtca tgnnnnnnnnn nnnnnctccg gtggttcggc 360

gtctcgattc tgctgagtcc tggttcgcgt cccagagcca aactgcctct ggcagcacct 420

agacggagac ttaagtgccg tagacggagg cttaagtccc caactgccta acaggcggtt 480

ggttctgatt caggaccagc ctgagtcacn nnnnnnncca gcctgagtca cgagagatat 540

ggggaaggtt cagagacttg acgggggtgg gtgaattcac tgctgctgca acaatataaa 600

tggggagaga tcctcttctt cttcnnnnnn nnntcttctt cttccaacaa ccaaaccaaa 660

ccacaactga acctcaaaca agacccacaa gctcttcaaa atgtcccatt cttctccctc 720

tcctgttctc gctaacggga gcgagtatgt cgtgagggat ggaggttcgt ctggtcaggc 780

ataaggaacg agaatgcagt ggcgtggttt gcttaagata aagtccgggc ttatgggaaa 840

ccatagg 847

<210> 341

<211> 365

<212> DNA

<213> Irregular arbuscular mycorrhizal fungi (Rhizophagus irregularis)

<220>

<223> Rhizophagus irregularis DAOM 181602, strain DAOM 197198, GLOINscaffold_4832_Cont4827, mitochondrial, whole genome shotgun sequence, AUPC01004827.1

<400> 341

ctatagtttt ataagccctg aagctataga tgtctatctg gctatatgct gggaaaccca 60

ctaactttct atttaagtta agaatatggt gaagtggaca atcagcaggt aaccctcctt 120

agcaaagtag ggaggctacc tcagagacta aacgccagag cctgcagtat gaattgcatt 180

ccctctgggc taaattggaa gggagtctgg gacactatct tgccgggtta atagaaggag 240

acggagctattattgtttcttctaaaataa ccttgttctttttgataaaa tcccggtaag 300

gtgagtcaaa gcatgctgtt caatctgcag gtaagatata gtccgatcca aatagtgatg 360

tttgg 365

<210> 342

<211> 311

<212> DNA

<213> Paludisphaera borealis

<220>

<223> Paludisphaera borealis PX4 strain, complete genome, CP019082.1

<400> 342

gcaggggact catcaaccaa aatggtggcg ccggagggcg accttcggat gcgaaccggg 60

tgaattgcgg gaaacctaaa cctctgtttt gaggcacggc gatccgcagc caagcctggc 120

cgggctttgg tggccaggaa ggttcagaga ctagcggggt gagtcccaac gataatcccc 180

gcctcgagcg cccggcctcc ctcgaatgct tcgaggcggt cacgtcaagc ggtccgtcaa 240

cgaccgccac gcaaccgttt cgatcgtcgc aggcgaggat gagatagtcc aagccccgtg 300

gaaacgcggg g 311

<210> 343

<211> 346

<212> DNA

<213> Common wheat (Triticum aestivum)

<220>

<223> Common wheat (Triticum aestivum) genome assembly, contig: Triticum_aestivum_CS42_TGACv1_scaffold_435076_5DL, FAOM01435076.1

<400> 343

attcatcgat tagctgctag ataatagcat gtgacatttt tagtcgctaa gtggtaactt 60

ccaaattcag agaaaccctg gaattaaaaa agggcaatcc tgagccaaat ccgtgttttg 120

agaaaacaag gggttctcga actagaatac aaaggagaag gataggtgta gagactcaat 180

ggaagctgtt ctaacgaatc gagttaatta cgttgtgttg ttagtggaat tccgaagtga 240

gtggcatcgt gccttctttt agagcgggtc acatccaatt tcgatatggc tcacctttga 300

atcacttgtt ggtaattatt ccatagaaat gacttattag gatacg 346

<210> 344

<211> 322

<212> DNA

<213> Exiguobacterium sibiricum

<220>

<223> Exiguobacterium sibiricum 255-15, complete genome, CP001022.1

<400> 344

gtaatttgat ttcaccgggc gtctgatcga gtaactgatc agagcatgac tgggtgaatt 60

gctggaactc cttagagcct tgatgtacga caacgtggct ggaaacggtg agcgtgaccg 120

ttctgaaaaa cgtcaaggat tggacaatca gcagccaagc acctgtaggg aaaccttggt 180

gaaggttcaa cgactaggat agacgaccta atggagactt ctgatggtta tgaaatccgt 240

actccgtaaa cggcggggga agcgcccagc tcctagtata cctaggatga agatatagtc 300

ttatcattag cgaaagttaa tg 322

<210> 345

<211> 349

<212> DNA

<213> Parasitella parasitica

<220>

<223> Parasitella parasitica CBS 412.66 genome assembly, contig: contig_1784, CCXP01001784.1

<400> 345

atatttgggt aaactataca cttgccccat attagttaat aactaatatg caaatctcac 60

tatatgctgg aaactcctta gagcttgcaa tacctagatc cctttgggta tcttacccca 120

ggcgctacgc gcccggggct agatggtgac aatttacaag attggacaat cagcaggaaa 180

ccaaaggaat attaatattc caagtttcgg cgggccctgc gggcccgccg tcaccgaacc 240

cgcgcgcttt gcgcgcggga aaagtaggat cttcagagac tacacgtgag acatcctata 300

gtatatttga cggatgatga tatagtccaa cctttattga aagatgaaa 349

<210> 346

<211> 306

<212> DNA

<213> Spizellomyces punctatus

<220>

<223> Spizellomyces punctatus DAOM BR117 chromosome unknown supercont1.30, whole genome shotgun sequence, KQ257479.1

<400> 346

aagaccatgt tatgcagtga tcagcacgtg cacttgcaaa gaaagtaaca tggataggat 60

cttctggctc aactgcgtgt ggcagagatc gtcaaattgt tcggggaagc ccttagagct 120

caagctacca accattggtt gaaagaccag tggggccctt cctagggatg gtaataatgc 180

tttgagattg ggtaatccgc agccaagctc ctaaaacttg cttagcaagt catggagaag 240

gttcaacgac tgtaaggcgt accgcgcaag cggaatatac agtctagccc cacgggaaac 300

tgtgcc 306

<210> 347

<211> 302

<212> DNA

<213> Unknown

<220>

<223> Lyngbya sp. PCC 8106 1099428180522, whole genome shotgun sequence, AAVU01000005.1

<400> 347

ggaaaatggt taatattagc cctttatatc agtaatgata taaatgcacc tcctgaattg 60

ctgggaaacc ctaaagctgt tttaacgaca acataactag aaatagtcag tgtgaacgtt 120

taaaaataaa acagatgaaa caatgggtga tcagcagccg agattctgtt aaatgaatca 180

ggttcaacga ctattccaaa cggaagtaca ctcaagcgag tggaagtagg aggtatcctg 240

tagtcaaatc tctaaattat tacaggataa agatatagtc tggtcttaca tgaaagtgta 300

ag 302

<210> 348

<211> 288

<212> DNA

<213> Sclerotinia sclerotiorum

<220>

<223> Sclerotinia sclerotiorum 1980 scaffold_35 genome scaffold, whole genome shotgun sequence, DS267914.1

<400> 348

gtaagagggg atgcgaatag cattccttta gtgatgagat cgcaacactg tcaaattgcg 60

gggagttcct aaagctcagg ctaccgcctc aggtgctgaa aagccctgaa ggcaccaagg 120

ttagcaacct tgggtatggt aataacgcct gtagatacta caatggatga tccgcagcca 180

agctctaaca atcttttcac gattcacgag cggggttcaa cgactagacg gcagtgggcc 240

tgcaaaacag gtttaagata tagtctgcgc ctagggaaaa atcccaag 288

<210> 349

<211> 305

<212> DNA

<213> Unknown

<220>

<223> Marssonina brunnea f. sp. 'multiple species' mitochondria, complete genome, JN204424.1

<400> 349

gtttgtgttt ttaaatggtg aatattgaat attacaatca actcctcgtg atataaataa 60

aaggtaatga cattagcccc ttcaaatctt tctatatgct ggaaactctt aaaggcttaa 120

gtactata aaattcatat ttaattttat aagtaaaaat cttaagtata tctagacaat 180

cagcaggaaa ccaacggata atatagattt attctagtag gatcctcaga gactacacga 240

aagagatggt atagcgtaaa gtctgtacca ttaagacata gtccaatttg tttgtaatgt 300

aacat 305

<210> 350

<211> 213

<212> DNA

<213> Unknown

<220>

<223> Parcubacteria (Yanofskybacteria) GW2011_GWA2_44_9 UW79_C0037, whole genome shotgun sequence, LCJR01000037.1

<400> 350

tcgggctcat aaataattgt gacctaccat agtaatgtgg catggaaaaa ctctctaaat 60

tgtctggaaa ccccactcgc ctatcagcga agggcaatca gcagcgaaac cttaagctca 120

tcgaaaggga cgttcagaga ctataatggg agcacccgta accgtaacaa aagttgggtg 180

atggtatagt ccgtcactgc aagtaattgc aga 213

<210> 351

<211> 269

<212> DNA

<213> Oscillatoria nigro-viridis

<220>

<223> Oscillatoria nigro-viridis PCC 7112, complete genome, CP003614.1

<400> 351

caaaagccct agtgacatag cagctctatc cggtaacggg tactgaaaaa tcgggtgaat 60

tcaaggaaac cgcagcactt cgggtggcga caatcttgag ccaagtctgg cgaaaggcag 120

cggttgcgat cgcaagtagc cggaaaggtg cagagactag agatgaggag cctaaccaat 180

aaatctcaca gcgcccgaca tccgacgaca gatcgcacaa atgatttgta gggatgatga 240

aatagtccgcccccttcggaaacgttggg 269

<210> 352

<211> 292

<212> DNA

<213> Pithovirus sibericum

<220>

<223> Pithovirus sibericum isolate P1084-T, complete genome, KF740664.1

<400> 352

tgacacgcat ttgatcttga atgtgtgttg agcaagaccc tcaaattcag ggaaacccct 60

aaagcttttg aataccaagc ttccagcgaa agttggaggt ggccgcgagt aaatctcgta 120

gggtatggtg aaaacgtcaa aagatatccg ggaaaccggt aatgggcaat cctgagccaa 180

gcaaccgaaa tgccgtatgg tagaggttga aggtgcaacg acttgacggg ggtcggtcag 240

aaacgacagt ttcaggctta aggtaaagtc tactccttag cgaaagttaa gg 292

<210> 353

<211> 221

<212> DNA

<213> Diplodia seriata

<220>

<223> Diplodia seriata DS_831_scaffold_v01_13, whole genome shotgun sequence, LAQI01000013.1

<400> 353

agaagcattt aactcagttg agcatatatt cccacataat gtgctcatta aaccaggctg 60

tttgctggga actctgccgc attaaaccgg ttgacaatca gcaggaacca aggggttttt 120

taaaatcctg atgggttctt cagagactat acgcctggcg cttaattatt aaagaaaaaa 180

attaaatgat gatatagtcc ttctactatt gaaaaattgt a 221

<210> 354

<211> 359

<212> DNA

<213> Rhizoctonia solani

<220>

<223> Complete mitochondrial genome of Rhizoctonia solani strain AG-1 IB, HF546977.1

<400> 354

gcacctcgat agtaacatgt cgagttaaat tagaaataat ttatgggaaa ttgggttaat 60

ttcaagaaaa tctttacact ccaaaatttt ttctatatgt taagcttcaa gcttaacaaa 120

cccacttacg gtgggttgcc tctacttttt cgggggtgcc cagcgaagct gggtcacccg 180

atggttaaaa atttttggag ttaaagacaa cttgaagcga agctagttct gacaataagc 240

taattatgga actagaacgt tcaacgacta gtgggtgagt tttgtcaaca ataatcccgc 300

cacgaatgcc caacaactaa agtcacagat agaatcggaa tttgtgattg aaacaatta 359

<210> 355

<211> 317

<212> DNA

<213> Chaetomium thermophilum

<220>

<223> Mitochondria of Chaetomium thermophilum var. thermophilum strain DSM1495, complete genome, JN007486.1

<400> 355

agaaggagttttctatggtc atccccatta agggactaac tgacattggc ctaaactgta 60

gtgaacctac ggttaaaaac catcaaattg cgggaaaccc ctaaaggaat cttaaccaag 120

taagtatggt aacataactt atggcacagg taatgactcg tggtatggta aaatcaagat 180

tcattattca atgggcaatc cgcagccaag tgccaaatat aaaatatttg gtatgcagtt 240

catcgactag acggtggttg gtattattag ttttaataat gcttaagata tagtcaacac 300

ccccctgaaa gggtgcg 317

<210> 356

<211> 216

<212> DNA

<213> Unknown

<220>

<223> Limnohabitans species 103DPR2, complete genome, CP011834.1

<400> 356

gcagaggact catatttctc aaatgtgcct tacacgtgga aactgtgtaa gggatggtgt 60

caaattcgat gaaacctaag tgtggcaaca catatggcaa tgtcgagcga agcttagtgc 120

gaaagcactt tgaacgtgta gagacttgac ggcacccacc taagtacagc gatgtatatg 180

gtgaaggcaa agtccagcga gtgatgaaag tcacac 216

<210> 357

<211> 334

<212> DNA

<213> Unknown

<220>

<223> Staphylococcus sp. HGB0015 genome scaffold aczIz-supercont1.1, whole genome shotgun sequence, KE150417.1

<400> 357

ttcagtgtgt agagaaatct gcacatcgtg acagtacgac tgtccaacaa agaaattgaa 60

ttgcttgaaa accctaaagc ctgcttgacc acaacgtaga gataatcaaa ctcaagcgtg 120

aaggttgcga aactgcagaa aaaataagca ggatgacata aggttaaaac ctaagtgttt 180

tttgcaatgg gcaactagca gccaagccta gaaataggaa ggttcaacga ctattcctct 240

tgagggaagt acacacaagc gtgtggaagt ggtttcgccg taatggataa tgccaacgga 300

aaagatatag tctgtgcttg tatgaaaata caag 334

<210> 358

<211> 383

<212> DNA

<213> Unknown

<220>

<223> Parcubacteria (Uhrbacteria) GW2011_GWC2_53_7UY82_C0027, whole genome shotgun sequence, LCRN01000027.1

<400> 358

atagcgacat tctgtataaa tcgtcttttc gtctaaaaat tgttcaatca tatgattgaa 60

ctcgaccgtg ctgtcataaa atctggctat atgctggaac atctggcatc tcccaaccat 120

caggagaatg ggagaattgga gaatctcaca ctacgtagta aactacttgt aaaagatacg 180

tgaaaatgtg ttgagtgcag ataaccagca gggaagacta agatatgaca gcgtcgattc 240

atccaaatct tgtttataca atgaacaagt ctggcaagta tcgacgttgt aaacatcatc 300

tatcttagaa ccctcagaga ctatacgccg gactccgatg tccatcggag aagatatagt 360

ccgaaccgca tggcgacatg cag 383

<210> 359

<211> 262

<212> DNA

<213> Unknown

<220>

<223> Leptolyngbya sp., Heron Island J, whole genome shotgun sequence, AWNH01000034.1

<400> 359

ctgcggactt agaaaactga gccttagtgg agaaatctgc taagtggaag ctctcaaact 60

cagggaaacc taagtcttgg ttggttactt gaccttctga gatatggcaa tcctgagcca 120

agcccttcaa aaggcgaaaa atagagggta aagttcatcc tttatctttt cgatttcatc 180

cttttgaagg gaaggtgcag aggcccgacg ggagctaccc taacgtcaag tcgagggtaa 240

agggagggtc caatcctcaa ag 262

<210> 360

<211> 339

<212> DNA

<213> Mortierella elongata

<220>

<223> Mortierella elongata AG-77 unplaced genome scaffold K457scaffold_276, whole genome shotgun sequence, KV442285.1

<400> 360

tcatatattc ataatattat gaatgtatat taatgattta attaggcatg gccgggtaat 60

atagtaatat attactttct tttcactatc tgctggaaca ccttaagagt atttaaaact 120

agttctgcat gctttcttta atgaaaagcg gtaggaaaca gtgacattta aataattagg 180

caatcagcag gaaaccaaag ataaaagggc ttaactttaa gcattaaaca cttttattga 240

gtaggatcct cagagactac acgtgaaata ccctattaag tgattattct taattattta 300

agggtaaaga tatagtccaa ccattaacga aagttaatg 339

<210> 361

<211> 209

<212> DNA

<213> Unknown

<220>

<223> Candidatus Gottesmanbacteria bacterium RIFCSPLOWO2_01_FULL_49_10 rifcsplowo2_01_scaffold_16705, whole genome shotgun sequence, MFJZ01000013.1

<400> 361

acagaaggct caacatatgg gctgtcctga gtttaatcga aggatagaat tcggctatat 60

cggtgaaacc ctaagaccaa cgtccgggta ataccgagga aagatcccga gcttgtcgag 120

ggaaatccgt agagactata cgccgaatcc ctccgaagct tttgagcgaa gggggaaaga 180

tatagtccga cactctcagt aatgggagg 209

<210> 362

<211> 258

<212> DNA

<213> Enterococcus dispar

<220>

<223> Enterococcus dispar ATCC 51266 genome scaffold acpMG-supercont1.1, whole genome shotgun sequence, KE136354.1

<400> 362

atattcggtt tgttgaaatc ccatattcaa tgaccgataa agaaatagaa aaagccatat 60

ttgaattaac tatgccaatc atgagccaag cctgtcggga aactgcagga aggtgcaacg 120

actagataaa ttaacctaag caaaagcagt catttatttg attgcttttt ttgtatggcg 180

aaatatccac gagcgcttga taccttaacg tttaaggcga aggtaatgat atagtctgaa 240

cttataggaa actataag 258

<210> 363

<211> 345

<212> DNA

<213> Aureococcus anophagefferens

<220>

<223> Aureococcus anophagefferens unplaced genome scaffold AURANscaffold_2, whole genome shotgun sequence, GL833121.1

<400> 363

ttagttgcgt gtctattgtg cgctagtcgc accgttccgc gaactgcacg ggaacggcgg 60

cggcaacatc atcgaattgc tgggaaacct cgataggccg gagctactaa aggctcgggg 120

aaacccgggt caaatcgagc ttagacgctc gaagtgaaaa tgcttcggat agaggcaatc 180

agcagccaag cgcctaaagc cgcgtgtatt acagtgtatt acagttgggt atacatgtat 240

tgcaagcggt cacggtgaag gtccagagac taagtggtga tgggtgtcgg cgcggttgac 300

cgcgccgatg cttaagatat agtccgcccc tcttgagaga gagct 345

<210> 364

<211> 272

<212> DNA

<213> Tuber melanosporum

<220>

<223> Tuber melanosporum whole genome shotgun sequence assembly, scaffold_368, strain Mel28, FN430284.1

<400> 364

caaaaagtat aggtaaaccc tctgctagtt cctaaaggga gcaaaaccat caaattgcgg 60

gaacatctta aagcaatttt taaccaagcg agaacggtaa cgtatttcgt ggcgcaggta 120

atgactcgcg gtaaggtaaa ataaaaattg atgtacgaaa ggaaatagaa aatccgcagc 180

caagttcgaa ataaaattcg aatgcagttc atcgactaaa tgttggttgg cgcaagctta 240

aaatatagtc agacctcaat cgaaagattt ag 272

<210> 365

<211> 203

<212> DNA

<213> Unknown

<220>

<223> Gracilibacteria bacteria tentative species CG1_02_38_174 cg_0.2_sub10_scaffold_1404_c, whole genome shotgun sequence, MNXD01000034.1

<400> 365

agatatgatt tctgtcaagg gctactagag aagtaatttt ctagtgaaaa tccgtcaaat 60

tcggggaaac cttcatttct agttatagaa atatggcaat cccgagccaa gcctattttat 120

aggaaggtgt agagactaga tggcggacat cctgatactc aggatgaagg gatagtccag 180

accacgaacc cgaaaggggc gat 203

<210> 366

<211> 207

<212> DNA

<213> Unknown

<220>

<223> Synechocystis sp. PCC 6803 DNA, complete genome, BA000022.2

<400> 366

cttggcatct catcttgcaa aaaggggctg cgcaaaagga aacttctgcg tgattatctc 60

tcaaattcgg ggaagccttt caaatggtaa tcccgagcca aacctaggaa tgcttggtgt 120

ttctgggaag gtgtagagac ttaatgggag acaccctaac agaaaagctg agggtgaaga 180

gaaagtccag accacaaact gacagag 207

<210> 367

<211> 355

<212> DNA

<213> Parcubacteria bacteria

<220>

<223> Parcubacteria bacteria GW2011_GWA2_46_9 UX68_C0001, whole genome shotgun sequence, LCND01000001.1

<400> 367

ataaatgcgttttattgtgc gcgaattgtc acagagaagt acatgctggt gtaatcgcag 60

ccccgcgtag gaatgcgcgg agaaaaactg ggtgaattcg gggaagccca gccccacttt 120

ttgataagat gccaccagta gataaataat tgtgtattta tttgctgacg ttagtactag 180

tcattatcgg ttttgtcaga aagtggggct gggtaatccc gagccaatac ctgacgggta 240

aaagtgtcag gaaaggtgta gagactagcg ggtgagtccc aacgataatc ccgccacgag 300

cgcccagcgc ctagaacagg cgatgagata gtccgtccct attggtaaca gtagg 355

<210> 368

<211> 92

<212> DNA

<213> Legionella feeleii

<220>

<223> Legionella feeleii strain WO-44C Lfee_ctg085, whole genome shotgun sequence, LNYB01000085.1

<400> 368

acgggtttac ccccgaatcg agccttgtgg cccttgccaa gcatcatgta tatgagctgc 60

tcgaataaca ctaaacacaa tcctgggtaa ac 92

<210> 369

<211> 96

<212> DNA

<213> Legionella shakespearei

<220>

<223> Legionella shakespearei DSM 23087 strain ATCC 49655Lsha_ctg016, whole genome shotgun sequence, LNYW01000016.1

<400> 369

cttgatttgc ctcatcattt cgagccttgc agcgcaagct ggatatcctc tttgagtgaa 60

tcgctcgatt aacactaaac caaagacagg gcaaat 96

<210> 370

<211> 96

<212> DNA

<213> Legionella waltersii

<220>

<223> Legionella waltersii ATCC 51914 strain Lwal_ctg060, whole genome shotgun sequence, LNZB01000060.1

<400> 370

attgatttgc cccccccgtt ggagccttgt ggcgtaagcc tggtatcgct tttgagtgag 60

ccgctcgatc aacactaaac caaagtcagg gcaagt 96

<210> 371

<211> 96

<212> DNA

<213> Legionella jamestowniensis

<220>

<223> Legionella jamestowniensis JA-26-G1-E2 strain Ljam_ctg012, whole genome shotgun sequence, LNYG01000012.1

<400> 371

actgatttgc ccctgaactg agccttgagg cactacgcct ggtactgcaa ccttgcaggc 60

cgctctacca acactaaaca aaataccagg gcaaat 96

<210> 372

<211> 96

<212> DNA

<213> Legionella fallonii

<220>

<223> Legionella fallonii LLAP-10 genome assembly, plasmid: III, LN614829.1

<400> 372

attgatttgc cccctctttg agcatttcgg cttttgccgg gtatcaattt tttggattga 60

gccgctcgac caacactaaa cacaaacagg gcaaat 96

<210> 373

<211> 126

<212> DNA

<213> Black flying fox (Pteropus alecto)

<220>

<223> Black flying fox (Pteropus alecto) contig 92670, whole genome shotgun sequence, ALWS01092670.1

<400> 373

caagaaatgtttcttgacca gttgcctgca gctgatgagc tccagtaaga gcgaaaccag 60

ttctcactcc actgaaacaa ttttgaagtg tgaattggtc ctgtagtact gtgtcagaaa 120

caactc 126

<210> 374

<211> 126

<212> DNA

<213> Yellow-throated Sandgrouse (Pterocles gutturalis)

<220>

<223> Yellow-throated sandgrouse (Pterocles gutturalis), contig 91464, whole genome shotgun sequence, JMFR01091464.1

<400> 374

aggtttatga tgttaaacca gttgcctaca gctgatgagt gccaggaaga gcgaaaccag 60

ttctgttctg tttcaacagt tatgaaaagt aaggactggt cctgtagtac tgtccagcat 120

caaaat 126

<210> 375

<211> 206

<212> DNA

<213> Anopheles dirus

<220>

<223> Anopheles dirus WRAIR2 strain unplaced genome scaffold supercont1.9, whole genome shotgun sequence, KB673645.1

<400> 375

tagaaatgca gcactggtac gggtacggat ccgacgcctc tcgtaggata cttaggctct 60

ccgtacccta ctcctactca aaacgtcccc gacgtacata ttcgtgtttc ttatcccgtt 120

tctctcgatt agtgatagcg tagtgatctg ttcactggca ccgataggta aaaatccttt 180

caaaatacta tacgaaacta aaagac 206

<210> 376

<211> 150

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.15 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663666.1

<400> 376

ttgtacttat gctctgcaat ggggtaggac ccggaacctt ttgaaggtta cacaggttct 60

cctattcaac tccttttcta ctacgtatcc aagcttggat acatgggcca tctacatccc 120

ctggagtggg cagaaacgaa actgggctac 150

<210> 377

<211> 192

<212> DNA

<213> Anopheles culicifacies

<220>

<223> Anopheles culicifacies strain species A-37_1 cont1.7520, whole genome shotgun sequence, KB663666.1

<400> 377

agtaaaattt cactggtaag ggatggatct gaaaacctat cgaaaatcaa caaaggctct 60

ccatattcta ctccgactca atagaagtcc ccgacgtata gaacggtaac ctgtctcact 120

aaatatctga gcttgggtat atggagaaac ccaacccttg ggaagatggg cggctagctt 180

cctttctatc ct 192

<210> 378

<211> 167

<212> DNA

<213> Anopheles funestus

<220>

<223> Unplaced genome scaffold supercont1.144 of Anopheles funestus FUMOZ strain, whole genome shotgun sequence, KB668664.1

<400> 378

agcaataccg cactgatata gatatggatt caaagtctct tgaaggataa tataggttct 60

ccgtcccgac cctactatac gtccatgtcg tatatacata tctctacaaa tatctgagct 120

tgggtatacg aggaaaccct ggagactaga tgttcctcat gccctgg 167

<210> 379

<211> 176

<212> DNA

<213> Anopheles sinensis

<220>

<223> Anopheles sinensis unplaced genome scaffold AS2_scf7180000696013, whole genome shotgun sequence, KE525305.1

<400> 379

ttgaccattt agtctgacca tgggtctgca aaggaactat aagctatcct cccccactcc 60

tactcaatgc gtccgcgaag tacagaacgg cagcttgtcg cttaaatatc caagcttggg 120

tacatgggga aacccaccccc cttgggcgaa tggccggcaa ggctgaattg agagga 176

<210> 380

<211> 162

<212> DNA

<213> Anopheles atroparvus

<220>

<223> Unplaced genome scaffold supercont1.22 of Anopheles atroparvus EBRO strain, whole genome shotgun sequence, KI421903.1

<400> 380

gtctgtgttg gtctgtgaat ggggcaggat ccgacgcctc ctgaaggcta cataggctct 60

cctatctaac tcatattctg gtatgtccaa gccatacaga ccgtgtacgg gttcaatccc 120

aaccccctgg gaggatgggg ttgcacggct aatgtagaag gg 162

<210> 381

<211> 186

<212> DNA

<213> Anopheles christyi

<220>

<223> Anopheles christyi ACHKN1017 strain cont1.4036, whole genome shotgun sequence, APCM01004036.1

<400> 381

agcaatactt cgctgacacg ggaatggatc cgaagcctcc agaaggctaa cataggctct 60

ccgttgccta ctcctactaa atattcacta cattcctaca gaacggcaac ttgtttctca 120

attatccaaa cttgatgcaa catgcaaccc cttgggaaga tggaaggaat ggcaaaatta 180

ggctgg 186

<210> 382

<211> 181

<212> DNA

<213> Anopheles dirus

<220>

<223> Unplaced genome scaffold supercont1.24 of Anopheles dirus WRAIR2 strain, whole genome shotgun sequence, KB672913.1

<400> 382

tttacattat gaaacacaaa tctacaatct tcacgcctgt cgaaggatgc acaggctctt 60

cttactctac tcctactcaa aacgttcccg actgtaacat gtctatccgc atatctgagc 120

ttgggtatac gaggaaacgc aaccccttgg gcgaatggat gatgtggcta atttgagtgg 180

a 181

<210> 383

<211> 164

<212> DNA

<213> Unknown

<220>

<223> Anopheles gambiae M scf_1925491374 genome scaffold, whole genome shotgun sequence, EQ090202.1

<400> 383

aataatgttt aaattcgaaa ctgacttgga aaaccctgta atatagactc tctcatccaa 60

cttctattct tagccgtcat tggtaacatg tgtcaccaca tatgagtact tagactagat 120

ccaaaccctt gggcggatgg tggcatatgg cgaaccagga gagg 164

<210> 384

<211> 201

<212> DNA

<213> Anopheles arabiensis

<220>

<223> Unplaced genome scaffold supercont1.17 of Anopheles arabiensis DONG5_A strain, whole genome shotgun sequence, KB704418.1

<400> 384

ttaagtagca aatgcaatcg gataggtttc gaagcctctc tgagggataa tagaggctct 60

actattcaac ttctaatcga acacgaccct attcgtgtag agtggtaaca tgtggattcg 120

gactagttcg aagggtccca aagggaacac ggactagttc caactcctcg cacagatggt 180

ggcatatggc gaatgaggcg a 201

<210> 385

<211> 167

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.186 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663706.1

<400> 385

atcaaatttc tgtgagttgg tgtggtagaa ataccgcagg agaatactcc tactcaatac 60

gtccccggcg tacagagtgg taacatgtct ctccaaatat ctgagcttgg gtatacggga 120

aaatccatcc tcttgggagg atgggtgata tggctaaatt gagagga 167

<210> 386

<211> 208

<212> DNA

<213> Anopheles melas

<220>

<223> Anopheles melas CM1001059_A strain cont2.23244, whole genome shotgun sequence, AXCO02023244.1

<400> 386

agcaccaaat tatctgcaaa tgagttaata tccgacacct ccttgaaggt taatatagac 60

tctcttactc tcttactctt tctcctatcc tgcgacgtcc gtttcgtata gtggtaacat 120

gtatcatagt atattcaagc atggctgcac gggcccagtc ccaacccctt gggcggatga 180

tggtacatac atggccaacc aggagggg 208

<210> 387

<211> 180

<212> DNA

<213> Anopheles christyi

<220>

<223> Anopheles christyi ACHKN1017 strain cont1.5619, whole genome shotgun sequence, APCM01005619.1

<400> 387

ctcattggct ggatcaggtg aagcgggact tgtcggagac ataggctctc ctattcaatt 60

cccatacgga cacgtcccag ttcgtgcaga gtagtaactt ggatcatcga atatccaagc 120

ttgggtacac gggcttgttc caaccccttg ggcggatggc tcatggcaaa tcaggagggc 180

<210> 388

<211> 140

<212> DNA

<213> Anopheles maculatus

<220>

<223> Anopheles maculatus maculatus3 strain cont1.9278, whole genome shotgun sequence, AXCL01009283.1

<400> 388

gtcagtataa cacactagta ttgatatggg tccgaagcct gtccaaggat aatataggct 60

ctccatgtac tccatatatc ttagatagag cataagggaa aactcttgga gagatgggtg 120

ttttagctaa attgagcggt 140

<210> 389

<211> 186

<212> DNA

<213> Unknown

<220>

<223> Anopheles gambiae M scf_1925491386 genome scaffold, whole genome shotgun sequence, EQ090214.1

<400> 389

attcttctgt gctctgcaat gggataggat ccgaagccct tctgagggat aatataggct 60

ctctttattta actcctactc ggacaagtcc ctgttcgtgc agagtggcaa catgtgtcat 120

cacatattca agcttgagtg cacggactag ttccaacccc tcgggaaaca gagtcgtaat 180

aaagga 186

<210> 390

<211> 148

<212> DNA

<213> Anopheles sinensis

<220>

<223> Anopheles sinensis unplaced genome scaffold AS2_scf7180000695538, whole genome shotgun sequence, KE524837.1

<400> 390

tgaatgagtt gttctgcaaa tggattgaat cacttgcccc tatcccaggg cagtatgaag 60

gcgtgttcat tcctagatcc tactcaacac gtccacgtcg tgcagaatgg tagcatttca 120

ttacgatgaa atgactaagt tgagaggg 148

<210> 391

<211> 169

<212> DNA

<213> Anopheles epiroticus

<220>

<223> Unplaced genome scaffold supercont1.178 of Anopheles epiroticus epiroticus2 strain, whole genome shotgun sequence, KB670480.1

<400> 391

actactacta gcttcttgaa gggttttgaa gccaggctct tctacttcta ctcttctaca 60

atttgtcctt atcgtacaga gcactaacat atattcaaat atctgagctt ggcaaaacgg 120

cgaaacccaa tcacaactcc atggatgaaa gacatggagt ttgagagggg 169

<210> 392

<211> 225

<212> DNA

<213> Anopheles gambiae

<220>

<223> Anopheles gambiae strain PEST chromosome 2L, whole genome shotgun sequence, CM000356.1

<400> 392

ctctttgcct caaaagtttg ccgaccgctg ctctagaagg ttaacatagg ctctccaccc 60

cctaccctta ctcaatacgt tcccgtcgta cggatgtccc tcaattattc agatgtccag 120

atatccagat gtacagatgt ccaatgtccc tcaattatcc aagcttgggt ataaggggaa 180

acctaacccc ttgggctgat ggattgcatg gctaaattaa gagga 225

<210> 393

<211> 175

<212> DNA

<213> Anopheles christyi

<220>

<223> Anopheles christyi ACHKN1017 strain cont1.3711, whole genome shotgun sequence, APCM01003711.1

<400> 393

agcaataccc cgctagtgcg ggaatggatc caaagcttcc agaagatgaa catacgctct 60

ccattccata ctcctactca atacgtcctc ggcgtacaga acaacaccat gtatctctat 120

tatccaagct tgggtaaatg gcgaaaatca tacaatttgg ggagatggga ggcag 175

<210> 394

<211> 173

<212> DNA

<213> Anopheles maculatus

<220>

<223> Anopheles maculatus maculatus3 strain cont1.28980, whole genome shotgun sequence, AXCL01028988.1

<400> 394

aaaaacaacc aattgggcca cgcaaaacga cttttgcaac atgatacatt acaaattgga 60

acataatacc tgtcgtacat aatactgaaa taaacatacc aaatatctga gcttgggtat 120

acggggaaac ccaacctcta gggaaaatgg gtgatatggc taaatttacc gaa 173

<210> 395

<211> 188

<212> DNA

<213> Anopheles melas

<220>

<223> Anopheles melas CM1001059_A strain cont2.8943, whole genome shotgun sequence, AXCO02008943.1

<400> 395

ggtagttcta ctggcaatgg ttggcagatt cgaaacctct agaaggttaa caaaggctct 60

ccatcaccga cttctactca atacgtcctt gtcgtacaga atggtaacat gtttcttaat 120

tatccaagct ttggtacacg gggaaaccca accccttgga cattggttgc atggctaaat 180

tgagagga 188

<210> 396

<211> 121

<212> DNA

<213> Anopheles stephensi

<220>

<223> Anopheles stephensi SDA-500 strain unplaced genome scaffold supercont1.383, whole genome shotgun sequence, KB664714.1

<220>

<221> Features not yet classified

<222> (105)..(121)

<223> n is a, c, g, or t

<400> 396

attaatctgg ctctgttaat ggggtaggaa ccgaagctcc tctcggggtt acacaggctc 60

tcctacccaa ctcctattcc gtcacgtcct cgtcgtacag agtgnnnnnn nnnnnnnnnn 120

n 121

<210> 397

<211> 175

<212> DNA

<213> Anopheles christyi

<220>

<223> Anopheles christyi ACHKN1017 strain cont1.2748, whole genome shotgun sequence, APCM01002748.1

<400> 397

tacttcatat gtattgcaat aagataagtt ccgtagcccc tttgagggat aatacaggct 60

ctccaattca actcctatcc gaaaacgtcc tagttcgtac aaagattcgt caccgctttt 120

cttgttgacc tgttctaacc ccttgggagg ttggcgcaag gctaatcagg agagt 175

<210> 398

<211> 167

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.2 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663721.1

<400> 398

tctagcaatg gtaagggaat ggatctggag cctctcgaag gataataaag gttctatata 60

tcatattact actcaacggt aacatgtatc gccaaatacc ctgagcttgg gaatatgaag 120

aaattcaacc actcggcagg atgaggaatg ttgtgaagct tggaaga 167

<210> 399

<211> 222

<212> DNA

<213> Anopheles stephensi

<220>

<223> Anopheles stephensi SDA-500 strain unplaced genome scaffold supercont1.505, whole genome shotgun sequence, KB664850.1

<400> 399

cctaaaagtt gctctgttaa tgaaatagga tccgagactc ctttcagggt tacacagggg 60

ggtaggagag agtttcaggg taggagagtc ctacccaact cctattccgt cacgtcctcg 120

tcgtacagag tggtaacttt tcccaccata tatctaagct tgggtacacggacctgtccc 180

aaccccttgg gcggatggtg gagaaaggct aaacaggagg ag 222

<210> 400

<211> 158

<212> DNA

<213> Anopheles dirus

<220>

<223> Unplaced genome scaffold supercont1.30 of Anopheles dirus WRAIR2 strain, whole genome shotgun sequence, KB672980.1

<400> 400

tagaaatgca gcactggtaa gggtacggat ccgacgcctc tcgaaggata cctaggctct 60

ccgtacccta ctcctactca aaacgtcccc gattttcctc ctgtctaacc taagacgcgt 120

tccgcgagag atcttagctt atggttagtt tggttggt 158

<210> 401

<211> 189

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.12 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663633.1

<400> 401

ttcacatagg ctctgcaata aggtagaact cggaacctat tgaaggtcac acaggctctc 60

ctactcaactccccattctgc aacgtcctcg tcgtgcagag tgacgagaac taccgtatat 120

ccaatattga gtacacggac tagctccaac cctttgatcg ggtagtgatg ctcggcaaaa 180

gaggagggc 189

<210> 402

<211> 193

<212> DNA

<213> Unknown

<220>

<223> Anopheles gambiae M scf_1925488698 genome scaffold, whole genome shotgun sequence, EQ087528.1

<400> 402

cccagtgtttttcctttttc ctgtttttcc agaaacccct cgagggatag taccggctct 60

tccatccaac tcctattccg acacgtcctc gtcgtgcaga gtggtagcat gtgccaccat 120

atatcctagg ttggataacac gggctagatc caacccttcg ggcggatggt ggcatatgac 180

gaaccaggag ggg 193

<210> 403

<211> 167

<212> DNA

<213> Anopheles sinensis

<220>

<223> Anopheles sinensis unplaced genome scaffold AS2_scf7180000696059, whole genome shotgun sequence, KE525351.1

<400> 403

tctagtagta gtcctttaac gggttggatc cggcgcctcg tgaaggctac tataggctct 60

ccttctcaaa atctattcga tgcgtccgcg acgtacagaa cggtaccttg tcgcccaaat 120

atccatcagt tcatcctcga gcggctgtcc tccatgttag gagcggc 167

<210> 404

<211> 189

<212> DNA

<213> Anopheles gambiae

<220>

<223> Anopheles gambiae strain PEST chromosome 2R, whole genome shotgun sequence, CM000357.1

<400> 404

agcaatacgt cgctgatatg gcagtggagc cgaagcctct agaaggttaa caaaggctca 60

ccatccttta ctcatattcc atacgtcctc aatgtacaga tcggtaacat gcctctcaat 120

tatctaagct tagctatacg aggttcttca tgactgttct ccatattagg agcggctccc 180

tccaagcgt 189

<210> 405

<211> 117

<212> DNA

<213> Anopheles arabiensis

<220>

<223> Anopheles arabiensis DONG5_A strain unplaced genome scaffold supercont1.5, whole genome shotgun sequence, KB704784.1

<400> 405

ctcgacttgc cgttcgaaca ccatgatcag gccacatata ggctaatttt ctatgcttag 60

atatacgggg aaacccaagc ccttgggcgg atgggttgta tggctaaatt gagtatt 117

<210> 406

<211> 155

<212> DNA

<213> Anopheles sinensis

<220>

<223> Anopheles sinensis unplaced genome scaffold AS2_scf7180000696013, whole genome shotgun sequence, KE525305.1

<400> 406

agtatcgtta aaaccacata ttgtcctttg caaaccttcc gtaccaaaat tacggagagt 60

acagaacggt agcctgttgc ctaaatattc aagcttgggt acacggggaa acccaccccc 120

ttgggcgaat gggcgacaag gccgaattga gacgg 155

<210> 407

<211> 136

<212> DNA

<213> Anopheles merus

<220>

<223> Unplaced genome scaffold supercont2.196 of Anopheles merus MAF strain, whole genome shotgun sequence, KI915351.1

<400> 407

ctgagacgag gatagatcca aaggtagatc cgaagaaggt taataaagga tctccaaccc 60

ctacaccaac taaatatgtc cccgtagtac ggcccggtaa catgtggtaa ccctctcagt 120

ggatgtacagacccga 136

<210> 408

<211> 119

<212> DNA

<213> Anopheles epiroticus

<220>

<223> Unplaced genome scaffold supercont1.208 of Anopheles epiroticus epiroticus2 strain, whole genome shotgun sequence, KB670814.1

<400> 408

tcaatcatgt gaaactgtta tgggatggga tccgaagcct ccagaagatt ccatctaact 60

cctgttccga cacgtccacg tcgtgcaggg tggggcaaca tggacctaac caggggggtc 119

<210> 409

<211> 208

<212> DNA

<213> Anopheles darlingi

<220>

<223> Anopheles darlingi Cont6653, whole genome shotgun sequence, ADMH02001348.1

<400> 409

gcaattggtt gctctacaaa tgaggttagg atccgaagca ctaggctaat ttaggctctc 60

ctcccctaac tcctactcgt cgcgtcctcg cggggctcat gggttctgct cgtgtagagc 120

ggtaacatgc ctcccacacg tctgagcttg ggtacacggg taacaccaac cccttgggaa 180

gatggggggt atggctgaga cgagaggg 208

<210> 410

<211> 172

<212> DNA

<213> Anopheles stephensi

<220>

<223> Anopheles stephensi SDA-500 strain unplaced genome scaffold supercont1.182, whole genome shotgun sequence, KB664491.1

<400> 410

cgtagcaata atgcactagt aaggtatgga tctgaagctt cttgagggtt aacaaaacct 60

ctccatacgc tacttcgact caatacgtcg tacgggcgta cagaactctc catacatatg 120

aacttgggtc atacggggaa acccaacccc ttgggaagag agatagcgag cg 172

<210> 411

<211> 193

<212> DNA

<213> Anopheles gambiae

<220>

<223> Anopheles gambiae strain PEST whole genome shotgun sequencing project, whole genome shotgun sequence, AAAB01008842.1

<400> 411

aacattttta gatcgtcagc atataataac aatttaacat gaggaatagc aaacacgaca 60

tcgttaataa atataataaa tagtaaaggt cctaatcacc acattgatat gtgtcaccac 120

atatccaagc ttgggtacac gggcttgacc caaccccttg ggcggatggt ggcacatggc 180

gaaccaggag ggg 193

<210> 412

<211> 153

<212> DNA

<213> Anopheles dirus

<220>

<223> Unplaced genome scaffold supercont1.20 of Anopheles dirus WRAIR2 strain, whole genome shotgun sequence, KB672869.1

<400> 412

tgcaacaacc atttcgtaca tgagggaatg aaaacaagaa gaggtggggt gacaaactta 60

aagaatttta acactatagt agatatctga gcttgggtat acggggaaac ccaacccctt 120

gggaggatgg aagacatggc taatttgaga gga 153

<210> 413

<211> 110

<212> DNA

<213> Anopheles culicifacies

<220>

<223> Anopheles culicifacies strain species A-37_1 cont1.7295, whole genome shotgun sequence, AXCM01007295.1

<400> 413

atcaatcgtt gctctgtaag taggtgtggt actgttaaga taagctctcc ataccgtact 60

cctactcaat acgtcctcgg cgtacagagc ggtaacatgt ctctccaaat 110

<210> 414

<211> 201

<212> DNA

<213> Anopheles dirus

<220>

<223> Anopheles dirus WRAIR2 strain unplaced genome scaffold supercont1.25, whole genome shotgun sequence, KB672924.1

<400> 414

cgtatctttc tactatataa tgggataaga tccaatacct tttgcaagct aacgcatgct 60

ctattattct cttctgtcgc gacacgtccc caacgtacag cttggtagca tatatgattt 120

attttccaag cttgggtagt ccggacaaat ggcaatctca acgttggaga agagctagac 180

gatatactca actacttcaa g 201

<210> 415

<211> 129

<212> DNA

<213> Anopheles culicifacies

<220>

<223> Anopheles culicifacies strain species A-37_1 cont1.8016, whole genome shotgun sequence, AXCM01008016.1

<400> 415

aaatggatcc gaagcctctc gaaggataat tgagggctac tcctacttac atgtctctac 60

aaatatctga gctcgggtat tcagggaaat ccaacccttt gggagtatga tacggctgaa 120

ttgagagga 129

<210> 416

<211> 129

<212> DNA

<213> Anopheles stephensi

<220>

<223> Anopheles stephensi SDA-500 strain unplaced genome scaffold supercont1.68, whole genome shotgun sequence, KB665043.1

<400> 416

gctagttctg cagctgttgt ggtagcatta ctgaagcctc ttgcaggtta acaaaggctc 60

tccataccct acctcgactc aatacgttcc cctgtcgtac aaaacggtag catgtctcaa 120

cggaaatga 129

<210> 417

<211> 190

<212> DNA

<213> Anopheles minimus

<220>

<223> Unplaced genome scaffold supercont1.11 of Anopheles minimus MINIMUS1 strain, whole genome shotgun sequence, KB663622.1

<400> 417

accataatat cctgctaacg gggtagaata tgaagtcgct taaaggttac acaggctctc 60

ctactcaacccttattcccc ctagcatcct cgtcgtgcag agcggcaact tgaaccatcg 120

catatccaag cttgggtacg cgggttagtt ccccttgggc ggatggtggt gtatgattaa 180

acaggaggga 190

<210> 418

<211> 183

<212> DNA

<213> Anopheles merus

<220>

<223> Unplaced genome scaffold supercont2.33 of Anopheles merus MAF strain, whole genome shotgun sequence, KI915188.1

<400> 418

ttcagatcat ctgctcagca atggaacagg atccgaagtt tccctgaatg ataacatagg 60

ttatccgatc taactcgtat ccagacaagt gtcagttcgt tacatgtgtc atcacatatc 120

caagcttatg tacacggacc tgttgcaacc gttcgggcgg attgttgcag tcatttgatt 180

gca 183

<210> 419

<211> 127

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. CAG:221 genome scaffold, scf67, FR883402.1

<400> 419

aactaagttg acgaggatga gatttatcga attttttcgg cggatatctc acgtaaatag 60

cactagcgtt aataattaac aaaactacaa agtaatttgt aggacaaatt taattatgtg 120

caatcta 127

<210> 420

<211> 159

<212> DNA

<213> Unknown

<220>

<223> Clostridium neopropionicum DSM-3847 strain CLNEO_contig000018, whole genome shotgun sequence, LRVM01000018.1

<400> 420

ttgaaaaatg aagcgccaga accgcagata gggcggttga cgaggtagaa gtgatcgaat 60

ttttcggcgg atgcttctcg cccattcgtt cagggacgca ggtctttcta caaataggag 120

aaggtaattc ttctgacaaa gggaaaggca acggacgaa 159

<210> 421

<211> 154

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. CAG:465 genome scaffold, scf33, FR891245.1

<400> 421

taacattaaa tagcgcatga acataccttt cgtatgtgac gaggatgata gttatcgaac 60

attcagcgga tactatcacg gtgatttagc atcgatatat atagtacaaa aagtaaaatc 120

tttaattact acaaaaacta tataatctaa atat 154

<210> 422

<211> 178

<212> DNA

<213> Atribacteria bacteria

<220>

<223> Atribacteria bacteria 34_128 MPI_scaffold_1295, whole genome shotgun sequence, LGGA01000028.1

<400> 422

aattgaatac aagcgccaga acttactcaa tttgaagggc taagtgtttt gataaggtaa 60

gttgacgagg aaggagttta tcgaaaattc ggcggatgct cctgggttgg ccagaccctt 120

agaaaacctg taaaacttgt gagtaattgc aaggacagag aggtttttat ggcaaaat 178

<210> 423

<211> 150

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. CAG:793 genome scaffold, scf49, HF993644.1

<400> 423

ttatttaagt aagcgccagg acattttttt aatgttgacg aggatagaac ttatcgaaat 60

tttcggcgga tggttctagg gaatgctact tcctaaaata ttgtcaaaaa ataatagcga 120

tattataaca aatcaatatt actagctgtg 150

<210> 424

<211> 171

<212> DNA

<213> Unknown

<220>

<223> Kurthia sp. 11kri321, complete genome, CP013217.1

<400> 424

aaaacagtta tagcgccagt actgaagaaa tcggacgaca agtatcttca gtagacgagg 60

tggagggagta tcgaaagttt cggcggatgc ctcccggtcg acagcccgat cgtaagttca 120

tctttaaaat agtgaagtga ttcattagac aaagagatga aatggcaaat c 171

<210> 425

<211> 148

<212> DNA

<213> Mycobacterium abscessus

<220>

<223> Mycobacterium abscessus strain PAP053 genome assembly, contigs: ERS075544SCcontig000014, CSXB01000014.1

<400> 425

caatatcgga tagcgccaga cctgaacgtt caggtgacga ggagagagct tatcgaagat 60

tcggcgggtg gctctaggga ctgcactcta cagataacaa agaaaaacta attgtgaagt 120

tagaacaaag cggttatcac gcaggtag 148

<210> 426

<211> 189

<212> DNA

<213> Shuttleworthia satelles

<220>

<223> Shuttleworthia satelles DSM 14600 genome scaffold Scfld0, whole genome shotgun sequence, GG665866.1

<400> 426

tcaaaaactt tagcgccatg tacccggccg cttttcatcc ctctgtattt ttggaggaac 60

gttttggccc gggttgacga ggtgtcaggt gatcgaagat tcggcggatg cctgatcgcg 120

gatgcggccg cgcatacagt tgacaaaaga tccggtaacg gagagacaaa gagactgtaa 180

ccgtatgga 189

<210> 427

<211> 324

<212> DNA

<213> Clostridium beijerinckii

<220>

<223> Clostridium beijerinckii NCIMB 8052, complete genome, CP000721.1

<400> 427

aatcaataaa aagcgccagg actaagtgga atttagttga aagtgaattt ttacagggtt 60

ggtttttata aaaggaattt agttaatagt aagctttgat ataagctacg cgaatagcta 120

atgaaatctttttaaaggaa actcgactca cgttcgttcg ctgagtaaat tcaactatcc 180

aaatcgaaga tttggagttt catttacgtc gacgaggttg gggagtatcg aaacttcggc 240

gggtgcccca cggtatcgca ctaccgtaaa cgactggtaa aactgtgaag tgattcacag 300

gacaaattca gtctggtgtt aaaa 324

<210> 428

<211> 147

<212> DNA

<213> Unknown

<220>

<223> Bacillus sp. CAG:988 genome scaffold, scf27, FR897768.1

<400> 428

gaattctttc tagcgccaga actttgaata gttgacgagg atagtagtga tcgaaaattc 60

ggcggatgct actacgtagg gaagatgcgt taggtatgtc taaaaagcaa aatcgacatt 120

gtaacaacag atgtatcact tttcaga 147

<210> 429

<211> 155

<212> DNA

<213> Desulfotomaculum reducens

<220>

<223> Desulfotomaculum reducens MI-1, complete genome, CP000612.1

<400> 429

taaataactg aagcgccaga accttttaca aagtaagggt tgacgaggag agggagtatc 60

gatgtttcgg cggatgccct ccggcccagt tgcggccgta aaagcagaac aaagctggaa 120

ggtaactttc ggtacaaaaa ctgcgggtga ctaaa 155

<210> 430

<211> 148

<212> DNA

<213> Mahella australiensis

<220>

<223> Mahella australiensis 50-1 BON, complete genome, CP002360.1

<400> 430

ttcgatataa aagcgccgga actcattttg agttgacgag gtcagggttt atcgattttt 60

cggcgggtgc cctgcggcat acggctgccg acaaaggttc cacaaaagca aaaagcgatt 120

tttgctacaa acgggactgg gccaaaat 148

<210> 431

<211> 202

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. CAG:7 genomic scaffold, scf260, HF990741.1

<400> 431

agtaaagcta tagcgccatg cacctgggtc gggtcgtgaa cagtaacgga tgaatgaata 60

ataacttact gctgacacag gttaacgagg tggagagaga atcgaacata ttcggcgggt 120

gctctcccat gcagtccggg cgtgttttat accagaaaaa tatgcgggta actgcaaaac 180

aaagctggta taaccggaca ga 202

<210> 432

<211> 173

<212> DNA

<213> Bacillus megaterium

<220>

<223> Bacillus megaterium QM B1551, complete genome, CP001983.1

<400> 432

agtgaataca aagcgcctga actaagtaaa gggacggaaa cgacttagtt gacgaggagg 60

aggtttatcg aagtatcggc ggatgcctcc cggttgttga ttatcacggc cgaaaacttg 120

atgtgaaaaa caatgaggtg acttattgga caaaagcatt gagatgataa tca 173

<210> 433

<211> 197

<212> DNA

<213> Caldicellulosiruptor saccharolyticus

<220>

<223> Caldicellulosiruptor saccharolyticus DSM 8903, complete genome, CP000679.1

<400> 433

ggttttttca aagcgccagg acctctggaa attatagcgc tattgccttt gcgcattttt 60

tccagaggtt gacgaggact ggggagaatc gaggttttcg gcgggtgccc cagcggggtt 120

ttgccttttt ccctcgcaac tttctgctac aaacccccgga aggcaacttc tgggacaaag 180

gcagaaagaa aaaaggc 197

<210> 434

<211> 159

<212> DNA

<213> Unknown

<220>

<223> Clostridium cellulosi genome assembly, chromosome: I, LM995447.1

<400> 434

ttaaatatta aagcgccagg accgatataa atcggttgac gaggtgggga gttatcgaaa 60

gattcggcgg gtgctcctcc ggccgttagt gttgcggtcg ttagctcatg ctacaaaaaa 120

cgcgggtaac cgcgcaaaaa aaggctgagc attcagcgc 159

<210> 435

<211> 152

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. CAG:245 genome scaffold, scf154, FR880072.1

<400> 435

agttcatttt tagcgccagg acagcgtttg tgctgttgac gaggttagag tttatcgaaa 60

tattcggcgg atgctctagg gctttctacg gtccttataa attagcaaaa cctagcagtg 120

atgttagaac agatataatt ttgagtagtt ta 152

<210> 436

<211> 157

<212> DNA

<213> Bacillus cereus

<220>

<223> Bacillus cereus cytotoxis subspecies NVH 391-98, complete genome, CP000764.1

<400> 436

agagtgatta aagcgccaga actacaaatt gtgtagttga cgaggaggag ttttatcgag 60

atttcggcgg atgactcccg gttattcatc ataaccgcaa gcttttattt aaatcactga 120

ggcgacttgg tggacaaaga taaaagtgtg atgagag 157

<210> 437

<211> 209

<212> DNA

<213> Staphylococcus aureus

<220>

<223> Staphylococcus aureus C0673 genome scaffold aedLz-supercont1.14, whole genome shotgun sequence, KK222758.1

<400> 437

aggaaactta tagcgcctga acaaagcgca tacacgattg tagaggcatg tataatcaga 60

tacatgctga atgagtgtta tgacctttgt tgacgaggag gatagttatc gaattttcgg 120

cggatgctat cccggatgtg gcccattcga agttcaatgt ttaaagcata taggtgactg 180

tatgtccaaa gacgttgaaa tagccataa 209

<210> 438

<211> 156

<212> DNA

<213> Clostridium grantii

<220>

<223> Clostridium grantii DSM 8605 genome assembly, contigs: EJ34DRAFT_scaffold00005.5, FQXM01000006.1

<400> 438

atattatagt tagcgccaga agtagtgggt aaagtgctac ttgacgagga tggggagtat 60

cgaaatttcg gcggatgccc cacggtataa cactatcgat aaacattggc aaagcaaaga 120

agtgattctttgtacaaatt caatggagtg tgaatc 156

<210> 439

<211> 153

<212> DNA

<213> Clostridium bartlettii

<220>

<223> Clostridium bartlettii CAG:1329 genome scaffold, scf11, HF999313.1

<400> 439

agtgtttata aagcgccaga acttaatttt ttaagttgac gaggtcagag ttaatcgaaa 60

tatcggcgga tgctctgcgg tgtgccacca tcgaaggatt tctacaaagg gtgaaagcaa 120

tttcactaca caaaaagaaa cccatgtgga ttg 153

<210> 440

<211> 154

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. SCN 57-10 ABT01_C0138, whole genome shotgun sequence, MEFT01000138.1

<400> 440

acacgttcaa aagcgccggg actgcgtgtg cagttgacga ggcggaggat gatcgaacca 60

ttcggcgggt gcctcctttt cgcgtcgcgc ggaaaagagc cgatgctcaa aacccgttcg 120

caaggacggc gacaaaagca cggcacgcga caca 154

<210> 441

<211> 178

<212> DNA

<213> Deinococcus radiodurans

<220>

<223> Deinococcus radiodurans R1 chromosome 1, complete sequence, AE000513.1

<400> 441

cttcttcggg cagcgcaagg ccccggcgac acgtgatgtc acaagccggg gagacgaggt 60

ggaggtcagc gacttttctg cggatgcctc caggccccgg tgaacgggcc tacccggcgc 120

gtgctttgcc gctctgagtc aaagactccg gcaggcagaa ccacgcgcaa gcccggcg 178

<210> 442

<211> 167

<212> DNA

<213> Fictibacillus macauensis

<220>

<223> Fictibacillus macauensis ZFHKF-1 contig 05, whole genome shotgun sequence, AKKV01000005.1

<400> 442

gcaataccac aagcgcctga actattttcg accggaatga aaatagttga cgaggaagag 60

gatcatcgag atttcggcgg atgcctctcg gatgacgtca catccgtaag cttttataca 120

aaccaggtaa ggtgacttcc tgtacaaaca taaaagtagt gacgaat 167

<210> 443

<211> 153

<212> DNA

<213> Clostridium methylpentosum

<220>

<223> Clostridium methylpentosum DSM 5476 Scfld6 genome scaffold, whole genome shotgun sequence, EQ973344.1

<400> 443

gggtacaacc aagcgccagg cccggataca gctccgggtg acgaggtgga gagtgatcga 60

acagaatcgg cggatgctct cccggtcccg gcaggaggat cgtcagatgg ttcacaaaag 120

cgcgttgcgc acaaaagagc caccccatcc cgc 153

<210> 444

<211> 158

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. CAG:470 genome scaffold, scf38, FR898135.1

<400> 444

gttatttttc aagcgccagg accactattt tcaggaggtt gacgaggtct agagttatcg 60

aaatattcgg cggatgctct agtggctttt acttaaggtc atcagtatta attaaaaact 120

aatagtaata ttagaacaga agttaatatt ttaagttt 158

<210> 445

<211> 159

<212> DNA

<213> Sporobacter termitidis

<220>

<223> Sporobacter termitidis DSM 10068 genome assembly, contigs: EK05DRAFT_scaffold00008.8, FQXV01000008.1

<400> 445

taaaatttag aagcgccaga cttcacgcag agcgcggagt tgacgagggc ggggtttatc 60

gaagtattcg gcgggtgccc cgtgctgcgg tccatcagca ttacaggttt tacaaatcct 120

caagcaattg acgggacaaa ataaacctga ttgggcctt 159

<210> 446

<211> 165

<212> DNA

<213> Listeria grayi

<220>

<223> Listeria grayi DSM 20601 genome scaffold SCAFFOLD1, whole genome shotgun sequence, GL538352.1

<400> 446

aatttaatag aagcgccaga actgatcgaa cggggtatca gttgacgagg aggagattaa 60

tcgagttttt tcggcgggag tctcccggtt attcatgtag ccgttatgtc tgagttacaa 120

aacaagcagg cgactgtttg gacagaaagc ttagacgcat gagtt 165

<210> 447

<211> 157

<212> DNA

<213> Unknown

<220>

<223> Oscillibacter sp. KLE 1745 genome scaffold 170, whole genome shotgun sequence, KI271673.1

<400> 447

cggcccatca cagcgccagc gcccgccgcc agcggcggga cgacgagggg aagggagtat 60

cgaaggattc ggcggatgcc cttccgtgcc cacgggcgcg tacacagccg gcaaatatgc 120

gggcaaccgc aaaacaaggg ggctgtgacc gtgaaga 157

<210> 448

<211> 155

<212> DNA

<213> Thermoanaerobacterium saccharolyticum

<220>

<223> Thermoanaerobacterium saccharolyticum JW SL-YS485, complete genome, CP003184.1

<400> 448

aactgaataa aagcgcctgg gcttagggga aactctaagt tgacgaggac aggggttaatc 60

gagttatcgg cggatgccct gcggcttcct gcggccgata gagaaccggg aaaaccatgg 120

gtgaccattg gcatagagcg gtttgagcag ggata 155

<210> 449

<211> 171

<212> DNA

<213> Gracilibacillus halophilus

<220>

<223> Gracilibacillus halophilus YIM-C55.5 contig_7, whole genome shotgun sequence, APML01000007.1

<400> 449

tattaatgga aagcgccagg gctatggtga acgaagaatc catagctgac gaggtggagg 60

ttttatcgag tttgatcggc ggatgcctcc cggttgttgc atctcaaccg tcacattttt 120

attcgaaaac atgaaggtaa ctttatgaac aagaataaaa ataagatgcc t 171

<210> 450

<211> 152

<212> DNA

<213> Salinicoccus alkaliphilus

<220>

<223> Salinicoccus alkaliphilus DSM 16010 genome assembly, contig: EJ97DRAFT_scaffold00003.3, FRCF01000004.1

<400> 450

aaaatcggaa tagcgcccgg gcagctttgc tgctgacgag gaggacggtc atcgatcatc 60

ggcggatacc gtcccggaca ctgaagtgtt cgttacaccg gatgttaaaa cccgcaagcg 120

attgcgggga cagagcaccg gtcgataatt gt 152

<210> 451

<211> 168

<212> DNA

<213> Butyricicoccus pullicaecorum

<220>

<223> Butyricicoccus pullicaecorum 1.2 genome scaffold acBRa-supercont1.1, whole genome shotgun sequence, KB976103.1

<400> 451

tgcatatcga tagcgccaga actgcggagc agcccgtttc gcagtggacg aggtaagggt 60

tgatcgaaaa ttcggcggat gacccttcgg ccgcgagtga gccggtcgtt agcaggacgt 120

gtaaaccccg ggagtgatcc cggaaacaga gcgtcttcgc atgaccca 168

<210> 452

<211> 198

<212> DNA

<213> Firmicutes bacteria

<220>

<223> Firmicutes bacteria CAG:176_63_11 Ley3_66761_scaffold_4747, whole genome shotgun sequence, MNSY01000086.1

<400> 452

tcctgcgctt ttgcgccttg tcttgacgaa aaatctctcg ccggcaggcc ggcggatatt 60

tttcttttcc gccgccgtgg gataacgagg tggggagcga tcgaaaattc ggcggatgct 120

cctccgcatc gtccggatgc gcacacagct cataaatatg cgggagaccg caaaacagag 180

gggctgtgac cggatgcg 198

<210> 453

<211> 168

<212> DNA

<213> Microgenomates

<220>

<223> Microgenomates bacteria OLB23 UZ22_OP11002CONTIG000039, whole genome shotgun sequence, LMZU01000039.1

<400> 453

cattgttata tagcgtgtgg cccccatctg cattggcagc ggggatacga ggagagggct 60

agcgaataat ctgcgggtac cctcaggccg aagccaagct gtcgacctta agtgtgctct 120

taaatccttc tggcaacaga agggacacgg gagcttgcat acagcata 168

<210> 454

<211> 174

<212> DNA

<213> Pyramidobacter piscolens

<220>

<223> Pyramidobacter piscolens W5455 contig 00008, whole genome shotgun sequence, ADFP01000071.1

<400> 454

gaacgaacgg aagcgccagg actgaaccgc ttctcactcc gcggcgacag tcgacgagga 60

cgaaagtgat cgaaccattc ggcggatgct ttcgtggcgg gcgaagggga gccatgaacc 120

tccgtcacaa aaccccgggg cgacccggga acaaacggcg gaggcccttc aatg 174

<210> 455

<211> 172

<212> DNA

<213> Bacillus sp. CHD6a

<220>

<223> Bacillus sp. CHD6a contig 17, whole genome shotgun sequence, LBMD01000017.1

<400> 455

tagatataca aagcgcctga actaagcgac ggacgaaacc atgcttagtt gacgaggagg 60

aggtttatcg atctatcggc ggatgcctcc cggttgccaa tcacaaccga ctttagggac 120

agtttaaagc ataaaggcaa ctttatgtac aaagactgtt actatgattg ca 172

<210> 456

<211> 147

<212> DNA

<213> Unknown

<220>

<223> Mycoplasma sp. CAG:472 genome scaffold, scf184, FR899424.1

<400> 456

atttttgtaa tagcgcatgg cctatttagg tgacgaggac ttacattgtc gatttatcag 60

cggatgtgta agcggagaag ttttacttcg taaaatacct taaaaaaata aaatcaaaat 120

tataacaaag aggtatttaa taaaaca 147

<210> 457

<211> 297

<212> DNA

<213> Clostridium novyi

<220>

<223> Clostridium novyi NT, complete genome, CP000382.1

<400> 457

aataatatta aagcgccaga acttaagttg agtaaatgag ggaataaagg aaactcaaac 60

tagacgtttg agtaagttcg actaactaaa tcatagattt aagaagttaa ggaaactcaa 120

actaaatgtt tgagtaagtt cgccttaacc aaatcgtaga tttggaggct cacttaagtt 180

gacgaggatg gggagtatcg agaattcggc ggatgtccca cggtatttat gtactaccga 240

taacagttag caaatctaaa aagcgatttt tagcacaaat ctaactaggt acatgat 297

<210> 458

<211> 228

<212> DNA

<213> Paenibacillus antarcticus

<220>

<223> Paenibacillus antarcticus strain CECT 5836 PBAT34, whole genome shotgun sequence, LVJI01000034.1

<400> 458

tgtatattaa aagcgccaga acttgactag cgcggggatgt aagatggacc gagggtatgg 60

atgagtacga tccgtttcgg tgcctgatta caacggcaga tcaagttgac gaggtggggg 120

tgttcgaaat gttcggcggg gacctcccgg tgatgcacca gagccgtgaa tcatatacgg 180

aaaataagcg ggcgactgct catacaacgt agtgatgggt gctaatta 228

<210> 459

<211> 224

<212> DNA

<213> Unknown

<220>

<223> Candidatus Gottesmanbacteria bacterium RIFCSPLOWO2_01_FULL_48_11 rifcsplowo2_01_scaffold_16357, whole genome shotgun sequence, MFJY01000009.1

<400> 459

cagagaacat aagcgtgtgg cccggctatc atgttcctcg ccgagctcgt cacaccagcc 60

gggcaagccc ggttgatgcg agcgaagcga gcataataac cgggatacga ggaaagggct 120

tatcggataa atctgcgggt gccctttggt agtactctct accacaacgg taatcagaaa 180

ctccaccggt aacggtggga acaaatgatt atcggcagag tcgc 224

<210> 460

<211> 144

<212> DNA

<213> Anaerococcus lactolyticus

<220>

<223> Anaerococcus lactolyticus ATCC 51172 genome scaffold SCAFFOLD12, whole genome shotgun sequence, GG666055.1

<400> 460

aaaaacaaca aagcgccaga tccctttggg atgacgaggg aggaagttat cgaaaattcg 60

gcggggagctt cctgggttca cagccttagt gttagacaaa agcagagagc aatttctgcg 120

acagagaaga cacagtggcc tcat 144

<210> 461

<211> 149

<212> DNA

<213> Enterococcus sp. 9D6_DIV0238

<220>

<223> Enterococcus sp. 9D6_DIV0238 scaffold00002, whole genome shotgun sequence, NIBQ01000002.1

<400> 461

aatataggaa tagcgccagg cctgttgttt caggtgacga ggagagagct tatcgaaaca 60

ttcggcggat agctctaggg gctgcactct acaagctgga aataaaaaat aattgcaaaa 120

ttataacaaa gattcagcta agcagaata 149

<210> 462

<211> 328

<212> DNA

<213> Clostridium amylolyticum

<220>

<223> Clostridium amylolyticum DSM 21864 genome assembly, contig: Ga0131114_103, FQZO01000003.1

<400> 462

tacaatataa aagcgccagg actagagtag agccaaatta aaatttggaa catatgatta 60

acttaccatg acgtatggag aagttggagc atatgataaa aggaaacttc ttctccgatg 120

ctaaagctta aaaaataaga aatatgaagc tttagcatct acgaagaaga aagcgaccgc 180

gccaaattaa aatttagagc ctctgcttta gttgacgagg atggggagta tcgagtcttc 240

ggcgggtgcc ccacggtagc gcactaccgt taacgattga taaagccggg aagtgatttc 300

tggaaacaac atcaatcagg tgttaaaa 328

<210> 463

<211> 84

<212> DNA

<213> Corchorus olitorius

<220>

<223> Corchorus olitorius cv. O-4 contig 22559, whole genome shotgun sequence, AWUE01022526.1

<400> 463

gcaataactc cgcctgtggc tgcactaaat acctacctct gcaaccacag ccggtcccaa 60

gcccggaaaa aggagggaggg ttgc 84

<210> 464

<211> 92

<212> DNA

<213> Upland cotton (Gossypium hirsutum)

<220>

<223> Chromosome 15 of Gossypium hirsutum cv. TM-1, whole genome shotgun sequence, CM003264.1

<400> 464

atgataactc cgcctgtgcc atattgaaac ctgagagtat atatcctttt cggcactgcc 60

ggtcccaagc ccggataaag gaggagggtc at 92

<210> 465

<211> 74

<212> DNA

<213> Mexican Characa (Astyanax mexicanus)

<220>

<223> Astyanax mexicanus chromosome 6, whole genome shotgun sequence, CM008305.1

<400> 465

ttagtaactc tgccaaagtg tctcttttaa ggtcactttg ccggtcccaa gcccggataa 60

aagaggaggg ttaa 74

<210> 466

<211> 81

<212> DNA

<213> Cephalotus follicularis

<220>

<223> Cephalotus follicularis DNA, scaffold: scaffold3557, isolate: St1, BDDD01003557.1

<400> 466

gtcataactc cgcctgtgta gcaatatgta taaccatgtg tacacagccg gtcccaagcc 60

cggatgaagg aggagggtga c 81

<210> 467

<211> 80

<212> DNA

<213> Macleaya cordata

<220>

<223> Macleaya cordata BLH2017 isolate scaffold525, whole genome shotgun sequence, MVGT01000217.1

<400> 467

tttttaactc cgccaatgca aatgttatgc cataacattt gcattgccgg tcccaagccc 60

gggtaaagga ggagggggaa 80

<210> 468

<211> 63

<212> DNA

<213> Macleaya cordata

<220>

<223> Macleaya cordata BLH2017 isolate scaffold7799, whole genome shotgun sequence, MVGT01000535.1

<400> 468

tttttaactc cgccaatgca tatgcattgc cggtctcaag cccgggtaaa ggaggaggga 60

gaa 63

<210> 469

<211> 57

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D6C49D_12908

<400> 469

gcacuaaugu agcucagacc ugugacaagc caaggcuaga aaaauacaga gucgugc 57

<210> 470

<211> 56

<212> RNA

<213> Unknown

<220>

<223> Unclassified sequence P1 type torsional ribozyme, URS0000D669BF_12908

<400> 470

ggccuaaugc agcauagucc ugucacaagc caggcugaaa aaugcagagu gaggca 56

<210> 471

<211> 85

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: In-R_005008, BABG01005008.1

<400> 471

gaaacccgct aggccgacag cctcaccgct gccgctggtg caagccccagc cgccccagac 60

cggggcgggc gctcatgggt aacag 85

<210> 472

<211> 82

<212> DNA

<213> Adelie Penguin (Pygoscelis adeliae)

<220>

<223> Adélie penguin (Pygoscelis adeliae) contig 107431, whole genome shotgun sequence, JMFP01107431.1

<400> 472

ttaggccgtt acctacagct gatgagctcc aagaagagcg aaacctttta agataggtcc 60

tgtagtattg gcctaacaat ct 82

<210> 473

<211> 77

<212> DNA

<213> Acanthisitta chloris

<220>

<223> Acanthisitta chloris contig 104940, whole genome shotgun sequence, JJRS01104940.1

<400> 473

attggctgtt agctgctgct cttgagctcc agaaagagca acactgctta ggtcctgcag 60

tactggccta caggtgt 77

<210> 474

<211> 109

<212> DNA

<213> Sea turtle (Chelonia mydas)

<220>

<223> Sea turtle (Chelonia mydas) contig 57739, whole genome shotgun sequence, AJIM01057739.1

<400> 474

ttaaccagtt acctacagct gatgagctcc aggaagagcg aaaccagttc cgccctgttt 60

cagtagttat gaaaagttcg gactggtcct gtagtactgt cctgcagca 109

<210> 475

<211> 82

<212> DNA

<213> Unknown

<220>

<223> African ostrich (Struthio camelus australis) contig 33602, whole genome shotgun sequence, JJRT01033602.1

<400> 475

ttaggctgtt acctatagct gatgagcttc aaaatgaatg aaaccactta aaataggtcc 60

tgtagaacta tccttagggc ca 82

<210> 476

<211> 77

<212> DNA

<213> Golden-collared Flycatcher (Manacus vitellinus)

<220>

<223> Golden-collared Flycatcher (Manacus vitellinus) contig 47454, whole genome shotgun sequence, JMFM02047454.1

<400> 476

tcaggccatt gcctataggt gatgagctcc atgaagagtg aaaccagtta ggtcctgtat 60

tgctagccta atgagca 77

<210> 477

<211> 83

<212> DNA

<213> Sea turtle (Chelonia mydas)

<220>

<223> Sea turtle (Chelonia mydas) contig 198956, whole genome shotgun sequence, AJIM01198956.1

<400> 477

ttgggctata gcctacagct aatgagcttc aaaaaggagc aaaagcattt aaaataggcc 60

ctgtagtatt agcctaatct aat 83

<210> 478

<211> 78

<212> DNA

<213> Sea turtle (Chelonia mydas)

<220>

<223> Sea turtle (Chelonia mydas) contig 141094, whole genome shotgun sequence, AJIM01141094.1

<400> 478

ttaagacatt gcctacagct gacaagctcc acaaagagca aaaccagtga ggatcctgta 60

acactgtcct acagagct 78

<210> 479

<211> 86

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 061484, whole genome shotgun sequence, AFYH01061484.1

<400> 479

acgttccagt tatctacagc tgatgagctc aaaggagagc gaaaccggac atcctgtccg 60

gtcttgtagt actggcttag ttgtct 86

<210> 480

<211> 77

<212> DNA

<213> Downy Woodpecker (Picoides pubescens)

<220>

<223> Downy Woodpecker (Picoides pubescens) contig 42547, whole genome shotgun sequence, JJRU01042547.1

<400> 480

ctgcaccgtt acctgcagcc gatgagctcc aaaaagagca aaaccagcca ggtcctgcag 60

tactggctgc agccttc 77

<210> 481

<211> 77

<212> DNA

<213> Mallard (Anas platyrhynchos)

<220>

<223> Mallard (Anas platyrhynchos) Pekin duck breed contig 108924, whole genome shotgun sequence, ADON01108924.1

<400> 481

aatgtctgtt acttgaagct gatgagctcc aaatagagca aaaccattta ggtcctatag 60

tactggcctg taagctt 77

<210> 482

<211> 111

<212> DNA

<213> Spot-tailed African Trogon (Apaloderma vittatum)

<220>

<223> Apaloderma vittatum, contig 91687, whole genome shotgun sequence, JMFV01091687.1

<400> 482

ttaaaccagt tacctacagc tgatgagctc cggaaagagc aaaaccagtt ctgttctatt 60

tcagcagtta tgaatgctaa gaactggtcc tgtagtaatg ttgaacatcg a 111

<210> 483

<211> 77

<212> DNA

<213> Yellow-throated Sandgrouse (Pterocles gutturalis)

<220>

<223> Yellow-throated sandgrouse (Pterocles gutturalis), contig 86319, whole genome shotgun sequence, JMFR01086319.1

<400> 483

ttaggccatt atctgcagct gatgagctcc aagaagagca aaatccgtta ggtcctacaa 60

tgctggccta atacaca 77

<210> 484

<211> 77

<212> DNA

<213> Jungle fowl (Gallus gallus)

<220>

<223> Gallus gallus isolate RJF #256 Red Junglefowl breed, inbred line UCD001 chromosome 10, whole genome shotgun sequence, CM000102.4

<400> 484

cttggccatt acctgcagct ggtgagctcc aaaaagagcg gtgccattta ggtcatgtca 60

ttctggccta tatgttt 77

<210> 485

<211> 77

<212> DNA

<213> White-collared Flycatcher (Ficedula albicollis)

<220>

<223> White-collared Flycatcher (Ficedula albicollis) OC2 isolate chromosome 10, whole genome shotgun sequence, CM001999.1

<400> 485

attgaatgtt acttgcagct gatgggctcc aaaaagagag aagcccctta ggtcctgtag 60

tactggactt gaaacat 77

<210> 486

<211> 77

<212> DNA

<213> Colius striatus

<220>

<223> Colius striatus contig 35117, whole genome shotgun sequence, JJRP01035117.1

<400> 486

attggctgtt acatgcagct agtgagctcc aaaaagagtg aaaccactta gctcctgtaa 60

ttctggcttataagtgt 77

<210> 487

<211> 77

<212> DNA

<213> Scarlet Hummingbird (Calypte anna)

<220>

<223> Vermilion Hummingbird (Calypte anna) contig 32988, whole genome shotgun sequence, JJRV01032988.1

<400> 487

attggccatt atctgcagct gatgagctcc aaaaagagca ggaccacttg gatcctttag 60

tactgccctataagtgt 77

<210> 488

<211> 85

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 209269, whole genome shotgun sequence, AFYH01209269.1

<400> 488

aatttcagtt atctgcagct gatgagctta aataaaagca aaactggaat taatttccag 60

tcctgcagta ctggttaata tgcct 85

<210> 489

<211> 78

<212> DNA

<213> Magenta Bee-eater (Merops nubicus)

<220>

<223> Magenta Bee-eater (Merops nubicus) contig 71858, whole genome shotgun sequence, JJRJ01071858.1

<400> 489

attgtccact acctgcagct gatgaacctc aaaaaagagc aaaactccct aggtcctgta 60

gtagtggcca gtaagtgt 78

<210> 490

<211> 80

<212> DNA

<213> Latimeria chalumnae

<220>

<223> Latimeria chalumnae contig 106573, whole genome shotgun sequence, AFYH01106573.1

<400> 490

caatccagtt atctacagct gacgagctct aatgagagcg aaactgggga accggtcctg 60

tggtactggc atgaaagaaa 80

<210> 491

<211> 77

<212> DNA

<213> Egret (Egretta garzetta)

<220>

<223> Egretta garzetta contig 72922, whole genome shotgun sequence, JJRC01072922.1

<400> 491

attggccact acccgcagcc ggtgagctct aaaaagagtg aaaccacttg ggtcttgtgg 60

tattggccga taagcgt 77

<210> 492

<211> 83

<212> DNA

<213> American alligator (Alligator mississippiensis)

<220>

<223> American alligator (Alligator mississippiensis) ScZkoYb_152, whole genome shotgun sequence, AKHW03006215.1

<400> 492

gcaggccatt acttgcagct aatgagttcc acagagaatg aaaccatttg aaattggtcc 60

ctgaagtact ggcctagaaa act 83

<210> 493

<211> 77

<212> DNA

<213> Echinops telfairi

<220>

<223> Echinops telfairi cont1.498693, whole genome shotgun sequence, AAIY01498693.1

<400> 493

ccgagccgtt gcctgcagct gatgagctcc aacaagagcg aaaccgaaca ggtcctgcag 60

tacgggtggggtcagca 77

<210> 494

<211> 52

<212> DNA

<213> Chicory yellow mottle virus

<220>

<223> Chicory yellow mottle virus satellite RNA gene for hypothetical protein, D00721.1

<400> 494

caacagcgaa gcgcgccagg gaaacacacc atgtgtggta tattatctgg ca 52

<210> 495

<211> 52

<212> DNA

<213> Arabias mosaic virus

<220>

<223> Arabis mosaic virus minisatellite RNA, complete genome, M21212.1

<400> 495

caacagcgaa gcggaacggc gaaacacacc ttgtgtggta tattacccgt tg 52

<210> 496

<211> 50

<212> DNA

<213> Tobacco ringspot virus

<220>

<223> Tobacco ringspot virus satellite RNA, M14879.1

<400> 496

aaacagagaa gtcaaccaga gaaacacacg ttgtggtata ttacctggta 50

<210> 497

<211> 70

<212> DNA

<213> Cyphomyrmex costatus

<220>

<223> Cyphomyrmex costatus contig 21873, whole genome shotgun sequence, LKEX01021873.1

<400> 497

gattctcaac aatcgtctac ctccccgtgg tgagaatcgg gaaacatttc aaataatggc 60

taaagacgat 70

<210> 498

<211> 73

<212> DNA

<213> Crassostrea gigas

<220>

<223> Crassostrea gigas strain 05x7-T-G4-1.051#20 contig28208, whole genome shotgun sequence, AFTI01028208.1

<400> 498

cctgctcaaa atcctacttc cacctccccg cggcgagcag ggggcaacgg acatttgtcc 60

ggcgaacgga aga 73

<210> 499

<211> 74

<212> DNA

<213> Clostridium saccharolyticum

<220>

<223> Draft genome of Clostridium saccharolyticum-like K10, FP929037.1

<400> 499

gctgctcgaa actttgcaca cctcttcgtg gtgagcagca ggcaacgatc ttatggtcgg 60

ctaagaatgc agag 74

<210> 500

<211> 75

<212> DNA

<213> Irregular arbuscular mycorrhizal fungi (Rhizophagus irregularis)

<220>

<223> Rhizophagus irregularis DAOM 197198wjcf7180003189428, whole genome shotgun sequence, JEMT01023831.1

<400> 500

cctgaacgaa gcttgccacc tctacgtggt gttcaggaga aacagttgta agttaataac 60

tggccaagag caagc 75

<210> 501

<211> 74

<212> DNA

<213> Florida Camponotus floridanus

<220>

<223> Camponotus floridanus CamFlo_1.0_4.contig2489, whole genome shotgun sequence, AEAB01026452.1

<400> 501

gattttccat tatatgttta cctccacgcg gtgaaaatcg ggcaacgtca aataaattgg 60

cggcaaaaga acgt 74

<210> 502

<211> 72

<212> DNA

<213> Tulasnella calospora

<220>

<223> Tulasnella calospora MUT 4182 unplaced genome scaffold_124, whole genome shotgun sequence, KN823065.1

<400> 502

ggggctcgat gatgcgcaca cctccccgtg gtgagccctg tcaacgtcgg caaggacggc 60

caagatgcgc at 72

<210> 503

<211> 72

<212> DNA

<213> Trichomalopsis sarcophagae

<220>

<223> Trichomalopsis sarcophagae Alberta strain scaffold26742, whole genome shotgun sequence, NNAY01026514.1

<400> 503

gatgttttga ctcattcacc tccacgcggt aagtatcggg atacgttgta catcaacggc 60

taagaaatga ga 72

<210> 504

<211> 69

<212> DNA

<213> Unknown

<220>

<223> Faecalibacterium cf. prausnitzii KLE1255 F_prausnitziiKLE1255.K95-1.0_Cont34.1, whole genome shotgun sequence, AECU01000025.1

<400> 504

gctgtccgaa aatgctgcct ctacgtggcg gacggcaggc aacggagcgt gtctccggct 60

aaagcatga 69

<210> 505

<211> 73

<212> DNA

<213> Hymenolepis nana

<220>

<223> Hymenolepis nana genome assembly, scaffold: HNAJ_contig0000132, LM398097.1

<400> 505

gggcaacgta tactcataca cctccacgtg gtgcaccctg ggcaacgtat attcatatgg 60

caaaaatgtc tat 73

<210> 506

<211> 75

<212> DNA

<213> Clostridiales bacteria

<220>

<223> Clostridiales bacteria 41_21_two_genomes Ley3_66761_scaffold_672, whole genome shotgun sequence, MNRE01000164.1

<400> 506

gctgtttgga taatcacaca ccgatgcgag gttagcagca ggcaacacag cggaagctat 60

ggcgaagatg caatg 75

<210> 507

<211> 75

<212> DNA

<213> Tulasnella calospora

<220>

<223> Tulasnella calospora MUT 4182 unplaced genome scaffold_99, whole genome shotgun sequence, KN823040.1

<400> 507

ggggttcgag ctgtacgcgt acctcctcgt ggtgaaccct gggcaacgct ctgacggagc 60

ggctgaatcg cgtac 75

<210> 508

<211> 68

<212> DNA

<213> Cerapachys biroi

<220>

<223> Unplaced genome scaffold 278 of Cerapachys biroi, whole genome shotgun sequence, KK107279.1

<400> 508

aactctaaaa cgtccacctc cacgtggtta gagttgggca acgttaaaca ttaacggcta 60

acggacga 68

<210> 509

<211> 69

<212> DNA

<213> Clostridium clostridioforme

<220>

<223> Clostridium clostridioforme CAG:132 genome scaffold, scf345, FR886101.1

<400> 509

gccgcccata ggtgctgcct ctgcgtggcg ggtggcaggc aacggaggag ttctccggct 60

aaagcactg 69

<210> 510

<211> 78

<212> DNA

<213> Intestinal parasitic nematodes (Heligmosomoides polygyrus)

<220>

<223> Genome assembly of the intestinal parasitic nematode Heligmosomoides polygyrus, scaffold: HPBE_contig0009563, LL216641.1

<400> 510

gcttcccgat gacggtgcca cctccacgtg gtgggaagcg ggcaacgggt ttggattggc 60

gcccggctaa gagcaccg 78

<210> 511

<211> 77

<212> DNA

<213> Nasonia vitripennis

<220>

<223> Nasonia vitripennis unplaced genome scaffold ChrUn_0243, whole genome shotgun sequence, GL341474.1

<400> 511

gggttttcaa tgaacgttca ccttcacgtg gtgaaacccg ggcaacgtta cattcagcag 60

cggctaagaa cgttcac 77

<210> 512

<211> 70

<212> DNA

<213> Unknown

<220>

<223> Ruminococcus sp. CAG:724 genome scaffold, scf297, HF994873.1

<400> 512

gttacgcaag atcaaagcct cctcgcggcg cgtagcgggc aacgaatttt cattcggctg 60

atttgatcga 70

<210> 513

<211> 79

<212> DNA

<213> Cyphomyrmex costatus

<220>

<223> Cyphomyrmex costatus contig 15289, whole genome shotgun sequence, LKEX01015289.1

<400> 513

agttttcgaa agtcgttcac ctcctcgtgg tgaaaactgg ataacgttta aataactgat 60

aaacggcaaa gaaacgaca 79

<210> 514

<211> 72

<212> DNA

<213> Unknown

<220>

<223> Ruminococcus sp. 18P13 draft genome, FP929052.1

<400> 514

gccgcacaaa atcaaagcct ccacgtggcg tgcggcggac aacggatgat tgatccggct 60

aagattgattga 72

<210> 515

<211> 77

<212> DNA

<213> Caenorhabditis tropicalis

<220>

<223> Unplaced genome scaffold 629 of Caenorhabditis tropicalis JU1373 strain, whole genome shotgun sequence, GL637601.1

<400> 515

cttctacacg tacttcgcct ctccgtggcg tagaagaggc aacactcctg ggcaaccaga 60

gtggctaaga agtacac 77

<210> 516

<211> 75

<212> DNA

<213> Hymenolepis nana

<220>

<223> Hymenolepis nana genome assembly, scaffold: HNAJ_contig0006064, LM407409.1

<400> 516

ggggtgctag atagacttac ctccacgcgg tgtaccctgg gcaacgtata ctcatcatac 60

ggcaaataag tcaat 75

<210> 517

<211> 75

<212> DNA

<213> Unknown

<220>

<223> Anaerotruncus sp. CAG:390 genome scaffold, scf127, FR897605.1

<400> 517

accgcacagg gtcaaagcct ccacgtggcg tgcggtgggc aacggacaac tcgctcgtcc 60

ggctgatttg accat 75

<210> 518

<211> 70

<212> DNA

<213> Faecalibacterium prausnitzii

<220>

<223> Faecalibacterium prausnitzii L2 6 draft genome, FP929045.1

<400> 518

gccgctcaga tatgctacct ctccgtggtg agcagcaggc aacgagagtt ttctctcggc 60

taaagcatat 70

<210> 519

<211> 75

<212> DNA

<213> Trichomalopsis sarcophagae

<220>

<223> Trichomalopsis sarcophagae Alberta strain scaffold35, whole genome shotgun sequence, NNAY01000035.1

<400> 519

ggttctcttt catcgttcac ctccccgtgg tgagaaccgg gcaacacaac atttcagagt 60

ggcaaagaaa cgatt 75

<210> 520

<211> 84

<212> DNA

<213> Trichomalopsis sarcophagae

<220>

<223> Trichomalopsis sarcophagae Alberta strain scaffold18563, whole genome shotgun sequence, NNAY01018372.1

<400> 520

gattctcaaa agcgttcacc tcctcgtggt gagaatcggg caactctgat gtttacgaat 60

aaatcagagg caaagaacgc gtga 84

<210> 521

<211> 82

<212> DNA

<213> Steinernema glaseri

<220>

<223> Unplaced genome scaffold GLAS_3282 of Steinernema glaseri NC strain, whole genome shotgun sequence, KN169778.1

<400> 521

ggaagacgac gagctacacc tccacgtggt gtcttccggg caacgttagg gcttctgggt 60

cctaacggca aagacagctc ta 82

<210> 522

<211> 83

<212> DNA

<213> Oesophagostomum dentatum

<220>

<223> Oesophagostomum dentatum strain OD-Hann O_dentatum-1.0_Cont728411.1, whole genome shotgun sequence, JOOK01112482.1

<400> 522

ggtcctcata gctgccacct ccacgtggtg aggaccgggc aacgttggtg cttctggagc 60

caccaacggc taaggcagcg tgg 83

<210> 523

<211> 71

<212> DNA

<213> Gonapodya prolifera

<220>

<223> Gonapodya prolifera JEL478 unplaced genome scaffold M427scaffold_56, whole genome shotgun sequence, KQ965786.1

<400> 523

ggagcgcgat ggcccgccca cctctacgtg gtgcgctcta gaaacaccag tttggtggct 60

gagaggcggg c 71

<210> 524

<211> 71

<212> DNA

<213> Gonapodya prolifera

<220>

<223> Gonapodya prolifera JEL478 unplaced genome scaffold M427scaffold_140, whole genome shotgun sequence, KQ965870.1

<400> 524

ggggtacacg gtgactgcct cctcgtggcg taccccgggc aacgttcgat tttcgaacgg 60

ctgaagtcac c 71

<210> 525

<211> 74

<212> DNA

<213> Trichomalopsis sarcophagae

<220>

<223> Trichomalopsis sarcophagae Alberta strain scaffold15944, whole genome shotgun sequence, NNAY01015791.1

<400> 525

gattctcaat gtttgctaac ctccacgtgg tgagaatcgg gcaacgttta tttataaacg 60

gcaaagaggc aata 74

<210> 526

<211> 82

<212> DNA

<213> Unknown

<220>

<223> Clostridium sp. C105KSO13 isolate C105KSO131 genome assembly, FBWL01000170.1

<400> 526

gctactcgga caaatcaaaa aattacacac ctcttcgtgg taagcagcag acaacgattt 60

tatgatcggc gaagatgtga ga 82

<210> 527

<211> 77

<212> DNA

<213> Clostridiales bacteria

<220>

<223> Clostridiales bacteria 41_21_two_genomes Ley3_66761_scaffold_1913, whole genome shotgun sequence, MNRE01000064.1

<400> 527

gttgctcgaa tgcgaatgaa tcacacacct ctccgtggtg agcagcaggc aatgaagtta 60

tatcataaaa tttttaa 77

<210> 528

<211> 76

<212> DNA

<213> Cyphomyrmex costatus

<220>

<223> Cyphomyrmex costatus contig 10795, whole genome shotgun sequence, LKEX01010795.1

<400> 528

ggttatcgat aagcgtccac ctcctcgcgg tgataaccgg gcaacgttga attcatcagc 60

ggcaaaggac gtctaa 76

<210> 529

<211> 74

<212> DNA

<213> Unknown

<220>

<223> Ruminococcus sp. CAG:353 genome scaffold, scf176, FR901357.1

<400> 529

gctgctcgaa aaatgcacac cgctacgagg tgagcagcag acaacacagc agagactgtg 60

gctaagaggc aaga 74

<210> 530

<211> 72

<212> DNA

<213> Hymenolepis nana

<220>

<223> Hymenolepis nana genome assembly, scaffold: HNAJ_scaffold0000733, LM398231.1

<400> 530

ggggtgtgag acagacttac ctcaacgtgg tacaccccag gcaacgtata tttatgcggc 60

aaaaaagttt at 72

<210> 531

<211> 75

<212> DNA

<213> Caenorhabditis brenneri

<220>

<223> Caenorhabditis brenneri strain PB2801 C_brenneri-6.0.1_Cont82.14, whole genome shotgun sequence, ABEG02002846.1

<400> 531

cttcttcgac ggtactaacc tctacgcggt gaagaagaga caacagtttc tgatgaaact 60

ggctaataag tacca 75

<210> 532

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-X_007529, BAAZ01007529.1

<400> 532

gctactcaaa aaaagacagc ctccacgcgg cgagcagcgg gcaacgggaa agacccggca 60

gatagtcttt a 71

<210> 533

<211> 80

<212> DNA

<213> Unknown

<220>

<223> Uncultured Faecalibacterium sp. TS29_contig04278, whole genome shotgun sequence, ADJT01005907.1

<400> 533

tccgctcgaa actttgcaca cctctacgcg gtgggcggca ggcaacacag tgtgtagatg 60

ctgtggcaaa gaatgcaaga 80

<210> 534

<211> 76

<212> DNA

<213> Unknown

<220>

<223> Ruminococcus sp. 5_1_39B_FAA cont1.60, whole genome shotgun sequence, ACII01000060.1

<400> 534

gctgctcaga aatgcacact gcgactggtg agcagtaggt gatgtttatc aaaggataag 60

cggctaagat gtagaa 76

<210> 535

<211> 75

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 1577600, whole genome shotgun sequence, AACY021109846.1

<400> 535

gggggacgaa gtcgaactga acacctccat cgtggtgtcc cccgggcaac gcttgcaaaa 60

gcggctaacg ttcag 75

<210> 536

<211> 89

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-V_032439, BAAX01032439.1

<400> 536

gctgctcgta ataatcacac acctctccgt ggtgagcagc aggcaacgat ttaagaatgt 60

atggttcgat gatcggcgaa gatgtgcga 89

<210> 537

<211> 71

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-X_004974, BAAZ01004974.1

<400> 537

actgcacaaa accaacagcc ttcacgcggc gtgcagtgag caacgtatag ttttatacgg 60

ccaatgttga a 71

<210> 538

<211> 76

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-W_003903, BAAY01003903.1

<400> 538

gccgctttat attttgtaca cctctacgtg gtaagcggca ggcaacgttt attttataga 60

cggctaagat gcaaaa 76

<210> 539

<211> 89

<212> DNA

<213> Coprococcus comes

<220>

<223> Coprococcus comes ATCC 27758 C_comes-1.0.1_Cont1600, whole genome shotgun sequence, ABVR01000037.1

<400> 539

gctgctcgaa tgaatcacac acctctttgt ggtgagtagc aggcaacgat ctaagaatca 60

gggatccggt gatcggctga gatgtgaag 89

<210> 540

<211> 78

<212> DNA

<213> Unknown

<220>

<223> Marine metagenome 1095527145240, whole genome shotgun sequence, AACY021449234.1

<400> 540

atctcacaac gttaatcgcc tcctcgtggc gtgagatgga aacagcatat ttgcaaatat 60

gttggctaag attaacag 78

<210> 541

<211> 45

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold1749_15, whole genome shotgun sequence, AMPZ01025371.1

<400> 541

tcccggctga cgagtctcaa acagaacgta atgcgcgtcc tggat 45

<210> 542

<211> 49

<212> DNA

<213> Schistosoma mattheei

<220>

<223> Genome assembly of Schistosoma mattheei, Zambia Denwood strain, scaffold: SMTD_contig0008514, LM184686.1

<400> 542

atccatctga tgaatcctaa aataggacga aacatgcgtc aaactggat 49

<210> 543

<211> 45

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_scaffold0000569, LL877594.1

<400> 543

atccaactga tgtgtcttag gtaaaacgaa acacgcgtcc tggat 45

<210> 544

<211> 45

<212> DNA

<213> Schistosoma curassoni

<220>

<223> Genome assembly of Schistosoma curassoni Dakar, Senegal, scaffold: SCUD_scaffold0001340, LM066427.1

<400> 544

accctaatga aaagtgccaa atagtacgaa acttaagtct agggt 45

<210> 545

<211> 216

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold839_8, whole genome shotgun sequence, AMPZ01016641.1

<400> 545

attcagctga cgagtcccaa tgtcgtgttt gaatgaacaa atgattcctt tgtacttgtt 60

gaatgcttga tttcgaattc taaatacagt acattcgctt gtgctttct actacttctt 120

gatccaatta cgttatttct ggaattctta gttcatacta taactcaaag actcctaatt 180

atcacacccg agtaggatga aacgcgcgtc ctgaat 216

<210> 546

<211> 68

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0004017, LL960995.1

<220>

<221> Features not yet classified

<222> (33)..(55)

<223> n is a, c, g, or t

<400> 546

atccagctga cgagtcccga ataggacaaa acnnnnnnnn nnnnnnnnnn nnnnngagcg 60

ttctggat 68

<210> 547

<211> 45

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0005707, LL962685.1

<400> 547

atccagacga cgagtccaag acaggaccaa acgcgctttt tgtat 45

<210> 548

<211> 45

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0002741, LL959719.1

<400> 548

acccagatga tgagtctcac ataaaacgaa acgtacgtct tagat 45

<210> 549

<211> 49

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0001662, LL001662.1

<400> 549

atccagatga cgagtcccag gtcgaacgaa atgcgcatcc tggctggat 49

<210> 550

<211> 46

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold572_14, whole genome shotgun sequence, AMPZ01012007.1

<400> 550

attctactga cgagtcccaa acaggacgag atggatttta tagaat 46

<210> 551

<211> 48

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0038465, LL038740.1

<400> 551

atccggatga cgagtccaaa atagggtgaa atacgcgtaa tcctggat 48

<210> 552

<211> 45

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold265_6, whole genome shotgun sequence, AMPZ01005699.1

<400> 552

accttgcgga cgagtaccaa atagcacgaa acccgggtcc agggt 45

<210> 553

<211> 45

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0003196, LL960174.1

<400> 553

atccatctga cgagccctaa atggggcgaa atgcacatcc tgcac 45

<210> 554

<211> 46

<212> DNA

<213> Schistosoma mansoni

<220>

<223> Schistosoma mansoni Puerto Rico strain chromosome 1, complete genome, HE601624.1

<400> 554

atccagccga agagttcaaa atttagacga aatgtgcgtc caggat 46

<210> 555

<211> 45

<212> DNA

<213> Schistosoma mansoni

<220>

<223> Schistosoma mansoni Puerto Rico strain chromosome 4, complete genome, HE601627.1

<400> 555

gtccagccga tgagttcgaa ataggatgaa acgcacgtcc tgaat 45

<210> 556

<211> 45

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_scaffold0001143, LL878569.1

<400> 556

attcaactga tgggttcaaa ataggacgga gctcgcgtcc tgaat 45

<210> 557

<211> 48

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_contig0000066, LL877199.1

<400> 557

atctagctga cgtgtctcaa atagggtgaa acgcgcatca aactggat 48

<210> 558

<211> 46

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0007499, LL964478.1

<400> 558

gtcttgctga ggagtcccac aattggacaa aacgatcgtc cagtac 46

<210> 559

<211> 45

<212> DNA

<213> Schistosoma mattheei

<220>

<223> Genome assembly of Schistosoma mattheei, Zambia Denwood strain, scaffold: SMTD_scaffold0000113, LM149431.1

<400> 559

atccagacga tgagtcgcaa tcaggacaaa acgcgtgtcc tgcat 45

<210> 560

<211> 314

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0002417, LL959395.1

<400> 560

tcactgctga ggagtcccac aacagggtga aacgaccatc cagtgctttc aggttctcca 60

tagtggtcca gcttcaatcg actcatgatt tcaactgtta aaatactaaa tctccacaaa 120

aacccttctg ataattcata atagatcaga ggggggtttg tggagaattt agtattttaa 180

cagttgaaat catgagtcga ttgaagctag accatcattg aaaacctgaa agcactggac 240

ggccatttcg ttctattatg ggaatcctca gcagtgcgca tccataataa taggacgaaa 300

cggccgtcca gtgc 314

<210> 561

<211> 45

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_scaffold0000011, LL876856.1

<400> 561

attcagctga cgagtgttga ataagacgga acgtgcatcc tgaat 45

<210> 562

<211> 51

<212> DNA

<213> Echinostoma caproni

<220>

<223> Genome assembly of Echinostoma caproni Egyptian strain, scaffold: ECPE_scaffold0005374, LL238470.1

<400> 562

gcactgctga cgagtcccag acaggacgaa acaacaacaa ctgtccagtg c 51

<210> 563

<211> 45

<212> DNA

<213> Schistosoma curassoni

<220>

<223> Genome assembly of Schistosoma curassoni Dakar, Senegal, scaffold: SCUD_contig0027497, LM120165.1

<400> 563

accttggtga cgagtgtcaa ataggacgaa acttaggtcc atgat 45

<210> 564

<211> 46

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0038963, LL039251.1

<400> 564

gtatcaccga agagtcccaa actaggacga aacagcagtc taatac 46

<210> 565

<211> 45

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0000311, LL957289.1

<400> 565

attcaactaa tgaatcccaa gtagaacgaa acgtacgtcc tgaat 45

<210> 566

<211> 141

<212> DNA

<213> Schistosoma mattheei

<220>

<223> Genome assembly of Schistosoma mattheei, Zambia Denwood strain, scaffold: SMTD_scaffold0017800, LM169888.1

<400> 566

attcagctga cgagtcccac ttagctattg agtcctgata attacttgct tgtgcaattt 60

ctgaagagaa catcaactct gggatgtagg cacatcctgc tgatgggtcg caaataggac 120

gaaacgcgcg tcaaaccgga t 141

<210> 567

<211> 45

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_contig0000349, LL878022.1

<400> 567

atctatctga caagtcctaa ataggactaa acgtgcgttc tgaat 45

<210> 568

<211> 46

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0003993, LL003993.1

<400> 568

gcaccgatga agagtcctaa aataggacga aacggctgtc tggcgc 46

<210> 569

<211> 45

<212> DNA

<213> Schistosoma curassoni

<220>

<223> Genome assembly of Schistosoma curassoni Dakar, Senegal, scaffold: SCUD_scaffold0002666, LM067904.1

<400> 569

acctagccga cgagtctgaa ataggacaaa acgtgtgtcc ttgat 45

<210> 570

<211> 46

<212> DNA

<213> Octopus bimaculoides

<220>

<223> Octopus bimaculoides Scaffold16004_contig_23, whole genome shotgun sequence, LGKD01170204.1

<400> 570

ttctggctga cgaaacacaa caggtcgaaa caccggtgtc ccagaa 46

<210> 571

<211> 45

<212> DNA

<213> Schistosoma mansoni

<220>

<223> Schistosoma mansoni Puerto Rico strain chromosome 7, complete genome, HE601630.1

<400> 571

atttagctga tgtatcccaa acaaaacgaa acacacgtca tgaat 45

<210> 572

<211> 45

<212> DNA

<213> Opisthorchis viverrini

<220>

<223> Opisthorchis viverrini opera_v5_148.27, whole genome shotgun sequence, JACJ01014299.1

<400> 572

gcactgctga tgagctctaa ttagagcgaa actcgagtcc agtgc 45

<210> 573

<211> 45

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold104_9, whole genome shotgun sequence, AMPZ01005908.1

<400> 573

attcaactga taagtcccaa acaggatgaa ataagcatct tgaat 45

<210> 574

<211> 49

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold15_47, whole genome shotgun sequence, AMPZ01001461.1

<400> 574

gcattgctga ggagtcccac aataagacga aacgtccgtc aaacaatac 49

<210> 575

<211> 214

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold555_12, whole genome shotgun sequence, AMPZ01011692.1

<400> 575

attgctgagg agtcccatac tagtatttta gtattttaga ttgaataaaa cttcataaac 60

aaagatggat agtggctagc agtggaatcc aggacacgcg tttcgtccta tttgtgactc 120

gttagctaga tggtcctgca tttcagagtt gatgttcact ctaggactcg aacccagtac 180

cgttcgctac aaggacgaaa cgcgcgtcct gaat 214

<210> 576

<211> 46

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_scaffold0000277, LL877183.1

<400> 576

gcactactga ggagtcccag aacaaaagga aacggccgtc tagtgt 46

<210> 577

<211> 46

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold631_7, whole genome shotgun sequence, AMPZ01013432.1

<400> 577

acactgctga agagtcctac aatgggacga aacagccgtc tggtat 46

<210> 578

<211> 45

<212> DNA

<213> Schistosoma haematobium

<220>

<223> Schistosoma haematobium scaffold313_14, whole genome shotgun sequence, AMPZ01007250.1

<400> 578

atccaactga caaatcccaa acaggatgaa acgcacgtcc tctat 45

<210> 579

<211> 45

<212> DNA

<213> Schistosoma rodhaini

<220>

<223> Genome assembly of Schistosoma rodhaini Burundi strain, scaffold: SROB_scaffold0000033, LL957011.1

<400> 579

atccaactga tgagtgtcaa ataggacaaa actctagttc tgtat 45

<210> 580

<211> 46

<212> DNA

<213> Schistosoma curassoni

<220>

<223> Genome assembly of Schistosoma curassoni Dakar, Senegal, scaffold: SCUD_scaffold0004111, LM069637.1

<400> 580

gtattgttga ggagtcgcat accagggcga aacggccgtc caatac 46

<210> 581

<211> 45

<212> DNA

<213> Schistosoma margrebowiei

<220>

<223> Schistosoma margrebowiei Zambian strain genome assembly, scaffold: SMRZ_contig0000159, LL877504.1

<400> 581

atccgtctga caagtcctag atagaacgag acgcgcgtct tggat 45

<210> 582

<211> 55

<212> DNA

<213> Schistosoma mansoni

<220>

<223> Schistosoma mansoni Puerto Rico strain chromosome W, complete genome, HE601631.1

<400> 582

atccaactga tgagtcccaa atagaaccaa ataggacgaa atgcatgtcc tggat 55

<210> 583

<211> 108

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0029912, LL030011.1

<220>

<221> Features not yet classified

<222> (21)..(85)

<223> n is a, c, g, or t

<400> 583

atccagctga tgagtcccaa nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60

nnnnnnnnnn nnnnnnnnnn nnnnnaggac gaaatgtgca tcttggat 108

<210> 584

<211> 46

<212> DNA

<213> Trichobilharzia regenti

<220>

<223> Trichobilharzia regenti genome assembly, scaffold: TRE_scaffold0035981, LL036185.1

<400> 584

atcgaaatga cgagtcccaa atggaacgaa acccgtgtct tttgat 46

<210> 585

<211> 151

<212> DNA

<213> Unknown

<220>

<223> Human intestinal metagenomic DNA, contig sequence: F2-X_000382, BAAZ01000382.1

<400> 585

aattcattcg caaagtaatt attctatgaa atgcaaatta tcttcatatg ttgtgaaaca 60

tagcttaacc acgttaaagt ataataatat aagttaggta tgcccttata aagacttagg 120

tagcgctaag gactatatta ttatacttct t 151

<210> 586

<211> 484

<212> PRT

<213> Pyrobaculum aerophilum

<400> 586

Met Arg Asn Ile Pro Ile Asn Lys Ile Asn Asp Tyr Val Trp Glu Ile

1 5 10 15

Pro Pro Gly Val Lys Pro Cys Gln Lys Val Pro Val Arg Ile Tyr Ala

20 25 30

Asp Ser Val Leu Leu Glu Lys Met Lys Ser Asp Met Thr Leu Glu Gln

35 40 45

Gly Ile Asn Val Gly Cys Leu Pro Gly Ile Tyr Arg Trp Ser Ile Val

50 55 60

Leu Pro Asp Ala His Gln Gly Tyr Gly Phe Pro Ile Gly Gly Val Ala

65 70 75 80

Ala Ile Asp Ala Glu Glu Gly Val Ile Ser Pro Gly Gly Ile Gly Tyr

85 90 95

Asp Ile Asn Cys Gly Val Arg Val Leu Arg Thr Asn Leu Thr Glu Glu

100 105 110

Asp Val Arg Pro Lys Leu Lys Glu Leu Val Asp Thr Ile Phe Arg Leu

115 120 125

Val Pro Pro Gly Val Gly Gly Thr Gly His Leu Arg Leu Ser Pro Ser

130 135 140

Glu Phe Glu Arg Val Leu Ala Glu Gly Val Glu Trp Ala Val Gln Lys

145 150 155 160

Gly Tyr Gly Trp Ala Glu Asp Met Glu Tyr Ile Glu Glu Arg Gly Ser

165 170 175

Trp Lys Leu Ala Asp Pro Ser Lys Val Ser Glu Lys Ala Lys Ala Arg

180 185 190

Gly Arg Asp Gln Leu Gly Thr Leu Gly Ser Gly Asn His Phe Leu Glu

195 200 205

Ile Gln Val Val Asp Lys Ile Tyr Asp Glu Lys Ile Ala Lys Leu Phe

210 215 220

Gly Ile Glu Arg Glu Gly Gln Val Val Val Met Ile His Thr Gly Ser

225 230 235 240

Arg Gly Phe Gly His Gln Val Ala Thr Asp Tyr Leu Leu Ile Met Glu

245 250 255

Arg Lys Met Arg Gln Trp Gly Leu Asn Leu Pro Asp Arg Glu Leu Ala

260 265 270

Ala Ala Pro Leu Lys Asp Lys Val Ala Glu Asp Tyr Ile Lys Ala Met

275 280 285

Ala Ser Ala Ala Asn Phe Ala Trp Thr Asn Arg His Ile Ile Met His

290 295 300

Trp Val Arg Glu Ala Phe Lys Lys Val Phe Gly Ser Ile Glu Lys Val

305 310 315 320

Gly Leu Glu Val Val Tyr Asp Val Ala His Asn Ile Ala Lys Leu Glu

325 330 335

Glu His Val Val Asp Glu Lys Gly Thr Val Arg Lys Val Trp Val His

340 345 350

Arg Lys Gly Ala Thr Arg Ala Phe Pro Pro Gly Arg Ser Glu Ile Pro

355 360 365

Ala Lys Tyr Arg Glu Val Gly Gln Pro Val Leu Ile Pro Gly Ser Met

370 375 380

Gly Thr Ala Ser Trp Ile Leu Val Gly Thr His Asp Ala Met Arg Leu

385 390 395 400

Thr Phe Gly Thr Ala Pro His Gly Ala Gly Arg Val Leu Ser Arg Glu

405 410 415

Ala Ala Ile Arg Met Tyr Pro Pro His Lys Val Gln Glu Glu Met Ala

420 425 430

Lys Arg Gly Ile Ile Val Arg Ser Ala Glu Thr Glu Val Ile Ser Glu

435 440 445

Glu Ala Pro Trp Ala Tyr Lys Asp Val Asp Arg Val Val Glu Ala Ala

450 455 460

His Gln Val Gly Phe Ala Lys Lys Val Val Arg Gln Arg Pro Ile Gly

465 470 475 480

Val Val Lys Gly

<210> 587

<211> 482

<212> PRT

<213> Sulfolobus acidocaldarius

<400> 587

Met Gln Thr Gln Ile Lys Arg Ile Gly Asn Tyr Glu Trp Arg Ile Glu

1 5 10 15

Lys Gly Ala Gln Glu Cys Met Lys Val Pro Val Thr Val Phe Ala Asp

20 25 30

Asp Val Leu Ile Asp Lys Met Lys Gln Asp Leu Thr Leu Arg Gln Ala

35 40 45

Thr Asn Val Ser Cys Leu Gln Gly Val Gln Glu Ser Val Tyr Val Leu

50 55 60

Pro Asp Gly His Gln Gly Tyr Gly Phe Pro Ile Gly Gly Ile Ala Ala

65 70 75 80

Ser Ala Ile Asp Glu Glu Gly Val Val Ser Pro Gly Gly Ile Gly Tyr

85 90 95

Asp Ile Asn Cys Gly Val Arg Leu Leu Arg Thr Asn Leu Asp Tyr Lys

100 105 110

Asp Val Lys Asp Lys Leu Lys Asp Leu Val Glu Glu Ile Tyr Arg Asn

115 120 125

Val Pro Ser Gly Val Gly Ser Glu Gly Arg Val Lys Leu Ser Tyr Gln

130 135 140

Gln Leu Asp Asn Val Leu Ser Glu Gly Val Lys Trp Ala Val Asp Asn

145 150 155 160

Gly Tyr Gly Trp Asn Arg Asp Met Glu His Ile Glu Gln Ser Gly Ser

165 170 175

Trp Asn Leu Ala Asp Pro Ser Lys Val Ser Pro Ile Ala Lys Gln Arg

180 185 190

Gly His Thr Gln Leu Gly Thr Leu Gly Ala Gly Asn His Phe Leu Glu

195 200 205

Ile Gln Val Val Asp Lys Ile Tyr Asp Glu Lys Val Ala Lys Ala Ile

210 215 220

Gly Ile Thr His Glu Gly Gln Ile Thr Val Met Val His Thr Gly Ser

225 230 235 240

Arg Gly Leu Gly His Gln Val Ala Ser Asp Tyr Leu Gln Val Met Glu

245 250 255

Arg Ala Met Lys Lys Tyr Asn Ile Lys Val Pro Asp Arg Glu Leu Ala

260 265 270

Ala Ile Pro Phe Asn Thr Arg Glu Ala Gln Asp Tyr Ile His Ala Met

275 280 285

Ser Ser Ala Ala Asn Phe Ala Trp Thr Asn Arg Gln Met Ile Thr His

290 295 300

Trp Ala Arg Glu Ser Phe Gly Arg Val Tyr Arg Ile Asp Pro Glu Lys

305 310 315 320

Leu Asp Leu Asn Ile Val Tyr Asp Val Ala His Asn Ile Ala Lys Ile

325 330 335

Glu Glu Tyr Asp Ile Asp Gly Lys Arg Lys Lys Val Leu Val His Arg

340 345 350

Lys Gly Ala Thr Arg Ala Phe Pro Pro Gly Ser Thr Glu Ile Pro Ala

355 360 365

Asp His Arg Asn Val Gly Gln Ile Val Leu Ile Pro Gly Ser Met Gly

370 375 380

Thr Ala Ser Tyr Ile Met Ala Gly Ile Pro Glu Gly Arg Arg Thr Trp

385 390 395 400

Phe Thr Ala Pro His Gly Ala Gly Arg Trp Met Ser Arg Glu Ala Ala

405 410 415

Val Arg Ser Tyr Pro Val Asn Ser Val Val Gln Asn Leu Glu Glu Lys

420 425 430

Gly Ile Ile Val Arg Ala Ala Thr Lys Arg Val Val Ala Glu Glu Ala

435 440 445

Pro Gly Ala Tyr Lys Asp Val Asp Arg Val Ala Lys Val Ala His Glu

450 455 460

Val Lys Ile Ala Lys Leu Val Ala Arg Leu Lys Pro Ile Gly Val Thr

465 470 475 480

Lys Gly

<210> 588

<211> 970

<212> PRT

<213> Pyrococcus furiosus

<400> 588

Met Ile Ile Leu Arg Val Val Asn Val Ala Val Pro Leu Lys Arg Ile

1 5 10 15

Asp Lys Ile Arg Trp Glu Ile Pro Lys Phe Asp Lys Arg Met Lys Val

20 25 30

Pro Gly Arg Val Tyr Ala Asp Asp Val Leu Ile Glu Lys Met Arg Gln

35 40 45

Asp Arg Thr Leu Glu Gln Ala Ala Asn Val Ala Met Leu Pro Gly Ile

50 55 60

Tyr Lys Tyr Ser Ile Val Met Pro Asp Gly His Gln Gly Tyr Gly Phe

65 70 75 80

Pro Ile Gly Gly Val Ala Ala Phe Asp Ile Lys Glu Gly Val Ile Ser

85 90 95

Pro Gly Gly Ile Gly Tyr Asp Ile Asn Cys Leu Ala Pro Gly Thr Lys

100 105 110

Val Leu Thr Glu His Gly Tyr Trp Leu Lys Ile Glu Glu Met Pro Glu

115 120 125

Lys Phe Lys Leu Gln Arg Leu Arg Leu Tyr Asn Ile Glu Glu Gly His

130 135 140

Asn Asp Phe Ser Arg Val Ala Phe Val Ala Glu Arg Asn Ile Glu Lys

145 150 155 160

Asp Glu Thr Ala Ile Arg Ile Val Thr Glu Thr Gly Thr Leu Ile Glu

165 170 175

Gly Ser Glu Asp His Pro Val Leu Thr Pro Gln Gly Tyr Val Tyr Leu

180 185 190

Lys Asn Ile Lys Glu Gly Asp Tyr Val Ile Val Tyr Pro Phe Glu Gly

195 200 205

Val Pro Tyr Glu Glu Lys Lys Gly Ile Ile Ile Asp Glu Ser Ala Phe

210 215 220

Glu Gly Glu Asp Pro Gln Val Ile Lys Phe Leu Lys Glu Arg Asn Leu

225 230 235 240

Leu Pro Leu Arg Trp Glu Asp Pro Lys Ile Gly Thr Leu Ala Arg Ile

245 250 255

Leu Gly Phe Ala Leu Gly Asp Gly His Leu Gly Glu Met Gly Gly Arg

260 265 270

Leu Val Leu Ala Phe Tyr Gly Arg Glu Glu Thr Leu Arg Glu Leu Lys

275 280 285

Lys Asp Leu Glu Ser Leu Gly Ile Lys Ala Asn Leu Tyr Val Arg Glu

290 295 300

Lys Asn Tyr Arg Ile Lys Thr Glu Ser Gly Glu Tyr Ser Gly Lys Thr

305 310 315 320

Val Leu Ala Glu Leu Arg Val Ser Ser Arg Ser Phe Ala Leu Leu Leu

325 330 335

Glu Lys Leu Gly Met Pro Arg Gly Glu Lys Thr Lys Lys Ala Tyr Arg

340 345 350

Ile Pro Val Trp Ile Met Glu Ala Pro Leu Trp Val Lys Arg Asn Phe

355 360 365

Leu Ala Gly Phe Phe Gly Ala Asp Gly Ser Ile Val Glu Phe Lys Gly

370 375 380

Thr Thr Pro Leu Pro Ile His Leu Thr Gln Ala Lys Asp Val Ala Leu

385 390 395 400

Glu Glu Asn Leu Lys Glu Phe Leu Tyr Asp Ile Ser Arg Ile Leu Glu

405 410 415

Glu Phe Gly Val Lys Thr Thr Ile Tyr Lys Val Asn Ser Lys Lys Ser

420 425 430

Val Thr Tyr Arg Leu Ser Ile Val Gly Glu Glu Asn Ile Arg Asn Phe

435 440 445

Leu Gly Lys Ile Asn Tyr Glu Tyr Asp Pro Lys Lys Lys Ala Lys Gly

450 455 460

Leu Ile Ala Tyr Ala Tyr Leu Lys Phe Lys Glu Ser Val Lys Lys Glu

465 470 475 480

Arg Arg Lys Ala Met Glu Ile Ser Lys Lys Ile Tyr Glu Glu Thr Gly

485 490 495

Asn Ile Asp Arg Ala Tyr Lys Ala Val Lys Asp Ile Val Asn Arg Arg

500 505 510

Phe Val Glu Arg Thr Ile Tyr Glu Gly Glu Arg Asn Pro Arg Val Pro

515 520 525

Lys Asn Phe Leu Thr Phe Glu Glu Phe Ala Lys Glu Arg Gly Tyr Glu

530 535 540

Gly Gly Phe Val Ala Glu Lys Val Val Lys Val Glu Arg Ile Lys Pro

545 550 555 560

Glu Tyr Asp Arg Phe Tyr Asp Ile Gly Val Tyr His Glu Ala His Asn

565 570 575

Phe Ile Ala Asn Gly Ile Val Val His Asn Cys Gly Val Arg Leu Ile

580 585 590

Arg Thr Asn Leu Thr Glu Lys Asp Val Arg Pro Lys Ile Lys Gln Leu

595 600 605

Val Asp Thr Leu Phe Lys Asn Val Pro Ser Gly Val Gly Ser Gln Gly

610 615 620

Lys Val Arg Leu His Trp Thr Gln Ile Asp Asp Val Leu Val Asp Gly

625 630 635 640

Ala Lys Trp Ala Val Asp Gln Gly Tyr Gly Trp Glu Arg Asp Leu Glu

645 650 655

Arg Leu Glu Glu Gly Gly Arg Met Glu Gly Ala Asp Pro Asp Ala Val

660 665 670

Ser Gln Arg Ala Lys Gln Arg Gly Ala Pro Gln Leu Gly Ser Leu Gly

675 680 685

Ser Gly Asn His Phe Leu Glu Val Gln Val Val Asp Lys Ile Phe Asp

690 695 700

Glu Glu Ile Ala Lys Ala Tyr Gly Leu Phe Glu Gly Gln Val Val Val

705 710 715 720

Met Val His Thr Gly Ser Arg Gly Leu Gly His Gln Val Ala Ser Asp

725 730 735

Tyr Leu Arg Ile Met Glu Arg Ala Ile Arg Lys Tyr Gly Ile Pro Trp

740 745 750

Pro Asp Arg Glu Leu Val Ser Val Pro Phe Gln Ser Glu Glu Gly Gln

755 760 765

Arg Tyr Phe Ser Ala Met Lys Ala Ala Ala Asn Phe Ala Trp Ala Asn

770 775 780

Arg Gln Met Ile Thr His Trp Val Arg Glu Ser Phe Gln Glu Val Phe

785 790 795 800

Arg Gln Asp Pro Glu Gly Asp Leu Gly Met Glu Ile Val Tyr Asp Val

805 810 815

Ala His Asn Ile Gly Lys Val Glu Glu His Glu Val Asp Gly Lys Lys

820 825 830

Val Lys Val Ile Val His Arg Lys Gly Ala Thr Arg Ala Phe Pro Pro

835 840 845

Gly His Glu Ala Ile Pro Lys Ile Tyr Arg Asp Val Gly Gln Pro Val

850 855 860

Leu Ile Pro Gly Ser Met Gly Thr Ala Ser Tyr Val Leu Ala Gly Thr

865 870 875 880

Glu Gly Ala Met Ala Glu Thr Phe Gly Ser Thr Cys His Gly Ala Gly

885 890 895

Arg Val Leu Ser Arg Ala Ala Ala Thr Arg Gln Tyr Arg Gly Asp Arg

900 905 910

Ile Arg Asp Glu Leu Leu Arg Arg Gly Ile Tyr Val Arg Ala Ala Ser

915 920 925

Met Arg Val Val Ala Glu Glu Ala Pro Gly Ala Tyr Lys Asn Val Asp

930 935 940

Asn Val Val Lys Val Val Ser Glu Ala Gly Ile Ala Lys Leu Val Ala

945 950 955 960

Arg Met Arg Pro Ile Gly Val Ala Lys Gly

965 970

<210> 589

<211> 444

<212> PRT

<213> Bacillus cereus

<400> 589

Met Asn Val Lys Leu Leu Met Asp Glu Ser Thr Lys Glu Leu Ser Ile

1 5 10 15

Tyr Leu Lys Gly Ile Glu Glu Phe Leu Asn Asn Phe Ser Glu Met Lys

20 25 30

His Ile Lys Lys Pro Ile Asn Ile Phe Pro Asp Ala Tyr Ile Lys Arg

35 40 45

Trp Gly Phe Pro Ser Gly Ile Thr Ile Ile Ser Glu Glu Asp Gly Leu

50 55 60

Val Phe Pro Ala Ala Ala Pro Asp Leu Gly Cys Gly Phe Arg Ile Ile

65 70 75 80

Lys Thr Asn Leu Asp Ile His Thr Phe Asn Asp Asp Leu Ala Lys Glu

85 90 95

Ile Leu Ile Gln Leu Glu Asp Met Ala Gly Val Asp Ser Lys Ile Arg

100 105 110

Met Lys Lys Val Ala Asn Leu Asp Lys Glu Arg Ile Phe Ser Gln Gly

115 120 125

Val Leu Tyr Leu Leu Glu Met Gly Ile Gly Ser Gln Glu Asp Leu Glu

130 135 140

Lys Ile Gln Gly Ile Ser Thr Asn Lys Ser Lys Lys Leu His Ile Ser

145 150 155 160

Glu Lys Asp Lys Asp Leu Leu Ile Glu Asn Phe Gly Ile Cys Ala Gly

165 170 175

His Phe Leu Glu Val Arg Tyr Val Thr Asp Ile Phe Asn Lys Thr Val

180 185 190

Gly Ser Lys Leu Asn Leu Ser Val Gly Gln Ile Ile Ile Ile Ile His

195 200 205

Ser Ser Ser Tyr Val Gly Lys Glu Ile Ile Leu Glu Asn Tyr Tyr Arg

210 215 220

Pro Ala Ile Glu Phe Met Leu Ser Lys Lys Leu Val Ser Asn Glu Gln

225 230 235 240

Leu Asn Arg Gly Ile Phe Gly Leu Pro Ile Lys Ser Glu Leu Gly Lys

245 250 255

Ala Tyr Ile Glu Ala Ser Asn Ala Leu Val Glu Tyr Ser Tyr Ala Ser

260 265 270

Arg His Phe Ala Gln Tyr Leu Val Asn Glu Val Leu Asn Asn Val Phe

275 280 285

Gly Asp Lys Val Glu Phe Glu Leu Ile Ser Asp Ile Cys His Ser Lys

290 295 300

Ile Glu Tyr Leu Asp Asn Gly Asp Val Leu His Gly Arg Gly Val Gln

305 310 315 320

Lys Ile Tyr Pro Ile Gly His Ala Asn Thr Leu Pro Tyr Tyr Ser Asp

325 330 335

Thr Gly Asp Val Ala Leu Leu Ala Gly Gln Lys Gly Thr Glu Ser His

340 345 350

Leu Ile Ile Pro Thr Ser Gln Ile Lys Glu Thr Ser Tyr Leu Cys Ser

355 360 365

His Gly Thr Gly Glu Phe Leu Val Glu Lys Asp Val His Asp Val Pro

370 375 380

Val Ser Val Arg Lys Glu Leu Glu Leu Cys Ser Phe Asp Thr Gln Tyr

385 390 395 400

Asp Glu Leu Asp Glu Phe Thr Leu Asp Tyr Phe Asn Thr Lys Met Cys

405 410 415

Leu Lys Glu Leu Glu Glu Asn Gln Lys Ile Ile Asn Lys Val Cys Arg

420 425 430

Leu Ala Pro Leu Ile Asn Tyr Trp Gly Asp Lys Glu

435 440

<210> 590

<211> 408

<212> PRT

<213> Escherichia coli

<400> 590

Met Asn Tyr Glu Leu Leu Thr Thr Glu Asn Ala Pro Val Lys Met Trp

1 5 10 15

Thr Lys Gly Val Pro Val Glu Ala Asp Ala Arg Gln Gln Leu Ile Asn

20 25 30

Thr Ala Lys Met Pro Phe Ile Phe Lys His Ile Ala Val Met Pro Asp

35 40 45

Val His Leu Gly Lys Gly Ser Thr Ile Gly Ser Val Ile Pro Thr Lys

50 55 60

Gly Ala Ile Ile Pro Ala Ala Val Gly Val Asp Ile Gly Cys Gly Met

65 70 75 80

Asn Ala Leu Arg Thr Ala Leu Thr Ala Glu Asp Leu Pro Glu Asn Leu

85 90 95

Ala Glu Leu Arg Gln Ala Ile Glu Thr Ala Val Pro His Gly Arg Thr

100 105 110

Thr Gly Arg Cys Lys Arg Asp Lys Gly Ala Trp Glu Asn Pro Pro Val

115 120 125

Asn Val Asp Ala Lys Trp Ala Glu Leu Glu Ala Gly Tyr Gln Trp Leu

130 135 140

Thr Gln Lys Tyr Pro Arg Phe Leu Asn Thr Asn Asn Tyr Lys His Leu

145 150 155 160

Gly Thr Leu Gly Thr Gly Asn His Phe Ile Glu Ile Cys Leu Asp Glu

165 170 175

Ser Asp Gln Val Trp Ile Met Leu His Ser Gly Ser Arg Gly Ile Gly

180 185 190

Asn Ala Ile Gly Thr Tyr Phe Ile Asp Leu Ala Gln Lys Glu Met Gln

195 200 205

Glu Thr Leu Glu Thr Leu Pro Ser Arg Asp Leu Ala Tyr Phe Met Glu

210 215 220

Gly Thr Glu Tyr Phe Asp Asp Tyr Leu Lys Ala Val Ala Trp Ala Gln

225 230 235 240

Leu Phe Ala Ser Leu Asn Arg Asp Ala Met Met Glu Asn Val Val Thr

245 250 255

Ala Leu Gln Ser Ile Thr Gln Lys Thr Val Arg Gln Pro Gln Thr Leu

260 265 270

Ala Met Glu Glu Ile Asn Cys His His Asn Tyr Val Gln Lys Glu Gln

275 280 285

His Phe Gly Glu Glu Ile Tyr Val Thr Arg Lys Gly Ala Val Ser Ala

290 295 300

Arg Ala Gly Gln Tyr Gly Ile Ile Pro Gly Ser Met Gly Ala Lys Ser

305 310 315 320

Phe Ile Val Arg Gly Leu Gly Asn Glu Glu Ser Phe Cys Ser Cys Ser

325 330 335

His Gly Ala Gly Arg Val Met Ser Arg Thr Lys Ala Lys Lys Leu Phe

340 345 350

Ser Val Glu Asp Gln Ile Arg Ala Thr Ala His Val Glu Cys Arg Lys

355 360 365

Asp Ala Glu Val Ile Asp Glu Ile Pro Met Ala Tyr Lys Asp Ile Asp

370 375 380

Ala Val Met Ala Ala Gln Ser Asp Leu Val Glu Val Ile Tyr Thr Leu

385 390 395 400

Arg Gln Val Val Cys Val Lys Gly

405

<210> 591

<211> 505

<212> PRT

<213> Caenorhabditis elegans

<400> 591

Met Pro Arg Thr Phe Glu Glu Glu Cys Asp Phe Ile Asp Arg Leu Thr

1 5 10 15

Asp Thr Lys Phe Arg Ile Lys Lys Gly Phe Val Pro Asn Met Asn Val

20 25 30

Glu Gly Arg Phe Tyr Val Asn Asn Ser Leu Glu Gln Leu Met Phe Asp

35 40 45

Glu Leu Lys Phe Ser Cys Asp Gly Gln Gly Ile Gly Gly Phe Leu Pro

50 55 60

Ala Val Arg Gln Ile Ala Asn Val Ala Ser Leu Pro Gly Ile Val Gly

65 70 75 80

His Ser Ile Gly Leu Pro Asp Ile His Ser Gly Tyr Gly Phe Ser Ile

85 90 95

Gly Asn Ile Ala Ala Phe Asp Val Gly Asn Pro Glu Ser Val Ile Ser

100 105 110

Pro Gly Gly Val Gly Phe Asp Ile Asn Cys Gly Val Arg Leu Leu Arg

115 120 125

Thr Asn Leu Phe Glu Glu Asn Val Lys Pro Leu Lys Glu Gln Leu Thr

130 135 140

Gln Ser Leu Phe Asp His Ile Pro Val Gly Val Gly Ser Arg Gly Ala

145 150 155 160

Ile Pro Met Leu Ala Ser Asp Leu Val Glu Cys Leu Glu Met Gly Met

165 170 175

Asp Trp Thr Leu Arg Glu Gly Tyr Ser Trp Ala Glu Asp Lys Glu His

180 185 190

Cys Glu Glu Tyr Gly Arg Met Leu Gln Ala Asp Ala Ser Lys Val Ser

195 200 205

Leu Arg Ala Lys Lys Arg Gly Leu Pro Gln Leu Gly Thr Leu Gly Ala

210 215 220

Gly Asn His Tyr Ala Glu Val Gln Val Val Asp Glu Ile Tyr Asp Lys

225 230 235 240

His Ala Ala Ser Thr Met Gly Ile Asp Glu Glu Gly Gln Val Val Val

245 250 255

Met Leu His Cys Gly Ser Arg Gly Leu Gly His Gln Val Ala Thr Asp

260 265 270

Ser Leu Val Glu Met Glu Lys Ala Met Ala Arg Asp Gly Ile Val Val

275 280 285

Asn Asp Lys Gln Leu Ala Cys Ala Arg Ile Asn Ser Val Glu Gly Lys

290 295 300

Asn Tyr Phe Ser Gly Met Ala Ala Ala Ala Asn Phe Ala Trp Val Asn

305 310 315 320

Arg Ser Cys Ile Thr Phe Cys Val Arg Asn Ala Phe Gln Lys Thr Phe

325 330 335

Gly Met Ser Ala Asp Asp Met Asp Met Gln Val Ile Tyr Asp Val Ser

340 345 350

His Asn Val Ala Lys Met Glu Glu His Met Val Asp Gly Arg Pro Arg

355 360 365

Gln Leu Cys Val His Arg Lys Gly Ala Thr Arg Ala Phe Pro Ala His

370 375 380

His Pro Leu Ile Pro Val Asp Tyr Gln Leu Ile Gly Gln Pro Val Leu

385 390 395 400

Ile Gly Gly Ser Met Gly Thr Cys Ser Tyr Val Leu Thr Gly Thr Glu

405 410 415

Gln Gly Leu Val Glu Thr Phe Gly Thr Thr Cys His Gly Ala Gly Arg

420 425 430

Ala Leu Ser Arg Ala Lys Ser Arg Arg Thr Ile Thr Trp Asp Ser Val

435 440 445

Ile Asp Asp Leu Lys Lys Lys Glu Ile Ser Ile Arg Ile Ala Ser Pro

450 455 460

Lys Leu Ile Met Glu Glu Ala Pro Glu Ser Tyr Lys Asn Val Thr Asp

465 470 475 480

Val Val Asp Thr Cys Asp Ala Ala Gly Ile Ser Lys Lys Ala Val Lys

485 490 495

Leu Arg Pro Ile Ala Val Ile Lys Gly

500 505

<210> 592

<211> 827

<212> PRT

<213> Saccharomyces cerevisiae

<400> 592

Met Pro Ser Pro Tyr Asp Gly Lys Arg Thr Val Thr Gln Leu Val Asn

1 5 10 15

Glu Leu Glu Lys Ala Glu Lys Leu Ser Gly Arg Gly Arg Ala Tyr Arg

20 25 30

Arg Val Cys Asp Leu Ser His Ser Asn Lys Lys Val Ile Ser Trp Lys

35 40 45

Phe Asn Glu Trp Asp Tyr Gly Lys Asn Thr Ile Thr Leu Pro Cys Asn

50 55 60

Ala Arg Gly Leu Phe Ile Ser Asp Asp Thr Thr Asn Pro Val Ile Val

65 70 75 80

Ala Arg Gly Tyr Asp Lys Phe Phe Asn Val Gly Glu Val Asn Phe Thr

85 90 95

Lys Trp Asn Trp Ile Glu Glu Asn Cys Thr Gly Pro Tyr Asp Val Thr

100 105 110

Ile Lys Ala Asn Gly Cys Ile Ile Phe Ile Ser Gly Leu Glu Asp Gly

115 120 125

Thr Leu Val Val Cys Ser Lys His Ser Thr Gly Pro Arg Ala Asp Val

130 135 140

Asp Arg Asn His Ala Glu Ala Gly Glu Lys Gln Leu Leu Arg Gln Leu

145 150 155 160

Ala Ala Met Asn Ile Asn Arg Ser Asp Phe Ala Arg Met Leu Tyr Thr

165 170 175

His Asn Val Thr Ala Val Ala Glu Tyr Cys Asp Asp Ser Phe Glu Glu

180 185 190

His Ile Leu Glu Tyr Pro Leu Glu Lys Ala Gly Leu Tyr Leu His Gly

195 200 205

Val Asn Val Asn Lys Ala Glu Phe Glu Thr Trp Asp Met Lys Asp Val

210 215 220

Ser Gln Met Ala Ser Lys Tyr Gly Phe Arg Cys Val Gln Cys Ile Thr

225 230 235 240

Ser Asn Thr Leu Glu Asp Leu Lys Lys Phe Leu Asp Asn Cys Ser Ala

245 250 255

Thr Gly Ser Phe Glu Gly Gln Glu Ile Glu Gly Phe Val Ile Arg Cys

260 265 270

His Leu Lys Ser Thr Glu Lys Pro Phe Phe Phe Lys Tyr Lys Phe Glu

275 280 285

Glu Pro Tyr Leu Met Tyr Arg Gln Trp Arg Glu Val Thr Lys Asp Tyr

290 295 300

Ile Ser Asn Lys Ser Arg Val Phe Lys Phe Arg Lys His Lys Phe Ile

305 310 315 320

Thr Asn Lys Tyr Leu Asp Phe Ala Ile Pro Ile Leu Glu Ser Ser Pro

325 330 335

Lys Ile Cys Glu Asn Tyr Leu Lys Gly Phe Gly Val Ile Glu Leu Arg

340 345 350

Asn Lys Phe Leu Gln Ser Tyr Gly Met Ser Gly Leu Glu Ile Leu Asn

355 360 365

His Glu Lys Val Ala Glu Leu Glu Leu Lys Asn Ala Ile Asp Tyr Asp

370 375 380

Lys Val Asp Glu Arg Thr Lys Phe Leu Ile Phe Pro Ile Ser Val Ile

385 390 395 400

Gly Cys Gly Lys Thr Thr Thr Ser Gln Thr Leu Val Asn Leu Phe Pro

405 410 415

Asp Ser Trp Gly His Ile Gln Asn Asp Asp Ile Thr Gly Lys Asp Lys

420 425 430

Ser Gln Leu Met Lys Lys Ser Leu Glu Leu Leu Ser Lys Lys Glu Ile

435 440 445

Lys Cys Val Ile Val Asp Arg Asn Asn His Gln Phe Arg Glu Arg Lys

450 455 460

Gln Leu Phe Glu Trp Leu Asn Glu Leu Lys Glu Asp Tyr Leu Val Tyr

465 470 475 480

Asp Thr Asn Ile Lys Val Ile Gly Val Ser Phe Ala Pro Tyr Asp Lys

485 490 495

Leu Ser Glu Ile Arg Asp Ile Thr Leu Gln Arg Val Ile Lys Arg Gly

500 505 510

Asn Asn His Gln Ser Ile Lys Trp Asp Glu Leu Gly Glu Lys Lys Val

515 520 525

Val Gly Ile Met Asn Gly Phe Leu Lys Arg Tyr Gln Pro Val Asn Leu

530 535 540

Asp Lys Ser Pro Asp Asn Met Phe Asp Leu Met Ile Glu Leu Asp Phe

545 550 555 560

Gly Gln Ala Asp Ser Ser Leu Thr Asn Ala Lys Gln Ile Leu Asn Glu

565 570 575

Ile His Lys Ala Tyr Pro Ile Leu Val Pro Glu Ile Pro Lys Asp Asp

580 585 590

Glu Ile Glu Thr Ala Phe Arg Arg Ser Leu Asp Tyr Lys Pro Thr Val

595 600 605

Arg Lys Ile Val Gly Lys Gly Asn Asn Asn Gln Gln Lys Thr Pro Lys

610 615 620

Leu Ile Lys Pro Thr Tyr Ile Ser Ala Lys Ile Glu Asn Tyr Asp Glu

625 630 635 640

Ile Ile Glu Leu Val Lys Arg Cys Ile Ala Ser Asp Ala Glu Leu Thr

645 650 655

Glu Lys Phe Lys His Leu Leu Ala Ser Gly Lys Val Gln Lys Glu Leu

660 665 670

His Ile Thr Leu Gly His Val Met Ser Ser Arg Glu Lys Glu Ala Lys

675 680 685

Lys Leu Trp Lys Ser Tyr Cys Asn Arg Tyr Thr Asp Gln Ile Thr Glu

690 695 700

Tyr Asn Asn Asn Arg Ile Glu Asn Ala Gln Gly Ser Gly Asn Asn Gln

705 710 715 720

Asn Thr Gln Val Lys Thr Thr Asp Lys Leu Asn Phe Arg Leu Glu Lys

725 730 735

Leu Cys Trp Asp Glu Lys Ile Ile Ala Ile Val Val Glu Leu Ser Lys

740 745 750

Asp Lys Asp Gly Cys Ile Ile Asp Glu Asn Asn Glu Lys Ile Lys Gly

755 760 765

Leu Cys Cys Gln Asn Lys Ile Pro His Ile Thr Leu Cys Lys Leu Glu

770 775 780

Ser Gly Val Lys Ala Val Tyr Ser Asn Val Leu Cys Glu Lys Val Glu

785 790 795 800

Ser Ala Glu Val Asp Glu Asn Ile Lys Val Val Lys Leu Asp Asn Ser

805 810 815

Lys Glu Phe Val Gly Ser Val Tyr Leu Asn Phe

820 825

<210> 593

<211> 1104

<212> PRT

<213> Arabidopsis thaliana

<400> 593

Met Asp Ala Pro Phe Glu Ser Gly Asp Ser Ser Ala Thr Val Val Ala

1 5 10 15

Glu Ala Val Asn Asn Gln Phe Gly Gly Leu Ser Leu Lys Glu Ser Asn

20 25 30

Thr Asn Ala Pro Val Leu Pro Ser Gln Thr Thr Ser Asn His Arg Val

35 40 45

Gln Asn Leu Val Trp Lys Pro Lys Ser Tyr Gly Thr Val Ser Gly Ser

50 55 60

Ser Ser Ala Thr Glu Val Gly Lys Thr Ser Ala Val Ser Gln Ile Gly

65 70 75 80

Ser Ser Gly Asp Thr Lys Val Gly Leu Asn Leu Ser Lys Ile Phe Gly

85 90 95

Gly Asn Leu Leu Glu Lys Phe Ser Val Asp Lys Ser Thr Tyr Cys His

100 105 110

Ala Gln Ile Arg Ala Thr Phe Tyr Pro Lys Phe Glu Asn Glu Lys Thr

115 120 125

Asp Gln Glu Ile Arg Thr Arg Met Ile Glu Met Val Ser Lys Gly Leu

130 135 140

Ala Thr Leu Glu Val Ser Leu Lys His Ser Gly Ser Leu Phe Met Tyr

145 150 155 160

Ala Gly His Lys Gly Gly Ala Tyr Ala Lys Asn Ser Phe Gly Asn Ile

165 170 175

Tyr Thr Ala Val Gly Val Phe Val Leu Ser Arg Met Phe Arg Glu Ala

180 185 190

Trp Gly Thr Lys Ala Pro Lys Lys Glu Ala Glu Phe Asn Asp Phe Leu

195 200 205

Glu Lys Asn Arg Met Cys Ile Ser Met Glu Leu Val Thr Ala Val Leu

210 215 220

Gly Asp His Gly Gln Arg Pro Leu Asp Asp Tyr Val Val Val Thr Ala

225 230 235 240

Val Thr Glu Leu Gly Asn Gly Lys Pro Gln Phe Tyr Ser Thr Ser Glu

245 250 255

Ile Ile Ser Phe Cys Arg Lys Trp Arg Leu Pro Thr Asn His Val Trp

260 265 270

Leu Phe Ser Thr Arg Lys Ser Val Thr Ser Phe Phe Ala Ala Phe Asp

275 280 285

Ala Leu Cys Glu Glu Gly Ile Ala Thr Ser Val Cys Arg Ala Leu Asp

290 295 300

Glu Val Ala Asp Ile Ser Val Pro Ala Ser Lys Asp His Val Lys Val

305 310 315 320

Gln Gly Glu Ile Leu Glu Gly Leu Val Ala Arg Ile Val Ser Ser Gln

325 330 335

Ser Ser Arg Asp Met Glu Asn Val Leu Arg Asp His Pro Pro Pro Pro

340 345 350

Cys Asp Gly Ala Asn Leu Asp Leu Gly Leu Ser Leu Arg Glu Ile Cys

355 360 365

Ala Ala His Arg Ser Asn Glu Lys Gln Gln Met Arg Ala Leu Leu Arg

370 375 380

Ser Val Gly Pro Ser Phe Cys Pro Ser Asp Val Glu Trp Phe Gly Asp

385 390 395 400

Glu Ser His Pro Lys Ser Ala Asp Lys Ser Val Ile Thr Lys Phe Leu

405 410 415

Gln Ser Gln Pro Ala Asp Tyr Ser Thr Ser Lys Leu Gln Glu Met Val

420 425 430

Arg Leu Met Lys Glu Lys Arg Leu Pro Ala Ala Phe Lys Cys Tyr His

435 440 445

Asn Phe His Arg Ala Glu Asp Ile Ser Pro Asp Asn Leu Phe Tyr Lys

450 455 460

Leu Val Val His Val His Ser Asp Ser Gly Phe Arg Arg Tyr His Lys

465 470 475 480

Glu Met Arg His Met Pro Ser Leu Trp Pro Leu Tyr Arg Gly Phe Phe

485 490 495

Val Asp Ile Asn Leu Phe Lys Ser Asn Lys Gly Arg Asp Leu Met Ala

500 505 510

Leu Lys Ser Ile Asp Asn Ala Ser Glu Asn Asp Gly Arg Gly Glu Lys

515 520 525

Asp Gly Leu Ala Asp Asp Asp Ala Asn Leu Met Ile Lys Met Lys Phe

530 535 540

Leu Thr Tyr Lys Leu Arg Thr Phe Leu Ile Arg Asn Gly Leu Ser Ile

545 550 555 560

Leu Phe Lys Asp Gly Ala Ala Ala Tyr Lys Thr Tyr Tyr Tyr Leu Arg Gln

565 570 575

Met Lys Ile Trp Gly Thr Ser Asp Gly Lys Gln Lys Glu Leu Cys Lys

580 585 590

Met Leu Asp Glu Trp Ala Ala Tyr Ile Arg Arg Lys Cys Gly Asn Asp

595 600 605

Gln Leu Ser Ser Ser Thr Tyr Leu Ser Glu Ala Glu Pro Phe Leu Glu

610 615 620

Gln Tyr Ala Lys Arg Ser Pro Lys Asn His Ile Leu Ile Gly Ser Ala

625 630 635 640

Gly Asn Leu Val Arg Thr Glu Asp Phe Leu Ala Ile Val Asp Gly Asp

645 650 655

Leu Asp Glu Glu Gly Asp Leu Val Lys Lys Gln Gly Val Thr Pro Ala

660 665 670

Thr Pro Glu Pro Ala Val Lys Glu Ala Val Gln Lys Asp Glu Gly Leu

675 680 685

Ile Val Phe Phe Pro Gly Ile Pro Gly Ser Ala Lys Ser Ala Leu Cys

690 695 700

Lys Glu Leu Leu Asn Ala Pro Gly Gly Phe Gly Asp Asp Arg Pro Val

705 710 715 720

His Thr Leu Met Gly Asp Leu Val Lys Gly Lys Tyr Trp Pro Lys Val

725 730 735

Ala Asp Glu Arg Arg Lys Lys Pro Gln Ser Ile Met Leu Ala Asp Lys

740 745 750

Asn Ala Pro Asn Glu Asp Val Trp Arg Gln Ile Glu Asp Met Cys Arg

755 760 765

Arg Thr Arg Ala Ser Ala Val Pro Ile Val Ala Asp Ser Glu Gly Thr

770 775 780

Asp Thr Asn Pro Tyr Ser Leu Asp Ala Leu Ala Val Phe Met Phe Arg

785 790 795 800

Val Leu Gln Arg Val Asn His Pro Gly Lys Leu Asp Lys Glu Ser Ser

805 810 815

Asn Ala Gly Tyr Val Leu Leu Met Phe Tyr His Leu Tyr Glu Gly Lys

820 825 830

Asn Arg Asn Glu Phe Glu Ser Glu Leu Ile Glu Arg Phe Gly Ser Leu

835 840 845

Ile Lys Met Pro Leu Leu Lys Ser Asp Arg Thr Pro Leu Pro Asp Pro

850 855 860

Val Lys Ser Val Leu Glu Glu Gly Ile Asp Leu Phe Asn Leu His Ser

865 870 875 880

Arg Arg His Gly Arg Leu Glu Ser Thr Lys Gly Thr Tyr Ala Ala Glu

885 890 895

Trp Thr Lys Trp Glu Lys Gln Leu Arg Asp Thr Leu Val Ala Asn Ser

900 905 910

Glu Tyr Leu Ser Ser Ile Gln Val Pro Phe Glu Ser Met Val His Gln

915 920 925

Val Arg Glu Glu Leu Lys Thr Ile Ala Lys Gly Asp Tyr Lys Pro Pro

930 935 940

Ser Ser Glu Lys Arg Lys His Gly Ser Ile Val Phe Ala Ala Ile Asn

945 950 955 960

Leu Pro Ala Thr Gln Val His Ser Leu Leu Glu Lys Leu Ala Ala Ala

965 970 975

Asn Pro Thr Met Arg Ser Phe Leu Glu Gly Lys Lys Lys Ser Ile Gln

980 985 990

Glu Lys Leu Glu Arg Ser His Val Thr Leu Ala His Lys Arg Ser His

995 1000 1005

Gly Val Ala Thr Val Ala Ser Tyr Ser Gln His Leu Asn Arg Glu

1010 1015 1020

Val Pro Val Glu Leu Thr Glu Leu Ile Tyr Asn Asp Lys Met Ala

1025 1030 1035

Ala Leu Thr Ala His Val Gly Ser Val Asp Gly Glu Thr Val Val

1040 1045 1050

Ser Lys Asn Glu Trp Pro His Val Thr Leu Trp Thr Ala Glu Gly

1055 1060 1065

Val Thr Ala Lys Glu Ala Asn Thr Leu Pro Gln Leu Tyr Leu Glu

1070 1075 1080

Gly Lys Ala Ser Arg Leu Val Ile Asp Pro Pro Val Ser Ile Ser

1085 1090 1095

Gly Pro Leu Glu Phe Phe

1100

<210> 594

<211> 334

<212> PRT

<213> Enterobacteria phage T4

<400> 594

Met Phe Lys Lys Tyr Ser Ser Leu Glu Asn His Tyr Asn Ser Lys Phe

1 5 10 15

Ile Glu Lys Leu Tyr Ser Leu Gly Leu Thr Gly Gly Glu Trp Val Ala

20 25 30

Arg Glu Lys Ile His Gly Thr Asn Phe Ser Leu Ile Ile Glu Arg Asp

35 40 45

Lys Val Thr Cys Ala Lys Arg Thr Gly Pro Ile Leu Pro Ala Glu Asp

50 55 60

Phe Phe Gly Tyr Glu Ile Ile Leu Lys Asn Tyr Ala Asp Ser Ile Lys

65 70 75 80

Ala Val Gln Asp Ile Met Glu Thr Ser Ala Val Val Ser Tyr Gln Val

85 90 95

Phe Gly Glu Phe Ala Gly Pro Gly Ile Gln Lys Asn Val Asp Tyr Cys

100 105 110

Asp Lys Asp Phe Tyr Val Phe Asp Ile Ile Val Thr Thr Glu Ser Gly

115 120 125

Asp Val Thr Tyr Val Asp Asp Tyr Met Met Glu Ser Phe Cys Asn Thr

130 135 140

Phe Lys Phe Lys Met Ala Pro Leu Leu Gly Arg Gly Lys Phe Glu Glu

145 150 155 160

Leu Ile Lys Leu Pro Asn Asp Leu Asp Ser Val Val Gln Asp Tyr Asn

165 170 175

Phe Thr Val Asp His Ala Gly Leu Val Asp Ala Asn Lys Cys Val Trp

180 185 190

Asn Ala Glu Ala Lys Gly Glu Val Phe Thr Ala Glu Gly Tyr Val Leu

195 200 205

Lys Pro Cys Tyr Pro Ser Trp Leu Arg Asn Gly Asn Arg Val Ala Ile

210 215 220

Lys Cys Lys Asn Ser Lys Phe Ser Glu Lys Lys Lys Ser Asp Lys Pro

225 230 235 240

Ile Lys Ala Lys Val Glu Leu Ser Glu Ala Asp Asn Lys Leu Val Gly

245 250 255

Ile Leu Ala Cys Tyr Val Thr Leu Asn Arg Val Asn Asn Val Ile Ser

260 265 270

Lys Ile Gly Glu Ile Gly Pro Lys Asp Phe Gly Lys Val Met Gly Leu

275 280 285

Thr Val Gln Asp Ile Leu Glu Glu Thr Ser Arg Glu Gly Ile Thr Leu

290 295 300

Thr Gln Ala Asp Asn Pro Ser Leu Ile Lys Lys Glu Leu Val Lys Met

305 310 315 320

Val Gln Asp Val Leu Arg Pro Ala Trp Ile Glu Leu Val Ser

325 330

<210> 595

<211> 832

<212> PRT

<213> Candida albicans

<400> 595

Met Lys Asp Ser Gln Ser Asp Ile Ile Glu Leu Cys Asn Lys Leu Asn

1 5 10 15

Glu Ala Thr Lys Leu Lys Arg Asn Gly Lys Ser Ile Lys Leu Thr Asn

20 25 30

Phe Val Ser Asn Thr Gln Ile Lys Leu Asp Ser Trp Lys Phe Leu Glu

35 40 45

Trp Asp Tyr Gly Lys Pro Ser Val Gln Leu Pro Ile Gln Ala Arg Gly

50 55 60

Leu Phe Thr Leu Asn Asn Asp Thr Ile Ala Val Arg Gly Tyr Asp Lys

65 70 75 80

Phe Phe Asn Val Glu Glu Lys Pro Phe Thr Lys Glu Thr Asn Leu Lys

85 90 95

Thr Ser Thr His Gly Pro Tyr Glu Val Thr Leu Lys Glu Asn Gly Cys

100 105 110

Ile Ile Phe Ile Ser Gly Leu Ser Thr Gly Asp Ile Val Val Cys Ser

115 120 125

Lys His Ser Thr Gly Asp Arg Ile Asp Asp Asn Glu Ser Asp Lys Thr

130 135 140

Thr Thr Ala Thr Ala Thr Ala Thr Ala Pro Thr Arg Asn His Ala Lys

145 150 155 160

Gln Gly Glu Phe Glu Leu Leu Gln Gln Phe Asp Gly Asp Gln Gln Lys

165 170 175

Val Lys Gln Leu Ala His Tyr Leu Tyr Glu Asn Asn Leu Thr Val Val

180 185 190

Ala Glu Leu Cys Asp Asp Glu Phe Glu Glu His Val Leu Pro Tyr Pro

195 200 205

Lys Asp Lys Ser Gly Leu Tyr Val His Gly Leu Asn Tyr Asn Thr Ile

210 215 220

Thr Phe Lys Thr Leu Pro Met Asp Gln Val Leu Gln Phe Ala Lys Glu

225 230 235 240

Trp Gly Phe Lys Tyr Val Ser Tyr Leu Thr Tyr Asp Asn Ala Asp Glu

245 250 255

Leu Phe Lys Phe Leu His Lys Cys Ser Glu Thr Gly Thr Tyr Asn Gly

260 265 270

Arg Glu Ile Glu Gly Phe Val Ile Arg Cys His Arg Gln Ser His Thr

275 280 285

Asn Gly Asp Thr Asp Gly Asp Cys Phe Phe Phe Lys Tyr Lys Phe Glu

290 295 300

Gln Pro Tyr Leu Leu Tyr Arg Gln Phe Arg Glu Val Thr Lys Gln Leu

305 310 315 320

Leu Asn Gly Thr Pro Ile Asn Ser Ile Lys Ile Lys Lys Asn Lys Pro

325 330 335

Ile Thr Lys Lys Tyr Leu Gln Phe Val Glu Lys Leu Phe Glu Gln Glu

340 345 350

Pro Glu Ile Ala Arg Asn Phe Glu Asn Gly Phe Asp Ile Ile Lys Val

355 360 365

Arg Gln Leu Phe Leu Gln Ser Leu Asn Glu Thr Asn Gly Met Asn Leu

370 375 380

Leu Ser Ile Asp Ser Glu Leu Ser Asp Gln Leu Lys Asn Leu Ala Leu

385 390 395 400

Ala Asn Gly Asn Glu Gly Leu Ser Thr Thr Thr Lys Tyr Ile Phe Val

405 410 415

Pro Ile Ala Thr Ile Gly Cys Gly Lys Thr Thr Val Phe Asn Thr Leu

420 425 430

Asn Asn Leu Phe Pro Gln Trp Thr His Ile Gln Asn Asp Asn Ile Ser

435 440 445

Lys Lys Ala Lys Leu Lys Ile Cys Asp Leu Thr Leu Leu Ala Leu Glu

450 455 460

Asp Asp Asp Gln Ser Val Val Leu Phe Asp Arg Asn Asn Ser Ala Ser

465 470 475 480

Arg Glu Arg Arg Gln Ile Phe Thr Thr Ile Asp Gln Lys Arg Asp Glu

485 490 495

His Leu Asp Asp Thr Val Asp Leu Lys Tyr Ile Ala Ile Asn Phe Ile

500 505 510

Pro Glu Asp Leu Ser Glu Glu Glu Leu Trp Asp Ile Thr Tyr Asn Arg

515 520 525

Val Ile Gln Arg Gly Asp Asn His Gln Ser Ile Lys Ser Gln Ser Asp

530 535 540

Glu Asn Leu Val Glu Ser Val Met Lys Gly Phe Ile Gln Arg Tyr Gln

545 550 555 560

Pro Ile Asn Thr Ser Arg Ser Pro Asp Asp Gln Phe Asp His Val Ile

565 570 575

His Leu Lys Leu Ser Lys Asp Glu Asn Ser Ser Lys Ser Ser Leu Glu

580 585 590

Asn Val Arg Ile Ile Ile Asp Asp Leu Val Gln Asn Phe Pro Asp Leu

595 600 605

Ile Lys Glu Lys Pro Ala Asp Glu Leu Ile Asn Glu Cys Phe Gln Lys

610 615 620

Ala Leu Asp Tyr Lys Pro Thr Phe Val Lys Asn Met Thr Ala Asn Thr

625 630 635 640

Ile Lys Lys Asp Pro Thr Tyr Tyr Gly Ile Ala Met His Tyr Ser Ser

645 650 655

Ile Leu Glu Asn Leu Glu Ile Val Ser His Asn Glu His Phe Gln Asn

660 665 670

Ile Lys Ser His Ile Gln Thr Glu Phe His Val Thr Leu Gly His Ile

675 680 685

Ala Ser Ser Lys Gln Asp Lys Ala Gly Arg Val Lys Trp Lys Lys Leu

690 695 700

Val Lys Thr Leu Gly Lys Gly Asp Pro Asn Lys Pro Lys Ser Ala Leu

705 710 715 720

Lys Phe Phe Ala Asp Val Lys Leu Leu Gln Ile Val Ile Asn Thr Asp

725 730 735

Lys Leu Ala Cys Ile Lys Val Glu Ile Leu Lys Ile Tyr Asp Thr Asn

740 745 750

Asp Val Leu Gln Ser Glu Ile Glu Pro Ile Asn Lys Gln Leu His Ile

755 760 765

Thr Ile Gly Cys Ile Pro Pro Ala Thr Ala Val Glu Ser Asn Ile Thr

770 775 780

Leu Glu Glu Leu Tyr Asp Asn Pro Asp Glu Gln Glu Leu Lys Pro Asp

785 790 795 800

Gly Thr Tyr Lys Cys Gly Asp Asp Thr Leu His Val Phe Asn Phe Asp

805 810 815

Asn Pro Asp Leu Lys Leu Phe Ser Gln Gln Leu Phe Val Ala Tyr Gln

820 825 830

<210> 596

<211> 469

<212> PRT

<213> Trypanosoma brucei brucei

<400> 596

Met Gln Leu Gln Arg Leu Gly Ala Pro Leu Leu Lys Arg Leu Val Gly

1 5 10 15

Gly Cys Ile Arg Gln Ser Thr Ala Pro Ile Met Pro Cys Val Val Val

20 25 30

Ser Gly Ser Gly Val Phe Leu Thr Pro Val Arg Thr Tyr Met Pro Leu

35 40 45

Pro Asn Asp Gln Ser Asp Phe Ser Pro Tyr Ile Glu Ile Asp Leu Pro

50 55 60

Ser Glu Ser Arg Ile Gln Ser Leu His Lys Ser Gly Leu Ala Ala Gln

65 70 75 80

Glu Trp Val Ala Cys Glu Lys Val His Gly Thr Asn Phe Gly Ile Tyr

85 90 95

Leu Ile Asn Gln Gly Asp His Glu Val Val Arg Phe Ala Lys Arg Ser

100 105 110

Gly Ile Met Asp Pro Asn Glu Asn Phe Phe Gly Tyr His Ile Leu Ile

115 120 125

Asp Glu Phe Thr Ala Gln Ile Arg Ile Leu Asn Asp Leu Leu Lys Gln

130 135 140

Lys Tyr Gly Leu Ser Arg Val Gly Arg Leu Val Leu Asn Gly Glu Leu

145 150 155 160

Phe Gly Ala Lys Tyr Lys His Pro Leu Val Pro Lys Ser Glu Lys Trp

165 170 175

Cys Thr Leu Pro Asn Gly Lys Lys Phe Pro Ile Ala Gly Val Gln Ile

180 185 190

Gln Arg Glu Pro Phe Pro Gln Tyr Ser Pro Glu Leu His Phe Phe Ala

195 200 205

Phe Asp Ile Lys Tyr Ser Val Ser Gly Ala Glu Glu Asp Phe Val Leu

210 215 220

Leu Gly Tyr Asp Glu Phe Val Glu Phe Ser Ser Lys Val Pro Asn Leu

225 230 235 240

Leu Tyr Ala Arg Ala Leu Val Arg Gly Thr Leu Asp Glu Cys Leu Ala

245 250 255

Phe Asp Val Glu Asn Phe Met Thr Pro Leu Pro Ala Leu Leu Gly Leu

260 265 270

Gly Asn Tyr Pro Leu Glu Gly Asn Leu Ala Glu Gly Val Val Ile Arg

275 280 285

His Val Arg Arg Gly Asp Pro Ala Val Glu Lys His Asn Val Ser Thr

290 295 300

Ile Ile Lys Leu Arg Cys Ser Ser Phe Met Glu Leu Lys His Pro Gly

305 310 315 320

Lys Gln Lys Glu Leu Lys Glu Thr Phe Ile Asp Thr Val Arg Ser Gly

325 330 335

Ala Leu Arg Arg Val Arg Gly Asn Val Thr Val Ile Ser Asp Ser Met

340 345 350

Leu Pro Gln Val Glu Ala Ala Ala Asn Asp Leu Leu Leu Asn Asn Val

355 360 365

Ser Asp Gly Arg Leu Ser Asn Val Leu Ser Lys Ile Gly Arg Glu Pro

370 375 380

Leu Leu Ser Gly Glu Val Ser Gln Val Asp Val Val Leu Met Leu Ala

385 390 395 400

Lys Asp Ala Leu Lys Asp Phe Leu Lys Glu Val Asp Ser Leu Val Leu

405 410 415

Asn Thr Thr Leu Ala Phe Arg Lys Leu Leu Ile Thr Asn Val Tyr Phe

420 425 430

Glu Ser Lys Arg Leu Val Glu Gln Lys Trp Lys Glu Leu Met Gln Glu

435 440 445

Glu Ala Ala Ala Gln Ser Glu Ala Ile Pro Pro Leu Ser Pro Ala Ala

450 455 460

Pro Thr Lys Gly Glu

465

<210> 597

<211> 416

<212> PRT

<213> Trypanosoma brucei brucei

<400> 597

Met Leu Arg Arg Leu Gly Val Arg His Phe Arg Arg Thr Pro Leu Leu

1 5 10 15

Phe Val Gly Gly Asp Gly Ser Ile Phe Glu Arg Tyr Thr Glu Ile Asp

20 25 30

Asn Ser Asn Glu Arg Arg Ile Asn Ala Leu Lys Gly Cys Gly Met Phe

35 40 45

Glu Asp Glu Trp Ile Ala Thr Glu Lys Val His Gly Ala Asn Phe Gly

50 55 60

Ile Tyr Ser Ile Glu Gly Glu Lys Met Ile Arg Tyr Ala Lys Arg Ser

65 70 75 80

Gly Ile Met Pro Asn Glu His Phe Phe Gly Tyr His Ile Leu Ile

85 90 95

Pro Glu Leu Gln Arg Tyr Val Thr Ser Ile Arg Glu Met Leu Cys Glu

100 105 110

Lys Gln Lys Lys Lys Leu His Val Val Leu Ile Asn Gly Glu Leu Phe

115 120 125

Gly Gly Lys Tyr Asp His Pro Ser Val Pro Lys Thr Arg Lys Thr Val

130 135 140

Met Val Ala Gly Lys Pro Arg Thr Ile Ser Ala Val Gln Thr Asp Ser

145 150 155 160

Phe Pro Gln Tyr Ser Pro Asp Leu His Phe Tyr Ala Phe Asp Ile Lys

165 170 175

Tyr Lys Glu Thr Glu Gly Gly Asp Tyr Thr Thr Leu Val Tyr Asp Glu

180 185 190

Ala Ile Glu Leu Phe Gln Arg Val Pro Gly Leu Leu Tyr Ala Arg Ala

195 200 205

Val Ile Arg Gly Pro Met Ser Lys Val Ala Ala Phe Asp Val Glu Arg

210 215 220

Phe Val Thr Thr Ile Pro Pro Leu Val Gly Met Gly Asn Tyr Pro Leu

225 230 235 240

Thr Gly Asn Trp Ala Glu Gly Leu Val Val Lys His Ser Arg Leu Gly

245 250 255

Met Ala Gly Phe Asp Pro Lys Gly Pro Thr Val Leu Lys Phe Lys Cys

260 265 270

Thr Ala Phe Gln Glu Ile Ser Thr Asp Arg Ala Gln Gly Pro Arg Val

275 280 285

Asp Glu Met Arg Asn Val Arg Arg Asp Ser Ile Asn Arg Ala Gly Val

290 295 300

Gln Leu Pro Asp Leu Glu Ser Ile Val Gln Asp Pro Ile Gln Leu Glu

305 310 315 320

Ala Ser Lys Leu Leu Leu Asn His Val Cys Glu Asn Arg Leu Lys Asn

325 330 335

Val Leu Ser Lys Ile Gly Thr Glu Pro Phe Glu Lys Glu Glu Met Thr

340 345 350

Pro Asp Gln Leu Ala Thr Leu Leu Ala Lys Asp Ala Leu Lys Asp Phe

355 360 365

Leu Lys Asp Thr Glu Pro Ser Ile Val Asn Ile Pro Val Leu Ile Arg

370 375 380

Lys Asp Leu Thr Arg Tyr Val Ile Phe Glu Ser Arg Arg Leu Val Cys

385 390 395 400

Ser Gln Trp Lys Asp Ile Leu Lys Arg Gln Ser Pro Asp Phe Ser Glu

405 410 415

<210> 598

<211> 374

<212> PRT

<213> Enterobacteria phage T4

<400> 598

Met Gln Glu Leu Phe Asn Asn Leu Met Glu Leu Cys Lys Asp Ser Gln

1 5 10 15

Arg Lys Phe Phe Tyr Ser Asp Asp Val Ser Ala Ser Gly Arg Thr Tyr

20 25 30

Arg Ile Phe Ser Tyr Asn Tyr Ala Ser Tyr Ser Asp Trp Leu Leu Pro

35 40 45

Asp Ala Leu Glu Cys Arg Gly Ile Met Phe Glu Met Asp Gly Glu Lys

50 55 60

Pro Val Arg Ile Ala Ser Arg Pro Met Glu Lys Phe Phe Asn Leu Asn

65 70 75 80

Glu Asn Pro Phe Thr Met Asn Ile Asp Leu Asn Asp Val Asp Tyr Ile

85 90 95

Leu Thr Lys Glu Asp Gly Ser Leu Val Ser Thr Tyr Leu Asp Gly Asp

100 105 110

Glu Ile Leu Phe Lys Ser Lys Gly Ser Ile Lys Ser Glu Gln Ala Leu

115 120 125

Met Ala Asn Gly Ile Leu Met Asn Ile Asn His His Arg Leu Arg Asp

130 135 140

Arg Leu Lys Glu Leu Ala Glu Asp Gly Phe Thr Ala Asn Phe Glu Phe

145 150 155 160

Val Ala Pro Thr Asn Arg Ile Val Leu Ala Tyr Gln Glu Met Lys Ile

165 170 175

Ile Leu Leu Asn Val Arg Glu Asn Glu Thr Gly Glu Tyr Ile Ser Tyr

180 185 190

Asp Asp Ile Tyr Lys Asp Ala Thr Leu Arg Pro Tyr Leu Val Glu Arg

195 200 205

Tyr Glu Ile Asp Ser Pro Lys Trp Ile Glu Glu Ala Lys Asn Ala Glu

210 215 220

Asn Ile Glu Gly Tyr Val Ala Val Met Lys Asp Gly Ser His Phe Lys

225 230 235 240

Ile Lys Ser Asp Trp Tyr Val Ser Leu His Ser Thr Lys Ser Ser Leu

245 250 255

Asp Asn Pro Glu Lys Leu Phe Lys Thr Ile Ile Asp Gly Ala Ser Asp

260 265 270

Asp Leu Lys Ala Met Tyr Ala Asp Asp Glu Tyr Ser Tyr Arg Lys Ile

275 280 285

Glu Ala Phe Glu Thr Thr Tyr Leu Lys Tyr Leu Asp Arg Ala Leu Phe

290 295 300

Leu Val Leu Asp Cys His Asn Lys His Cys Gly Lys Asp Arg Lys Thr

305 310 315 320

Tyr Ala Met Glu Ala Gln Gly Val Ala Lys Gly Ala Gly Met Asp His

325 330 335

Leu Phe Gly Ile Ile Met Ser Leu Tyr Gln Gly Tyr Asp Ser Gln Glu

340 345 350

Lys Val Met Cys Glu Ile Glu Gln Asn Phe Leu Lys Asn Tyr Lys Lys

355 360 365

Phe Ile Pro Glu Gly Tyr

370

<210> 599

<211> 694

<212> PRT

<213> Autographa californica nuclear polyhedrosis virus

<400> 599

Met Leu His Val Ser Arg Leu Leu Ala Asn Gly Gly Val Lys Asn Leu

1 5 10 15

Cys Asp Lys Phe Lys Val Lys Ile Lys Asn Tyr Thr Glu His Asp Leu

20 25 30

Met Val Leu Asn Tyr Glu Ser Phe Glu Arg Asp Arg Asp His Pro Val

35 40 45

Val Val Glu Cys Arg Gly Leu Ile Leu Asn Ser Arg Thr Tyr Ala Val

50 55 60

Val Ser Arg Ser Phe Asp Arg Phe Phe Asn Phe Gln Glu Leu Leu Gln

65 70 75 80

Asn Ile Gly Gly Glu Asp Ala His His Lys Leu Phe Gln Ser Lys Glu

85 90 95

Asn Phe Lys Phe Tyr Glu Lys Ile Asp Gly Ser Leu Ile Lys Ile Tyr

100 105 110

Lys Tyr Asn Gly Glu Trp His Ala Ser Thr Arg Gly Ser Ala Phe Ala

115 120 125

Glu Asn Leu Cys Val Ser Asp Val Thr Phe Lys Arg Leu Val Leu Gln

130 135 140

Ala Leu Gln Leu Asp Glu Ala His Asn Gln Phe Gln Ala Leu Cys Asn

145 150 155 160

Glu Tyr Leu Asp Cys Ala Ser Thr His Met Phe Glu Leu Thr Ser Lys

165 170 175

His Asn Arg Ile Val Thr Val Tyr Asp Glu Gln Pro Thr Leu Trp Tyr

180 185 190

Leu Ala Ser Arg Asn Asn Glu Thr Gly Asp Tyr Phe Tyr Cys Ser Asn

195 200 205

Leu Pro Phe Cys Lys Tyr Pro Lys Cys Tyr Glu Phe Thr Ser Val Gln

210 215 220

Glu Cys Val Glu His Ala Ala Gln Leu Lys Asn Leu Glu Glu Gly Phe

225 230 235 240

Val Val Tyr Asp Lys Asn Asn Ala Pro Leu Cys Lys Ile Lys Ser Asp

245 250 255

Val Tyr Leu Asn Met His Lys Asn Gln Ser Arg Ala Glu Asn Pro Thr

260 265 270

Lys Leu Ala Gln Leu Val Ile Asn Gly Glu His Asp Asp Phe Leu Ala

275 280 285

Leu Phe Pro His Leu Lys Ser Val Ile Lys Pro Tyr Val Asp Ala Arg

290 295 300

Asn Thr Phe Thr Asn Glu Ser Thr Ile Asn Ile Met Val Ser Gly Leu

305 310 315 320

Thr Leu Asn Gln Gln Arg Phe Asn Glu Leu Val Gln Thr Leu Pro Trp

325 330 335

Lys Cys Leu Ala Tyr Arg Cys Arg Lys Ala Gln Thr Ile Asp Val Glu

340 345 350

Ser Glu Phe Leu Lys Leu Thr Glu Pro Glu Lys Ile Lys Met Ile Lys

355 360 365

Asn Ile Ile Lys Phe Val Ser Thr Lys Gln Ala Leu Asn Asn Lys Leu

370 375 380

Ala Pro Thr Ile Lys Leu Pro Ser Ser Lys Gln Leu Leu Val Leu Ile

385 390 395 400

Gly Ile Ser Gly Ser Gly Lys Ser Thr Tyr Tyr Ala Lys Ser Leu Lys Gly

405 410 415

Tyr Thr Glu Ile Asn Arg Asp Asp Val Arg Val Lys Leu Phe Leu Asn

420 425 430

Gly Asp Tyr Thr Lys Leu Asn Ala Phe Tyr Asn Gln Ser Arg Lys Cys

435 440 445

Arg Gln Thr Lys Glu Glu Gln Ile Thr Lys Met Cys Ile Glu Gln Phe

450 455 460

Leu Lys Ala Ala Lys Cys Gly Ala Asn Val Val Val Ser Asp Thr Asn

465 470 475 480

Leu Asn Thr Gln Ser Val Asp Met Trp Gln Lys Met Ala Ala Thr His

485 490 495

Asn Tyr His Phe Leu Thr Arg Leu Met Asp Val Ser Leu Glu Thr Ala

500 505 510

Leu Glu Arg Asn Tyr Lys Arg Ser Asp Lys Phe Pro Leu Asn Pro Glu

515 520 525

Thr Ile Lys Lys Gln Tyr Lys Lys Phe Leu Lys Val Asn Asn Phe Glu

530 535 540

Tyr Tyr Val Pro Val Gly Asp Lys Phe Pro Arg Ala Val Leu Cys Asp

545 550 555 560

Leu Asp Gly Thr Val Ala Leu Pro Thr Asn Arg Ser Phe Tyr Asp Phe

565 570 575

Asp Asn Arg Val Ala Gln Asp Glu Ala Arg Leu Asp Val Ile Thr Cys

580 585 590

Val Lys Tyr Leu Ala Asn Cys His Asp Ala Ile Ile Val Phe Met Ser

595 600 605

Gly Arg Ser Val Ile Cys Glu Gln Pro Thr Arg Asn Trp Ile Glu Lys

610 615 620

Tyr Phe Asp Ile Lys Ser Tyr Lys Leu Phe Met Arg Pro Ser Asp Asp

625 630 635 640

Thr Cys Lys Asp Tyr Leu Leu Lys Leu Lys Leu Phe Asn Asn Tyr Ile

645 650 655

Arg Gly Lys Tyr Asn Val Ile Ala Val Phe Asp Asp Arg Pro Cys Val

660 665 670

Val Arg Met Trp Gln Asp Leu Lys Ile Pro Thr Val Phe Asn Val Cys

675 680 685

Arg Asp Tyr Leu Glu Phe

690

<210> 600

<211> 184

<212> PRT

<213> Pyrococcus furiosus

<400> 600

Met Arg Ala Phe Ile Ala Ile Asp Val Ser Glu Ser Val Arg Asp Ala

1 5 10 15

Leu Val Arg Ala Gln Asp Tyr Ile Gly Ser Lys Glu Ala Lys Ile Lys

20 25 30

Phe Val Glu Arg Glu Asn Phe His Ile Thr Leu Lys Phe Leu Gly Glu

35 40 45

Ile Thr Glu Glu Gln Ala Glu Glu Ile Lys Lys Ile Leu Glu Lys Ile

50 55 60

Ala Lys Lys Tyr Lys Lys His Glu Val Asn Val Arg Gly Ile Gly Val

65 70 75 80

Phe Pro Asn Pro Asn Tyr Val Arg Val Ile Trp Ala Gly Val Glu Asn

85 90 95

Asp Glu Ile Ile Lys Lys Ile Ala Lys Glu Ile Asp Asp Glu Leu Ala

100 105 110

Lys Leu Gly Phe Lys Lys Glu Gly Asn Phe Val Ala His Ile Thr Leu

115 120 125

Gly Arg Val Lys Phe Val Lys Asp Lys Leu Gly Leu Ala Met Lys Leu

130 135 140

Lys Glu Leu Ala Asn Glu Asp Phe Gly Ser Phe Ile Val Glu Ala Ile

145 150 155 160

Glu Leu Lys Lys Ser Thr Leu Thr Pro Lys Gly Pro Ile Tyr Glu Thr

165 170 175

Leu Ala Arg Phe Glu Leu Ser Glu

180

<210> 601

<211> 176

<212> PRT

<213> Escherichia coli

<400> 601

Met Ser Glu Pro Gln Arg Leu Phe Phe Ala Ile Asp Leu Pro Ala Glu

1 5 10 15

Ile Arg Glu Gln Ile Ile His Trp Arg Ala Thr His Phe Pro Pro Glu

20 25 30

Ala Gly Arg Pro Val Ala Ala Asp Asn Leu His Leu Thr Leu Ala Phe

35 40 45

Leu Gly Glu Val Ser Ala Glu Lys Glu Lys Ala Leu Ser Leu Leu Ala

50 55 60

Gly Arg Ile Arg Gln Pro Gly Phe Thr Leu Thr Leu Asp Asp Ala Gly

65 70 75 80

Gln Trp Leu Arg Ser Arg Val Val Trp Leu Gly Met Arg Gln Pro Pro

85 90 95

Arg Gly Leu Ile Gln Leu Ala Asn Met Leu Arg Ser Gln Ala Ala Arg

100 105 110

Ser Gly Cys Phe Gln Ser Asn Arg Pro Phe His Pro His Ile Thr Leu

115 120 125

Leu Arg Asp Ala Ser Glu Ala Val Thr Ile Pro Pro Pro Gly Phe Asn

130 135 140

Trp Ser Tyr Ala Val Thr Glu Phe Thr Leu Tyr Ala Ser Ser Phe Ala

145 150 155 160

Arg Gly Arg Thr Arg Tyr Thr Pro Leu Lys Arg Trp Ala Leu Thr Gln

165 170 175

<210> 602

<211> 183

<212> PRT

<213> Bacillus subtilis

<400> 602

Met Pro Asp Ile Arg Pro His Tyr Phe Ile Gly Val Pro Ile Pro Glu

1 5 10 15

Gly Ile Ala Asn Pro Ile Tyr Gln Ala Ala Lys Asn Glu Pro Ile Leu

20 25 30

Thr Phe Gln Lys Trp Val His Pro Leu Asp Tyr His Ile Thr Leu Ile

35 40 45

Phe Leu Gly Ala Ala Asp Glu Thr Gln Ile Lys Lys Leu Glu Gly Ser

50 55 60

Leu Ala Glu Ile Ala Ser Glu Ile Asp Pro Phe Ser Ile Lys Phe Gly

65 70 75 80

Lys Ile Asp Val Phe Gly Asp Arg Arg Lys Pro Arg Val Leu His Leu

85 90 95

Glu Pro Lys Lys Asn Lys Thr Leu Asp Arg Leu Arg Glu His Thr Lys

100 105 110

Gln Ala Val Leu Gln Ala Gly Phe Gln Val Glu Lys Arg Pro Tyr His

115 120 125

Pro His Met Thr Leu Ala Arg Lys Trp Thr Gly Glu Asp Gly Phe Pro

130 135 140

Ala His Val Pro Phe Glu Ser Gly Glu Val Ser Met Met Ala Glu Arg

145 150 155 160

Phe Ser Leu Phe Gln Ile His Leu Asn Gln Ser Pro Lys Tyr Glu Glu

165 170 175

Ile Phe Lys Phe Gln Leu Ser

180

<210> 603

<211> 392

<212> RNA

<213> Artificial

<220>

<223> Synthetic

<400> 603

gggaauuccu agggaacccg gucccaagcc cggauaaaau ccgagggggc gggaaaccgc 60

cuaaggaugu guucccuagg aggggugggug uaccucuuuu ggaccaaucg uggcgugucg 120

gccugcuucg gcaggcacug gcgccgucca ggagagagca caacauuuca accagaaaca 180

cuagccgaag caaauccauu ccacaagcac cuggugggau caucucauca ucagaaacca 240

agagagagau uccguguccg cuuguuguag uagauuguga ggacugagga ccgagaagca 300

gccacaccucucccccuccc agguacuauccccuuucaacacugccaaug ccggucccaa 360

gcccggauaa aaguggaggg aaaggggaua gu 392

<210> 604

<211> 245

<212> RNA

<213> Artificial

<220>

<223> Synthetic

<400> 604

gggaauuccu agggaacccg gucccaagcc cggauaaaau ccgagggggc gggaaaccgc 60

cuaaggaugu guucccuagg aggggugggug uaccucuuuu ggaccaaucg uggcgugucg 120

gccugcuucg gcaggcacug gcgccgucca ggagagacac cucucccccu cccagguacu 180

auccccuuuc aacacugcca augccggucc caagccccgga uaaaagugga gggaaagggg 240

auagu 245

<210> 605

<211> 557

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 605

tactccaaga atatcaaaga tacagtctca gaagaccaaa gggctattga gacttttcaa 60

caaagggtaa tatcgggaaa cctcctcgga ttccattgcc cagctatctg tcacttcatc 120

aaaaggacag tagaaaagga aggtggcacc tacaaatgcc atcattgcga taaaggaaag 180

gctatcgttc aagatgcctc tgccgacagt ggtcccaaag atggaccccc acccacaagg 240

agcatcgtgg aaaaagaaga cgttccaacc acgtcttcaa agcaagtgga ttgatgtgat 300

atctccactg acgtaaggga tgacgcacaa tcccactatc cttcgcccca agcttgggcc 360

caagcttggg tcgcgcccca cggatggtat aagaataaag gcattccgcg tgcaggattc 420

acccgttcgc ctctcacctt ttcgctgtac tctctcgcca cacacacccc ctctccagct 480

ccgttggagc tccggacagc agcaggcgcg gggcggtcac gtagtaagca gctctcggct 540

ccctctcccc ttgctcc 557

<210> 606

<211> 49

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 606

tttcccctga tgagtccgtg aggacgaaac gagtaagctc gtcgggaaa 49

<210> 607

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 607

gggaaaaaaa tgccgtcggt 20

<210> 608

<211> 53

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 608

ttggaccaat cgtggcgtgt cggcctgctt cggcaggcac tggcgccgtc cag 53

<210> 609

<211> 464

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 609

gagggcccgg aaacctggcc ctgtcttctt gacgagcatt cctaggggtc tttcccctct 60

cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa gcagttcctc tggaagcttc 120

ttgaagacaa acaacgtctg tagcgacct ttgcaggcag cggaaccccc cacctggcga 180

caggtgcctc tgcggccaaa agccacgtgt ataagataca cctgcaaagg cggcacaacc 240

ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc aaatggctct cctcaagcgt 300

attcaacaag gggctgaagg atgcccagaa ggtaccccat tgtatggggat ctgatctggg 360

gcctcggtgc acatgcttta catgtgttta gtcgaggtta aaaaaacgtc taggcccccc 420

gaaccacggg gacgtggttt tcctttgaaa aacacgatga taat 464

<210> 610

<211> 516

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 610

atggtcttca cactcgaaga tttcgttggg gactggcgac agacagccgg ctacaacctg 60

gaccaagtcc ttgaacaggg aggtgtgtcc agtttgtttc agaatctcgg ggtgtccgta 120

actccgatcc aaaggattgt cctgagcggt gaaaatgggc tgaagatcga catccatgtc 180

atcatcccgt atgaaggtct gagcggcgac caaatgggcc agatcgaaaa aatttttaag 240

gtggtgtacc ctgtggatga tcatcacttt aaggtgatcc tgcactatgg cacactggta 300

atcgacgggg ttacgccgaa catgatcgac tatttcggac ggccgtatga aggcatcgcc 360

gtgttcgacg gcaaaaagat cactgtaaca gggaccctgt ggaacggcaa caaaattatc 420

gacgagcgcc tgatcaaccc cgacggctcc ctgctgttcc gagtaaccat caacggagtg 480

accggctggc ggctgtgcga acgcattctg gcgtaa 516

<210> 611

<211> 20

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 611

accgacggca aaaaaaaaaa 20

<210> 612

<211> 68

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 612

ggccggcatg gtcccagcct cctcgctggc gccggctggg caacatgctt cggcatggcg 60

aatgggac 68

<210> 613

<211> 250

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 613

cgttcaaaca tttggcaata aagtttctta agattgaatc ctgttgccgg tcttgcgatg 60

attatcatat atatttctgt tgattacgtt aagcatgtaa taattaacat gtaatgcatg 120

acgttattta tgagatgggt ttttatgatt agagtcccgc aattatacat ttaatacgcg 180

atagaaaaca aaatatagcg cgcaaactag gataaattat cgcgcgcggt gtcatctatg 240

ttactagatc 250

<210> 614

<211> 451

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 614

acggattaga agccgccgag cgggtgacag ccctccgaag gaagactctc ctccgtgcgt 60

cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc actgctccga 120

acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat tggcagtaac 180

ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat gataatgcga 240

ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt tttgatctat 300

taacagatat ataaatgcaa aaactgcata accactttaa ctaatacttt caacattttc 360

ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt gttaatatac 420

ctctatactt taacgtcaag gagaaaaaac c 451

<210> 615

<211> 60

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 615

atggacgccc cctttgaatc tggcgacagc agcgccaccg tcgtcgctga ggctgtcaac 60

<210> 616

<211> 249

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 616

atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 60

gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 120

ttagtattaa gaacgttattatatttcaa atttttcttt tttttctgta cagacgcgtg 180

tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 240

ttaatttgc 249

<210> 617

<211> 2442

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 617

atggcagaac aagatgtgac agagcttgtg aaaagactcg aagaagcttc tcatttgaag 60

aagcatggta aagctactaa gatcacatgt tctatatttg atagacccga tgtgaagttg 120

gacagttgga aattcaatga atgggactat ggcaaatcta aaagagtgtt accttgtagt 180

gccagaggtt tatttatcca aaatgctgat tcagagccca ggatagttac tagaggatac 240

gataagttct ttaacatcga cgaagtgcaa agaactagct ggaggtcaat tcaagataac 300

actgaggggc catatgagat tactgtcaaa gagaacggat gtataatttt gattggagga 360

ttggaagatg gtactgttgt agtatgttcg aaacatagca ctgggcccag agatgacatc 420

aacagaaacc attcacaagc tggtcagcag ttcttggaac aacaattgaa ggagaaaaac 480

cttggattga aggatcttgg acggtattta tatgaagcca attgtactgc tattgcagaa 540

tattgcgatg atacctttga agaacatatt ttggaatata acagagacaa tgcaggtcta 600

tacctacatg gtttaaacta caatactgta gggttcagca cttttccaat ggcaaaagta 660

gctgaatttg caaacgcatg gggatttaag catattgatt actttactac ggaagatagt 720

tcatcattga agacattcct tactgagtgc gaaaaagctg gtcattacaa taatcaagag 780

attgaagggt ttgtgataag atgtaaggat aaatcaactg gtgggacatt ctttttcaag 840

tacaagttta aagaacctta tttgatgtat cgacaatgga gggaggttac aagagagtac 900

atatctacca aacaacgtgt cttcagatac cgctcgcata actatatcac caacaaatat 960

atggactttg tgattccctt gttggatcgt gacccaaaac tagctgatga tttcatgaac 1020

gggaagggta taataaaatt gagaaaactc tttctagaag attacggtat gtcaggccta 1080

gagatcctca atttggacaa gattaaagaa ttagaagagg cagaaaatca tgtcgaaaat 1140

gtaatcgatg aaaatacaaa atttttacta gtgacaattg ctacaatcgg atgtggtaaa 1200

tctacagttt ctctcacgct taatgagtta ttccctgagt catggggact agttgtgaac 1260

gacaatataa cctctaataa aacagactac attaaatcag ccttgcaatt attcaaagac 1320

gggaaacaat ttgtgcttgc tgataaaaac aatcatcagt tccgtgaaag ggcagctgtt 1380

tttgaatgga tcaatcaata tagggattcg tatattccat acaactgtaa tttacaagtc 1440

atagcattgt gttttgtaga cgaggtatcc ccagaaatga gagcattaac catcgacagg 1500

gttatgaaaa gaggtgacaa tcatcaaagc atcaaatctg aaagtgacga tcagcagaag 1560

gttttgaaaa ttatgcaagg attcatgaac agattccaac cattccttcc ttccaaagat 1620

cctgataaca aatttgactt ccatatacaa ttggaagttg gcaagaactc gtcgttgaaa 1680

aacactatta ccgttctcaa agagttacag cgtaattatg gagacgttat tccctcgatc 1740

ccagatgatt ctatcgtaca tcaggctttt gaaagggctc tcaattacaa accaaccata 1800

acgaaaatca ttaaaggtgg gaataaaatt gacaaaaagc agcataaacc tgtatatttc 1860

tcagctaatg taattgacac agacctttta ttgcaacatg tcagaaaaac cattgaaaca 1920

catgcatcag agtacccttc tttgcttaaa agtctggatt caactccttt caaagacgct 1980

ttgcatataa ccctatacca taagtctcaa atcaggtctg ttggtataaa agctaaacaa 2040

atgtgggcca aatacttgga tagatacaaa gagtatctct caaaagaaaa taactcagag 2100

gctaccaaaa acattatcgc tacggaggat agtgtctctt tcaaattgag agatttaatc 2160

tgggataagc acgtaattct agctaccgtt tccctgctag atgataccca tccaattgtt 2220

atgccagatg gtactcattt ggaccatttg acttgcctta acaaagttcc tcacataaca 2280

cttgcattgt tttccaatga gaaaacagcg aagtattctg gtgaattggc taaagaagtg 2340

tatgagttcg gtatacaaga atcggatact gatacaggta tgatcagtct agggacaact 2400

gacaaccttc agtttagcgg gaaagtctgt attaatttat aa 2442

<210> 618

<211> 351

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 618

ttggtcatgc gaaacacgca cggcgcgcgc acgcagctta gcacaaacgc gtcgttgcac 60

gcgcccaccg ctaaccgcag gccaatcggt cggccggcct catatccgct caccagccgc 120

gtcctatcgg gcgcggcttc cgcgcccatt ttgaataaat aaacgataac gccgttggtg 180

gcgtgaggca tgtaaaaggg ttacatcatt atcttgttcg ccatccggtt ggtataaata 240

gacgttcatg ttggtttttg tttcagttgc aagttggctg cggcgcgcgc agcacctttg 300

ccgggatctg ccgggctgca gcacgtgttg acaattaatc atcggcatag t 351

<210> 619

<211> 43

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 619

taatacgact cactataggg aattgtgagc ggataacaat tcc 43

<210> 620

<211> 307

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 620

ttatacatat attttgaatt taattaatta tacatatatt ttatattatt tttgtctttt 60

attatcgagg ggccgttgtt ggtgtggggt tttgcataga aataacaatg ggagttggcg 120

acgttgctgc gccaacacca cctcccttcc ctcctttcat catgtatctg tagataaaat 180

aaaatattaa acctaaaaac aagaccgcgc ctatcaacaa aatgataggc attaacttgc 240

cgctgacgct gtcactaacg ttggacgatt tgccgactaa accttcatcg cccagtaacc 300

aatctag 307

<210> 621

<211> 166

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 621

aaccagataa gtgaaatcta gttccaaact attttgtcat ttttaatttt cgtattagct 60

tacgacgcta cacccagttc ccatctattt tgtcactctt ccctaaataa tccttaaaaa 120

ctccatttcc acccctccca gttcccaact attttgtccg cccaca 166

<210> 622

<211> 92

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 622

atcatggaga taattaaaat gataaccatc tcgcaaataa ataagtattt tactgttttc 60

gtaacagttt tgtaataaaa aaacctataa at 92

<210> 623

<211> 135

<212> DNA

<213> Simian Virus 40

<400> 623

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 120

tatcatgtct ggatc 135

<210> 624

<211> 225

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 624

tgtgggcgga caataaagtc ttaaactgaa caaaatagat ctaaactatg acaataaagt 60

cttaaactag acagaatagt tgtaaactga aatcagtcca gttatgctgt gaaaaagcat 120

actggacttt tgttatggct aaagcaaact cttcattttc tgaagtgcaa attgcccgtc 180

gtattaaaga ggggcgtggc caagggcatg gtaaagacta tattc 225

<210> 625

<211> 701

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 625

gtcacaagtt tgtacaaaaa agcaggctct atgaattacg aattactgac cactgaaaat 60

gccccggtaa aaatgtggac caaaggcgtg ccggtagagg ccgatgcgcg tcagcaactt 120

attaatacgg cgaagatgcc gtttattttc aaacatattg cggtaatgcc tgatgtacac 180

ctgggtaaag gttccaccat tggtagcgtg atcccgacca aaggggcgat tattccggcg 240

gcggtgggcg tggatattgg ctgtggaatg aacgcgctgc gtaccgcgtt aacggcggaa 300

gacctgcctg aaaacctggc agagctgcgt caggcgattg aaacggccgt gccgcacggg 360

cgtaccactg gccgttgtaa acgtgataaa ggtgcctggg aaaatccacc tgttaacgtc 420

gatgctaaat gggctgagct tgaagccggt tatcagtggt taacgcaaaa atatccccgt 480

ttcctgaata ccaataacta taaacacctg ggaacgctgg gaaccggtaa ccactttatt 540

gaaatctgcc ttgatgagtc ggaccaggtg tggattatgc tgcactccgg ttcacgcgga 600

attggtaacg ccatcgggac ttactttatc gatctggcac aaaaagagat gcaggaaacg 660

cttgagacgt tgccgtcgcg tgatctggcg tactttatgg a 701

<210> 626

<211> 655

<212> DNA

<213> Unknown

<220>

<223> Cytomegalovirus

<400> 626

cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc 60

aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt 120

aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta 180

tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg actatttacg 240

gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 300

cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt 360

tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg 420

gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc 480

cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg 540

taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat 600

aagcagagct ctctggctaa ctagagaacc cactgcttac tggctttatcg aaatt 655

<210> 627

<211> 60

<212> DNA

<213> Simian Virus 40

<400> 627

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 60

<210> 628

<211> 66

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 628

gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa ggatgacgat 60

gacaag 66

<210> 629

<211> 379

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 629

gagtttactc cctatcagtg atagagaacg tatgaagagt ttactcccta tcagtgatag 60

agaacgtatg cagactttac tccctatcag tgatagagaa cgtataagga gtttatccc 120

tatcagtgat agagaacgta tgaccagttt actccctatc agtgatagag aacgtatcta 180

cagtttactc cctatcagtg atagagaacg tatatccagt ttactcccta tcagtgatag 240

agaacgtata agctttaggc gtgtacggtg ggcgcctata aaagcagagc tcgtttagtg 300

aaccgtcaga tcgcctggag caattccaca acacttttgt cttataccaa ctttccgtac 360

cacttcctac cctcgtaaa 379

<210> 630

<211> 25

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 630

aaggatgtgt tccctaggag ggtgg 25

<210> 631

<211> 26

<212> DNA

<213> Artificial

<220>

<223> Synthetic

<400> 631

gaaagggggat agtacctggg agggggg 26

Claims (51)

1. A eukaryotic system for cyclizing polyribonucleotides, the eukaryotic system comprising a eukaryotic cell, the eukaryotic cell comprising: (a) Linear polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein element (A) , (B), (C), (D), and (E) are operably connected, and wherein: (A) Contains 5' self-cleaving ribozyme; (B) Contains a 5' annealing region containing a 5' complementary region; (C) Contains a polyribonucleotide payload; (D) Contains a 3' annealing region containing a 3' complementary region; and (E) Contains a 3' self-cleaving ribozyme; wherein the 5' complementary region and the 3' complementary region have a binding free energy of less than -5 kcal/mol, and/or wherein the 5' complementary region and the 3' complementary region have a binding Tm of at least 10°C; as well as (b) RNA ligase; wherein cleavage by the 5' self-cleaving ribozyme produces a free 5'-hydroxyl group on the 5' end of the linear polyribonucleotide, and wherein cleavage by the 3' self-cleaving ribozyme generates a free 5'-hydroxyl group on the 5' end of the linear polyribonucleotide A free 2',3'-cyclic phosphate group is generated on the 3' end of the nucleotide to obtain a ligase-compatible linear polyribonucleotide; And wherein the 5' and 3' ends of the ligase-compatible linear polyribonucleotide are connected by the RNA ligase, thereby producing a cyclic polyribonucleotide. 2. The eukaryotic system of claim 1, wherein the 5' self-cleaving ribozyme is a ribozyme selected from the group consisting of: hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme (HDV), Varkud satellite (VS) ribozyme, glmS ribozyme, twist ribozyme, twist sister ribozyme, axon ribozyme, and pistol ribozyme. 3. The eukaryotic system of claim 1, wherein the 3' self-cleaving ribozyme is a ribozyme selected from the group consisting of: hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme (HDV), Varkud satellite (VS) ribozyme, glmS ribozyme, twist ribozyme, twist sister ribozyme, axon ribozyme, and pistol ribozyme. 4. The eukaryotic system of claim 1, wherein the 5' complementary region has between 5 and 50 ribonucleotides and the 3' complementary region has between 5 and 50 ribonucleotides acid. 5. The eukaryotic system of claim 1, wherein the 5' complementary region and the 3' complementary region have sequence complementarity between 50% and 100%, and optionally wherein the 5' complementary region and The 3' complementary region includes no more than 10 mismatches between them. 6. The eukaryotic system of claim 1, wherein the 5' annealing region further comprises a 5' non-complementary region having between 5 and 50 ribonucleotides and located 5' of the 5' complementary region. ; and wherein the 3' annealing region further comprises a 3' non-complementary region having between 5 and 50 ribonucleotides and located 3' of the 3' complementary region; and wherein: (a) The 5' non-complementary region and the 3' non-complementary region have sequence complementarity between 0% and 50%; and/or (b) The 5' non-complementary region and the 3' non-complementary region have a binding free energy greater than -5kcal/mol; and/or (c) The 5' non-complementary region and the 3' non-complementary region have a binding Tm below 10°C. 7. The eukaryotic system of claim 1, wherein the 3' annealing region and the 5' annealing region promote association of the 3' and 5' ends of the linear polyribonucleotide. 8. The eukaryotic system of claim 1, wherein the RNA ligase is a tRNA ligase, optionally wherein the tRNA ligase is (a) a ligase selected from the group consisting of: T4 ligase, RtcB Ligase, TRL-1 ligase, and Rnl1 ligase, Rnl2 ligase, LIG1 ligase, LIG2 ligase, PNK/PNL ligase, PF0027 ligase, thpR ligT ligase, and ytlPor ligase; or (b) A ligase selected from the group consisting of plant RNA ligase, chloroplast RNA ligase, RNA ligase from archaea, bacterial RNA ligase, eukaryotic RNA ligase, viral RNA ligase, and mitochondrial RNA ligase. 9. The eukaryotic system of claim 1, wherein the polyribonucleotide cargo comprises: (a) at least one coding sequence encoding a polypeptide; or (b) at least one non-coding sequence; or (c) A combination of at least one coding sequence encoding a polypeptide and at least one non-coding sequence. 10. The eukaryotic system of claim 1, wherein the polyribonucleotide load comprises at least one coding sequence encoding a polypeptide, and wherein the polypeptide is contained in the genome of a vertebrate, invertebrate, plant or microorganism The encoded amino acid sequence, and/or wherein the polypeptide comprises a therapeutic polypeptide, a plant-modifying polypeptide, or an agricultural polypeptide; and optionally, wherein the encoding sequence is codon-optimized for expression in a subject. 11. The eukaryotic system of claim 1, wherein the polyribonucleotide cargo comprises at least one coding sequence encoding a polypeptide, and further comprises additional elements selected from the group consisting of: (a) An internal ribosome entry site (IRES) or 5'UTR sequence located 5' to and operably linked to the coding sequence, optionally between the IRES or 5'UTR sequence and the coding sequence With inserted ribonucleotides in between; (b) a 3'UTR sequence located 3' to and operably linked to the coding sequence, optionally with an intervening ribonucleotide between the 3'UTR and the coding sequence; and (c) Both (a) and (b). 12. The eukaryotic system of claim 1, wherein the polyribonucleotide cargo comprises at least one non-coding sequence, and wherein the at least one non-coding RNA sequence comprises: (a) At least one RNA selected from the group consisting of: RNA aptamer, long non-coding RNA (lncRNA), transfer RNA-derived fragment (tRF), transfer RNA (tRNA), ribosomal RNA (rRNA), small RNA Nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and Piwi-interacting RNA (piRNA); or fragments of any of these RNAs; and/or (b) At least one RNA selected from the group consisting of: small interfering RNA (siRNA) or its precursor, double-stranded RNA (dsRNA) or at least partially double-stranded RNA; hairpin RNA (hpRNA), microRNA (miRNA) , or its precursor; phasic small interfering RNA (phasiRNA) or its precursor; heterochromatic small interfering RNA (hcsiRNA) or its precursor; and natural antisense short interfering RNA (natsiRNA) or its precursor; and/or (c) Guide RNA (gRNA) or its precursor; and/or (d) Ribozymes or riboswitches. 13. The eukaryotic system of claim 1, wherein the polyribonucleotide cargo comprises at least one non-coding sequence, and wherein the at least one non-coding RNA sequence comprises a regulatory RNA that trans-regulates a target sequence, optionally wherein the target sequence comprises a nucleotide sequence of a gene of the subject genome, and wherein the modulation of the target sequence is (a) an up-regulation of the expression of the target sequence, or (b) a down-regulation of the expression of the target sequence, or ( c) Inducible expression of the target sequence. 14. The eukaryotic system of claim 1, wherein the ligase: (a) is endogenous to the eukaryotic cell, or (b) Is heterologous to the eukaryotic cell. 15. The eukaryotic system of claim 1, wherein the linear polynucleotide is provided to the eukaryotic cell by: (a) providing an exogenous polyribonucleotide comprising the linear polynucleotide to the eukaryotic cell; (b) transcribing in the eukaryotic cell an exogenous recombinant DNA molecule transiently provided to the eukaryotic cell, the exogenous recombinant DNA molecule comprising DNA encoding the linear polyribonucleotide and optionally comprising and encoding A heterologous promoter to which the DNA of the linear polyribonucleotide is operably linked; or (c) a recombinant DNA molecule transcribed in the eukaryotic cell and incorporated into the genome of the eukaryotic cell, the recombinant DNA molecule comprising DNA encoding the linear polyribonucleotide and optionally comprising and encoding the linear polyribonucleotide Nucleotide DNA is operably linked to a heterologous promoter. 16. The eukaryotic system of claim 1, wherein the eukaryotic cell is: (a) a unicellular eukaryotic cell, optionally wherein the unicellular eukaryotic cell is selected from the group consisting of a unicellular fungal cell, an oomycete cell, a unicellular animal cell, a unicellular plant cell, a unicellular algal cell, protist cells, and protozoan cells; (b) A cell of a multicellular eukaryote, optionally wherein the multicellular eukaryote is selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular oomycetes, multicellular algae, and multicellular algae. Cellular plants. 17. A formulation comprising the eukaryotic system of claim 1, optionally wherein the formulation is a pharmaceutical, veterinary, or agricultural formulation. 18. A cyclic polyribonucleotide produced by the eukaryotic system of claim 1, optionally wherein the cyclic polyribonucleotide is purified. 19. A formulation comprising the cyclic polyribonucleotide of claim 18, optionally wherein the formulation is a pharmaceutical, veterinary, or agricultural formulation. 20. A method of producing circular RNA, the method comprising: (a) Bringing (i) into contact with (ii) in a eukaryotic cell: (i) Linear polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein element (A) , (B), (C), (D), and (E) are operably connected, and wherein: (A) Contains 5' self-cleaving ribozyme; (B) Contains a 5' annealing region containing a 5' complementary region; (C) Contains a polyribonucleotide payload; (D) Contains a 3' annealing region containing a 3' complementary region; and (E) Contains a 3' self-cleaving ribozyme; wherein the 5' complementary region and the 3' complementary region have a binding free energy of less than -5 kcal/mol, and/or wherein the 5' complementary region and the 3' complementary region have a binding Tm of at least 10°C; wherein cleavage by the 5' self-cleaving ribozyme produces a free 5'-hydroxyl group on the 5' end of the linear polyribonucleotide, and wherein cleavage by the 3' self-cleaving ribozyme generates a free 5'-hydroxyl group on the 5' end of the linear polyribonucleotide A free 2',3'-cyclic phosphate group is generated on the 3' end of the nucleotide to obtain a ligase-compatible linear polyribonucleotide; and (ii) RNA ligase; whereby the 5' and 3' ends of the ligase-compatible linear polyribonucleotide are ligated by the RNA ligase, thereby producing a cyclic polyribonucleotide; and (b) Optionally, purifying the cyclic polyribonucleotide. 21. The method of claim 20, wherein the linear polynucleotide is provided to the eukaryotic cell by: (a) providing an exogenous polyribonucleotide comprising the linear polynucleotide to the eukaryotic cell; (b) transcribing in the eukaryotic cell an exogenous recombinant DNA molecule transiently provided to the eukaryotic cell, the exogenous recombinant DNA molecule comprising DNA encoding the linear polyribonucleotide and optionally comprising and encoding A heterologous promoter to which the DNA of the linear polyribonucleotide is operably linked; or (c) a recombinant DNA molecule transcribed in the eukaryotic cell and incorporated into the genome of the eukaryotic cell, the recombinant DNA molecule comprising DNA encoding the linear polyribonucleotide and optionally comprising and encoding the linear polyribonucleotide Nucleotide DNA is operably linked to a heterologous promoter. 22. The method of claim 20, wherein the 5' self-cleaving ribozyme is a ribozyme selected from the group consisting of: hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme (HDV ), Varkud satellite (VS) ribozyme, glmS ribozyme, twist ribozyme, twist sister ribozyme, ax ribozyme, and pistol ribozyme. 23. The method of claim 20, wherein the 3' self-cleaving ribozyme is a ribozyme selected from the group consisting of: hammerhead ribozyme, hairpin ribozyme, hepatitis delta virus ribozyme (HDV ), Varkud satellite (VS) ribozyme, glmS ribozyme, twist ribozyme, twist sister ribozyme, ax ribozyme, and pistol ribozyme. 24. The method of claim 20, wherein the 5' complementary region has between 5 and 50 ribonucleotides and the 3' complementary region has between 5 and 50 ribonucleotides. 25. The method of claim 20, wherein the 5' complementary region and the 3' complementary region have sequence complementarity between 50% and 100%, and optionally wherein the 5' complementary region and the 3' complementary region 'Complementary regions include no more than 10 mismatches between them. 26. The method of claim 20, wherein the 5' annealing region further comprises a 5' non-complementary region having between 5 and 50 ribonucleotides and located 5' of the 5' complementary region; and wherein the 3' annealing region further comprises a 3' non-complementary region having between 5 and 50 ribonucleotides and located 3' of the 3' complementary region; and wherein: (a) The 5' non-complementary region and the 3' non-complementary region have sequence complementarity between 0% and 50%; and/or (b) The 5' non-complementary region and the 3' non-complementary region have a binding free energy greater than -5kcal/mol; and/or (c) The 5' non-complementary region and the 3' non-complementary region have a binding Tm below 10°C. 27. The method of claim 20, wherein the 3' annealing region and the 5' annealing region promote association of the 3' and 5' ends of the linear polyribonucleotide. 28. The method of claim 20, wherein the RNA ligase is a tRNA ligase, optionally wherein the tRNA ligase is (a) a ligase selected from the group consisting of: T4 ligase, RtcB ligase , TRL-1 ligase, and Rnl1 ligase, Rnl2 ligase, LIG1 ligase, LIG2 ligase, PNK/PNL ligase, PF0027 ligase, thpR ligT ligase, and ytlPor ligase; or (b) selected from Ligase from the group consisting of: plant RNA ligase, chloroplast RNA ligase, RNA ligase from archaea, bacterial RNA ligase, eukaryotic RNA ligase, viral RNA ligase, and mitochondrial RNA ligase. 29. The method of claim 20, wherein the polyribonucleotide cargo comprises: (a) at least one coding sequence encoding a polypeptide; or (b) at least one non-coding sequence; or (c) A combination of at least one coding sequence encoding a polypeptide and at least one non-coding sequence. 30. The method of claim 20, wherein the polyribonucleotide load comprises at least one coding sequence encoding a polypeptide, and wherein the polypeptide comprises a sequence encoded in the genome of a vertebrate, invertebrate, plant or microorganism. The amino acid sequence, and/or wherein the polypeptide comprises a therapeutic polypeptide, a plant-modifying polypeptide, or an agricultural polypeptide; and optionally, wherein the coding sequence is codon-optimized for expression in a subject. 31. The method of claim 20, wherein the polyribonucleotide cargo comprises at least one coding sequence encoding a polypeptide, and further comprises additional elements selected from the group consisting of: (a) An internal ribosome entry site (IRES) or 5'UTR sequence located 5' to and operably linked to the coding sequence, optionally between the IRES or 5'UTR sequence and the coding sequence With inserted ribonucleotides in between; (b) a 3'UTR sequence located 3' to and operably linked to the coding sequence, optionally with an intervening ribonucleotide between the 3'UTR and the coding sequence; and (c) Both (a) and (b). 32. The method of claim 20, wherein the polyribonucleotide payload comprises at least one non-coding sequence, and wherein the at least one non-coding RNA sequence comprises: (a) At least one RNA selected from the group consisting of: RNA aptamer, long non-coding RNA (lncRNA), transfer RNA-derived fragment (tRF), transfer RNA (tRNA), ribosomal RNA (rRNA), small RNA Nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and Piwi-interacting RNA (piRNA); or fragments of any of these RNAs; and/or (b) At least one RNA selected from the group consisting of: small interfering RNA (siRNA) or its precursor, double-stranded RNA (dsRNA) or at least partially double-stranded RNA; hairpin RNA (hpRNA), microRNA (miRNA) , or its precursor; phasic small interfering RNA (phasiRNA) or its precursor; heterochromatic small interfering RNA (hcsiRNA) or its precursor; and natural antisense short interfering RNA (natsiRNA) or its precursor; and/or (c) Guide RNA (gRNA) or its precursor; and/or (d) Ribozymes or riboswitches. 33. The method of claim 20, wherein the polyribonucleotide cargo comprises at least one non-coding sequence, and wherein the at least one non-coding RNA sequence comprises a regulatory RNA that trans-regulates a target sequence, optionally wherein The target sequence comprises a nucleotide sequence of a gene of the subject genome, and wherein the modulation of the target sequence is (a) up-regulation of the expression of the target sequence, or (b) down-regulation of the expression of the target sequence, or (c) Inducible expression of the target sequence. 34. The method of claim 20, wherein the eukaryotic cell is: (a) a unicellular eukaryotic cell, optionally wherein the unicellular eukaryotic cell is selected from the group consisting of a unicellular fungal cell, a unicellular animal cell, a unicellular plant cell, a unicellular algal cell, an oomycete cell, Protist cells, and protozoan cells; (b) A cell of a multicellular eukaryote, optionally wherein the multicellular eukaryote is selected from the group consisting of vertebrates, invertebrates, multicellular fungi, multicellular oomycetes, multicellular algae, and multicellular algae. Cellular plants. 35. A cyclic polynucleotide produced by the method of claim 20. 36. The method of claim 20, wherein the cyclic polynucleotide is purified and formulated for delivery to a subject, optionally to treat a condition in the subject, and further optionally wherein the formulation is Pharmaceutical preparations, veterinary preparations, or agricultural preparations. 37. The method of claim 36, wherein the subject is a human, non-human vertebrate, invertebrate, or plant. 38. A eukaryotic cell comprising: (a) Linear polyribonucleotide having the formula 5'-(A)-(B)-(C)-(D)-(E)-3', wherein element (A) , (B), (C), (D), and (E) are operably connected, and wherein: (A) Contains 5' self-cleaving ribozyme; (B) Contains a 5' annealing region containing a 5' complementary region; (C) Contains a polyribonucleotide payload; (D) Contains a 3' annealing region containing a 3' complementary region; and (E) Contains a 3' self-cleaving ribozyme; wherein the 5' complementary region and the 3' complementary region have a binding free energy of less than -5 kcal/mol, and/or wherein the 5' complementary region and the 3' complementary region have a binding Tm of at least 10°C; wherein cleavage by the 5' self-cleaving ribozyme produces a free 5'-hydroxyl group on the 5' end of the linear polyribonucleotide, and wherein cleavage by the 3' self-cleaving ribozyme generates a free 5'-hydroxyl group on the 5' end of the linear polyribonucleotide A free 2',3'-cyclic phosphate group is generated on the 3' end of the nucleotide to obtain a ligase-compatible linear polyribonucleotide; as well as (b) RNA ligase, wherein the RNA ligase is capable of ligating the 5' end and the 3' end of a linear polyribonucleotide compatible with the ligase to produce a circular RNA. 39. The eukaryotic cell of claim 38, wherein the 5' annealing region further comprises a 5' non-complementary region having between 5 and 50 ribonucleotides and located 5' of the 5' complementary region ; and wherein the 3' annealing region further comprises a 3' non-complementary region having between 5 and 50 ribonucleotides and located 3' of the 3' complementary region; and wherein: (a) The 5' non-complementary region and the 3' non-complementary region have sequence complementarity between 0% and 50%; and/or (b) The 5' non-complementary region and the 3' non-complementary region have a binding free energy greater than -5kcal/mol; and/or (c) The 5' non-complementary region and the 3' non-complementary region have a binding Tm below 10°C. 40. The eukaryotic cell of claim 38, wherein the polyribonucleotide cargo comprises: (a) at least one coding sequence encoding a polypeptide; or (b) at least one non-coding sequence; or (c) A combination of at least one coding sequence encoding a polypeptide and at least one non-coding sequence. 41. The eukaryotic cell of claim 38, wherein the polyribonucleotide load comprises at least one coding sequence encoding a polypeptide, and wherein the polypeptide is comprised in the genome of a vertebrate, invertebrate, plant or microorganism The encoded amino acid sequence, and/or wherein the polypeptide comprises a therapeutic polypeptide, a plant-modifying polypeptide, or an agricultural polypeptide; and optionally, wherein the encoding sequence is codon-optimized for expression in a subject. 42. The eukaryotic cell of claim 38, wherein the polyribonucleotide cargo comprises at least one coding sequence encoding a polypeptide, and further comprises additional elements selected from the group consisting of: (a) An internal ribosome entry site (IRES) or 5'UTR sequence located 5' to and operably linked to the coding sequence, optionally between the IRES or 5'UTR sequence and the coding sequence With inserted ribonucleotides in between; (b) a 3'UTR sequence located 3' to and operably linked to the coding sequence, optionally with an intervening ribonucleotide between the 3'UTR and the coding sequence; and (c) Both (a) and (b). 43. The eukaryotic cell of claim 38, wherein the polyribonucleotide cargo comprises at least one non-coding sequence, and wherein the at least one non-coding RNA sequence comprises: (a) At least one RNA selected from the group consisting of: RNA aptamer, long non-coding RNA (lncRNA), transfer RNA-derived fragment (tRF), transfer RNA (tRNA), ribosomal RNA (rRNA), small RNA Nuclear RNA (snRNA), small nucleolar RNA (snoRNA), and Piwi-interacting RNA (piRNA); or fragments of any of these RNAs; and/or (b) At least one RNA selected from the group consisting of: small interfering RNA (siRNA) or its precursor, double-stranded RNA (dsRNA) or at least partially double-stranded RNA; hairpin RNA (hpRNA), microRNA (miRNA) , or its precursor; phasic small interfering RNA (phasiRNA) or its precursor; heterochromatic small interfering RNA (hcsiRNA) or its precursor; and natural antisense short interfering RNA (natsiRNA) or its precursor; and/or (c) Guide RNA (gRNA) or its precursor; and/or (d) Ribozymes or riboswitches. 44. The eukaryotic cell of claim 38, wherein the RNA ligase is (a) endogenous to the eukaryotic cell, or (b) heterologous to the eukaryotic cell. 45. The eukaryotic cell of claim 38, wherein the RNA ligase is a tRNA ligase, optionally wherein the tRNA ligase is (a) a ligase selected from the group consisting of: T4 ligase, RtcB Ligase, TRL-1 ligase, and Rnl1 ligase, Rnl2 ligase, LIG1 ligase, LIG2 ligase, PNK/PNL ligase, PF0027 ligase, thpRligT ligase, and ytlPor ligase; or (b) option Ligase from the group consisting of: plant RNA ligase, chloroplast RNA ligase, RNA ligase from archaea, bacterial RNA ligase, eukaryotic RNA ligase, viral RNA ligase, and mitochondrial RNA ligase. 46. The eukaryotic cell of claim 38, further comprising the circular RNA. 47. A method of providing circular RNA to a subject, the method comprising providing the eukaryotic cell of claim 38 to the subject, optionally wherein the eukaryotic cell is lysed, dried or frozen, And further optionally wherein the eukaryotic cell is provided in a pharmaceutical formulation, a veterinary formulation, or an agricultural formulation. 48. The method of claim 47, wherein the subject is a human, non-human vertebrate, invertebrate, or plant. 49. A formulation comprising the eukaryotic cell of claim 38, optionally wherein the eukaryotic cell is lysed, dried or frozen, and further optionally wherein the formulation is a pharmaceutical formulation , veterinary preparations, or agricultural preparations. 50. A method of treating a disorder in a subject in need thereof, the method comprising providing the formulation of claim 49 to the subject. 51. The method of claim 50, wherein the subject is a human, non-human vertebrate, invertebrate, or plant.