NOVEL CRISPR DNA TARGETING ENZYMES AND SYSTEMS

Abstract

The disclosure describes novel systems, methods, and compositions for the manipulation of nucleic acids in a targeted fashion. The disclosure describes non-naturally occurring, engineered CRISPR systems, components, and methods for targeted modification of nucleic acids such as DNA. Each system includes one or more protein components and one or more nucleic acid components that together target nucleic acids.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No. 17/139,678, filed Dec. 31, 2020, which is a continuation of U.S. application Ser. No. 17/020,215, filed Sep. 14, 2020, which is a continuation of U.S. application Ser. No. 16/680,104, filed Nov. 11, 2019, which is a continuation of International Application No. PCT/US2019/022375, filed Mar. 14, 2019, which claims the benefit of priority of U.S. Application No. 62/642,919, filed Mar. 14, 2018; U.S. Application No. 62/666,397, filed May 3, 2018; U.S. Application No. 62/672,489, filed May 16, 2018; U.S. Application No. 62/679,628, filed Jun. 1, 2018; U.S. Application No. 62/703,857, filed Jul. 26, 2018; U.S. Application No. 62/740,856, filed Oct. 3, 2018; U.S. Application No. 62/746,528, filed Oct. 16, 2018; U.S. Application No. 62/772,038, filed Nov. 27, 2018; and U.S. Application No. 62/775,885, filed Dec. 5, 2018. The content of each of the foregoing applications is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 29, 2019, is named 45138-0011WO1_SL.txt and is 185,394 bytes in size.

FIELD OF THE INVENTION

[0003] The present disclosure relates to systems, methods, and compositions used for the control of gene expression involving sequence targeting and nucleic acid editing, which uses vector systems related to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.

BACKGROUND

[0004] Recent application of advances in genome sequencing technologies and analysis have yielded significant insights into the genetic underpinning of biological activities in many diverse areas of nature, ranging from prokaryotic biosynthetic pathways to human pathologies. To fully understand and evaluate the vast quantities of information produced by genetic sequencing technologies, equivalent increases in the scale, efficacy, and ease of technologies for genome and epigenome manipulation are needed. These novel genome and epigenome engineering technologies will accelerate the development of novel applications in numerous areas, including biotechnology, agriculture, and human therapeutics.

[0005] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and the CRISPR-associated (Cas) genes, collectively known as the CRISPR-Cas or CRISPR/Cas systems, are currently understood to provide immunity to bacteria and archaea against phage infection. The CRISPR-Cas systems of prokaryotic adaptive immunity are an extremely diverse group of proteins effectors, non-coding elements, as well as loci architectures, some examples of which have been engineered and adapted to produce important biotechnologies.

[0006] The components of the system involved in host defense include one or more effector proteins capable of modifying DNA or RNA and an RNA guide element that is responsible to targeting these protein activities to a specific sequence on the phage DNA or RNA. The RNA guide is composed of a CRISPR RNA (crRNA) and may require an additional trans-activating RNA (tracrRNA) to enable targeted nucleic acid manipulation by the effector protein(s). The crRNA consists of a direct repeat responsible for protein binding to the crRNA and a spacer sequence that is complementary to the desired nucleic acid target sequence. CRISPR systems can be reprogrammed to target alternative DNA or RNA targets by modifying the spacer sequence of the crRNA.

[0007] CRISPR-Cas systems can be broadly classified into two classes: Class 1 systems are composed of multiple effector proteins that together form a complex around a crRNA, and Class 2 systems consist of a single effector protein that complexes with the RNA guide to target DNA or RNA substrates. The single-subunit effector composition of the Class 2 systems provides a simpler component set for engineering and application translation, and have thus far been an important source of programmable effectors. Thus, the discovery, engineering, and optimization of novel Class 2 systems may lead to widespread and powerful programmable technologies for genome engineering and beyond.

[0008] CRISPR-Cas systems are adaptive immune systems in archaea and bacteria that defend the species against foreign genetic elements. The characterization and engineering of Class 2 CRISPR-Cas systems, exemplified by CRISPR-Cas9, have paved the way for a diverse array of biotechnology applications in genome editing and beyond. Nevertheless, there remains a need for additional programmable effectors and systems for modifying nucleic acids and polynucleotides (i.e., DNA, RNA, or any hybrid, derivative, or modification) beyond the current CRISPR-Cas systems that enable novel applications through their unique properties.

[0009] Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

SUMMARY

[0010] This disclosure provides non-naturally-occurring, engineered systems and compositions for new single-effector Class 2 CRISPR-Cas systems, together with methods for computational identification from genomic databases, development of the natural loci into an engineered system, and experimental validation and application translation. These new effectors are divergent in sequence to orthologs and homologs of existing Class 2 CRISPR effectors, and also have unique domain organizations. They provide additional features that include, but are not limited to, 1) novel DNA/RNA editing properties and control mechanisms, 2) smaller size for greater versatility in delivery strategies, 3) genotype triggered cellular processes such as cell death, and 4) programmable RNA-guided DNA insertion, excision, and mobilization. Adding the novel DNA-targeting systems described herein to the toolbox of techniques for genome and epigenome manipulation enables broad applications for specific, programmed perturbations.

[0011] In general, this disclosure relates to new CRISPR-Cas systems including newly discovered enzymes and other components used to create minimal systems that can be used in non-natural environments, e.g., in bacteria other than those in which the system was initially discovered.

[0012] In one aspect, the disclosure provides engineered, non-naturally occurring CRISPR-Cas systems that include: i) one or more Type V-I (CLUST.029130) RNA guides or one or more nucleic acids encoding the one or more Type V-I RNA guides, wherein a Type V-I RNA guide includes or consists of a direct repeat sequence and a spacer sequence capable of hybridizing to a target nucleic acid; and ii) a Type V-I (CLUST.029130) CRISPR-Cas effector protein or a nucleic acid encoding the Type V-I CRISPR-Cas effector protein, wherein the Type V-I CRISPR-Cas effector protein is capable of binding to a Type V-I RNA guide and of targeting the target nucleic acid sequence complementary to the spacer sequence, wherein the target nucleic acid is a DNA. As used herein, the Type V-I (CLUST.029130) CRISPR-Cas effector proteins are also referred to as Cas12i effector proteins, and these two terms are used interchangeably in this disclosure.

[0013] In some embodiments of any of the systems described herein, the Type V-I CRISPR-Cas effector protein is about 1100 amino acids or less in length (excluding any amino acid signal sequence or peptide tag fused thereto) and includes at least one RuvC domain. In some embodiments, none, one, or more of the RuvC domains are catalytically inactivated. In some embodiments, the Type V-I CRISPR-Cas effector protein includes or consists of the amino acid sequence X.sub.1SHX.sub.4DX.sub.6X.sub.7 (SEQ ID NO: 200), wherein X.sub.1 is S or T, X.sub.4 is Q or L, X.sub.6 is P or S, and X.sub.7 is F or L.

[0014] In some embodiments, the Type V-I CRISPR-Cas effector protein includes or consists of the amino acid sequence X.sub.1XDXNX.sub.6X.sub.7XXXX.sub.11 (SEQ ID NO: 201), wherein X.sub.1 is A or G or S, X is any amino acid, X.sub.6 is Q or I, X.sub.7 is T or S or V, and X.sub.10 is T or A. In some embodiments, the Type V-I CRISPR-Cas effector protein includes or consists of the amino acid sequence X1X2X3E (SEQ ID NO: 210), wherein X1 is C or F or I or L or M or P or V or W or Y, X2 C or F or I or L or M or P or R or V or W or Y, and X3 C or F or G or I or L or M or P or V or W or Y.

[0015] In some embodiments, the Type V-I CRISPR-Cas effector protein includes more than one sequence from the set SEQ ID NO: 200, SEQ ID NO: 201, and SEQ ID NO: 210. In some embodiments, the Type V-I CRISPR-Cas effector protein includes or consists of an amino acid sequence that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to an amino acid sequence provided in Table 4 (e.g., SEQ ID NOs: 1-5, and 11-18).

[0016] In some embodiments of any of the systems described herein, the Type V-I CRISPR-Cas effector protein includes or consists of an amino acid sequence that is at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identical to the amino acid sequence of Cas12i1 (SEQ ID NO: 3) or Cas12i2 (SEQ ID NO: 5). In some embodiments, the Type V-I CRISPR-Cas effector protein is Cas12i1 (SEQ ID NO: 3) or Cas12i2 (SEQ ID NO: 5).

[0017] In some embodiments, the Type V-I CRISPR-Cas effector protein is capable of recognizing a protospacer adjacent motif (PAM), and the target nucleic acid includes or consists of a PAM including or consisting of the nucleic acid sequence 5'-TTN-3' or 5'-TTH-3' or 5'-TTY-3' or 5'-TTC-3'.

[0018] In some embodiments of any of the systems described herein, the Type V-I CRISPR-Cas effector protein includes one or more amino acid substitutions within at least one of the RuvC domains. In some embodiments, the one or more amino acid substitutions include a substitution, e.g., an alanine substitution, at an amino residue corresponding to D647 or E894 or D948 of SEQ ID NO: 3. In some embodiments, the one or more amino acid substitutions include an alanine substitution at an amino residue corresponding to D599 or E833 or D886 of SEQ ID NO: 5. In some embodiments, the one or more amino acid substitutions result in a reduction of the nuclease activity of the Type V-I CRISPR-Cas effector protein as compared to the nuclease activity of the Type V-I CRISPR-Cas effector protein without the one or more amino acid substitutions.

[0019] In some embodiments of any of the systems described herein, the Type V-I RNA guide includes a direct repeat sequence that includes a stem-loop structure proximal to the 3' end (immediately adjacent to the spacer sequence). In some embodiments, the Type V-I RNA guide direct repeat includes a stem loop proximal to the 3' end where the stem is 5 nucleotides in length. In some embodiments, the Type V-I RNA guide direct repeat includes a stem loop proximal to the 3' end where the stem is 5 nucleotides in length and the loop is 7 nucleotides in length. In some embodiments, the Type V-I RNA guide direct repeat includes a stem loop proximal to the 3' end where the stem is 5 nucleotides in length and the loop is 6, 7, or 8 nucleotides in length.

[0020] In some embodiments, the Type V-I RNA guide direct repeat includes the sequence 5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO: 202) proximal to the 3' end, wherein N refers to any nucleobase. In some embodiments, the Type V-I RNA guide direct repeat includes the sequence 5'-GUGCCNNNNNNUGGCAC-3' (SEQ ID NO: 203) proximal to the 3' end, wherein N refers to any nucleobase.

[0021] In some embodiments, the Type V-I RNA guide direct repeat includes the sequence 5'-GUGUCN.sub.5-6UGACAX.sub.1-3' (SEQ ID NO: 204) proximal to the 3' end, wherein Ns-6 refers to a contiguous sequence of any 5 or 6 nucleobases, and X.sub.1 refers to C or T or U. In some embodiments, the Type V-I RNA guide direct repeat includes the sequence 5'-UCX.sub.3UX.sub.5X.sub.6X.sub.7UUGACGG-3' (SEQ ID NO: 205) proximal to the 3' end, wherein X.sub.3 refers to C or T or U, X.sub.5 refers to A or T or U, X.sub.6 refers to A or C or G, and X.sub.7 refers to A or G. In some embodiments, the Type V-I RNA guide direct repeat includes the sequence 5'-CCX.sub.3X.sub.4X.sub.5CX.sub.7UUGGCAC-3' (SEQ ID NO: 206) proximal to the 3' end, wherein X.sub.3 refers to C or T or U, X.sub.4 refers to A or T or U, X.sub.5 refers to C or T or U, and X.sub.7 refers to A or G.

[0022] In some embodiments, the Type V-I RNA guide includes a direct repeat sequence including or consisting of a nucleotide sequence that is at least 80% identical, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, to a nucleotide sequence provided in Table 5A (e.g., SEQ ID NOs: 6-19, and 19-24).

[0023] In some embodiments, the Type V-I RNA guide includes or consists of a nucleotide sequence or subsequence thereof provided in Table 5B (e.g., SEQ ID Nos: 150-163). In some embodiments, the Type V-I RNA guide includes or consists of a nucleotide sequence constructed by the concatenation of a direct repeat, spacer, direct repeat sequence wherein the direct repeat sequence is provided in Table 5A and the length of the spacer is provided in the Spacer Lens 1 column in Table 5B. In some embodiments, the Type V-I RNA guide includes or consists of a nucleotide sequence constructed by the concatenation of a direct repeat, spacer, direct repeat sequence wherein the direct repeat sequence is provided in Table 5A and the length of the spacer is provided in the Spacer Lens 2 column in Table 5B. In some embodiments, the Type V-I RNA guide includes or consists of a nucleotide sequence constructed by the concatenation of a direct repeat, spacer, direct repeat sequence wherein the direct repeat sequence is provided in Table 5A and the length of the spacer is provided in the Spacer Lens 3 column in Table 5B.

[0024] In some embodiments of any of the systems described herein, the spacer sequence of the Type V-I RNA guide includes or consists of between about 15 to about 34 nucleotides (e.g., 16, 17, 18, 19, 20, 21, or 22 nucleotides). In some embodiments of any of the systems described herein, the spacer is between 17 nucleotides and 31 nucleotides in length.

[0025] In some embodiments of any of the systems provided herein, the target nucleic acid is a DNA. In some embodiments of any of the systems described herein, the target nucleic acid includes a protospacer adjacent motif (PAM), e.g., a PAM including or consisting of the nucleic acid sequence 5'-TTN-3' or 5'-TTH-3' or 5'-TTY-3' or 5'-TTC-3'.

[0026] In certain embodiments of any of the systems provided herein, the targeting of the target nucleic acid by the Type V-I CRISPR-Cas effector protein and RNA guide results in a modification (e.g., a single-stranded or a double-stranded cleavage event) in the target nucleic acid. In some embodiments, the modification is a deletion event. In some embodiments, the modification is an insertion event. In some embodiments, the modification results in cell toxicity and/or cell death.

[0027] In some embodiments, the Type V-I CRISPR-Cas effector protein has non-specific (i.e., "collateral") nuclease (e.g., DNase) activity. In certain embodiments of any of the systems provided herein, the system further includes a donor template nucleic acid (e.g., a DNA or a RNA).

[0028] In some embodiments of any of the systems provided herein, the system is within a cell (e.g., a eukaryotic cell (e.g., a mammalian cell) or a prokaryotic cell (e.g., a bacterial cell)).

[0029] In another aspect, the disclosure provides methods of targeting and editing a target nucleic acid, wherein the methods include contacting the target nucleic acid with any of the systems described herein. These can be carried out ex vivo or in vitro methods. In some embodiments, the methods described herein do not modify the germ line genetic identity of a human being.

[0030] In other aspects, the disclosure provides methods of targeting the insertion of a payload nucleic acid at a site of a target nucleic acid, wherein the methods include contacting the target nucleic acid with any of the systems described herein.

[0031] In yet another aspect, the disclosure provides methods of targeting the excision of a payload nucleic acid from a site at a target nucleic acid, wherein the methods include contacting the target nucleic acid with any of the systems described herein.

[0032] In another aspect, the disclosure provides methods of targeting and nicking a non-target strand (non-spacer complementary strand) of a double-stranded target DNA upon recognition of a target strand (spacer complementary strand) of the double-stranded target DNA. The method includes contacting the double-stranded target DNA with any of the systems described herein.

[0033] In yet another aspect, the disclosure provides methods of targeting and cleaving a double-stranded target DNA, the method including contacting the double-stranded target DNA with any of the systems described herein.

[0034] In some embodiments of the methods of targeting and cleaving a double-stranded target DNA, a non-target strand (non-spacer complementary strand) of the double-stranded target DNA is nicked before a target strand (spacer complementary strand) of the double-stranded target nucleic acid is nicked.

[0035] In yet another aspect, the disclosure provides methods of specifically editing a double-stranded nucleic acid, the methods including: contacting (a) a Type V-I effector protein and one other enzyme with sequence-specific nicking activity; (b) a Type V-I RNA guide that guides the Type V-I effector protein to nick the opposing strand relative to the activity of the other sequence-specific nickase; and (c) the double-stranded nucleic acid, wherein the method results in reduced likelihood of off-target modification.

[0036] In some embodiments, the Type V-I effector protein further includes a linker sequence. In some embodiments, the Type V-I effector protein includes one or more mutations or amino acid substitutions that render the CRISPR-associated protein unable to cleave DNA.

[0037] In yet another aspect, the disclosure provides methods of base editing a double-stranded nucleic acid, the method including: contacting (a) a fusion protein comprising a Type V-I effector protein and a protein domain with DNA modifying activity (e.g., cytidine deamination); (b) a Type V-I RNA guide targeting the double-stranded nucleic acid, and (c) the double-stranded nucleic acid. The Type V-I effector of the fusion protein can be modified to nick non-target strand of the double-stranded nucleic acid. In some embodiments, the Type V-I effector of the fusion protein can be modified to be nuclease deficient. zzz

[0038] In another aspect, the disclosure provides methods of modifying a DNA molecule, the methods including contacting the DNA molecule with a system described herein.

[0039] In some embodiments of any of the methods described herein (and compositions for use in such methods), the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell. In some embodiments, the cell is a cancer cell (e.g., a tumor cell). In some embodiments, the cell is an infectious agent cell or a cell infected with an infectious agent. In some embodiments, the cell is a bacterial cell, a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.

[0040] In another aspect, the disclosure provides methods of treating a condition or disease in a subject in need thereof and compositions for use in such methods. The methods include administering to the subject a system described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease, wherein the Type V-I CRISPR-Cas effector protein associates with the RNA guide to form a complex, wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence, and wherein upon binding of the complex to the target nucleic acid sequence the Type V-I CRISPR-Cas effector protein cleaves or silences the target nucleic acid, thereby treating the condition or disease in the subject.

[0041] In some embodiments of the methods described herein (and compositions for use in such methods), the condition or disease is a cancer or an infectious disease. In some embodiments, the condition or disease is cancer, wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer.

[0042] In some embodiments, the Type V-I effector protein includes or consists of at least one (e.g., two, three, four, five, six, or more) nuclear localization signal (NLS). In some embodiments, the Type V-I effector protein includes or consists of at least one (e.g., two, three, four, five, six, or more) nuclear export signal (NES). In some embodiments, the Type V-I effector protein includes at least one (e.g., two, three, four, five, six, or more) NLS and at least one (e.g., two, three, four, five, six, or more) NES.

[0043] In some embodiments, the systems described herein include a nucleic acid encoding one or more RNA guides. In some embodiments, the nucleic acid encoding the one or more RNA guides is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).

[0044] In some embodiments, the systems described herein include a nucleic acid encoding a target nucleic acid (e.g., a target DNA). In some embodiments, the nucleic acid encoding the target nucleic acid is operably linked to a promoter (e.g., a constitutive promoter or an inducible promoter).

[0045] In some embodiments, the systems described herein include a nucleic acid encoding a Type V-I CRISPR-Cas effector protein in a vector. In some embodiments, the system further includes one or more nucleic acids encoding an RNA guide present in the vector.

[0046] In some embodiments, the vectors included in the systems are viral vectors (e.g., retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated vectors, and herpes simplex vectors. In some embodiments, the vectors included in the system are phage vectors.

[0047] In some embodiments, the systems provided herein are in a delivery system. In some embodiments, the delivery system is a nanoparticle, a liposome, an exosome, a microvesicle, and a gene-gun.

[0048] The disclosure also provides a cell (e.g., a eukaryotic cell or a prokaryotic cell (e.g., a bacterial cell)) comprising a system described herein. In some embodiments, the eukaryotic cell is a mammalian cell (e.g., a human cell) or a plant cell. The disclosure also provides animal models (e.g., rodent, rabbit, dog, monkey, or ape models) and plant model that include the cells.

[0049] In some embodiments, the methods are used to treat a subject, e.g., a mammal, such as a human patient. The mammalian subject can also be a domesticated mammal, such as a dog, cat, horse, monkey, rabbit, rat, mouse, cow, goat, or sheep

[0050] In yet another aspect, the disclosure provides methods of detecting a target nucleic acid (e.g., DNA or RNA) in a sample, the methods including: (a) contacting the sample with a system provided herein and a labeled reporter nucleic acid, wherein hybridization of the crRNA to the target nucleic acid causes cleavage of the labeled reporter nucleic acid; and (b) measuring a detectable signal produced by cleavage of the labeled reporter nucleic acid, thereby detecting the presence of the target nucleic acid in the sample.

[0051] In some embodiments, the methods of detecting a target nucleic acid can also include comparing a level of the detectable signal with a reference signal level, and determining an amount of target nucleic acid in the sample based on the level of the detectable signal.

[0052] In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, or semiconductor based-sensing.

[0053] In some embodiments, the labeled reporter nucleic acid can include a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluorophore pair, wherein cleavage of the labeled reporter nucleic acid by the effector protein results in an increase or a decrease of the amount of signal produced by the labeled reporter nucleic acid.

[0054] Turning to another aspect, the disclosure includes methods of modifying a target DNA, which include contacting the target DNA with a complex comprising a Cas12i effector protein and an engineered Type V-I RNA guide, which is designed to hybridize with (e.g., is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to) a target sequence of the target DNA, and the system is distinguished by (a) the lack of a tracrRNA in the system, and (b) the Cas12i effector protein and Type V-I RNA guide form a complex that associates with the target DNA, thereby modifying the target DNA.

[0055] In certain embodiments, modifying the target DNA includes cleaving at least one strand of the target DNA (e.g., creating a single-strand break or "nick," or creating a double strand break). Alternatively, or additionally, modification of the target DNA includes either (i) binding to the target DNA, thereby preventing the target DNA from associating with another biomolecule or complex, or (ii) unwinding a portion of the target DNA. In some instances, the target DNA includes a protospacer adjacent motif (PAM) sequence that is recognized by the Cas12i effector protein, such as 5'-TTN-3' or 5'-TTH-3' or 5'-TTY-3' or 5'-TTC-3'. The Cas12 effector protein is, in certain embodiments, a Cas12i1 effector protein or a Cas12i2 effector protein.

[0056] Continuing with this aspect of the disclosure, in certain embodiments the contacting of the target DNA with the complex occurs in a cell, for instance by (a) contacting the cell with the complex, which complex is formed in vitro, or (b) contacting the cell with one or more nucleic acids encoding the Cas12i effector protein and the Type V-I RNA guide, which are then expressed by the cell and which form the complex within the cell. In some cases, the cell is a prokaryotic cell; in other cases, it is a eukaryotic cell.

[0057] In another aspect, this disclosure relates to methods of altering a target DNA, including contacting the target DNA within the cell with a genome editing system including a Cas12i protein and a Type V-I RNA guide (e.g., a crRNA, guide RNA or like structure, optionally comprising one or more nucleotide, nucleobase or backbone modifications) comprising a 15-24 nucleotide spacer sequence having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a sequence in the target DNA, but which system does not comprise a tracrRNA. In various embodiments, the Cas12i protein includes or consists of an amino acid sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, sequence identity to SEQ ID NO: 3 and the Type V-I RNA guide comprises a direct repeat sequence with at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOS: 7 or 24; or the Cas12i protein includes or consists of an amino acid sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, sequence identity to SEQ ID NO: 5 and the Type V-I RNA guide comprises a direct repeat sequence with at least 95% e.g., 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOS: 9 or 10. The target DNA is optionally a cellular DNA, and the contacting optionally occurs within a cell such as a prokaryotic cell or a eukaryotic cell (e.g., a mammalian cell, a plant cell, or a human cell).

[0058] In some embodiments, the Type V-I CRISPR-Cas effector protein comprises an amino acid sequence having at least 90%, or at least 95%, sequence identity to one of SEQ ID NOs: 1-5 or 11-18. According to certain embodiments, the Type V-I CRISPR-Cas effector protein comprises an amino acid sequence given by SEQ ID NO: 3, or an amino acid sequence given by SEQ ID NO: 5. The total length of the CRISPR-Cas effector protein according to certain embodiments is less than 1100 amino acids, excluding any amino acid signal sequence or peptide tag fused thereto. In some cases, the CRISPR-Cas effector protein comprises an amino acid substitution, for instance a substitution at an amino acid residue corresponding to D647, E894, or D948 of SEQ ID NO: 3 or a substitution at an amino acid residue corresponding to D599, E833, or D886 of SEQ ID NO: 5. The substitution is optionally an alanine.

[0059] In yet another aspect, this disclosure relates to an engineered, non-naturally occurring CRISPR-Cas systems, including or consisting of a Cas12i effector protein, and an engineered Type V-I RNA guide (e.g., a crRNA, guide RNA or like structure, optionally including one or more nucleotide, nucleobase or backbone modifications) having a 15-34 nucleotide spacer sequence that is at least 80%, e.g., 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, complementary to a target sequence. The systems do not include a tracrRNA, and the Cas12i effector protein and the Type V-I RNA guide form a complex that associates with the target sequence. In some instances, the complex of the Cas12i effector protein and Type V-I RNA guide causes cleavage of at least one strand of a DNA comprising the target sequence. The target sequence can include a protospacer adjacent motif (PAM) sequence recognized by the Cas12i effector protein, which PAM sequence is optionally 5'-TTN-3', 5'-TTY-3' or 5'-TTH-3' or 5'-TTC-3'. The Type V-I RNA guide can include a direct repeat sequence having at least 95%, e.g., 96%, 97%, 98%, 99%, or 100%, sequence identity to one of SEQ ID NOS: 7, 9, 10, 24, 100, or 101.

[0060] In certain embodiments, the Cas12i effector protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 3 and the direct repeat sequence has at least 95% sequence identity to SEQ ID NO: 100, or the Cas12i effector protein comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 5 and the direct repeat sequence has at least 95% sequence identity to SEQ ID NO: 101. Alternatively, or additionally, the Cas12i effector protein comprises an amino acid substitution (optionally, an alanine substitution) selected from the group consisting of (a) a substitution at an amino acid residue corresponding to D647, E894, or D948 of SEQ ID NO: 3; and (b) a substitution at an amino acid residue corresponding to D599, E833, or D886 of SEQ ID NO: 5.

[0061] In still another aspect, this disclosure relates to a composition comprising one or more nucleic acids encoding a CRISPR-Cas system (or a genome editing system) according to one of the aspects of the disclosure. And in another aspect, the disclosure relates to a viral vector encoding a CRISPR-Cas system (or a genome editing system) according to one of the aspects of the disclosure.

[0062] The disclosure also includes methods of targeting and nicking a non-spacer complementary strand of a double-stranded target DNA upon recognition of a spacer complementary strand of the double-stranded target DNA, the method comprising contacting the double-stranded target DNA with any of the systems described herein.

[0063] In another aspect, the disclosure includes methods of targeting and cleaving a double-stranded target DNA, the method comprising contacting the double-stranded target DNA with a system as described herein. In these methods, a non-spacer complementary strand of the double-stranded target DNA is nicked before a spacer complementary strand of the double-stranded target nucleic acid is nicked.

[0064] In other embodiments, the disclosure includes methods of detecting a target nucleic acid in a sample, the method comprising: (a) contacting the sample with a system as described herein and a labeled reporter nucleic acid, wherein hybridization of the crRNA to the target nucleic acid causes cleavage of the labeled reporter nucleic acid; and (b) measuring a detectable signal produced by cleavage of the labeled reporter nucleic acid, thereby detecting the presence of the target nucleic acid in the sample. These methods can further include comparing a level of the detectable signal with a reference signal level, and determining an amount of target nucleic acid in the sample based on the level of the detectable signal. In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, or semiconductor based-sensing. In some embodiments, the labeled reporter nucleic acid comprises a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluorophore pair, wherein cleavage of the labeled reporter nucleic acid by the effector protein results in an increase or a decrease of the amount of signal produced by the labeled reporter nucleic acid.

[0065] In another aspect, the methods herein include specifically editing a double-stranded nucleic acid, the method comprising contacting, under sufficient conditions and for a sufficient amount of time, (a) a Type V-I CRISPR-Cas effector and one other enzyme with sequence-specific nicking activity, and a crRNA that guides the the Type V-I CRISPR-Cas effector to nick the opposing strand relative to the activity of the other sequence-specific nickase; and (b) the double-stranded nucleic acid; wherein the method results in the formation of a double-stranded break.

[0066] Another aspect includes methods of editing a double-stranded nucleic acid, the method comprising contacting, under sufficient conditions and for a sufficient amount of time, (a) a fusion protein comprising a the Type V-I CRISPR-Cas effector and a protein domain with DNA modifying activity and an RNA guide targeting the double-stranded nucleic acid; and (b) the double-stranded nucleic acid; wherein the the Type V-I CRISPR-Cas effector of the fusion protein is modified to nick a non-target strand of the double-stranded nucleic acid.

[0067] Another aspect includes methods of inducing genotype-specific or transcriptional-state-specific cell death or dormancy in a cell, the method comprising contacting a cell, e.g., a prokaryotic or eukaryotic cell, with any system disclosed herein, wherein the RNA guide hybridizing to the target DNA causes a collateral DNase activity-mediated cell death or dormancy. For example, the cell can be a mammalian cell, e.g., a cancer cell. The cell can be an infectious cell or a cell infected with an infectious agent, e.g., a cell infected with a virus, a cell infected with a prion, a fungal cell, a protozoan, or a parasite cell.

[0068] In another aspect, the disclosure provides methods of treating a condition or disease in a subject in need thereof, the method comprising administering to the subject any of the systems described herein, wherein the spacer sequence is complementary to at least 15 nucleotides of a target nucleic acid associated with the condition or disease; wherein the Type V-I CRISPR-Cas effector protein associates with the RNA guide to form a complex; wherein the complex binds to a target nucleic acid sequence that is complementary to the at least 15 nucleotides of the spacer sequence; and wherein upon binding of the complex to the target nucleic acid sequence the Type V-I CRISPR-Cas effector protein cleaves the target nucleic acid, thereby treating the condition or disease in the subject. For example, the condition or disease can be a cancer or an infectious disease. For example, the condition or disease can be cancer, and wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer.

[0069] The disclosure also includes the systems or cells as described herein for use as a medicament, or for use in the treatment or prevention of a cancer or an infectious disease, e.g., wherein the cancer is selected from the group consisting of Wilms' tumor, Ewing sarcoma, a neuroendocrine tumor, a glioblastoma, a neuroblastoma, a melanoma, skin cancer, breast cancer, colon cancer, rectal cancer, prostate cancer, liver cancer, renal cancer, pancreatic cancer, lung cancer, biliary cancer, cervical cancer, endometrial cancer, esophageal cancer, gastric cancer, head and neck cancer, medullary thyroid carcinoma, ovarian cancer, glioma, lymphoma, leukemia, myeloma, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, and urinary bladder cancer.

[0070] The disclosure also provides the use of the systems or cells as described herein in vitro or ex vivo methods of:

[0071] a) targeting and editing a target nucleic acid;

[0072] b) non-specifically degrading single-stranded DNA upon recognition of a DNA target nucleic acid;

[0073] c) targeting and nicking a non-spacer complementary strand of a double-stranded target DNA upon recognition of a spacer complementary strand of the double-stranded target DNA;

[0074] d) targeting and cleaving a double-stranded target DNA;

[0075] e) detecting a target nucleic acid in a sample;

[0076] f) specifically editing a double-stranded nucleic acid;

[0077] g) base editing a double-stranded nucleic acid;

[0078] h) inducing genotype-specific or transcriptional-state-specific cell death or dormancy in a cell.

[0079] i) creating an indel in a double-stranded target DNA;

[0080] j) inserting a sequence into a double-stranded target DNA, or

[0081] k) deleting or inverting a sequence in a double-stranded target DNA.

[0082] In another aspect, the disclosure provides the use of the systems or cells described herein in methods of:

[0083] a) targeting and editing a target nucleic acid;

[0084] b) non-specifically degrading single-stranded DNA upon recognition of a DNA target nucleic acid;

[0085] c) targeting and nicking a non-spacer complementary strand of a double-stranded target DNA upon recognition of a spacer complementary strand of the double-stranded target DNA;

[0086] d) targeting and cleaving a double-stranded target DNA;

[0087] e) detecting a target nucleic acid in a sample;

[0088] f) specifically editing a double-stranded nucleic acid;

[0089] g) base editing a double-stranded nucleic acid;

[0090] h) inducing genotype-specific or transcriptional-state-specific cell death or dormancy in a cell;

[0091] i) creating an indel in a double-stranded target DNA;

[0092] j) inserting a sequence into a double-stranded target DNA, or

[0093] k) deleting or inverting a sequence in a double-stranded target DNA,

[0094] wherein the method does not comprise a process for modifying the germ line genetic identity of a human being and does not comprise a method of treatment of the human or animal body.

[0095] In the methods described herein, cleaving the target DNA or target nucleic acid results in the formation of an indel, or wherein cleaving the target DNA or target nucleic acid results in the insertion of a nucleic acid sequence, or, wherein cleaving the target DNA or target nucleic acid comprises cleaving the target DNA or target nucleic acid in two sites, and results in the deletion or inversion of a sequence between the two sites.

[0096] The various systems described herein can lack a tracrRNA. In some embodiments, the Type V-I CRISPR-Cas effector protein and Type V-I RNA guide form a complex that associates with the target nucleic acid, thereby modifying the target nucleic acid.

[0097] In some embodiments of the systems described herein, the spacer sequence is between 15 and 47 nucleotides in length, e.g., between 20 and 40 nucleotides in length, or between 24 and 38 nucleotides in length.

[0098] In another aspect, the disclosure provides eukaryotic cells, e.g., mammalian cells, e.g., human cells, comprising a modified target locus of interest, wherein the target locus of interest has been modified according to a method or via use of a composition of any one of the preceding claims. For example, the modification of the target locus of interest can result in:

[0099] (i) the eukaryotic cell comprising altered expression of at least one gene product;

[0100] (ii) the eukaryotic cell comprising altered expression of at least one gene product, wherein the expression of the at least one gene product is increased;

[0101] (iii) the eukaryotic cell comprising altered expression of at least one gene product, wherein the expression of the at least one gene product is decreased; or

[0102] (iv) the eukaryotic cell comprising an edited genome.

[0103] In another aspect, the disclosure provides a eukaryotic cell line of or comprising the eukaryotic cells described herein, or progeny thereof, or a multicellular organism comprising one or more eukaryotic cells described herein.

[0104] The disclosure also provides plant or animal models comprising one or more cells as described herein.

[0105] In another aspect, the disclosure provides methods of producing a plant, having a modified trait of interest encoded by a gene of interest, the method comprising contacting a plant cell with any of the systems described herein, thereby either modifying or introducing said gene of interest, and regenerating a plant from the plant cell.

[0106] The disclosure also provides methods of identifying a trait of interest in a plant, wherein the trait of interest is encoded by a gene of interest, the method comprising contacting a plant cell with any of the systems described herein, thereby identifying the gene of interest. For example, the method can further comprising introducing the identified gene of interest into a plant cell or plant cell line or plant germ plasm and generating a plant therefrom, whereby the plant contains the gene of interest. The method can include having the plant exhibit the trait of interest.

[0107] The disclosure also includes methods of targeting and cleaving a single-stranded target DNA, the method comprising contacting the target nucleic acid with any of the systems described herein. The methods can include the condition or disease being infectious, and wherein the infectious agent is selected from the group consisting of human immunodeficiency virus (HIV), herpes simplex virus-1 (HSV1), and herpes simplex virus-2 (HSV2).

[0108] In some of the method described herein, both strands of target DNA can be cleaved at different sites, resulting in a staggered cut. In other embodiments, both strands of target DNA are cleaved at the same site, resulting in a blunt double-strand break (DSB).

[0109] In some of the therapeutic methods described herein, the condition or disease is selected from the group consisting of Cystic Fibrosis, Duchenne Muscular Dystrophy, Becker Muscular Dystrophy, Alpha-1-antitrypsin Deficiency, Pompe Disease, Myotonic Dystrophy, Huntington Disease, Fragile X Syndrome, Friedreich's ataxia, Amyotrophic Lateral Sclerosis, Frontotemporal Dementia, Hereditary Chronic Kidney Disease, Hyperlipidemia, Hypercholesterolemia, Leber Congenital Amaurosis, Sickle Cell Disease, and Beta Thalassemia.

[0110] The term "cleavage event," as used herein, refers to a DNA break in a target nucleic acid created by a nuclease of a CRISPR system described herein. In some embodiments, the cleavage event is a double-stranded DNA break. In some embodiments, the cleavage event is a single-stranded DNA break.

[0111] The term "CRISPR-Cas system," "Type V-I CRISPR-Cas system," or "Type V-I system" as used herein refers to a Type V-I CRISPR-Cas effector protein (i.e., Cas12i effector protein) and one or more Type V-I RNA guides, and/or nucleic acids encoding the Type V-I CRISPR-Cas effector protein or the one or more Type V-I RNA guides, and optionally promoters operably linked to the expression of the CRISPR effector or to the RNA guide or to both.

[0112] The term "CRISPR array" as used herein refers to the nucleic acid (e.g., DNA) segment that includes CRISPR repeats and spacers, starting with the first nucleotide of the first CRISPR repeat and ending with the last nucleotide of the last (terminal) CRISPR repeat. Typically, each spacer in a CRISPR array is located between two repeats. The terms "CRISPR repeat," or "CRISPR direct repeat," or "direct repeat," as used herein, refer to multiple short direct repeating sequences, which show very little or no sequence variation within a CRISPR array. Suitably, a Type V-I direct repeat may form a stem-loop structure.

[0113] A "stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides that are known or predicted to form a double strand (stem portion) that is linked on one side by a region of predominantly single-stranded nucleotides (loop portion). The terms "hairpin" and "fold-back" structures are also used herein to refer to stem-loop structures. Such structures are well known in the art and these terms are used consistently with their known meanings in the art. As is known in the art, a stem-loop structure does not require exact base-pairing. Thus, the stem may include one or more base mismatches. Alternatively, the base-pairing may be exact, i.e., not include any mismatches. The predicted stem loop structures of some Type V-I direct repeats are illustrated in FIG. 3. The stem for the Type V-I direct repeat contained within the RNA guide is composed of 5 complementary nucleobases that hybridize to each other, and the loop is 6, 7, or 9 nucleotides in length.

[0114] The term "CRISPR RNA" or "crRNA" as used herein refers to an RNA molecule comprising a guide sequence used by a CRISPR effector to target a specific nucleic acid sequence. Typically, crRNAs contains a spacer sequence that mediates target recognition and a direct repeat sequence (referred to herein as a direct repeat or "DR" sequence) that forms a complex with a CRISPR-Cas effector protein.

[0115] The term "donor template nucleic acid," as used herein refers to a nucleic acid molecule that can be used by one or more cellular proteins to alter the structure of a target nucleic acid after a CRISPR enzyme described herein has altered a target nucleic acid. In some embodiments, the donor template nucleic acid is a double-stranded nucleic acid. In some embodiments, the donor template nucleic acid is a single-stranded nucleic acid. In some embodiments, the donor template nucleic acid is linear. In some embodiments, the donor template nucleic acid is circular (e.g., a plasmid). In some embodiments, the donor template nucleic acid is an exogenous nucleic acid molecule. In some embodiments, the donor template nucleic acid is an endogenous nucleic acid molecule (e.g., a chromosome).

[0116] The term "CRISPR-Cas effector," "CRISPR effector," "effector," "CRISPR-associated protein," or "CRISPR enzyme," "Type V-I CRISPR-Cas effector protein," "Type V-I CRISPR-Cas effector," "Type V-I effector," or Cas12i effector protein" as used herein refers to a protein that carries out an enzymatic activity or that binds to a target site on a nucleic acid specified by an RNA guide. A CRISPR-Cas Type V-I effector protein associated within a Type V-I CRISPR-Cas system can also be referred to herein as "Cas12i" or "Cas12i enzyme." A Cas12i enzyme can recognize a short motif associated in the vicinity of a target DNA called a Protospacer Adjacent Motif (PAM). Suitably, a Cas12i enzyme of the present disclosure can recognize a PAM comprising or consisting of TTN, wherein N denotes any nucleotide. For example, the PAM may be TTN, TTH, TTY or TTC.

[0117] In some embodiments, a Type V-I CRISPR-Cas effector protein has endonuclease activity, nickase activity, and/or exonuclease activity.

[0118] The terms "CRISPR effector complex," "effector complex," "binary complex," or "surveillance complex" as used herein refer to a complex containing a Type V-I CRISPR-Cas effector protein and a Type V-I RNA guide.

[0119] The term "RNA guide" as used herein refers to any RNA molecule that facilitates the targeting of a protein described herein to a target nucleic acid. Exemplary "RNA guides" include, but are not limited to, crRNAs, pre-crRNAs (e.g. DR-spacer-DR), and mature crRNAs (e.g. mature_DR-spacer, mature DR-spacer-mature_DR).

[0120] As used herein, the term "targeting" refers to the ability of a complex including a CRISPR-associated protein and an RNA guide, such as a crRNA, to preferentially or specifically bind to, e.g., hybridize to, a specific target nucleic acid compared to other nucleic acids that do not have the same or similar sequence as the target nucleic acid.

[0121] As used herein, the term "target nucleic acid" refers to a specific nucleic acid substrate that contains a nucleic acid sequence complementary to the entirety or a part of the spacer in an RNA guide. In some embodiments, the target nucleic acid comprises a gene or a sequence within a gene. In some embodiments, the target nucleic acid comprises a non-coding region (e.g., a promoter). In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded.

[0122] The terms "activated CRISPR complex," "activated complex." or "ternary complex" as used herein refer to a CRISPR effector complex after it has bound to or has modified a target nucleic acid.

[0123] The terms "collateral RNA" or "collateral DNA" as used herein refer to a nucleic acid substrate that is cleaved non-specifically by an activated CRISPR complex.

[0124] The term "collateral DNase activity," as used herein in reference to a CRISPR enzyme, refers to non-specific DNase activity of an activated CRISPR complex.

[0125] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0126] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

BRIEF FIGURE DESCRIPTION

[0127] The figures include a series of schematics and nucleic acid and amino acid sequences that represent the results of locus analysis of various protein clusters.

[0128] FIGS. 1A-B together depict a classification tree of Type V effectors (Cas12 proteins). The corresponding CRISPR-Cas loci organization is shown for each branch, with the need for a tracrRNA depicted by a white rectangle adjacent to a CRISPR array. CLUST.029130 (Type V-I) systems are depicted as Cas12i.

[0129] FIG. 2A is a schematic representation of the functional domains of the CLUST.029130 (Type V-I) effector, designated Cas12i. The solid grey shading indicates the location of the C-terminal RuvC domain, with the catalytic residues in the three conserved sequence motifs (I, II and III) indicated and shown to scale. The location of the bridge helix domain is indicated with the superscript h.

[0130] FIG. 2B is a schematic representation of a multiple sequence alignment of Cas12i effector proteins, with the relative locations of the conserved catalytic residues of the RuvC domain denoted by RuvC I/I/III.

[0131] FIG. 3 is a group of schematic diagrams that show predicted secondary structure of the RNA transcript of examples of Type V-I direct repeat sequences.

[0132] FIG. 4A is a schematic representation of the design of in vivo screen Effector and Non-coding Plasmids. CRISPR array libraries were designed including non-repetitive spacers uniformly sampled from both strands of pACYC184 or E. coli essential genes flanked by two DRs and expressed by J23119.

[0133] FIG. 4B is a schematic representation of the negative selection screening workflow; 1) CRISPR array libraries were cloned into the Effector Plasmid, 2) the Effector Plasmid and, when present, the Non-coding Plasmid were transformed into E. coli followed by outgrowth for negative selection of CRISPR arrays conferring interference against DNA or RNA transcripts from pACYC184 or E. coli essential genes, 3) Targeted sequencing of the Effector Plasmid was used to identify depleted CRISPR arrays and small RNA sequencing was used to identify mature crRNAs and tracrRNAs.

[0134] FIGS. 5A-B and FIGS. 5C-D are graphic representations that show the density of depleted and non-depleted targets for Cas12i1 and Cas12i2, respectively. Strongly depleted spacers targeting both pACYC184 and E. coli essential genes are depicted in separate plots. Targets on the top strand and bottom strand are shown separately, and in relation to the orientation of the annotated genes.

[0135] FIGS. 6A and 6B are scatter plots that show the effect of mutating the RuvC-I catalytic residue aspartate (in location 647 for Cas12i1, and 599 for Cas12i2) to alanine. Each point represents a spacer, and the value indicates the fold depletion under the condition specified for the axis (wild type vs mutant). Higher values indicate stronger depletion (i.e. fewer surviving colonies).

[0136] FIGS. 7A and 7B are scatter plots that show the effect of adding or removing the non-coding sequences to the Type V-I CRISPR-Cas system being screened. Each point represents a spacer, and the value indicates the fold depletion under the condition specified for the axis (wild type vs mutant). Higher values indicate stronger depletion (i.e., fewer surviving colonies).

[0137] FIGS. 8A and 8B are heatmaps of the aggregate screening results for Cas12i1 and Cas12i2, respectively. The heatmap is decomposed into dependencies such as the orientation of the direct repeat, necessity of noncoding sequence, as well as the requirement of the intact RuvC domain (where dCas12i refers to a point mutant in a catalytically active residue of the RuvC-I domain). The Y-axis decomposes the library targets into the constituent features of targeting pACYC184. E. coli essential genes (E. coli EG), or strandedness of targeting (S, sense; AS, antisense). Cas12i1 and Cas12i2 in vivo screens were run in Endura Stbl3 and E. Cloni.RTM. competent cell strains, respectively. CRISPR arrays strongly depleted in negative controls without Cas12i1 or Cas12i2 effectors are subtracted from the respective analyses.

[0138] FIGS. 9A and 9B are weblogos of 5' PAM motifs identified from sequences flanking targets for strongly depleted spacers from Cas12i1 and Cas12i2 in vivo screens, respectively.

[0139] FIGS. 10A and 10B are violin plots of bit scores for all possible permutations of target and flanking nucleotides, confirming that Cas12i1 and Cas12i2 each have a preference for only a single 2-nt PAM motif at the 2nd and 3rd positions 5' of spacer targets.

[0140] FIGS. 11A and 11B depict the read mapping of small RNA sequencing of in vivo screening samples of the minimal Cas12i systems, revealing the mature crRNA of Cas12i1 and Cas12i2 systems respectively.

[0141] FIG. 12 is a denaturing gel showing pre-crRNA processing by Cas12i1 effector protein. Magnesium independent processing of pre-crRNA expressed from a minimal CRISPR array (repeat-spacer-repeat-spacer-repeat) with a 24 nt repeat and 28 nt spacer by Cas12i1. pre-crRNA was incubated with Cas12i1 for 30 minutes at 37.degree. C. and analyzed on a 15% TBE-Urea gel.

[0142] FIG. 13 is a representation of a gel that show the manipulation of IR800 dye-labeled target (left) or non-target (right) ssDNA by increasing doses of Cas12i1 binary complex. Samples were analyzed by 15% TBE-urea denaturing gel electrophoresis.

[0143] FIG. 14 is a representation of a gel that shows the manipulation of IR800 dye-labeled collateral ssDNA (with no sequence similarity to the target) in the presence of unlabeled target (left) or non-target (right) ssDNA by increasing doses of Cas12i1 binary complex. Samples were analyzed by 15% TBE-urea denaturing gel electrophoresis.

[0144] FIG. 15 is a representation of a gel that shows the manipulation of IR800 dye-labeled target (left) or non-target (right) dsDNA by increasing doses of Cas12i1 binary complex. Samples were analyzed by 15% TBE-urea denaturing gel electrophoresis.

[0145] FIG. 16 is a representation of a gel that shows the manipulation of IR800 dye-labeled target dsDNA by increasing doses of Cas12i1 binary complex and quenched directly (left) or treated with S1 nuclease before quenching (right). Samples were analyzed by 4-20% TBE non-denaturing gel electrophoresis.

[0146] FIGS. 17A and 17B are representations of gels that show the asymmetric cleavage efficiency of dsDNA target strand (spacer complementary; "SC") versus non-target strand (non-spacer complementary; "NSC"). FIG. 17A is a denaturing gel imaged by IR800 (only labeled DNA), while FIG. 17B is a denaturing gel imaged by SYBR stain (total DNA). Each gel depicts cleavage or nicking activity on dsDNA with 5' IR800-labeled NSC strand (left), or 5' IR800-labeled SC strand (right), with increasing concentrations of Cas12i1 binary complex. Cas12i1 binary complex was formed by pre-incubating Cas12i1 with pre-crRNA for 10 minutes at 37.degree. C. prior to adding to the substrates and incubating for 1 hour at 37.degree. C.

[0147] FIG. 18A is a schematic representation of the design of an in vitro assay to detect gene silencing. In a one pot reaction (depicted by the outer boundary), linear DNA templates encoding the Cas12i effector, RNA guide, and sigma factor 28 are combined with a reconstituted IVTT (in vitro transcription and translation) reagent and E. coli RNA polymerase core enzyme (denoted by RNAPc). A DNA plasmid encoding GFP targeted by the RNA guide is included, as is a non-target linear DNA template expressing RFP as an internal control. Both GFP and RFP are expressed from the sigma factor 28 promoter (fliC), and the GFP and RFP fluorescence is measured every 5 minutes for up to 12 hours.

[0148] FIG. 18B is a schematic representation of the design of the GFP-encoding plasmid used as a substrate in the in vitro gene silencing assay. The plasmid encodes GFP under the sig28 promoter, and engineered RNA guides are designed to target both strands of the promoter region and the GFP gene (denoted by short chevrons in both orientations).

[0149] FIGS. 19A and 19B are graphs that show the GFP fluorescence fold depletion (y-axis) over 12 hours (720 minutes, x-axis) with the Type V-I effector as indicated in a complex with a guide containing a sequence complementary to the template strand (FIG. 19A) and coding strand (FIG. 19B) of the substrate GFP-coding region. GFP fluorescence fold depletion is calculated as the ratio of the normalized GFP fluorescence with the Type V-I effector in a complex with a non-target RNA guide over that of the Type V-I effector in a complex with a GFP-targeting RNA guide. Cas12i1 (solid line) shows greater depletion (gene silencing) compared to the activity of each of the mutant forms Cas12i1 D647A or Cas12i1 E894A or Cas12i1 D948A.

[0150] FIG. 20 shows the different forms of protein and/or RNAs in the in vitro reconstitution of the CRISPR-Cas system used in in vitro pooled screening. Transcriptional directions are indicated by the orientation of the T7 promoter arrow.

[0151] FIG. 21 shows one embodiment of the ssDNA and dsDNA substrates for in vitro pooled screening. The target sequence is flanked by 6 degenerate bases ("N") on both the 5' and 3' side, which are adjacent to a common region used as a fiducial mark for downstream data analysis following next generation sequencing. In the dsDNA substrate, the second strand synthesis is completed using a DNA polymerase I fill-in after annealing a primer to the 3' fiducial mark.

[0152] FIG. 22 displays a schematic of the unidirectional sequencing library preparation of the ssDNA fragments post incubation with the reconstituted CRISPR-Cas system.

[0153] FIG. 23 displays a schematic of the bidirectional sequencing library preparation possible with the dsDNA fragments post incubation with the reconstituted CRISPR-Cas systems. The sequencing adaptor can be ligated to both cut fragments, and then selected for using a combination of primers common to the adaptor and common to the dsDNA substrate.

[0154] FIGS. 24A-B show the forms of the full length and cleaved products captured by the next generation sequencing library preparation and readout using A) I5/P5 ligation adapter and 3' fiducial for targeted amplification and addition of I7/P7, or B) I7/P7 ligation adapter and 5' fiducial for targeted amplification and addition of I5/P5.

[0155] FIGS. 25A-B show a schematic for A) ssDNA target length mapping and B) substrate length mapping, respectively.

[0156] FIGS. 26A-B show the distribution of dsDNA substrate lengths for IVTT-expressed Cas12i1 in complex with a top-strand (active orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0157] FIGS. 27A-B show the distribution of dsDNA target lengths for IVTT-expressed Cas12i1 in complex with a top-strand (active orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0158] FIGS. 28A-B show the distribution of dsDNA substrate lengths (X) vs target lengths (Y) for IVTT-expressed Cas12i1 in complex with a top-strand (active orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0159] FIG. 29 shows a weblogo indicating a 5' TTN PAM motif (left of the target sequence) for Cas12i1 associated with non-target strand cleavage between the +24/+25 nucleotides relative to the PAM. No PAM sequence requirement is observed on the right side of the Cas12i1 target.

[0160] FIG. 30 shows a 5 nt 3' overhang associated with double stranded DNA cleavage by Cas12i1 indicated by cleavage observed between the +24/+25 nucleotides of the non-target strand relative to the PAM and cleavage between the +19/+20 nucleotides of the target strand relative to the PAM.

[0161] FIGS. 31A-B show the distribution of dsDNA substrate lengths for IVTT-expressed Cas12i1 in complex with a non-target crRNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0162] FIGS. 32A-B show the distribution of dsDNA substrate lengths for IVTT-expressed Cas12i1 in complex with a bottom-strand (inactive orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0163] FIGS. 33A-B show the distribution of dsDNA substrate lengths for IVTT-expressed Cas12i2 in complex with a top-strand (active orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0164] FIGS. 34A-B show the distribution of dsDNA target lengths for IVTT-expressed Cas12i2 in complex with a top-strand (active orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0165] FIGS. 35A-B show the distribution of dsDNA substrate lengths (X) vs target lengths (Y) for IVTT-expressed Cas12i2 in complex with a top-strand (active orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0166] FIG. 36 shows a weblogo indicating a 5' TTN PAM motif (left of the target sequence) for Cas12i2 associated with non-target strand cleavage between the +24/+25 nucleotides relative to the PAM. No PAM sequence requirement is observed on the right side of the Cas12i2 target.

[0167] FIG. 37 shows a blunt cut associated with double stranded DNA cleavage by Cas12i2 indicated by cleavage observed between the +24/+25 nucleotides of the non-target strand relative to the PAM and cleavage between the +24/+25 nucleotides of the target strand relative to the PAM.

[0168] FIGS. 38A-B show the distribution of dsDNA substrate lengths for IVTT-expressed Cas12i2 in complex with a non-target crRNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0169] FIGS. 39A-B show the distribution of dsDNA substrate lengths for IVTT-expressed Cas12i2 in complex with a bottom-strand (inactive orientation) crRNA targeting dsDNA (red) vs. apo (effector-only) controls (blue). (A) Next generation sequencing libraries for readout were prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of the substrate (and containing I7/P7 sequences). (B) Next generation sequencing libraries for readout were prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences).

[0170] FIG. 40 is a schematic of the constructs used for mammalian validation of the Type V-I CRISPR systems as described herein. The effector is mammalian codon optimized and a nucleoplasmin nuclear localization sequence (npNLS) is appended to the C-terminus of the protein. Mammalian expression from the plasmid uses a EF1alpha-short promoter (EFS) and a polyA sequence from bGH (bGHpA). The RNA guide is expressed from a linear dsDNA fragment, driven by a RNA polymerase III promoter (U6). The schematic describes different implementations, with the RNA guide expressed as either a pre-crRNA bearing a single target, mature crRNA, or multiplexed with multiple targets in the shown configuration.

[0171] FIG. 41A is a bar graph that shows indel activity induced by the Cas12i2 CRISPR effector targeted to the VEGFA locus in the 293T cell line 72 hours post transient transfection of effector and RNA guide constructs described in FIG. 40. Different RNA guide designs were assayed and display varying degrees of efficacy. The error bars represent the S.E.M., with 3 replicates.

[0172] FIG. 41B is a representation of representative indels from next generation sequencing. Labeled are the TTC PAM sequence, and the representative indels occurring .gtoreq.20 bp downstream of the PAM.

DETAILED DESCRIPTION

[0173] The broad natural diversity of CRISPR-Cas defense systems contain a wide range of activity mechanisms and functional elements that can be harnessed for programmable biotechnologies. In a natural system, these mechanisms and parameters enable efficient defense against foreign DNA and viruses while providing self vs. non-self discrimination to avoid self-targeting. In an engineered system, the same mechanisms and parameters also provide a diverse toolbox of molecular technologies and define the boundaries of the targeting space. For instance, systems Cas9 and Cas13a have canonical DNA and RNA endonuclease activity and their targeting spaces are defined by the protospacer adjacent motif (PAM) on targeted DNA and protospacer flanking sites (PFS) on targeted RNA, respectively.

[0174] The methods described herein have been used to discover additional mechanisms and parameters within single subunit Class 2 effector systems that can expand the capabilities of RNA-programmable nucleic acid manipulation.

[0175] In one aspect, the disclosure relates to the use of computational methods and algorithms to search for and identify novel protein families that exhibit a strong co-occurrence pattern with certain other features within naturally occurring genome sequences. In certain embodiments, these computational methods are directed to identifying protein families that co-occur in close proximity to CRISPR arrays. However, the methods disclosed herein are useful in identifying proteins that naturally occur within close proximity to other features, both non-coding and protein-coding (e.g., fragments of phage sequences in non-coding areas of bacterial loci; or CRISPR Cas1 proteins). It is understood that the methods and calculations described herein may be performed on one or more computing devices.

[0176] In some embodiments, a set of genomic sequences is obtained from genomic or metagenomic databases. The databases comprise short reads, or contig level data, or assembled scaffolds, or complete genomic sequences of organisms. Likewise, the database may comprise genomic sequence data from prokaryotic organisms, or eukaryotic organisms, or may include data from metagenomic environmental samples. Examples of database repositories include the National Center for Biotechnology Information (NCBI) RefSeq. NCBI GenBank, NCBI Whole Genome Shotgun (WGS), and the Joint Genome Institute (JGI) Integrated Microbial Genomes (IMG).

[0177] In some embodiments, a minimum size requirement is imposed to select genome sequence data of a specified minimum length. In certain exemplary embodiments, the minimum contig length may be 100 nucleotides, 500 nt, 1 kb, 1.5 kb, 2 kb, 3 kb, 4 kb, 5 kb, 10 kb, 20 kb, 40 kb, or 50 kb.

[0178] In some embodiments, known or predicted proteins are extracted from the complete or a selected set of genome sequence data. In some embodiments, known or predicted proteins are taken from extracting coding sequence (CDS) annotations provided by the source database. In some embodiments, predicted proteins are determined by applying a computational method to identify proteins from nucleotide sequences. In some embodiments, the GeneMark Suite is used to predict proteins from genome sequences. In some embodiments. Prodigal is used to predict proteins from genome sequences. In some embodiments, multiple protein prediction algorithms may be used over the same set of sequence data with the resulting set of proteins de-duplicated.

[0179] In some embodiments, CRISPR arrays are identified from the genome sequence data. In some embodiments, PILER-CR is used to identify CRISPR arrays. In some embodiments, CRISPR Recognition Tool (CRT) is used to identify CRISPR arrays. In some embodiments, CRISPR arrays are identified by a heuristic that identifies nucleotide motifs repeated a minimum number of times (e.g. 2, 3, or 4 times), where the spacing between consecutive occurrences of a repeated motif does not exceed a specified length (e.g. 50, 100, or 150 nucleotides). In some embodiments, multiple CRISPR array identification tools may be used over the same set of sequence data with the resulting set of CRISPR arrays de-duplicated.

[0180] In some embodiments, proteins in close proximity to CRISPR arrays are identified. In some embodiments, proximity is defined as a nucleotide distance, and may be within 20 kb, 15 kb, or 5 kb. In some embodiments, proximity is defined as the number of open reading frames (ORFs) between a protein and a CRISPR array, and certain exemplary distances may be 10, 5, 4, 3, 2, 1, or 0 ORFs. The proteins identified as being within close proximity to a CRISPR array are then grouped into clusters of homologous proteins. In some embodiments, blastclust is used to form protein clusters. In certain other embodiments, mmseqs2 is used to form protein clusters.

[0181] To establish a pattern of strong co-occurrence between the members of a protein cluster with CRISPR arrays, a BLAST search of each member of the protein family may be performed over the complete set of known and predicted proteins previously compiled. In some embodiments, UBLAST or mmseqs2 may be used to search for similar proteins. In some embodiments, a search may be performed only for a representative subset of proteins in the family.

[0182] In some embodiments, the clusters of proteins within close proximity to CRISPR arrays are ranked or filtered by a metric to determine co-occurrence. One exemplary metric is the ratio of the number of elements in a protein cluster against the number of BLAST matches up to a certain E value threshold. In some embodiments, a constant E value threshold may be used. In other embodiments, the E value threshold may be determined by the most distant members of the protein cluster. In some embodiments, the global set of proteins is clustered and the co-occurrence metric is the ratio of the number of elements of the CRISPR associated cluster against the number of elements of the containing global cluster(s).

[0183] In some embodiments, a manual review process is used to evaluate the potential functionality and the minimal set of components of an engineered system based on the naturally occurring locus structure of the proteins in the cluster. In some embodiments, a graphical representation of the protein cluster may assist in the manual review, and may contain information including pairwise sequence similarity, phylogenetic tree, source organisms/environments, predicted functional domains, and a graphical depiction of locus structures. In some embodiments, the graphical depiction of locus structures may filter for nearby protein families that have a high representation. In some embodiments, representation may be calculated by the ratio of the number of related nearby proteins against the size(s) of the containing global cluster(s). In certain exemplary embodiments, the graphical representation of the protein cluster may contain a depiction of the CRISPR array structures of the naturally occurring loci. In some embodiments, the graphical representation of the protein cluster may contain a depiction of the number of conserved direct repeats versus the length of the putative CRISPR array, or the number of unique spacer sequences versus the length of the putative CRISPR array. In some embodiments, the graphical representation of the protein cluster may contain a depiction of various metrics of co-occurrence of the putative effector with CRISPR arrays predict new CRISPR-Cas systems and identify their components.

Pooled-Screening

[0184] To efficiently validate the activity of the engineered novel CRISPR-Cas systems and simultaneously evaluate in an unbiased manner different activity mechanisms and functional parameters, a new pooled-screening approach is used in E. coli. First, from the computational identification of the conserved protein and noncoding elements of the novel CRISPR-Cas system, DNA synthesis and molecular cloning is used to assemble the separate components into a single artificial expression vector, which in one embodiment is based on a pET-28a+ backbone. In a second embodiment, the effectors and noncoding elements are transcribed on a single mRNA transcript, and different ribosomal binding sites are used to translate individual effectors.

[0185] Second, the natural crRNA and targeting spacers are replaced with a library of unprocessed crRNAs containing non-natural spacers targeting a second plasmid, pACYC184. This crRNA library is cloned into the vector backbone containing the protein effectors and noncoding elements (e.g. pET-28a+), and then subsequently transformed the library into E. coli along with the pACYC184 plasmid target. Consequently, each resulting E. coli cell contains no more than one targeting spacer. In an alternate embodiment, the library of unprocessed crRNAs containing non-natural spacers additionally target E. coli essential genes, drawn from resources such as those described in Baba et al. (2006) Mol. Syst. Biol. 2: 2006.0008; and Gerdes et al. (2003) J. Bacteriol. 185(19): 5673-84, the entire contents of each of which are incorporated herein by reference. In this embodiment, positive, targeted activity of the novel CRISPR-Cas systems that disrupts essential gene function results in cell death or growth arrest. In some embodiments, the essential gene targeting spacers can be combined with the pACYC184 targets to add another dimension to the assay. In other embodiments, the non-coding sequences flanking the CRISPR array, putative effector or accessory open reading frames, and predicted anti-repeats indicative of tracrRNA elements were concatenated together and cloned into pACYC184 and expressed by lac and IPTG-inducible T7 promoters

[0186] Third, the E. coli are grown under antibiotic selection. In one embodiment, triple antibiotic selection is used: kanamycin for ensuring successful transformation of the pET-28a+ vector containing the engineered CRISPR-Cas effector system, and chloramphenicol and tetracycline for ensuring successful co-transformation of the pACYC184 target vector. Since pACYC184 normally confers resistance to chloramphenicol and tetracycline, under antibiotic selection, positive activity of the novel CRISPR-Cas system targeting the plasmid will eliminate cells that actively express the effectors, noncoding elements, and specific active elements of the crRNA library. Examining the population of surviving cells at a later time point compared to an earlier time point typically provides a depleted signal compared to the inactive crRNAs. In some embodiments, double antibiotic selection is used. For example, withdrawal of either chloramphenicol or tetracycline to remove selective pressure can provide novel information about the targeting substrate, sequence specificity, and potency. In some embodiments, only kanamycin is used to ensure successful transformation of the pET-28a+ vector containing the engineered CRISPR-Cas effector system. This embodiment is suitable for libraries containing spacers targeting E. coli essential genes, as no additional selection beyond kanamycin is needed to observe growth alterations. In this embodiment, chloramphenicol and tetracycline dependence is removed, and their targets (if any) in the library provides an additional source of negative or positive information about the targeting substrate, sequence specificity, and potency.

[0187] Since the pACYC184 plasmid contains a diverse set of features and sequences that may affect the activity of a CRISPR-Cas system, mapping the active crRNAs from the pooled screen onto pACYC184 provides patterns of activity that can be suggestive of different activity mechanisms and functional parameters in a broad, hypothesis-agnostic manner. In this way, the features required for reconstituting the novel CRISPR-Cas system in a heterologous prokaryotic species can be more comprehensively tested and studied.

[0188] Certain important advantages of the in vivo pooled-screen described herein include:

[0189] (1) Versatility--plasmid design allows multiple effectors and/or noncoding elements to be expressed; library cloning strategy enables both transcriptional directions of the computationally predicted crRNA to be expressed;

[0190] (2) Comprehensive tests of activity mechanisms and functional parameters can be used to evaluate diverse interference mechanisms, including DNA or RNA cleavage; to examine co-occurrence of features such as transcription, plasmid DNA replication; and flanking sequences for a crRNA library to reliably determine PAMs with complexity equivalence of 4N's;

[0191] (3) Sensitivity--pACYC184 is a low copy plasmid, enabling high sensitivity for CRISPR-Cas activity, because even modest interference rates can eliminate the antibiotic resistance encoded by the plasmid; and

[0192] (4) Efficiency--the pooled-screening includes optimized molecular biology steps that enable greater speed and throughput for RNA-sequencing and the protein expression samples can be directly harvested from the surviving cells in the screen.

[0193] As discussed in more detail in the Examples below, the novel CRISPR-Cas families described herein were evaluated using this in vivo pooled-screen to evaluate their operational elements, mechanisms and parameters, as well as their ability to be active and reprogrammed in an engineered system outside of their natural cellular environment.

In Vitro Pooled Screening

[0194] In vitro pooled screening approaches can also be used and are complementary to in vivo pooled screens. In vitro pooled screens enable rapid biochemical characterization and reduction of a CRISPR system to the essential components necessary for the system's activity. In one embodiment, a cell-free in vitro transcription and translation (IVTT) system is used to directly synthesize RNA and protein from DNA encoding the noncoding and effector proteins of the CRISPR system, thus enabling a faster and higher throughput method to evaluate a larger number of distinct separate CRISPR-Cas effector systems than conventional biochemical assays reliant on FPLC-purified proteins. In addition to enabling greater throughput and efficiency of biochemical reactions, the in vitro screening has several advantages that make it complementary to the in vivo pooled screening approach described above.

[0195] (1) Direct observation of both enrichment and depletion signals--in vitro pooled screening enables a readout of both cleavage enrichment, in which the cleavage products can be directly captured and sequenced to identify specific cut sites, cleavage patterns, and sequence motifs for active effector systems, as well as target depletion, in which the negative signal from the depletion of specific targets within the uncleaved population is used as a proxy for activity. As the in vivo pooled screen utilizes a target depletion readout, the enrichment mode offers additional insight into the effector activity.

[0196] (2) Greater control of the reaction components and environment--the well-defined components and activity of the proprietary IVTT enables precise control of the reaction components to identify the minimal components necessary for further activity translation, as compared to the complex E. coli cellular milieu for an in vivo screen. Additionally, non-natural modifications may be made to reaction components for enhanced activity or easier readout; for instance, adding phosphorothioated bonds onto the ssDNA and dsDNA substrates to reduce noise by limiting exonuclease degradation of substrates.

[0197] (3) Robustness to toxic/growth inhibiting proteins--for proteins that may be toxic to E. coli cell growth, the in vitro pooled screen enables functional screening without being subject to the growth constraints of a live cell. This ultimately enables greater versatility in protein selection and screening.

[0198] The novel CRISPR-Cas families described herein were evaluated using a combination of in vivo and in vitro pooled-screens to evaluate their operational elements, mechanisms and parameters, as well as their ability to be active and reprogrammed in an engineered system outside of their natural cellular environment.

Class 2 CRISPR-Cas Effectors Having a RuvC Domain

[0199] In one aspect, the disclosure provides Class 2 CRISPR-Cas systems referred to herein as CLUST.029130 (Type V-I) CRISPR-Cas systems. These Class 2 CRISPR-Cas systems include an isolated CRISPR-associated protein having a RuvC domain and an isolated crRNA, also referred to as an RNA guide, guide RNA, or gRNA, comprising a spacer sequence that is complementary to a target nucleic acid sequence such as a DNA sequence.

[0200] Suitably, a CRISPR-Cas effector protein having a RuvC domain may include one or motifs from the set of: the RuvC III motif, X.sub.1SHX.sub.4DX.sub.6X.sub.7(SEQ ID NO: 200), wherein X.sub.1 is S or T, X.sub.4 is Q or L, X.sub.6 is P or S, and X.sub.7 is F or L; the RuvC I motif, X.sub.1XDXNX.sub.6X.sub.7XXXX.sub.11 (SEQ ID NO: 201), wherein X.sub.1 is A or G or S, X is any amino acid, X.sub.6 is Q or I, X.sub.7 is T or S or V, and X.sub.11 is T or A; and the RuvC II motif, X.sub.1X.sub.2X.sub.3E (SEQ ID NO: 210), wherein X.sub.1 is C or F or I or L or M or P or V or W or Y, X.sub.2 is C or F or I or L or M or P or R or V or W or Y, and X.sub.3 is C or F or G or I or L or M or P or V or W or Y.

[0201] Suitably, a Type V-I CRISPR-Cas system includes a CRISPR-Cas effector having a RuvC domain and a Type V-I crRNA. Suitably, the Cas12i effector is about 1100 amino acids or less in length, and includes a functional PAM interacting domain that recognizes the PAM in the target DNA. Type V-I CRISPR-Cas effector proteins are capable of binding to a Type V-I RNA guide to form a Type V-I CRISPR-Cas system, wherein the Type V-I RNA guide includes a stem-loop structure with a 5-nucleotide stem and a loop of 6, 7, or 8 nucleotides. Type V-I CRISPR-Cas systems are capable of targeting and binding to sequence-specific DNA without the presence of a tracrRNA.

[0202] In some embodiments, the Type V-I CRISPR-Cas effector protein and the Type V-I RNA guide form a binary complex that may include other components. The binary complex is activated upon binding to a nucleic acid substrate that is complementary to a spacer sequence in the RNA guide (i.e., a sequence-specific substrate or target nucleic acid). In some embodiments, the sequence-specific substrate is a double-stranded DNA. In some embodiments, the sequence-specific substrate is a single-stranded DNA. In some embodiments, the sequence-specificity requires a complete match of the spacer sequence in the RNA guide (e.g., crRNA) to the target substrate. In other embodiments, the sequence specificity requires a partial (contiguous or non-contiguous) match of the spacer sequence in the RNA guide (e.g., crRNA) to the target substrate. Sequence specificity in certain embodiments further requires a complete match between a protospacer adjacent motif ("PAM") sequence proximate to the spacer sequence, and a canonical PAM sequence recognized by the CRISPR-associated protein. In some instances, a complete PAM sequence match is not required, and a partial match is sufficient for sequence-specific association of the binary complex and the DNA substrate.

[0203] In some embodiments, the target nucleic acid substrate is a double stranded DNA (dsDNA). In some embodiments, the target nucleic acid substrate is a dsDNA and includes a PAM. In some embodiments, the binary complex modifies the target sequence-specific dsDNA substrate upon binding to it. In some embodiments, the binary complex preferentially nicks the non-target strand of the target dsDNA substrate. In some embodiments, the binary complex cleaves both strands of the target dsDNA substrate it. In some embodiments, the binary complex cleaves both strands of target dsDNA substrate with a staggered cut. In some embodiments, the binary complex creates a blunt double-stranded break (DSB) on the target dsDNA substrate.

[0204] In some embodiments, the target nucleic acid substrate is a single stranded DNA (ssDNA). In some embodiments, the target nucleic acid substrate is a ssDNA and does not include a PAM. In some embodiments, the binary complex modifies the target sequence-specific ssDNA substrate upon binding to it. In some embodiments, the binary complex cleaves the target ssDNA substrate.

[0205] In some embodiments, the binary complex becomes activated upon binding to the target substrate. In some embodiments, the activated complex exhibits "multiple turnover" activity, whereby upon acting on (e.g., cleaving) the target substrate the activated complex remains in an activated state. In some embodiments, the binary complex exhibits "single turnover" activity, whereby upon acting on the target substrate the binary complex reverts to an inactive state. In some embodiments, the activated complex exhibits non-specific (i.e., "collateral") cleavage activity whereby the activated complex cleaves nucleic acids with no sequence similarity to the target. In some embodiments, the collateral nucleic acid substrate is a ssDNA.

CRISPR Enzyme Modifications

Nuclease-Deficient CRISPR Enzymes

[0206] Where the CRISPR enzymes described herein have nuclease activity, the CRISPR enzymes can be modified to have diminished nuclease activity, e.g., nuclease inactivation of at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, or 100% as compared with the wild type CRISPR enzymes. The nuclease activity can be diminished by several methods, e.g., introducing mutations into the nuclease or PAM interacting domains of the CRISPR enzymes. In some embodiments, catalytic residues for the nuclease activities are identified, and these amino acid residues can be substituted by different amino acid residues (e.g., glycine or alanine) to diminish the nuclease activity. Examples of such mutations for Cas12i1 include D647A or E894A or D948A. Examples of such mutations for Cas12i2 include D599A or E833A or D886A.

[0207] The inactivated CRISPR enzymes can comprise (e.g., via fusion protein, linker peptides, Gly4Ser (GS) peptide linkers, etc.) or be associated (e.g., via co-expression of multiple proteins) with one or more functional domains. These functional domains can have various activities, e.g., methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, DNA cleavage activity, nucleic acid binding activity, and switch activity (e.g., light inducible). In some embodiments, the functional domains are Kruppel associated box (KRAB), VP64, VP16, Fok1, P65, HSF1, MyoDi, and biotin-APEX.

[0208] The positioning of the one or more functional domains on the inactivated CRISPR enzymes allows for correct spatial orientation for the functional domain to affect the target with the attributed functional effect. For example, if the functional domain is a transcription activator (e.g., VP16, VP64, or p65), the transcription activator is placed in a spatial orientation that allows it to affect the transcription of the target. Likewise, a transcription repressor is positioned to affect the transcription of the target, and a nuclease (e.g., Fok1) is positioned to cleave or partially cleave the target. In some embodiments, the functional domain is positioned at the N-terminus of the CRISPR enzyme. In some embodiments, the functional domain is positioned at the C-terminus of the CRISPR enzyme. In some embodiments, the inactivated CRISPR enzyme is modified to comprise a first functional domain at the N-terminus and a second functional domain at the C-terminus.

Split Enzymes

[0209] The present disclosure also provides a split version of the CRISPR enzymes described herein. The split version of the CRISPR enzymes may be advantageous for delivery. In some embodiments, the CRISPR enzymes are split to two parts of the enzymes, which together substantially comprises a functioning CRISPR enzyme.

[0210] The split can be done in a way that the catalytic domain(s) are unaffected. The CRISPR enzymes may function as a nuclease or may be inactivated enzymes, which are essentially RNA-binding proteins with very little or no catalytic activity (e.g., due to mutation(s) in their catalytic domains).

[0211] In some embodiments, the nuclease lobe and .alpha.-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the RNA guide recruits them into a complex that recapitulates the activity of full-length CRISPR enzymes and catalyzes site-specific DNA cleavage. The use of a modified RNA guide abrogates split-enzyme activity by preventing dimerization, allowing for the development of an inducible dimerization system. The split enzyme is described, e.g., in Wright, Addison V., et al. "Rational design of a split-Cas9 enzyme complex," Proc. Nat'l. Acad. Sci., 112.10 (2015): 2984-2989, which is incorporated herein by reference in its entirety.

[0212] In some embodiments, the split enzyme can be fused to a dimerization partner, e.g., by employing rapamycin sensitive dimerization domains. This allows the generation of a chemically inducible CRISPR enzyme for temporal control of CRISPR enzyme activity. The CRISPR enzymes can thus be rendered chemically inducible by being split into two fragments and rapamycin-sensitive dimerization domains can be used for controlled reassembly of the CRISPR enzymes.

[0213] The split point is typically designed in silico and cloned into the constructs. During this process, mutations can be introduced to the split enzyme and non-functional domains can be removed. In some embodiments, the two parts or fragments of the split CRISPR enzyme (i.e., the N-terminal and C-terminal fragments), can form a full CRISPR enzyme, comprising, e.g., at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% of the sequence of the wild-type CRISPR enzyme.

Self-Activating or Inactivating Enzymes

[0214] The CRISPR enzymes described herein can be designed to be self-activating or self-inactivating. In some embodiments, the CRISPR enzymes are self-inactivating. For example, the target sequence can be introduced into the CRISPR enzyme coding constructs. Thus, the CRISPR enzymes can cleave the target sequence, as well as the construct encoding the enzyme thereby self-inactivating their expression. Methods of constructing a self-inactivating CRISPR system is described, e.g., in Epstein, Benjamin E., and David V. Schaffer. "Engineering a Self-Inactivating CRISPR System for AAV Vectors," Mol. Ther., 24 (2016): 550, which is incorporated herein by reference in its entirety.

[0215] In some other embodiments, an additional RNA guide, expressed under the control of a weak promoter (e.g., 7SK promoter), can target the nucleic acid sequence encoding the CRISPR enzyme to prevent and/or block its expression (e.g., by preventing the transcription and/or translation of the nucleic acid). The transfection of cells with vectors expressing the CRISPR enzyme, RNA guides, and RNA guides that target the nucleic acid encoding the CRISPR enzyme can lead to efficient disruption of the nucleic acid encoding the CRISPR enzyme and decrease the levels of CRISPR enzyme, thereby limiting the genome editing activity.

[0216] In some embodiments, the genome editing activity of the CRISPR enzymes can be modulated through endogenous RNA signatures (e.g., miRNA) in mammalian cells. The CRISPR enzyme switch can be made by using a miRNA-complementary sequence in the 5'-UTR of mRNA encoding the CRISPR enzyme. The switches selectively and efficiently respond to miRNA in the target cells. Thus, the switches can differentially control the genome editing by sensing endogenous miRNA activities within a heterogeneous cell population. Therefore, the switch systems can provide a framework for cell-type selective genome editing and cell engineering based on intracellular miRNA information (Hirosawa, Moe et al. "Cell-type-specific genome editing with a microRNA-responsive CRISPR-Cas9 switch," Nucl. Acids Res., 2017 Jul. 27; 45(13): el18).

Inducible CRISPR Enzymes

[0217] The CRISPR enzymes can be inducible, e.g., light inducible or chemically inducible. This mechanism allows for activation of the functional domain in the CRISPR enzymes with a known trigger. Light inducibility can be achieved by various methods known in the art, e.g., by designing a fusion complex wherein CRY2 PHR/CIBN pairing is used in split CRISPR Enzymes (see, e.g., Konermann et al. "Optical control of mammalian endogenous transcription and epigenetic states," Nature, 500.7463 (2013): 472). Chemical inducibility can be achieved, e.g., by designing a fusion complex wherein FKBP/FRB (FK506 binding protein/FKBP rapamycin binding domain) pairing is used in split CRISPR Enzymes. Rapamycin is required for forming the fusion complex, thereby activating the CRISPR enzymes (see, e.g., Zetsche, Volz, and Zhang, "A split-Cas9 architecture for inducible genome editing and transcription modulation," Nature Biotech., 33.2 (2015): 139-142).

[0218] Furthermore, expression of the CRISPR enzymes can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone inducible gene expression system (e.g., an ecdysone inducible gene expression system), and an arabinose-inducible gene expression system. When delivered as RNA, expression of the RNA targeting effector protein can be modulated via a riboswitch, which can sense a small molecule like tetracycline (see, e.g., Goldfless, Stephen J. et al. "Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction," Nucl. Acids Res., 40.9 (2012): e64-e64).

[0219] Various embodiments of inducible CRISPR enzymes and inducible CRISPR systems are described, e.g., in U.S. Pat. No. 8,871,445, US20160208243, and WO2016205764, each of which is incorporated herein by reference in its entirety.

Functional Mutations

[0220] Various mutations or modifications can be introduced into CRISPR enzymes as described herein to improve specificity and/or robustness. In some embodiments, the amino acid residues that recognize the Protospacer Adjacent Motif (PAM) are identified. The CRISPR enzymes described herein can be modified further to recognize different PAMs, e.g., by substituting the amino acid residues that recognize PAM with other amino acid residues. In some embodiments, the CRISPR enzymes can recognize alternative PAMs, e.g., as described herein.

[0221] In some embodiments, the CRISPR-associated proteins include at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Localization Signal (NLS) attached to the N-terminal or C-terminal of the protein. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 300); the NLS from nucleoplasmin (e.g., the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 301)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 302) or RQRRNELKRSP (SEQ ID NO: 303); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 304); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 305) of the IBB domain from importin-alpha; the sequences VSRKRPRP (SEQ ID NO: 306) and PPKKARED (SEQ ID NO: 307) of the myoma T protein; the sequence PQPKKKPL (SEQ ID NO: 308) of human p53; the sequence SALIKKKKKMAP (SEQ ID NO: 309) of mouse c-abl IV; the sequences DRLRR (SEQ ID NO: 310) and PKQKKRK(SEQ ID NO: 311) of the influenza virus NS1; the sequence RKLKKKIKKL (SEQ ID NO: 312) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 313) of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 314) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 315) of the human glucocorticoid receptor. In some embodiments, the CRISPR-associated protein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) Nuclear Export Signal (NES) attached the N-terminal or C-terminal of the protein. In a preferred embodiment, a C-terminal and/or N-terminal NLS or NES is attached for optimal expression and nuclear targeting in eukaryotic cells, e.g., human cells.

[0222] In some embodiments, the CRISPR enzymes described herein are mutated at one or more amino acid residues to alter one or more functional activities. For example, in some embodiments, the CRISPR enzyme is mutated at one or more amino acid residues to alter its helicase activity. In some embodiments, the CRISPR enzyme is mutated at one or more amino acid residues to alter its nuclease activity (e.g., endonuclease activity or exonuclease activity). In some embodiments, the CRISPR enzyme is mutated at one or more amino acid residues to alter its ability to functionally associate with a RNA guide. In some embodiments, the CRISPR enzyme is mutated at one or more amino acid residues to alter its ability to functionally associate with a target nucleic acid.

[0223] In some embodiments, the CRISPR enzymes described herein are capable of cleaving a target nucleic acid molecule. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid molecule. However, in some embodiments, the CRISPR enzyme is mutated at one or more amino acid residues to alter its cleaving activity. For example, in some embodiments, the CRISPR enzyme may comprise one or more mutations which render the enzyme incapable of cleaving a target nucleic acid. In other embodiments, the CRISPR enzyme may comprise one or more mutations such that the enzyme is capable of cleaving a single strand of the target nucleic acid (i.e., nickase activity). In some embodiments, the CRISPR enzyme is capable of cleaving the strand of the target nucleic acid that is complementary to the strand that the RNA guide hybridizes to. In some embodiments, the CRISPR enzyme is capable of cleaving the strand of the target nucleic acid that the RNA guide hybridizes to.

[0224] In some embodiments, a CRISPR enzyme described herein may be engineered to comprise a deletion in one or more amino acid residues to reduce the size of the enzyme while retaining one or more desired functional activities (e.g., nuclease activity and the ability to interact functionally with a RNA guide). The truncated CRISPR enzyme may be advantageously used in combination with delivery systems having load limitations.

[0225] In one aspect, the present disclosure provides nucleic acid sequences that are at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic sequences described herein. In another aspect, the present disclosure also provides amino acid sequences that are at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequences described herein.

[0226] In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that are the same as the sequences described herein.

[0227] In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from the sequences described herein.

[0228] In some embodiments, the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as the sequences described herein. In some embodiments, the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from the sequences described herein.

[0229] To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In general, the length of a reference sequence aligned for comparison purposes should be at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100% of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0230] Beyond the biochemical and diagnostic applications described herein, programmable Type V-I CRISPR-Cas systems described herein have important applications in eukaryotic cells such as therapeutic modification of the genome, with examples of modifications including, but not limited to; genotype correction, gene knockout, genetic sequence insertion/deletion (by homology directed repair or otherwise), single nucleotide modification, or gene regulation. These gene modification modalities can use the nuclease activity of Cas12i, double nicking, or programmable DNA binding of catalytically inactive Cas12i fused to additional effector domains.

[0231] In some embodiments, the CRISPR-associated proteins and accessory proteins described herein can be fused to one or more peptide tags, including a His-tag, GST-tag, FLAG-tag, or myc-tag. In some embodiments, the CRISPR-associated proteins or accessory proteins described herein can be fused to a detectable moiety such as a fluorescent protein (e.g., green fluorescent protein or yellow fluorescent protein). And in some embodiments, CRISPR-associated proteins or accessory proteins of this disclosure are fused to a peptide or non-peptide moiety that allows these proteins to enter or localize to a tissue, a cell, or a region of a cell. For instance, a CRISPR-associated protein or accessory protein of this disclosure (such as Cas12i) may comprise a nuclear localization sequence (NLS) such as an SV40 (simian virus 40) NLS, c-Myc NLS, or other suitable monopartite NLS. The NLS may be fused to an N-terminal and/or a C-terminal of the CRISPR-associated protein or accessory protein, and may be fused singly (i.e., a single NLS) or concatenated (e.g., a chain of 2, 3, 4, etc. NLS).

[0232] In those embodiments where a tag is fused to a CRISPR-associated protein, such tag may facilitate affinity-based or charge-based purification of the CRISPR-associated protein, e.g., by liquid chromatography or bead separation utilizing an immobilized affinity or ion-exchange reagent. As a non-limiting example, a recombinant CRISPR-associated protein of this disclosure (such as Cas12i) comprises a polyhistidine (His) tag, and for purification is loaded onto a chromatography column comprising an immobilized metal ion (e.g. a Zn.sup.2+, Ni.sup.2+, Cu.sup.2+ ion chelated by a chelating ligand immobilized on the resin, which resin may be an individually prepared resin or a commercially available resin or ready to use column such as the HisTrap FF column commercialized by GE Healthcare Life Sciences, Marlborough, Massachusetts). Following the loading step, the column is optionally rinsed, e.g., using one or more suitable buffer solutions, and the His-tagged protein is then eluted using a suitable elution buffer. Alternatively or additionally, if the recombinant CRISPR-associated protein of this disclosure utilizes a FLAG-tag, such protein may be purified using immunoprecipitation methods known in the industry. Other suitable purification methods for tagged CRISPR-associated proteins or accessory proteins of this disclosure will be evident to those of skill in the art.

[0233] The proteins described herein (e.g., CRISPR-associated proteins or accessory proteins) can be delivered or used as either nucleic acid molecules or polypeptides. When nucleic acid molecules are used, the nucleic acid molecule encoding the CRISPR-associated proteins can be codon-optimized, as discussed in further detail below. The nucleic acid can be codon optimized for use in any organism of interest, in particular human cells or bacteria. For example, the nucleic acid can be codon-optimized for any non-human eukaryote including mice, rats, rabbits, dogs, livestock, or non-human primates. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at World Wide Web address kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura et al. Nucl. Acids Res. 28:292 (2000), which is incorporated herein by reference in its entirety. Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa.).

[0234] In some instances, nucleic acids of this disclosure which encode CRISPR-associated proteins or accessory proteins for expression in eukaryotic (e.g., human, or other mammalian cells) cells include one or more introns, i.e., one or more non-coding sequences comprising, at a first end (e.g., a 5' end), a splice-donor sequence and, at second end (e.g., the 3' end) a splice acceptor sequence. Any suitable splice donor/splice acceptor can be used in the various embodiments of this disclosure, including without limitation simian virus 40 (SV40) intron, beta-globin intron, and synthetic introns. Alternatively or additionally, nucleic acids of this disclosure encoding CRISPR-associated proteins or accessory proteins may include, at a 3' end of a DNA coding sequence, a transcription stop signal such as a polyadenylation (polyA) signal. In some instances, the polyA signal is located in close proximity to, or adjacent to, an intron such as the SV40 intron.

RNA Guides

[0235] In some embodiments, the CRISPR systems described herein include at least one Type V-I RNA guide. The architecture of many RNA guides is known in the art (see, e.g., International Publication Nos. WO 2014/093622 and WO 2015/070083, the entire contents of each of which are incorporated herein by reference). In some embodiments, the CRISPR systems described herein include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, or more RNA guides).

[0236] In some embodiments, the CRISPR systems described herein include at least one Type V-I RNA guide or a nucleic acid encoding at least one Type V-I RNA guide. In some embodiments, the RNA guide includes a crRNA. Generally, the crRNAs described herein include a direct repeat sequence and a spacer sequence. In certain embodiments, the crRNA includes, consists essentially of, or consists of a direct repeat sequence linked to a guide sequence or spacer sequence. In some embodiments, the crRNA includes a direct repeat sequence, a spacer sequence, and a direct repeat sequence (DR-spacer-DR), which is typical of precursor crRNA (pre-crRNA) configurations in other CRISPR systems. In some embodiments, the crRNA includes a truncated direct repeat sequence and a spacer sequence, which is typical of processed or mature crRNA. In some embodiments, the CRISPR-Cas effector protein forms a complex with the RNA guide, and the spacer sequence directs the complex to a sequence-specific binding with the target nucleic acid that is complementary to the spacer sequence.

[0237] Suitably, CRISPR systems described herein comprise at least one Type V-I RNA guide or nucleic acids encoding a Type V-I RNA guide, wherein the RNA guide comprises a direct repeat. Suitably, the Type V-I RNA guide may form a secondary structure such as a stem loop structure, e.g., as described herein.

[0238] The direct repeat can include two stretches of nucleotides that may be complementary to one another, separated by intervening nucleotides such that the direct repeat can hybridize to form the double stranded RNA duplex (dsRNA duplex) resulting in a stem-loop structure where the two complementary stretches of nucleotides form a stem and the intervening nucleotides form a loop or hair-pin (FIG. 3). For example, the intervening nucleotides that form the "loop" have a length of from about 6 nucleotides to about 8 nucleotides, or about 7 nucleotides. In different embodiments, the stem can include at least 2, at least 3, at least 4, or 5 base pairs.

[0239] Suitably, the direct repeat can include two complementary stretches of nucleotides that are about 5 nucleotides in length separated by about seven intervening nucleotides.

[0240] Some exemplary direct repeats of Type V-I systems are illustrated in FIG. 3, suitably when departing from naturally occurring Type V-I direct repeats, the skilled person may mimic the structure of such direct repeats illustrated in FIG. 3.

[0241] The direct repeat can include or consist of about 22 to 40 nucleotides, or about 23 to 38 nucleotides or about 23 to 36 nucleotides.

[0242] In some embodiments, the CRISPR systems described herein include a plurality of RNA guides (e.g., 2, 3, 4, 5, 10, 15, or more) or a plurality of nucleic acids encoding a plurality of RNA guides.

[0243] In some embodiments, the CRISPR system described herein includes an RNA guide or a nucleic acid encoding the RNA guide. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'-CCGUCNNNNNNNGACGG-3' (SEQ ID NO: 202) proximal to its 3' end and adjacent to the spacer sequence. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'-GUGCCNNNNNNNGGCAC-3' (SEQ ID NO: 203) proximal to its 3' end and adjacent to the spacer sequence. In some embodiments, the RNA guide comprises or consists of a direct repeat sequence and a spacer sequence capable of hybridizing (e.g., hybridizes under appropriate conditions) to a target nucleic acid, wherein the direct repeat sequence comprises 5'-GUGUCN.sub.5-6UGACAX.sub.1-3' (SEQ ID NO: 204) proximal to the 3' end and adjacent to the spacer sequence, wherein N.sub.5-6 refers to a contiguous sequence of any 5 or 6 nucleobases, and X.sub.1 refers to C or T or U.

[0244] Examples of RNA guide direct repeat sequences and effector protein pairs are provided in Table 5A. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid sequence listed in Table 5A (e.g., SEQ ID NOs: 6-10, 19-24). In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the initial three 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the initial four 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the initial five 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the initial six 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the initial seven 5' nucleotides. In some embodiments, the direct repeat sequence comprises or consists of a nucleic acid having a nucleic acid sequence listed in Table 5A with a truncation of the initial eight 5' nucleotides.

Multiplexing RNA Guides

[0245] CLUST.029130 (Type V-I) CRISPR-Cas effectors have been demonstrated to employ more than one RNA guide, thus enabling the ability of these effectors, and systems and complexes that include them, to target multiple different nucleic acid targets. In some embodiments, the CRISPR systems described herein include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more RNA guides). In some embodiments, the CRISPR systems described herein include a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem. The single RNA strand can include multiple copies of the same RNA guide, multiple copies of distinct RNA guides, or combinations thereof.

[0246] In some embodiments, the CLUST.029130 (Type V-I) CRISPR-Cas effector proteins are delivered complexed with multiple RNA guides directed to different target nucleic acids. In some embodiments, the CLUST.029130 (Type V-I) CRISPR-Cas effector proteins can be co-delivered with multiple RNA guides, each specific for a different target nucleic acid. Methods of multiplexing using CRISPR-associated proteins are described, for example, in U.S. Pat. No. 9,790,490, and EP 3009511, the entire contents of each of which are expressly incorporated herein by reference.

RNA Guide Modifications

Spacer Lengths

[0247] The spacer length of RNA guides can range from about 15 to 50 nucleotides. In some embodiments, the spacer length of an RNA guide is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides. In some embodiments, the spacer length is from 15 to 17 nucleotides, from 15 to 23 nucleotides, from 16 to 22 nucleotides, from 17 to 20 nucleotides, from 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides), from 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides), from 24 to 27 nucleotides, from 27 to 30 nucleotides, from 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 40, or 45 nucleotides), from 30 or 35 to 40 nucleotides, from 41 to 45 nucleotides, from 45 to 50 nucleotides, or longer. In some embodiments, the spacer length of an RNA guide is 31 nucleotides. In some embodiments, the direct repeat length of the RNA guide is at least 21 nucleotides, or is from 21 to 37 nucleotides (e.g., 23, 24, 25, 30, 35, or 36 nucleotides). In some embodiments, the direct repeat length of the RNA guide is 23 nucleotides.

[0248] The RNA guide sequences can be modified in a manner that allows for formation of the CRISPR effector complex and successful binding to the target, while at the same time not allowing for successful nuclease activity (i.e., without nuclease activity/without causing indels). These modified guide sequences are referred to as "dead guides" or "dead guide sequences." These dead guides or dead guide sequences may be catalytically inactive or conformationally inactive with regard to nuclease activity. Dead guide sequences are typically shorter than respective guide sequences that result in active RNA cleavage. In some embodiments, dead guides are 5%, 10%, 20%, 30%, 40%, or 50%, shorter than respective RNA guides that have nuclease activity. Dead guide sequences of RNA guides can be from 13 to 15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length), from 15 to 19 nucleotides in length, or from 17 to 18 nucleotides in length (e.g., 17 nucleotides in length).

[0249] Thus, in one aspect, the disclosure provides non-naturally occurring or engineered CRISPR systems including a functional CRISPR enzyme as described herein, and a RNA guide (gRNA) wherein the gRNA comprises a dead guide sequence whereby the gRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a genomic locus of interest in a cell without detectable cleavage activity.

[0250] A detailed description of dead guides is described, e.g., in WO 2016094872, which is incorporated herein by reference in its entirety.

Inducible Guides

[0251] RNA guides can be generated as components of inducible systems. The inducible nature of the systems allows for spatiotemporal control of gene editing or gene expression. In some embodiments, the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and/or thermal energy.

[0252] In some embodiments, the transcription of RNA guide can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems), hormone inducible gene expression systems (e.g., ecdysone inducible gene expression systems), and arabinose-inducible gene expression systems. Other examples of inducible systems include, e.g., small molecule two-hybrid transcription activations systems (FKBP, ABA, etc.), light inducible systems (Phytochrome, LOV domains, or cryptochrome), or Light Inducible Transcriptional Effector (LITE). These inducible systems are described, e.g., in WO 2016205764 and U.S. Pat. No. 8,795,965, both of which are incorporated herein by reference in their entirety.

Chemical Modifications

[0253] Chemical modifications can be applied to the RNA guide's phosphate backbone, sugar, and/or base. Backbone modifications such as phosphorothioates modify the charge on the phosphate backbone and aid in the delivery and nuclease resistance of the oligonucleotide (see, e.g., Eckstein, "Phosphorothioates, essential components of therapeutic oligonucleotides," Nucl. Acid Ther., 24 (2014), pp. 374-387); modifications of sugars, such as 2'-O-methyl (2'-OMe), 2'-F, and locked nucleic acid (LNA), enhance both base pairing and nuclease resistance (see, e.g., Allerson et al. "Fully 2'-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA," J. Med. Chem., 48.4 (2005): 901-904). Chemically modified bases such as 2-thiouridine or N6-methyladenosine, among others, can allow for either stronger or weaker base pairing (see, e.g., Bramsen et al., "Development of therapeutic-grade small interfering RNAs by chemical engineering," Front. Genet., 2012 Aug. 20; 3:154). Additionally, RNA is amenable to both 5' and 3' end conjugations with a variety of functional moieties including fluorescent dyes, polyethylene glycol, or proteins.

[0254] A wide variety of modifications can be applied to chemically synthesized RNA guide molecules. For example, modifying an oligonucleotide with a 2'-OMe to improve nuclease resistance can change the binding energy of Watson-Crick base pairing. Furthermore, a 2'-OMe modification can affect how the oligonucleotide interacts with transfection reagents, proteins or any other molecules in the cell. The effects of these modifications can be determined by empirical testing.

[0255] In some embodiments, the RNA guide includes one or more phosphorothioate modifications. In some embodiments, the RNA guide includes one or more locked nucleic acids for the purpose of enhancing base pairing and/or increasing nuclease resistance.

[0256] A summary of these chemical modifications can be found, e.g., in Kelley et al., "Versatility of chemically synthesized guide RNAs for CRISPR-Cas9 genome editing," J. Biotechnol. 2016 Sep. 10; 233:74-83; WO 2016205764; and U.S. Pat. No. 8,795,965 B2; each which is incorporated by reference in its entirety.

Sequence Modifications

[0257] The sequences and the lengths of the RNA guides and crRNAs described herein can be optimized. In some embodiments, the optimized length of RNA guide can be determined by identifying the processed form of the crRNA, or by empirical length studies for RNA guides, of crRNAs.

[0258] The RNA guides can also include one or more aptamer sequences. Aptamers are oligonucleotide or peptide molecules that can bind to a specific target molecule. The aptamers can be specific to gene effectors, gene activators, or gene repressors. In some embodiments, the aptamers can be specific to a protein, which in turn is specific to and recruits/binds to specific gene effectors, gene activators, or gene repressors. The effectors, activators, or repressors can be present in the form of fusion proteins. In some embodiments, the RNA guide has two or more aptamer sequences that are specific to the same adaptor proteins. In some embodiments, the two or more aptamer sequences are specific to different adaptor proteins. The adaptor proteins can include, e.g., MS2, PP7, Q.beta., F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KUl, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, .PHI.Cb5, .PHI.Cb8r, .PHI.Cb12r, .PHI.Cb23r, 7s, and PRR1. Accordingly, in some embodiments, the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein. In some embodiments, the aptamer sequence is a MS2 loop. A detailed description of aptamers can be found, e.g., in Nowak et al., "Guide RNA engineering for versatile Cas9 functionality," Nucl. Acid. Res., 2016 Nov. 16; 44(20):9555-9564; and WO 2016205764, which are incorporated herein by reference in their entirety.

Guide: Target Sequence Matching Requirements

[0259] In classic CRISPR systems, the degree of complementarity between a guide sequence and its corresponding target sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%. In some embodiments, the degree of complementarity is 100%. The RNA guides can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.

[0260] To reduce off-target interactions, e.g., to reduce the guide interacting with a target sequence having low complementarity, mutations can be introduced to the CRISPR systems so that the CRISPR systems can distinguish between target and off-target sequences that have greater than 80%, 85%, 90%, or 95% complementarity. In some embodiments, the degree of complementarity is from 80% to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (for example, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2, or 3 mismatches). Accordingly, in some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%. In some embodiments, the degree of complementarity is 100%.

[0261] It is known in the field that complete complementarity is not required provided that there is sufficient complementarity to be functional. Modulations of cleavage efficiency can be exploited by introduction of mismatches, e.g., one or more mismatches, such as 1 or 2 mismatches between spacer sequence and target sequence, including the position of the mismatch along the spacer/target. The more central (i.e., not at the 3' or 5' ends) a mismatch, e.g., a double mismatch, is located; the more cleavage efficiency is affected. Accordingly, by choosing mismatch positions along the spacer sequence, cleavage efficiency can be modulated. For example, if less than 100% cleavage of targets is desired (e.g., in a cell population), 1 or 2 mismatches between spacer and target sequence can be introduced in the spacer sequences.

Optimization of CRISPR Systems for use in Select Organisms

Codon-Optimization

[0262] The invention contemplates all possible variations of nucleic acids, such as cDNA, that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide encoding naturally occurring variant, and all such variations are to be considered as being specifically disclosed. Nucleotide sequences encoding type V-I CRISPR-Cas-associated effector protein variants that have been codon-optimized for expression in bacteria (e.g., E. coli) and in human cells are disclosed herein. For example, the codon-optimized sequences for human cells can be generated by substituting codons in the nucleotide sequence that occur at lower frequency in human cells for codons that occur at higher frequency in human cells. The frequency of occurrence for codons can be computationally determined by methods known in the art. An example of a calculation of these codon frequencies for various host cells (e.g., E. coli, yeast, insect, C. elegans, D. melanogaster, human, mouse, rat, pig, P. pastoris, A. thalian, maize, and tobacco) have been published or made available by sources such as the GenScript.RTM. Codon Usage Frequence Table Tool (example codon usage tables for E. coli and Humans are included below.

TABLE-US-00001 TABLE 1 E. coli Codon Usage Table Amino Triplet acid Fraction Number TTT F 0.58 80995 TTC F 0.42 58774 TTA L 0.14 52382 TTG L 0.13 47500 TAT Y 0.59 63937 TAC Y 0.41 44631 TAA * 0.61 7356 TAG * 0.09 989 CTT L 0.12 43449 CTC L 0.1 37347 CTA L 0.04 15409 CTG L 0.47 177210 CAT H 0.57 45879 CAC H 0.43 34078 CAA Q 0.34 53394 CAG Q 0.66 104171 ATT I 0.49 109072 ATC I 0.39 86796 ATA I 0.11 24984 ATG M 1 96695 AAT N 0.49 75436 AAC N 0.51 78443 AAA K 0.74 129137 AAG K 0.26 45459 GTT V 0.28 72584 GTC V 0.2 52439 GTA V 0.17 42420 GTG V 0.35 89265 GAT D 0.63 119939 GAC D 0.37 70394 GAA E 0.68 143353 GAG E 0.32 68609 TCT S 0.17 38027 TCC S 0.15 33430 TCA S 0.14 32715 TCG S 0.14 31146 TGT C 0.46 19138 TGC C 0.54 22188 TGA * 0.3 3623 TGG W 1 50991 CCT P 0.18 27340 CCC P 0.13 19666 CCA P 0.2 31534 CCG P 0.49 76644 CGT R 0.36 73197 CGC R 0.36 72212 CGA R 0.07 13844 CGG R 0.11 21552 ACT T 0.19 37842 ACC T 0.4 80547 ACA T 0.17 33910 ACG T 0.25 50269 AGT S 0.16 36097 AGC S 0.25 55551 AGA R 0.07 13152 AGG R 0.04 7607 GCT A 0.18 62479 GCC A 0.26 88721 GCA A 0.23 77547 GCG A 0.33 110308 GGT G 0.35 93325 GGC G 0.37 99390 GGA G 0.13 34799 GGG G 0.15 41277

TABLE-US-00002 TABLE 2 Human Codon Usage Table Amino Triplet acid Fraction Number TTT F 0.45 336562 TTC F 0.55 406571 TTA L 0.07 143715 TTG L 0.13 249879 TAT Y 0.43 239268 TAC Y 0.57 310695 TAA * 0.28 14322 TAG V 0.2 10915 CTT L 0.13 253795 CTC L 0.2 386182 CTA L 0.07 138154 CTG L 0.41 800774 CAT H 0.41 207826 CAC H 0.59 297048 CAA Q 0.25 234785 CAG Q 0.75 688316 ATT I 0.36 313225 ATC I 0.48 426570 ATA I 0.16 140652 ATG M 1 443795 AAT N 0.46 331714 AAC N 0.54 387148 AAA K 0.42 476554 AAG K 0.58 654280 GTT V 0.18 216818 GTC V 0.24 290874 GTA V 0.11 139156 GTG V 0.47 575438 GAT D 0.46 443369 GAC D 0.54 517579 GAA E 0.42 577846 GAG E 0.58 810842 TCT S 0.18 291040 TCC S 0.22 346943 TCA S 0.15 233110 TCG S 0.06 89429 TGT C 0.45 197293 TGC C 0.55 243685 TGA * 0.52 25383 TGG W 1 255512 CCT P 0.28 343793 CCC P 0.33 397790 CCA P 0.27 331944 CCG P 0.11 139414 CGT R 0.08 93458 CGC R 0.19 217130 CGA R 0.11 126113 CGG R 0.21 235938 ACT T 0.24 255582 ACC T 0.36 382050 ACA T 0.28 294223 ACG T 0.12 123533 AGT S 0.15 237404 AGC S 0.24 385113 AGA R 0.2 228151 AGG R 0.2 227281 GCT A 0.26 370873 GCC A 0.4 567930 GCA A 0.23 317338 GCG A 0.11 150708 GGT G 0.16 215544 GGC G 0.34 453917 GGA G 0.25 325243 GGG G 0.25 326879

Methods of Using CRISPR Systems

[0263] The CRISPR systems described herein have a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, or activating) a target polynucleotide in a multiplicity of cell types. The CRISPR systems have a broad spectrum of applications in, e.g., DNA/RNA detection (e.g., specific high sensitivity enzymatic reporter unlocking (SHERLOCK)), tracking and labeling of nucleic acids, enrichment assays (extracting desired sequence from background), detecting circulating tumor DNA, preparing next generation library, drug screening, disease diagnosis and prognosis, and treating various genetic disorders. Without wishing to be bound by any particular theory, CRISPR systems including a Cas12i protein may exhibit increased activity or may be preferentially active when targeting in certain environments, such as DNA plasmids, supercoiled DNA, or transcriptionally-active genomic loci.

[0264] Genome Editing Systems Generally

[0265] The term "genome editing system" refers to an engineered CRISPR system of the present disclosure having RNA-guided DNA editing activity. Genome editing systems of the present disclosure include at least two components of the CRISPR systems described above: an RNA guide and a cognate CRISPR effector protein. In certain embodiments of this disclosure the effector is a Cas12i protein and the RNA guide is a cognate Type V-I RNA guide. As described above, these two components form a complex that is capable of associating with a specific nucleic acid sequence and editing the DNA in or around that nucleic acid sequence, for instance by making one or more of a single strand break (an SSB or nick), a double strand break (a DSB), a nucleobase modification, a DNA methylation or demethylation, a chromatin modification, etc.

[0266] In certain embodiments, a genome editing system is transiently active (e.g., incorporating an inducible CRISPR effector as discussed above), while in other embodiments the system is constitutively (e.g., encoded by nucleic acids in which expression of CRISPR system components is controlled by one or more strong promoters).

[0267] Genome editing systems of the present disclosure, when introduced into cells, may alter (a) endogenous genomic DNA (gDNA) including, without limitation, DNA encoding e.g., a gene target of interest, an exonic sequence of a gene, an intronic sequence of a gene, a regulatory element of a gene or group of genes, etc.; (b) endogenous extra-genomic DNA such as mitochondrial DNA (mtDNA); and/or (c) exogenous DNA such as a non-integrated viral genome, a plasmid, an artificial chromosome, etc. Throughout this disclosure, these DNA substrates are referred to as "target DNA."

[0268] In instances where a genome editing operates by generating SSBs or DSBs, alterations caused by the system may take the form of short DNA insertions or deletions, which are collectively referred to as "indels." These indels may be formed within or proximate to a predicted cleavage site that is typically proximate to the PAM sequence and/or within a region of complementarity to the spacer sequence, though in some cases indels may occur outside of such predicted cleavage site. Without wishing to be bound by any theory, it is believed that indels are often the result of the repair of an SSB or DSB by "error-prone" DNA damage repair pathways, such as non-homologous end joining (NHEJ).

[0269] In some cases, a genome editing is used to generate two DSBs within 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1250, 1500, 1750, or 2000 base pairs of one another, which results in one or more outcomes, including the formation of an indels at one or both sites of cleavage, as well as deletion or inversion of a DNA sequence disposed between the DSBs.

[0270] Alternatively, genome editing systems of this disclosure may alter target DNA via integration of new sequences. These new sequences may be distinct from the existing sequence of the target DNA (as a non-limiting example, integrated by NHEJ by ligation of blunt-ends) or the may correspond to a DNA template having one or more regions that are homologous to a region of the targeted DNA. Integration of templated homologous sequences is also referred to as "homology-directed repair" or "HDR." Template DNA for HDR may be endogenous to the cell, including without limitation in the form of a homologous sequence located on another copy of the same chromosome as the target DNA, a homologous sequence from the same gene cluster as the target DNA, etc. Alternatively, or additionally, the template DNA may be provided exogenously, including without limitation as a free linear or circular DNA, as a DNA bound (covalently or non-covalently) to one or more genome editing system components, or as part of a vector genome.

[0271] In some instances, editing comprises a temporary or permanent silencing of a gene by CRISPR-mediated interference, as described by Matthew H. Larson et al. "CRISPR interference (CRISPRi) for sequence-specific control of gene expression," Nature Protocols 8, 2180-2196 (2013), which is incorporated by reference in its entirety and for all purposes.

[0272] Genome editing systems may include other components, including without limitation one or more heterologous functional domains which mediate site specific nucleobase modification, DNA methylation or demethylation, or chromatin modification. In some cases, the heterologous functional domain covalently bound to a CRISPR-associated protein such as a Cas12i, for instance by means of a direct peptide bond or an intervening peptide linker. Fusions of this type are described in greater detail below. In some embodiments, the heterologous functional domain is covalently bound to the crRNA, for instance by means of a chemical cross-link. And in some embodiments, one or more functional groups may be non-covalently associated with a CRISPR associated protein and/or a crRNA. This is done, variously, by means of an aptamer appended to the crRNA and/or the heterologous functional group, a peptide motif fused to the CRISPR-associated protein and a binding domain configured to bind such motif fused to the heterologous functional domain, or vice versa.

[0273] Genome editing system designs and genome editing outcomes are described in greater detail elsewhere in this specification.

DNA/RNA Detection

[0274] In one aspect, the CRISPR-Cas system described herein can be used in DNA/RNA detection by DNA sensing. Single effector RNA-guided DNases can be reprogrammed with RNA guides to provide a platform for specific single-stranded DNA (ssDNA) sensing. Upon recognition of its DNA target, an activated CRISPR Type V-I effector protein engages in "collateral" cleavage of nearby ssDNA with no sequence similarity to the target sequence. This RNA-programmed collateral cleavage activity allows the CRISPR systems to detect the presence of a specific DNA by nonspecific degradation of labeled ssDNA.

[0275] The collateral ssDNase activity can be combined with a reporter in DNA detection applications such as a method called the DNA Endonuclease-Targeted CRISPR trans reporter (DETECTR) method, which when combined with amplification achieves attomolar sensitivity for DNA detection (see, e.g., Chen et al., Science, 360(6387):436-439, 2018), which is incorporated herein by reference in its entirety. One application of using the enzymes described herein is to degrade non-target ssDNA in an in vitro environment. A "reporter" ssDNA molecule linking a fluorophore and a quencher can also be added to the in vitro system, along with an unknown sample of DNA (either single-stranded or double-stranded). Upon recognizing the target sequence in the unknown piece of DNA, the surveillance complex containing a Type V-I effector cleaves the reporter ssDNA resulting in a fluorescent readout.

[0276] In other embodiments, the SHERLOCK method (Specific High Sensitivity Enzymatic Reporter UnLOCKing) also provides an in vitro nucleic acid detection platform with attomolar (or single-molecule) sensitivity based on nucleic acid amplification and collateral cleavage of a reporter ssDNA, allowing for real-time detection of the target. Methods of using CRISPR in SHERLOCK are described in detail, e.g., in Gootenberg, et al. "Nucleic acid detection with CRISPR-Cas13a/C2c2," Science, 356(6336):438-442 (2017), which is incorporated herein by reference in its entirety.

[0277] In some embodiments, the CRISPR systems described herein can be used in multiplexed error-robust fluorescence in situ hybridization (MERFISH). These methods are described in, e.g., Chen et al., "Spatially resolved, highly multiplexed RNA profiling in single cells," Science, 2015 Apr. 24; 348(6233):aaa6090, which is incorporated herein by reference in its entirety.

[0278] In some embodiments, the CRISPR systems described herein can be used to detect a target DNA in a sample (e.g., a clinical sample, a cell, or a cell lysate). The collateral DNase activity of the CLUST.029130 (Type V-I) CRISPR-Cas effector proteins described herein is activated when the effector proteins bind to a target nucleic acid. Upon binding to the target DNA of interest, the effector protein cleaves a labeled detector ssDNA to generate or change a signal (e.g., an increased signal or a decreased signal) thereby allowing for the qualitative and quantitative detection of the target DNA in the sample. The specific detection and quantification of DNA in the sample allows for a multitude of applications including diagnostics.

[0279] In some embodiments, the methods include a) contacting a sample with: (i) an RNA guide (e.g., crRNA) and/or a nucleic acid encoding the RNA guide, wherein the RNA guide consists of a direct repeat sequence and a spacer sequence capable of hybridizing to the target RNA; (ii) a CLUST.029130 (Type V-I) CRISPR-Cas effector protein and/or a nucleic acid encoding the effector protein; and (iii) a labeled detector ssDNA; wherein the effector protein associates with the RNA guide to form a surveillance complex; wherein the surveillance complex hybridizes to the target DNA; and wherein upon binding of the surveillance complex to the target DNA, the effector protein exhibits collateral DNase activity and cleaves the labeled detector ssDNA; and b) measuring a detectable signal produced by cleavage of the labeled detector ssDNA, wherein said measuring provides for detection of the target DNA in the sample.

[0280] In some embodiments, the methods further include comparing the detectable signal with a reference signal and determining the amount of target DNA in the sample. In some embodiments, the measuring is performed using gold nanoparticle detection, fluorescence polarization, colloid phase transition/dispersion, electrochemical detection, and semiconductor based-sensing. In some embodiments, the labeled detector ssDNA includes a fluorescence-emitting dye pair, a fluorescence resonance energy transfer (FRET) pair, or a quencher/fluorophore pair. In some embodiments, upon cleavage of the labeled detector ssDNA by the effector protein, an amount of detectable signal produced by the labeled detector ssDNA is decreased or increased. In some embodiments, the labeled detector ssDNA produces a first detectable signal prior to cleavage by the effector protein and a second detectable signal after cleavage by the effector protein.

[0281] In some embodiments, a detectable signal is produced when the labeled detector ssDNA is cleaved by the effector protein. In some embodiments, the labeled detector ssDNA includes a modified nucleobase, a modified sugar moiety, a modified nucleic acid linkage, or a combination thereof.

[0282] In some embodiments, the methods include the multi-channel detection of multiple independent target DNAs in a sample (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more target RNAs) by using multiple CLUST.029130 (Type V-I) CRISPR-Cas systems, each including a distinct orthologous effector protein and corresponding RNA guides, allowing for the differentiation of multiple target DNAs in the sample. In some embodiments, the methods include the multi-channel detection of multiple independent target DNAs in a sample, with the use of multiple instances of CLUST.029130 (Type V-I) CRISPR-Cas systems, each containing an orthologous effector protein with differentiable collateral ssDNase substrates. Methods of detecting a DNA in a sample using CRISPR-associated proteins are described, for example, in U.S. Patent Publication No. 2017/0362644, the entire contents of which are incorporated herein by reference.

Tracking and Labeling of Nucleic Acids

[0283] Cellular processes depend on a network of molecular interactions among proteins, RNAs, and DNAs. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques employ an affinity tag combined with, a reporter group, e.g., a photoactivatable group, to label polypeptides and DNAs in the vicinity of a protein or DNA of interest in vitro. After UV irradiation, the photoactivatable groups react with proteins and other molecules that are in close proximity to the tagged molecules, thereby labelling them. Labelled interacting molecules can subsequently be recovered and identified. The DNA targeting effector proteins can for instance be used to target probes to selected DNA sequences. These applications can also be applied in animal models for in vivo imaging of diseases or difficult-to culture cell types. The methods of tracking and labeling of nucleic acids are described, e.g., in U.S. Pat. No. 8,795,965; WO 2016205764; and WO 2017070605; each of which is incorporated herein by reference in its entirety.

Genome Editing Using Paired CRISPR Nickases

[0284] The CRISPR systems described herein can be used in tandem such that two Cas12i nicking enzymes, or one Cas12i enzyme and one other CRISPR Cas enzyme with nicking activity, targeted by a pair of RNA guides to opposite strands of a target locus, can generate a double-strand break with overhangs. This method may reduce the likelihood of off-target modifications, because a double-strand break is expected to occur only at loci where both enzymes generate a nick, thereby increasing genome editing specificity. This method is referred to as a `double nicking` or `paired nickase` strategy and is described, e.g., in Ran et al., "Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity," Cell, 2013 Sep. 12; 154(6):1380-1389, and in Mali et al., "CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering," Nature Biotechnology, 2013 Aug. 1; 31:833-838, which are both incorporated herein by reference in their entireties.

[0285] The first applications of paired nickases demonstrated the utility of this strategy in mammalian cell lines. Applications of paired nickases have been described in the model plant Arabidopsis (e.g., in Fauser et al., "Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana," The Plant Journal 79(2):348-59 (2014), and Shiml et al., "The CRISPR/Cas system can be used as nuclease for in planta gene targeting and as paired nickases for directed mutagenesis in Arabidopsis resulting in heritable progeny," The Plant Journal 80(6):1139-50 (2014); in crops such as in rice (e.g., in Mikami et al., "Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice," Plant and Cell Physiology 57(5):1058-68 (2016) and in wheat (e.g., in ermak et al., "A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants," Plant Cell 29: 1196-1217 (2017); in bacteria (e.g., in Standage-Beier et al., "Targeted Large-Scale Deletion of Bacterial Genomes Using CRISPR-Nickases," ACS Synthetic Biology 4(11):1217-25 (2015); and in primary human cells for therapeutic purposes (e.g., in Dabrowska et al., "Precise Excision of the CAG Tract from the Huntingtin Gene by Cas9 Nickases," Frontiers in Neuroscience 12:75 (2018), and in Kocher et al., "Cut and Paste: Efficient Homology-Directed Repair of a Dominant Negative KRT14 Mutation via CRISPR/Cas9 Nickases," Molecular Therapy 25(11):2585-2598 (2017)), all of which are incorporated herein by reference in their entireties.

[0286] The CRISPR systems described herein can also be used as paired nickases to detect splice junctions as described e.g., in Santo & Paik, "A splice junction-targeted CRISPR approach (spJCRISPR) reveals human FOXO3B to be a protein-coding gene," Gene 673:95-101 (2018).

[0287] The CRISPR systems described herein can also be used as paired nickases to insert DNA molecules into target loci as described in e.g., Wang et al, "Therapeutic Genome Editing for Myotonic Dystrophy Type 1 Using CRISPR/Cas9," Molecular Therapy 26(11):2617-2630 (2018). The CRISPR systems described herein can also be used as single nickases to insert genes as described in e.g., Gao et al, "Single Cas9 nickase induced generation of NRAMPI knockin cattle with reduced off-target effects," Genome Biology 18(1):13 (2017).

Enhancing Base Editing using CRISPR Nickases

[0288] The CRISPR systems described herein can be used to augment the efficiency of CRISPR base editing. In base editing, a protein domain with DNA nucleotide modifying activity (e.g., cytidine deamination) is fused to a programmable CRISPR Cas enzyme that has been deactivated by mutation so as to no longer possess double-strand DNA cleavage activity. In some embodiments, using a nickase as the programmable Cas protein has been shown to improve the efficiency of base editing as described e.g., in Komor et al., "Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage," Nature 533:420-424 (2016), and Nishida et al., "Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems," Science 353 (6305): aaf8729 (2016), both of which are incorporated herein by reference in their entirety. A nickase that nicks the non-edited strand of the target locus is hypothesized to stimulate endogenous DNA repair pathways-such as mismatch repair or long-patch base excision repair, which preferentially resolves a mismatch generated by base editing to a desired allele--or to provide better accessibility of the catalytic editing domain to the target DNA.

Targeted Mutagenesis and DNA Labeling with Nickases and DNA Polymerases

[0289] The CRISPR systems described herein can be used in conjunction with proteins that act on nicked DNA. One such class of proteins is nick-translating DNA polymerases, such as E. coli DNA polymerase I or Taq DNA polymerase.

[0290] In some embodiments, the CRISPR system (e.g., a CRISPR nickase) can be fused to an error-prone DNA polymerase I. This fusion protein can be targeted with an RNA guide to generate a nick at a target DNA site. The DNA polymerase then initiates DNA synthesis at the nick, displacing downstream nucleotides, and, because an error-prone polymerase is used, resulting in mutagenesis of the target locus. Polymerase variants with varying processivity, fidelity, and misincorporation biases may be used to influence characteristics of the mutants that are generated. This method, called EvolvR, is described in detail, e.g., in Halperin et al., "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window," Nature 560, 248-252 (2018), which is incorporated herein by reference in its entirety.

[0291] In some embodiments, a CRISPR nickase can be used in a nick translation DNA labeling protocol. Nick translation, first described by Rigby et al in 1977, involves incubating DNA with a DNA nicking enzyme, such as DNase I, which creates one or more nicks in the DNA molecule.

[0292] Next, a nick-translating DNA polymerase, such as DNA polymerase I, is used to incorporate labeled nucleic acid residues at the nicked sites. Methods of harnessing the programmability of CRISPR nickases to covalently tag telomeric repeats with fluorescent dyes, using a variant of a classical nick translation labeling protocol, are described in detail e.g., in McCaffery et al., "High-throughput single-molecule telomere characterization," Genome Research 27:1904-1915 (2017), which is incorporated herein by reference in its entirety. This method enables haplotype-resolved analysis of telomere lengths at the single-molecule level.

Tracking and Labeling of Nucleic Acids

[0293] Cellular processes depend on a network of molecular interactions among proteins, RNAs, and DNAs. Accurate detection of protein-DNA and protein-RNA interactions is key to understanding such processes. In vitro proximity labeling techniques employ an affinity tag combined with, a reporter group, e.g., a photoactivatable group, to label polypeptides and RNAs in the vicinity of a protein or RNA of interest in vitro. After UV irradiation, the photoactivatable groups react with proteins and other molecules that are in close proximity to the tagged molecules, thereby labelling them. Labelled interacting molecules can subsequently be recovered and identified. The RNA targeting effector proteins can for instance be used to target probes to selected RNA sequences. These applications can also be applied in animal models for in vivo imaging of diseases or difficult-to culture cell types. The methods of tracking and labeling of nucleic acids are described, e.g., in U.S. Pat. No. 8,795,965; WO 2016205764; and WO 2017070605; each of which is incorporated herein by reference in its entirety.

High-Throughput Screening

[0294] The CRISPR systems described herein can be used for preparing next generation sequencing (NGS) libraries. For example, to create a cost-effective NGS library, the CRISPR systems can be used to disrupt the coding sequence of a target gene, and the CRISPR enzyme transfected clones can be screened simultaneously by next-generation sequencing (e.g., on the Ion Torrent PGM system). A detailed description regarding how to prepare NGS libraries can be found, e.g., in Bell et al., "A high-throughput screening strategy for detecting CRISPR-Cas9 induced mutations using next-generation sequencing," BMC Genomics, 15.1 (2014): 1002, which is incorporated herein by reference in its entirety.

Engineered Microorganisms

[0295] Microorganisms (e.g., E. coli, yeast, and microalgae) are widely used for synthetic biology. The development of synthetic biology has a wide utility, including various clinical applications. For example, the programmable CRISPR systems described herein can be used to split proteins of toxic domains for targeted cell death, e.g., using cancer-linked RNA as target transcript. Further, pathways involving protein-protein interactions can be influenced in synthetic biological systems with e.g. fusion complexes with the appropriate effectors such as kinases or enzymes.

[0296] In some embodiments, RNA guide sequences that target phage sequences can be introduced into the microorganism. Thus, the disclosure also provides methods of vaccinating a microorganism (e.g., a production strain) against phage infection.

[0297] In some embodiments, the CRISPR systems provided herein can be used to engineer microorganisms, e.g., to improve yield or improve fermentation efficiency. For example, the CRISPR systems described herein can be used to engineer microorganisms, such as yeast, to generate biofuel or biopolymers from fermentable sugars, or to degrade plant-derived lignocellulose derived from agricultural waste as a source of fermentable sugars. More particularly, the methods described herein can be used to modify the expression of endogenous genes required for biofuel production and/or to modify endogenous genes, which may interfere with the biofuel synthesis. These methods of engineering microorganisms are described e.g., in Verwaal et al., "CRISPR/Cpf1 enables fast and simple genome editing of Saccharomyces cerevisiae," Yeast, 2017 Sep. 8. doi: 10.1002/yea.3278; and Hlavova et al., "Improving microalgae for biotechnology--from genetics to synthetic biology," Biotechnol. Adv., 2015 Nov. 1; 33:1194-203, both of which are incorporated herein by reference in their entirety.

[0298] In some embodiments, the CRISPR systems described herein can be used to engineer microorganisms that have defective repair pathways, such as the mesophilic cellulolytic bacterium Clostridium cellylolyticum, a model organism for bioenergy research. In some embodiments, a CRISPR nickase can be used to introduce single nicks at a target locus, which may result in insertion of an exogenously provided DNA template by homologous recombination. A detailed method regarding how to use a CRISPR nickase to edit repair-defective microbes is described e.g., in Xu et al., "Efficient Genome Editing in Clostridium cellulolyticum via CRISPR-Cas9 Nickase," Appl Environ Microbiology 81:4423-4431 (2015), which is incorporated herein in its entirety.

[0299] In some embodiments, the CRISPR systems provided herein can be used to induce death or dormancy of a cell (e.g., a microorganism such as an engineered microorganism). These methods can be used to induce dormancy or death of a multitude of cell types including prokaryotic and eukaryotic cells, including, but not limited to, mammalian cells (e.g., cancer cells, or tissue culture cells), protozoans, fungal cells, cells infected with a virus, cells infected with an intracellular bacteria, cells infected with an intracellular protozoan, cells infected with a prion, bacteria (e.g., pathogenic and non-pathogenic bacteria), protozoans, and unicellular and multicellular parasites. For instance, in the field of synthetic biology it is highly desirable to have mechanisms of controlling engineered microorganisms (e.g., bacteria) to prevent their propagation or dissemination. The systems described herein can be used as "kill-switches" to regulate and/or prevent the propagation or dissemination of an engineered microorganism. Further, there is a need in the art for alternatives to current antibiotic treatments.

[0300] The systems described herein can also be used in applications where it is desirable to kill or control a specific microbial population (e.g., a bacterial population). For example, the systems described herein may include an RNA guide (e.g., a crRNA) that targets a nucleic acid (e.g., a DNA) that is genus-, species-, or strain-specific, and can be delivered to the cell. Upon complexing and binding to the target nucleic acid, the nuclease activity of the CLUST.029130 (Type V-I) CRISPR-Cas effector proteins disrupts essential functions within the microorganisms, ultimately resulting in dormancy or death. In some embodiments, the methods comprise contacting the cell with a system described herein including a CLUST.029130 (Type V-I) CRISPR-Cas effector proteins or a nucleic acid encoding the effector protein, and a RNA guide (e.g., a crRNA) or a nucleic acid encoding the RNA guide, wherein the spacer sequence is complementary to at least 15 nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides) of a target nucleic acid.

[0301] Without wishing to be bound by any particular theory, the nuclease activity of the CLUST.029130 (Type V-I) CRISPR-Cas effector proteins can induce programmed cell death, cell toxicity, apoptosis, necrosis, necroptosis, cell death, cell cycle arrest, cell anergy, a reduction of cell growth, or a reduction in cell proliferation. For example, in bacteria, the cleavage of DNA by the CLUST.029130 (Type V-I) CRISPR-Cas effector proteins can be bacteriostatic or bactericidal.

Application in Plants

[0302] The CRISPR systems described herein have a wide variety of utility in plants. In some embodiments, the CRISPR systems can be used to engineer genomes of plants (e.g., improving production, making products with desired post-translational modifications, or introducing genes for producing industrial products). In some embodiments, the CRISPR systems can be used to introduce a desired trait to a plant (e.g., with or without heritable modifications to the genome), or regulate expression of endogenous genes in plant cells or whole plants. Plants that can be edited using CRISPR systems of this disclosure (e.g., Cas12i systems) can be monocots or dicots and include, without limitation safflower, maize, cannabis, rice, sugarcane, canola, sorghum, tobacco, rye, barley, wheat, millet, oats, peanut, potato, switchgrass, turfgrass, soybean, alfalfa, sunflower, cotton, and Arabidopsis. The present disclosure also encompasses a plant having a trait made according to a method of the disclosure and/or utilizing a CRISPR system of the disclosure.

[0303] In some embodiments, the CRISPR systems can be used to identify, edit, and/or silence genes encoding specific proteins, e.g., allergenic proteins (e.g., allergenic proteins in peanuts, soybeans, lentils, peas, green beans, and mung beans). A detailed description regarding how to identify, edit, and/or silence genes encoding proteins is described, e.g., in Nicolaou et al., "Molecular diagnosis of peanut and legume allergy," Curr. Opin. Allergy Clin. Immunol., 11(3):222-8 (2011), and WO 2016205764 A1; both of which are incorporated herein by reference in their entirety.

Gene Drives

[0304] Gene drive is the phenomenon in which the inheritance of a particular gene or set of genes is favorably biased. The CRISPR systems described herein can be used to build gene drives. For example, the CRISPR systems can be designed to target and disrupt a particular allele of a gene, causing the cell to copy the second allele to fix the sequence. Because of the copying, the first allele will be converted to the second allele, increasing the chance of the second allele being transmitted to the offspring. A detailed method regarding how to use the CRISPR systems described herein to build gene drives is described, e.g., in Hammond et al., "A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae," Nat. Biotechnol., 2016 January; 34(1):78-83, which is incorporated herein by reference in its entirety.

Pooled-Screening

[0305] As described herein, pooled CRISPR screening is a powerful tool for identifying genes involved in biological mechanisms such as cell proliferation, drug resistance, and viral infection. Cells are transduced in bulk with a library of RNA guide (gRNA)-encoding vectors described herein, and the distribution of gRNAs is measured before and after applying a selective challenge. Pooled CRISPR screens work well for mechanisms that affect cell survival and proliferation, and they can be extended to measure the activity of individual genes (e.g., by using engineered reporter cell lines). Arrayed CRISPR screens, in which only one gene is targeted at a time, make it possible to use RNA-seq as the readout. In some embodiments, the CRISPR systems as described herein can be used in single-cell CRISPR screens. A detailed description regarding pooled CRISPR screenings can be found, e.g., in Datlinger et al., "Pooled CRISPR screening with single-cell transcriptome read-out," Nat. Methods., 2017 March; 14(3):297-301, which is incorporated herein by reference in its entirety.

Saturation Mutagenesis ("Bashing")

[0306] The CRISPR systems described herein can be used for in situ saturating mutagenesis. In some embodiments, a pooled RNA guide library can be used to perform in situ saturating mutagenesis for particular genes or regulatory elements. Such methods can reveal critical minimal features and discrete vulnerabilities of these genes or regulatory elements (e.g., enhancers). These methods are described, e.g., in Canver et al., "BCL11A enhancer dissection by Cas9-mediated in situ saturating mutagenesis," Nature, 2015 Nov. 12; 527(7577):192-7, which is incorporated herein by reference in its entirety.

Therapeutic Applications

[0307] The CRISPR systems described herein that have activity in a mammalian cellular context (e.g., Cas12i2) can have a diverse range of therapeutic applications. Moreover, each nuclease ortholog may have unique properties (e.g., size, PAM, etc.) that render it advantaged for certain targeting, treatment, or delivery modalities, so the ortholog selection is important in allocating the nuclease that provides maximum therapeutic benefit.

[0308] There are numerous factors that influence the suitability of gene editing as a therapeutic for a particular disease. With nuclease-based gene therapies, the primary approaches to therapeutic editing have been gene disruption and gene correction. In the former, gene disruption generally occurs with an event (such as a nuclease-induced, targeted double stranded break) that activates the endogenous non homologous end joining DNA repair mechanism of the target cell, yielding indels that often result in a loss of function mutation that is intended to benefit the patient. The latter, gene correction utilizes the nuclease activity to induce alternative DNA repair pathways (such as homology directed repair, or HDR) with the help of a template DNA (whether endogenous or exogenous, single stranded or double stranded). The templated DNA can either be an endogenous correction of a disease-causing mutation, or otherwise the insertion of a therapeutic transgene into an alternate locus (commonly safe harbor loci such as AAVS1). Methods of designing exogenous donor template nucleic acids are described, for example, in PCT Publication No. WO 2016094874 A1, the entire contents of which are expressly incorporated herein by reference. A requisite of therapies that use either of these editing modalities is an understanding of the genetic modulators of a certain disease; the diseases do not necessarily have to be monogenic, but insight into how mutations can effect the disease progress or outcome are important to providing guidance as to the potential efficacy of a gene therapy.

[0309] Without wishing to be limited, the CRISPR systems described herein can be utilized to treat the following diseases, wherein the specific gene targets are identified, in addition to the relevant references to aid in the adaption of the Type V-I CRISPR systems to specific disease areas; Cystic fibrosis by targeting CFTR (WO2015157070A2), Duchenne Muscular Dystrophy and Becker Muscular Dystrophy by targeting Dystrophin (DMD) (WO2016161380A1), Alpha-1-antitrypsin deficiency by targeting Alpha-1-antitrypsin (A1AT) (WO2017165862A1), lysosomal storage disorders such as Pompe Disease aka Glycogen storage disease type II by targeting acid alpha-glucosidase (GAA), myotonic dystrophy by targeting DMPK, Huntington disease by targeting HTT, Fragile X by targeting FMR1, Friedreich's ataxia by targeting Frataxin, amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) by targeting C9orf72, hereditary chronic kidney disease by targeting ApoL1, cardiovascular disease and hyperlipidemia by targeting PCSK9, APOC3, ANGPTL3, LPA (Nature 555, S23-S25 (2018)), and congenital blindness such as Leber Congenital Amaurosis Type 10 (LCA10) by targeting CEP290 (Maeder et al., Nat Med. 2019 February; 25(2):229-233). The majority of the aforementioned diseases are best treated with an in vivo gene editing approach, in which the cell types and tissues involved in the disease need to be edited in situ with a sufficient dose and efficiency to yield a therapeutic benefit. Some challenges of in vivo delivery are described in the "Delivery of CRISPR Systems" section below, though in general the smaller gene size of the Type V-I CRISPR effectors enables more versatile packaging into viral vectors with a payload restriction, such as adeno-associated viruses.

[0310] Ex vivo editing, in which cells are removed from the patient's body and then edited prior to transplantation back into the patient, present a prime therapeutic opportunity for gene editing technologies. The ability to manipulate cells outside the body presents multiple advantages, ranging from the ability to use technologies for high efficiency delivery of protein, DNA, and RNA into cells such as electroporation and nucleofection that are not amenable in an in vivo context, to being able to evaluate toxicity (such as from off-target effects), then further select and expand successfully edited cells to yield a population that provides a therapeutic advantage. These advantages are counterbalanced by the relatively few cell types and populations that can be successfully harvested, processed, and then returned to the body while preserving functionality. Without wishing to be limited, there nevertheless are serious diseases that are amenable to ex vivo genome editing using the systems described herein. For example, sickle cell disease (SCD) as referenced in WO2015148863A2, and beta-thalassemia as referenced in WO2015148860A1, both are examples of diseases in which the understanding of the pathophysiology has enabled a number of different editing modalities in hematopoietic stem cells for disease treatment. Beta thalassemia and SCD can both be treated with the disruption of the BCL11A erythroid enhancer to increase the levels of fetal hemoglobin (as illustrated using Zinc Finger Nucleases by Psatha et al. Mol Ther Methods Clin Dev. 2018 Sep. 21). In addition, methods of gene correction can be used to reverse the deleterious mutations in SCD and beta thalassemia. In another instance, the addition of a beta globin expressed from a safe harbor locus provides another alternative therapeutic strategy for ex vivo gene editing.

[0311] As a corollary of ex vivo editing of hematopoietic stem cells, immune cells can also be edited. In cancer immunotherapy, one therapeutic mode is to modify immune cells such as T-cells to recognize and fight cancer, as referenced in WO2015161276A2. To increase the efficacy and availability while decreasing cost, the creation of `off-the-shelf` allogeneic T-cell therapies is attractive, and gene editing has the potential to modify surface antigens to minimize any immunological side effects (Jung et al., Mol Cell. 2018 Aug. 31).

[0312] In another embodiment, the invention be used to target viruses or other pathogens with a double stranded DNA intermediate stage of their life cycle. Specifically, targeting viruses whose initial infection leaves a latent infection that persists permanently would be of significant therapeutic value. In the following examples, the Type V-I CRISPR systems can be used to directly target the viral genome (such as with HSV-1, HSV-2 or HIV), or used to edit the host cells to reduce or eliminate the receptors enabling infection to make them impervious to the virus (HIV), as referenced for HSV-1 and HSV-2 in WO2015153789A1, WO2015153791A1, and WO2017075475A1, and for HIV in WO2015148670A1 and WO2016183236A1.

[0313] In another aspect, the CRISPR systems described herein can be engineered to enable additional functions that utilize enzymatically inactive Cas12i as a chassis on top of which protein domains can be attached to confer activities such as transcriptional activation, repression, base editing, and methylation/demethylation.

[0314] Thus, this disclosure provides CRISPR-Cas systems and cells for use in the treatment or prevention of any of the disease disclosed herein.

Delivery of CRISPR Systems

[0315] The CRISPR systems described herein, or components thereof, nucleic acid molecules thereof, or nucleic acid molecules encoding or providing components thereof, can be delivered by various delivery systems such as vectors, e.g., plasmids, viral delivery vectors, such as adeno-associated viruses (AAV), lentiviruses, adenoviruses, and other viral vectors, or methods, such as nucleofection or electroporation of ribonucleoprotein complexes consisting of Type V-I effectors and their cognate RNA guide or guides. The proteins and one or more RNA guides can be packaged into one or more vectors, e.g., plasmids or viral vectors. For bacterial applications, the nucleic acids encoding any of the components of the CRISPR systems described herein can be delivered to the bacteria using a phage. Exemplary phages, include, but are not limited to, T4 phage, Mu, .lamda. phage, T5 phage, T7 phage, T3 phage, .PHI.29, M13, MS2, Q.beta., and .PHI.X174.

[0316] In some embodiments, the vectors, e.g., plasmids or viral vectors, are delivered to the tissue of interest by, e.g., intramuscular injection, intravenous administration, transdermal administration, intranasal administration, oral administration, or mucosal administration. Such delivery may be either via a single dose or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.

[0317] In certain embodiments, the delivery is via adeno-associated viruses (AAV), e.g., AAV2, AAV8, or AAV9, which can be administered in a single dose containing at least 1.times.10.sup.5 particles (also referred to as particle units, pu) of adenoviruses or adeno-associated viruses. In some embodiments, the dose is at least about 1.times.10.sup.6 particles, at least about 1.times.10.sup.7 particles, at least about 1.times.10.sup.8 particles, or at least about 1.times.10.sup.9 particles of the adeno-associated viruses. The delivery methods and the doses are described, e.g., in WO 2016205764 and U.S. Pat. No. 8,454,972, both of which are incorporated herein by reference in their entirety. Due to the limited genomic payload of recombinant AAV, the smaller size of the Type V-I CRISP-Cas effector proteins described herein enables greater versatility in packaging the effector and RNA guides with the appropriate control sequences (e.g., promoters) required for efficient and cell-type specific expression.

[0318] In some embodiments, the delivery is via a recombinant adeno-associated virus (rAAV) vector. For example, in some embodiments, a modified AAV vector may be used for delivery. Modified AAV vectors can be based on one or more of several capsid types, including AAV1, AV2, AAV5, AAV6, AAV8, AAV8.2. AAV9, AAV rhlO, modified AAV vectors (e.g., modified AAV2, modified AAV3, modified AAV6) and pseudotyped AAV (e.g., AAV2/8, AAV2/5 and AAV2/6). Exemplary AAV vectors and techniques that may be used to produce rAAV particles are known in the art (see, e.g., Aponte-Ubillus et al. (2018) Appl. Microbiol. Biotechnol. 102(3): 1045-54; Zhong et al. (2012) J. Genet. Syndr. Gene Ther. S1: 008; West et al. (1987) Virology 160: 38-47 (1987); Tratschin et al. (1985) Mol. Cell. Biol. 5: 3251-60); U.S. Pat. Nos. 4,797,368 and 5,173,414; and International Publication Nos. WO 2015/054653 and WO 93/24641, each of which is incorporated by reference).

[0319] In some embodiments, the delivery is via plasmids. The dosage can be a sufficient number of plasmids to elicit a response. In some cases, suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg. Plasmids will generally include (i) a promoter; (ii) a sequence encoding a nucleic acid-targeting CRISPR enzymes, operably linked to the promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). The plasmids can also encode the RNA components of a CRISPR-Cas system, but one or more of these may instead be encoded on different vectors. The frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or a person skilled in the art.

[0320] In another embodiment, the delivery is via liposomes or lipofectin formulations and the like, and can be prepared by methods known to those skilled in the art. Such methods are described, for example, in WO 2016205764 and U.S. Pat. Nos. 5,593,972; 5,589,466; and 5,580,859; each of which is incorporated herein by reference in its entirety.

[0321] In some embodiments, the delivery is via nanoparticles or exosomes. For example, exosomes have been shown to be particularly useful in the delivery of RNA.

[0322] Further means of introducing one or more components of the new CRISPR systems into cells is by using cell penetrating peptides (CPP). In some embodiments, a cell penetrating peptide is linked to the CRISPR enzymes. In some embodiments, the CRISPR enzymes and/or RNA guides are coupled to one or more CPPs to transport them inside cells effectively (e.g., plant protoplasts). In some embodiments, the CRISPR enzymes and/or RNA guide(s) are encoded by one or more circular or non-circular DNA molecules that are coupled to one or more CPPs for cell delivery.

[0323] CPPs are short peptides of fewer than 35 amino acids derived either from proteins or from chimeric sequences capable of transporting biomolecules across cell membrane in a receptor independent manner. CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, peptides having proline-rich and anti-microbial sequences, and chimeric or bipartite peptides. Examples of CPPs include, e.g., Tat (which is a nuclear transcriptional activator protein required for viral replication by HIV type 1), penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin .beta.3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide. CPPs and methods of using them are described, e.g., in Hallbrink et al., "Prediction of cell-penetrating peptides," Methods Mol. Biol., 2015; 1324:39-58; Ramakrishna et al., "Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and guide RNA," Genome Res., 2014 June; 24(6):1020-7; and WO 2016205764 A1; each of which is incorporated herein by reference in its entirety.

[0324] Delivery of the Type V-I CRISPR system as a ribonucleoprotein complex by electroporation or nucleofection, in which purified Cas12i protein is pre-incubated with a RNA guide and electroporated (or nucleofected) into cells of interest, is another method of efficiently introducing the CRISPR system to cells for gene editing. This is particularly useful for ex vivo genome editing and the development of cellular therapies, and such methods are described in Roth et al. "Reprogramming human T cell function and specificity with non-viral genome targeting," Nature, 2018 July; 559(7714): 405-409.

[0325] Various delivery methods for the CRISPR systems described herein are also described, e.g., in U.S. Pat. No. 8,795,965, EP 3009511, WO 2016205764, and WO 2017070605; each of which is incorporated herein by reference in its entirety

Kits

[0326] This disclosure also encompasses kits for carrying out the various methods of the disclosure utilizing the CRISPR systems described herein. One exemplary kit of the present disclosure comprises (a) one or more nucleic acids encoding a CRISPR-associated protein and a cognate crRNA, and/or (b) a ribonucleoprotein complex of a CRISPR-associated protein and a cognate crRNA. In some embodiments, the kit comprises a Cas12i protein and a Cas12i guide RNA. As described above, a complex of the protein and guide RNA has an editing activity such as SSB formation, DSB formation, CRISPR interference, nucleobase modification, DNA methylation or demethylation, chromatin modification, etc. In certain embodiments, the CRISPR-associated protein is a variant, such as a variant having reduced endonuclease activity.

[0327] Kits of this disclosure also optionally include additional reagents, including one or more of a reaction buffer, a wash buffer, one or more control materials (e.g., a substrate or a nucleic acid encoding a CRISPR system component), etc. A kit of the present disclosure also optionally includes instructions for performing a method of this disclosure using materials provided in the kit. The instructions are provided in physical form, e.g., as a printed document physically packaged with another item of the kit, and/or in digital form, e.g., a digitally published document downloadable from a website or provided on computer readable media.

EXAMPLES

[0328] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Identification of Minimal Components for the CLUST.029130 (Type V-I) CRISPR-Cas System (FIGS. 1-3)

[0329] This protein family describes a large single effector associated with CRISPR systems found in uncultured metagenomic sequences collected from freshwater environments (Table 3). CLUST.029130 (Type V-I) effectors, designated Cas12i, include the exemplary proteins detailed in Tables 3 and 4. Exemplary direct repeat sequences for these systems are shown in Table 5.

[0330] Genome and metagenome sequences were downloaded from NCBI (Benson et al. (2013) GenBank. Nucleic Acids Res. 41, D36-42; Pruitt et al. (2012) NCBI Reference Sequences (RefSeq): current status, new features and genome annotation policy. Nucleic Acids Res. 40, D130-135), NCBI whole genome sequencing (WGS), and DOE JGI Integrated Microbial Genomes (Markowitz et al. (2012) IMG: the Integrated Microbial Genomes database and comparative analysis system. Nucleic Acids Res. 40, D115-122) and compiled to construct a database of 293,985 putative CRISPR-Cas systems within which we identified novel nuclease systems. This approach to pipeline engineering performs minimal filtering in the intermediate stages to expand the search space for novel CRISPR effector discovery and reduce biases.

[0331] The classification tree depicted in FIGS. 1A-1B was constructed by comparing sequence profiles extracted from multiple alignments of groups of readily alignable Cas12 proteins. Profile-profile comparisons were performed using HHsearch (Soding et al. (2005) Protein homology detection by HMM-HMM comparison. Bioinforma. Oxf. Engl. 21, 951-960); scores between two profiles were normalized by the minimum of the self-scores and converted to a distance matrix on the natural log scale. The UPGMA dendrogram was reconstructed from the distance matrix. The tree at the depth of 2 distance unites (corresponding to the pairwise HHsearch score of e.sup.-2D=0.02 relative to the self-score) typically reliably recovers profile similarity and can serve as a guide for subtype classification (Shmakov et al., 2017).

[0332] The domain architecture of Cas12i, depicted in FIGS. 2A and 2B indicate that the effector contains the active catalytic residues of the RuvC nuclease domain. Additionally, the predicted secondary structure of the most prevalent direct repeat for Type V-I loci, depicted in FIG. 3, indicates a stem-loop structure that is conserved in the crRNA of many exemplary Type V-I CRISPR-Cas systems.

TABLE-US-00003 TABLE 3 Representative CLUST.029130 (Type V-I) Effector Proteins # effector species Cas12i accession spacers cas1 cas2 size SRR1522973 (SRR1522973) SRR1522973_megahit_k177_1081830_2|M 9 N N 1098 SRR1522973 (SRR1522973) SRR1522973_megahit_k177_427371_1|M 20 N N 1088 SRR2179954 (SRR2179954) SRR2179954_megahit_k177_1417524_4|M 7 N N 1074 SRR6475631 (SRR6475631) SRR6475631_megahit_k177_2773783_7|M 22 N N 1031 SRR6837575 (SRR6837575) SRR6837575_megahit_k177_919599_7|M 4 N N 1066 SRR6837577 (SRR6837577) SRR6837577_megahit_k177_410843_33|P 20 N N 1066 3300020508 3300020508|Ga0208225_1000010_34|M 10 N N 1093 (3300020508|Ga0208225_1000010) aquatic-freshwater 3300002408|release|scaffold05697_22|M 13 N N 1091 (3300002408|release|scaffold05697) aquatic-freshwater 3300002408|release|scaffold05697_22|P 13 N N 1046 (3300002408|release|scaffold05697) aquatic-freshwater 3300002408|release|scaffold08426_1|P 6 N N 1093 (3300002408|release|scaffold08426) aquatic-freshwater 3300028569|Ga0247843_1000055_230|M 12 N N 1080 (3300028569|Ga0247843_1000055) aquatic-freshwater 3300028569|Ga0247843_1000055_232|P 12 N N 1046 (3300028569|Ga0247843_1000055) aquatic-freshwater 3300028571|Ga0247844_1000101_90|M 12 N N 1080 (3300028571|Ga0247844_1000101) aquatic-freshwater 3300028571|Ga0247844_1000101_88|P 12 N N 1046 (3300028571|Ga0247844_1000101) aquatic-freshwater-freshwater lake 3300009183|Ga0114974_10028552_1|M 7 N N 1033 (3300009183|Ga0114974_10028552) aquatic-freshwater-freshwater lake 3300010885|Ga0133913_10053227_5|M 26 N N 1046 (3300010885|Ga0133913_10053227) aquatic-freshwater-freshwater lake 3300020193|Ga0194131_10013618_4|P 5 N N 1054 (3300020193|Ga0194131_10013618) aquatic-freshwater-freshwater lake 3300020214|Ga0194132_10015959_3|M 8 N N 1054 (3300020214|Ga0194132_10015959)

TABLE-US-00004 TABLE 4 Amino Acid Sequences of Representative CLUST.029130 (Type V-I) Effector Proteins >SRR1522973_megahit_k177_1081830_2|M [SRR1522973] MSISNNNILPYNPKLLPDDRKHKMLVDTFNQLDLIRNNLHDMIIALYGALKYDNIKQFASKEKPHISADALCSI- NW FRLVKTNERKPAIESNQIISKFIQYSGHTPDKYALSHITGNHEPSHKWIDCREYAINYARIMHLSFSQFQDLAT- AC LNCKILILNGTLTSSWAWGANSALFGGSDKENFSVKAKILNSFIENLKDEMNTTKFQVVEKVCQQIGSSDAADL- FD LYRSTVKDGNRGPATGRNPKVMNLFSQDGEISSEQREDFIESFQKVMQEKNSKQIIPHLDKLKYHLVKQSGLYD- IY SWAAAIKNANSTIVASNSSNLNTILNKTEKQQTFEELRKDEKIVACSKILLSVNDTLPEDLHYNPSTSNLGKNL- DV FFDLLNENSVHTIENKEEKNKIVKECVNQYMEECKGLNKPPMPVLLTFISDYAHKHQAQDFLSAAKMNFIDLKI- KS IKVVPTVHGSSPYTWISNLSKKNKDGKMIRTPNSSLIGWIIPPEEIHDQKFAGQNPIIWAVLRVYCNNKWEMHH- FP FSDSRFFTEVYAYKPNLPYLPGGENRSKREGYRHSTNLSNESRQILLDKSKYAKANKSVLRCMENMTHNVVFDP- KT SLNIRIKTDKNNSPVLDDKGRITFVMQINHRILEKYNNTKIEIGDRILAYDQNQSENHTYAILQRTEEGSHAHQ- FN GWYVRVLETGKVTSIVQGLSGPIDQLNYDGMPVTSHKENCWQADRSAFVSQFASLKISETETFDEAYQAINAQG- AY TWNLFYLRILRKALRVCHMENINQFREEILAISKNRLSPMSLGSLSQNSLKMIRAFKSIINCYMSRMSFVDELQ- KK EGDLELHTIMRLTDNKLNDKRVEKINRASSFLTNKAHSMGCKMIVGESDLPVADSKTSKKQNVDRMDWCARALS- HK VEYACKLMGLAYRGIPAYMSSHQDPLVHLVESKRSVLRPRFVVADKSDVKQHHLDNLRRMLNSKTKVGTAVYYR- EA VELMCEELGIHKTDMAKGKVSLSDFVDKFIGEKAIFPQRGGRFYMSTKRLTTGAKLICYSGSDVWLSDADEIAA- IN IGMFVVCDQTGAFKKKKKEKLDDEECDILPFRPM (SEQ ID NO: 14) >SRR1522973_megahit_k177_427371_1|4 [SRR1522973] MSSQVVRPYNAKFLPDDRKHKMLTDTINQLDKISSKHFDLLVAFYGSIQHKHVSINDKQEEHITPDSVCAINWF- RP MSKDYAKYQVKIDSMITNFKEYAGHIPDKYAIEYMGSNIDTDRFVWVDCRNFAKDYVRNMDMSFSEFQNLVDAL- VF CKILALNESTSTNWAWGAISAIYGGGDKEDSQFKAKVLNTFVKALNDENNKTKFDVINKVCSDLGYNDHLSLIE- DF RSTIDENGNKKSASGSPPAIAKFTEDGEISDNYRRACISSFSKTAKEKQDKKSIPHLDILKTHMIAMCGEYNTY- AW TEAIKNANTDITSRNTRNMTFIKEKIESRNSLKIYDTEENMKAAKILNGINHKLTPDLHYTPAPKHLGKNLKDL- FE MLEEKNILAQNEKEKKAALDECIKQYIDDCKGLNQQPIASLLAHISNYHKEITAENFLDGAKLLVLLQKINRQK- AH PSVFSPKAYTWGSKLEKNRRAANSALLGWIVPPEEKHKDRHAGQHPVMWVTMTLLNNGKWEKHHVPFTNSRFFS- EV YAYQPELPYKEGGYARNSKTATKPSQIMLPAYAESMRHHIATKGNGHKKSEKIVLRALSNIRHNVRFDPSTSFF- VR IMRDKKGNHRLDTKGRITFGLQINHRITVGKTKSEINIGDRLLAFDQNQSENHTFAIMQRVEENTPNSHQFNGW- NI RVLETGKVVSMTKGIESYYDQLSYDGVPYETKKFEDWRNERKAFVKKNKDIVIKEEKTFGQMFAEIKKSSLYKW- NL SYLKILRMAIRAKSGDTVSLFREELISIAKNRFGPLGLGSLSASSLKMLGAFCGVIQSYFSVLNCLDDKDKSNF- DS ELYFYLVSAFEKRVFKRNEKTSRASSFIMAMAYNHGCKMIVCEDDLPTAGAGANKRQNSDRMDWCARSLAQKIK- TG CEAMSIAYRAIPAYMSSHQDPLVHLADGKTSVLCPRFALVSKDDIKQYQLDGMRRMLNSKSKIGTAVYYRAAVE- LL CKELGINKTDIAKGKLSVSQFADIVNGEILLPQRGGRVYLATKELTNGAKLVSYNGSDVWLSNADEIAAINIGM- FV VCTQTGVFGKKKKKDEQDGDIEIA (SEQ ID NO: 15) >SRR2179954_megahit_k177_1417524_4|M [SRR2179954] MASISRPYGTKLRPDARKKEMLDKFFNTLTKGQRVFADLALCIYGSLTLEMAKSLEPESDSELVCAIGWFRLVD- KT IWSKDGIKQENLVKQYEAYSGKEASEVVKTYLNSPSSDKYVWIDCRQKFLRFQRELGTRNLSEDFECMLFEQYI- RL TKGEIEGYAAISNMFGNGEKEDRSKKRMYATRMKDWLEANENITWEQYREALKNQLNAKNLEQVVANYKGNAGG- AD PFFKYSFSKEGMVSKKEHAQQLDKFKTVLKNKARDLNFPNKEKLKQYLEAEIGIPVDANVYSQMFSNGVSEVQP- KT TRNMSFSNEKLDLLTELKDLNKGDGFEYAREVLNOFFDSELHTTEDKFNITSRYLGODKSNRLSKLYKIWKKEG- VD CEEGIQQFCEAVKDKMGQIPIRNVLKYLWQFRETVSAEDFEAAAKANHLEEKISRVKAHPIVISNRYWAFGTSA- LV GNIMPADKRHQGEYAGQNFKMWLEAELHYDGKKAKHHLPFYNARFFEEVYCYHPSVAEITPFKTKQFGCEIGKD- IP DYVSVALKDNPYKKATKRILRAIYNPVANTTGVDKTTNCSFMIKRENDEYKLVINRKISVDRPKRIEVGRTIMG- YD RNQTASDTYWIGRLVPPGTRGAYRIGEWSVQYIKSGPVLSSTQGVNNSTTDQLVYNGMPSSSERFKAWKKARMA- FI RKLIRQLNDEGLESKGQDYIPENPSSFDVRGETLYVFNSNYLKALVSKHRKAKKPVEGILDEIEAWTSKDKDSC- SL MRLSSLSDASMQGIASLKSLINSYFNKNGCKTIEDKEKFNPVLYAKLVEVEQRRTNKRSEKVGRIAGSLEQLAL- LN GVEVVIGEADLGEVEKOKSKKQNSRNMDWCAKQVAQRLEYKLAFHGIGYFGVNPMYTSHQDPFEHRRVADHIVM- RA RFEEVNVENIAEWHVRNFSNYLRADSGTGLYYKQATMDFLKHYGLEEHAEGLENKKIKFYDFRKILEDKNLTSV- II PKRGGRIYMATNPVTSDSTPITYAGKTYNRCNADEVAAANIVISVLAPRSKKNEEQDDIPLITKKAESKSPPKD- RK RSKTSQLPQK (SEQ ID NO: 16) >SRR6475631_megahit_k177_2773783_7|M [SRR6475631] MVSDSTIRPYTSKLAPNDPKRKMLNDTFNWLDHAYKVFFDVSVALFGGIDYEAAEELIDEKSTFDADLLCAIMW- FR LEEKSNNPOPLQTTEQRTRLFQKYSGHEPSSFAQEYIKGNTDTEKYEWVDCRLKFADLARNIHTTQESLKTDAY- TL FMNKLIPVSKDDEFNAYGFISQLFGTGKKEDRSVKASMLEEISNIIEDKKPNTWEEYQDLIKKTFNVSNYKELK- EK LSAGSSGRDGSLVIDLKEEKTGLLQPNFIKNRIVKFREDADKKRTVFSLPNRMKLREFISSQIGPFEQNSWSAV- LN RSMAAIQSKNSSNILYTNQKQERNNEIQELLKEDILSAASILNDFRRGEFNSSVVSKNHLGSRLNELFEMWQAL- KM NDGIEKYTDLCKDNFSRRPVSALLQYIYPYFDKITAKQFLDAASYNTLVETNNRKKIHPTVTGPTVCNWGPKST- IN GSITPPNQMVKDRPAGSHGMIWVTMTVRDNGRWVKHHLPFHNSRYYEEHYCYREGLPTKNQPRTKQLGTQVGSI- IS APSLAILKSQEEQDRRNDRKSRFKAHKSIIRSQENIKYNVAFDKSTNFDVTRKNGEFFITISSRVTTPKYSHKL- NV GDIIMGLDNNQTAPCTYSIWRIVEKDTEGSFFHNKIWLQLVTDGKITSIVDNNRQVDQLSYAGVEYSNFAEWRK- DR RQFLRSINEDYVKKSDNWLNMNLYQWNAEYSRLLLGVMKDNKDKNIQNTFRAEIEELICGKFGIRLGSLSHHSL- QF LTNCKSLISSYFMLNNKKEEHDQESFDSDFFRLMRSIDDKRIRKRKEKSSRISSSVLQIARENNVKSLCVEGDL- PT ATKKTKPKQNQKSIDWCARAVVKKLNDOCKVLGINLQAIDPRDTSHLDPFVYYGKKSTKVGKEARYVIVEPSNI- KE YMTKKFTDWHRGVSKKSKKGDVQTSTTAPLYQEALKQFADHYKLDFDSLPKMKFYELAKILEDHKQVIIPCROG- RA YLSTYPITKDSSKINFNGRERWYNQSDVVAAVNIVLROIRDEN (SEQ ID NO: 17) >SRR6837575_megahit_k177_919599_7|M [SRR6837575] MPDPIKSYKSPIIIDPNNAHDVEKLDFLRETFVYLSNGTKCFMHVFLSLLGGMNETLAKKIVSLETPKKEKKKK- SN KPSHKIELFLAICWFRLVKISKNESSVLPALLGNRFEKYFGAKATPEVMEYFSANYDEATYAWKDMREEFVSLK- SK LKVSEKDLISDIGSMINERYIGLKFGKPWGIISGLFGEGKKVDRSLKVELLKNVLEEIEKNPPKTKDQLAKMIL- KC ADCKNGQEIHAKCGKIGRMSSVSNWADEVGSEKEIVLSFVKSKISQDLAKQSNERNWKCVNALKSYILSEIGNC- FD QSSWSEMLNNSLSVIQSKTTRNYNFCIEQLEEKKNLNQNHRKFGTMIEDYFSSRFFTGENKFIICNFHVGDKDK- VS ALLASCEGLSEEELEEKIQNFCESQKQESKMPIPALLMYLNSLKDSITVDQMFQGILYNKIRDKIERQKLHPIV- PN NDSFDWGMSSKINGRIISPKEKAKHNAQNNRSLYDSGIWIEISVLKNKEWAKHHYKISNTRFVEEFYYPSSNDE- NS LDQVFRTGRNGFNNPAKNNLSLEQVSNIKNAPKNRRRAIKRQMRVEAAHQQNVLPHVKWDDNYCITISKYGDKF- VT FISKKFKSKKSKEYVVFLGFDQNQTASHTFAAVQICDSKDENVIPYCGLFVKPLECGHITSVQKVKDRSIDQLS- YS GLPWKDFISWSQERKEFVSKWRMVEVKTRNGEKLDDLTVKINKLDENKHGLYAYNSKYFWYLKSIMRKKTKDEL- FE IRKELLTVIKTGRLCVLRLSSLNHSSFLMLKNAKSAISCYFNNLLKGVSNDQEKYEADPEMFELRREVEAKRQN- KC MSKKNLISSQIVSKAIELRGNYGSVAIIGEDLSDYVPDKGKKSTQNANLLDWLSRGVANKVKQIANMHDNISFK- DV SPQWTSHQDSFVDRNPNSALRVRFGSCDPEEMYEKDFESLIKFLKEDCGHYTNSMNDFLSHYGVSRKDMLEIKF- SA FKILMKNILNKTGEKSLLYPKRGGRLYLATHKLGQCTRRTYNGVDFWECDADCVAAFNIALSGIRKYYGIKSEA- VS PV (SEQ ID NO: 18) >SRR6837577_megahit_k177_410843_33|P [SRR6837577] MPDPIKSYKSPIIIDPNNAHDVEKLDFLRETFVYLSNGTKCFMHVFLSLLGGMNETLAKKIVSLETPKKEKKKK- SN KPSHKIELFLAICWFRLVKISKNESSVLPALLGNRFEKYFGAKATPEVMEYFSANYDEATYAWKDMREEFVSLK- SK LKVSEKDLISDIGSMINERYIGLKFGKPWGIISGLFGEGKKVDRSLKVELLKNVLEEIEKNPPKTKDQLAKMIL- KC ADCKNGQEIHAKCGKIGRMSSVSNWADEVGSEKEIVLSFVKSKISQDLAKQSNERNWKCVNALKSYILSEIGNC- FD QSSWSEMLNNSLSVIQSKTTRNYNFCIEQLEEKKNLNQNHRKFGTMIEDYFSSRFFTGENKFIICNFHVGDKDK- VS ALLASCEGLSEEELEEKIQNFCESQKQESKMPIPALLMYLNSLKDSITVDQMFQGILYNKIRDKIERQKLHPIV- PN NDSFDWGMSSKINGRIISPKEKAKHNAQNNRSLYDSGIWIEISVLKNKEWAKHHYKISNTRFVEEFYYPSSNDE- NS LDQVFRTGRNGFNNPAKNNLSLEQVSNIKNAPKNRRRAIKRQMRVEAAHQQNVLPHVKWDDNYCITISKYGDKF- VT FISKKFKSKKSKEYVVFLGFDQNQTASHTFAAVQICDSKDENVIPYCGLFVKPLECGHITSVQKVKDRSIDQLS- YS GLPWKDFISWSQERKEFVSKWRMVEVKTRNGEKLDDLTVKINKLDENKHGLYAYNSKYFWYLKSIMRKKTKDEL- FE IRKELLTVIKTGRLCVLRLSSLNHSSFLMLKNAKSAISCYFNNLLKGVSNDQEKYEADPEMFELRREVEAKRQN- KC MSKKNLISSQIVSKAIELRGNYGSVAIIGEDLSDYVPDKGKKSTQNANLLDWLSRGVANKVKQIANMHDNISFK- DV SPQWTSHQDSFVDRNPNSALRVRFGSCDPEEMYEKDFESLIKFLKEDCGHYTNSMNDFLSHYGVSRKDMLEIKF- SA FKILMKNILNKTGEKSLLYPKRGGRLYLATHKLGQCTRRTYNGVDFWECDADCVAAFNIALSGIRKYYGIKSEA- VS PV (SEQ ID NO: 18) >3300020508|Ga0208225_1000010_34|M [3300020508] MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISGTANKDKISDDLL- LA VNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQAQEYFASNFDTEKHQWKDMRVEYERLLAELQLSRSDMH- HD LKLMYKEKCIGLSLSTAHYITSVMFGTGAKNNRQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSYRK- LR IRCSRKGATPSILKIVQDYELGTNHDDEVNVPSLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYS- AM LENALSPIKGMTTKNCKFVLKQIDAKNDIKYENEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNIKTLWE- DL NAIHSKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRYLYSRKDDIAVDKIIDGITF- LS KKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNLKCNRNQVDNYIWIEIKVLNTKTMRWEKHHYA- LS STRFLEEVYYPATSENPPDALAARFRTKTNGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPR- YT WGKDFNINICKRGNNFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYNDLPVKPIESG- FV TVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGTMKDNRGNNIQIDFMKDFEAIADDETSLYYFNM- KY CKLLQSSIRNHSSQAKEYREEIFELLRDGKLSVLKLSSLSNLSFVMFKVAKSLIGTYFGHLLKKPKNSKSDVKA- PP ITDEDKQKADPEMFALRLALEEKRLNKVKSKKEVIANKIVAKALELRDKYGPVLIKGENISDTTKKGKKSSTNS- FL MDWLARGVANKVKEMVMMHQGLEFVEVNPNFTSHQDPFVHKNPENTFRARYSRCTPSELTEKNRKEILSFLSDK- PS KRPTNAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYKLDADATSVN- WN GKQFWVCNADLVAAYNVGLVDIQKDFKKK (SEQ ID NO: 3) >3300002408|release|scaffold05697_22|M [aquatic-freshwater] MFTLLLSDISQQNFNKFLKNFFFTRNKTVVHCSSEIRHKGYRSNVMVSESTIRPYTSKLAPNDPKLKMLNDTFN- WL DHAYKVFFDVSVALFGAIEHETAQELIGEKSKFDADLLCAIMWFRLEEKSDNPGPLQTVEQRMRLFQKYSGHEP- SS FTQEYIKGNIDSEKYQWVDCRLKFIDLARNINTTQESLKIDAYTLFMNKLIPVSKDDEFNAYGLISQLFGTGKK- ED RSIKASMLEEISNIIEDKKPNTWEEYHDLIKKTFNVDNYKELKEKLSAGSSGRDSSLVIDLKEEKTGLLQPNFI- KN RIVKFREDADKKRTVFLLPNRMKLREFIASQIGPFEQNSWSAVLNRSMAAIQSKNSSNILYTNEKEERNNEIQE- LL KKDILSAASILGDFRRGEFNRSVVSKNHLGARLNELFEIWQELTMDDGIKKYVDLCKDKFSRRPVKALLQYIYP- YF DKINAKQFLDAASYNTLVETNNRKKIHPTVTGPTVCNWGPKSTINGSITPPNQMVKGRPAGSHGMIWVTMTVID- NG RWIKHHLPFHNSRYYEEHYCYREGLPTKNKPRTKQLGTQVGSTISAPSLAILKSQEEQDRRNDRKNRFKAHKSI- IR SQENIEYNVAFDKSTNFDVTRKNGEFFITISSRVATPKYSYKLNIGDMIMGLDNNQTAPCTYSIWRVVEKDTEG- SF FHNKIWLQLVTDGKVTSIVDNNRQVDQLSYAGIEYSNFAEWRKDRRQFLRSINEDYVKKSDNWRNMNLYQWNAE- YS

RLLLDVMKENKGKNIQNTFRAEIEELICGKFGIRLGSLFHHSLQFLTNCKSLISSYFMLNNKKEEYDQELFDSD- FF RLMKSIGDKRVRKRKEKSSRISSTVLQIARENNVKSLCVEGYLPTSTKKTKPKQNQKSIDWCARAVVKKLNDGC- KV LGINLQAIDPRDTSHLDPFVYYGKKSTKVGKEARYTIVEPSNIKEYMTNRFDDWHRGVTKKSKKGDVQTSTTVL- LY QEALRQFASHYKLDFDSLPKMKFYELAKILGDHEKVIIPCRGGRAYLSTYPVTKDSSKITFNGRERWYNESDVV- AA VNIVLRGIIDEDEQPDGAKKQALARTK (SEQ ID NO: 2) >3300002408|release|scaffold05697_22|P [aquatic-freshwater] MVSESTIRPYTSKLAPNDPKLKMLNDTFNWLDHAYKVFFDVSVALFGAIEHETAQELIGEKSKFDADLLCAIMW- FR LEEKSDNPGPLQTVEQRMRLFQKYSGHEPSSFTQEYIKGNIDSEKYQWVDCRLKFIDLARNINTTQESLKIDAY- TL FMNKLIPVSKDDEFNAYGLISQLFGTGKKEDRSIKASMLEEISNIIEDKKPNTWEEYHDLIKKTFNVDNYKELK- EK LSAGSSGRDSSLVIDLKEEKTGLLQPNFIKNRIVKFREDADKKRTVFLLPNRMKLREFIASQIGPFEQNSWSAV- LN RSMAAIQSKNSSNILYTNEKEERNNEIQELLKKDILSAASILGDFRRGEFNRSVVSKNHLGARLNELFEIWQEL- TM DDGIKKYVDLCKDKFSRRPVKALLQYIYPYFDKINAKQFLDAASYNTLVETNNRKKIHPTVTGPTVCNWGPKST- IN GSITPPNQMVKGRPAGSHGMIWVTMTVIDNGRWIKHHLPFHNSRYYEEHYCYREGLPTKNKPRTKQLGTQVGST- IS APSLAILKSQEEQDRRNDRKNRFKAHKSIIRSQENIEYNVAFDKSTNFDVTRKNGEFFITISSRVATPKYSYKL- NI GDMIMGLDNNQTAPCTYSIWRVVEKDTEGSFFHNKIWLQLVTDOKVTSIVDNNRQVDQLSYAGIEYSNFAEWRK- DR RQFLRSINEDYVKKSDNWRNMNLYQWNAEYSRLLLDVMKENKGKNIQNTFRAEIEELICGKFGIRLGSLFHHSL- QF LTNCKSLISSYFMLNNKKEEYDQELFDSDFFRLMKSIGDKRVRKRKEKSSRISSTVLQIARENNVKSLCVEGYL- PT STKKTKPKQNQKSIDWCARAVVKKLNDGCKVLGINLQAIDPRDTSHLDPFVYYGKKSTKVGKEARYTIVEPSNI- KE YMTNRFDDWHRGVTKKSKKGDVQTSTTVLLYQEALRQFASHYKLDFDSLPKMKFYELAKILGDHEKVIIPCRGG- RA YLSTYPVTKDSSKITFNGRERWYNESDVVAAVNIVLRGIIDEDEQPDGAKKQALARTK (SEQ ID NO: 1) >3300002408|release|scaffold08426_1|P [aquatic-freshwater] MSNKEKNASETRKAYTTKMIPRSHDRMKLLGNFMDYLMDGTPIFFELWNQFGGGIDRDIISGTANKDKISDDLL- LA VNWFKVMPINSKPQGVSPSNLANLFQQYSGSEPDIQAQEYFASNFDTEKHQWKDMRVEYERLLAELQLSRSDMH- HD LKLMYKEKCIGLSLSTAHYITSVMFGTGAKNNRQTKHQFYSKVIQLLEESTQINSVEQLASIILKAGDCDSYRK- LR IRCSRKGATPSILKIVQDYELGTNHDDEVNVPSLIANLKEKLGRFEYECEWKCMEKIKAFLASKVGPYYLGSYS- AM LENALSPIKGMTTKNCKFVLKQIDAKNDIKYENEPFGKIVEGFFDSPYFESDTNVKWVLHPHHIGESNIKTLWE- DL NAIHSKYEEDIASLSEDKKEKRIKVYQGDVCQTINTYCEEVGKEAKTPLVQLLRYLYSRKDDIAVDKIIDGITF- LS KKHKVEKQKINPVIQKYPSFNFGNNSKLLGKIISPKDKLKHNLKCNRNQVDNYIWIEIKVLNTKTMRWEKHHYA- LS STRFLEEVYYPATSENPPDALAARFRTKTNGYEGKPALSAEQIEQIRSAPVGLRKVKKRQMRLEAARQQNLLPR- YT WGKDFNINICKRGNNFEVTLATKVKKKKEKNYKVVLGYDANIVRKNTYAAIEAHANGDGVIDYNDLPVKPIESG- FV TVESQVRDKSYDQLSYNGVKLLYCKPHVESRRSFLEKYRNGTMKDNRGNNIQIDFMKDFEAIADDETSLYYFNM- KY CKLLQSSIRNHSSQAKEYREEIFELLRDGKLSVLKLSSLSNLSFVMFKVAKSLIGTYFGHLLKKPKNSKSDVKA- PP ITDEDKQKADPEMFALRLALEEKRLNKVKSKKEVIANKIVAKALELRDKYGPVLIKGENISDTTKKGKKSSTNS- FL MDWLARGVANKVKEMVMMHQGLEFVEVNPNFTSHQDPFVHKNPENTFRARYSRCTPSELTEKNRKEILSFLSDK- PS KRPTNAYYNEGAMAFLATYGLKKNDVLGVSLEKFKQIMANILHQRSEDQLLFPSRGGMFYLATYKLDADATSVN- WN GKQFWVCNADLVAAYNVGLVDIQKDFKKK (SEQ ID NO: 3) >3300028569|Ga0247843_1000055_230|M [aquatic-freshwater] MPRNYFLGIFSLQKNKSVVHCSVEIRHKGYRSSVMVSDSTIRPYASKLAPNDPKLKMLNDTFNWLDHAYKVFFD- VS VALFGAIEHETAQELIGEKSKFDADLICAIMWFRLEEKSDNPGPLQTVEQRMRLFQKYSGHEPSSFTQEYIKGN- ID SEKYEWVDCRLKFIDLARNINTTQESLKIDAYTLFMNKLIPVSKDDEFNAYGLISQLFGTGKKEDRSIKAAMLE- EI SNILADKKPDTWEEYHDLIKKNFNVDNYKELKEKLSAGSSGRDSSLVIDLKEEKTGLLQPNFIKNRIVKFREDA- DK KKTVFLLPNRMKLREFIASQIGPFEQNSWSAVLNRSMAAIQSKNSSNILYTNEKEERNNEIQELLKKDILSAAS- IL GDFRRGEFNRSVVSKNHLGARLNELFEIWQDLTMDDGIRKYVDLCKDKFSRRPVKALLQYIYPYFDKITAKQFL- DA ASYNTLVETNNRKKIHPTVTGPTVCNWGPKSTINGSITPPNQMVKGRPAGSHGMIWVTMTVIDNGRWIKHHLPF- YN SRYYEEHYCYREGLPTKNQPRTKQLGTQVGSTISATSLAALKSQEEQDRRNDRKNRFKAHKSIIRSQENIEYNV- AF DKSTNFDVTRKNGEFFITISSRVATPKYSYKLNIGDMIMGLDNNQTAPCTYSIWRVVEKDTEGSFFHNKIWLQL- VT DGKITSIVDNNRQVDQLSYAGIEYSNFAEWRKDRRQFLRSINEDYVKKSDNWRNMNLYQWNAEYSRLLLDVMKE- NK GKNIQNTFRAEIEELICGKFGIRLGSLFHHSLQFLTNCKSLISSYFMLNNKKEEYDQELFDSDFFRLMKSIGDK- RV RKRKEKSSRISSTVLQIARENNIKSLCVEGDLPTATKKTKPKQNQKSIDWCARAVVKKLNDGCKVLGINLQAID- PR DTSHLDPFVYYGKKSTKVGKEARYTIVEPSNIKEYMTNRFDDWHRGVTKKSKKGDVQTSTTVLLYQEALRQFAS- HY ELDFDSLPKMKFYDLAKRLGDHEKVIIPCRGGRAYLSTYPVTKDSSKITFNGRERWYNESDVVAAVNIVLRGIR- DE DEQPDDAKKQALARTK (SEQ ID NO: 11) >3300028569|Ga0247843_1000055_232|P [aquatic-freshwater] MVSDSTIRPYASKLAPNDPKLKMLNDTFNWLDHAYKVFFDVSVALFGAIEHETAQELIGEKSKFDADLICAIMW- FR LEEKSDNPGPLQTVEQRMRLFQKYSGHEPSSFTQEYIKGNIDSEKYEWVDCRLKFIDLARNINTTQESLKIDAY- TL FMNKLIPVSKDDEFNAYGLISQLFGTGKKEDRSIKAAMLEEISNILADKKPDTWEEYHDLIKKNFNVDNYKELK- EK LSAGSSGRDSSLVIDLKEEKTGLLQPNFIKNRIVKFREDADKKKTVFLLPNRMKLREFIASQIGPFEQNSWSAV- LN RSMAAIQSKNSSNILYTNEKEERNNEIQELLKKDILSAASILGDFRRGEFNRSVVSKNHLGARLNELFEIWQDL- TM DDGIRKYVDLCKDKFSRRPVKALLQYIYPYFDKITAKQFLDAASYNTLVETNNRKKIHPTVTGPTVCNWGPKST- IN GSITPPNQMVKGRPAGSHGMIWVTMTVIDNGRWIKHHLPFYNSRYYEEHYCYREGLPTKNQPRTKQLGTQVGST- IS ATSLAALKSQEEQDRRNDRKNRFKAHKSIIRSQENIEYNVAFDKSTNFDVTRKNGEFFITISSRVATPKYSYKL- NI GDMIMGLDNNQTAPCTYSIWRVVEKDTEGSFFHNKIWLQLVTDGKITSIVDNNRQVDQLSYAGIEYSNFAEWRK- DR RQFLRSINEDYVKKSDNWRNMNLYQWNAEYSRLLLDVMKENKGKNIQNTFRAEIEELICGKFGIRLGSLFHHSL- QF LTNCKSLISSYFMLNNKKEEYDQELFDSDFFRLMKSIGDKRVRKRKEKSSRISSTVLQIARENNIKSLCVEGDL- PT ATKKTKPKQNQKSIDWCARAVVKKLNDGCKVLGINLQAIDPRDTSHLDPFVYYGKKSTKVGKEARYTIVEPSNI- KE YMTNRFDDWHRGVTKKSKKGDVQTSTTVLLYQEALRQFASHYELDFDSLPKMKFYDLAKRLGDHEKVIIPCRGG- RA YLSTYPVTKDSSKITFNGRERWYNESDVVAAVNIVLRGIRDEDEQPDDAKKQALARTK (SEQ ID NO: 12) >3300028571|Ga0247844_1000101_90|M [aquatic-freshwater] MPRNYFLGIFSLQKNKSVVHCSVEIRHKGYRSSVMVSDSTIRPYASKLAPNDPKLKMLNDTFNWLDHAYKVFFD- VS VALFGAIEHETAQELIGEKSKFDADLICAIMWFRLEEKSDNPGPLQTVEQRMRLFQKYSGHEPSSFTQEYIKGN- ID SEKYEWVDCRLKFIDLARNINTTQESLKIDAYTLFMNKLIPVSKDDEFNAYGLISQLFGTGKKEDRSIKAAMLE- EI SNILADKKPDTWEEYHDLIKKNFNVDNYKELKEKLSAGSSGRDSSLVIDLKEEKTGLLQPNFIKNRIVKFREDA- DK KKTVFLLPNRMKLREFIASQIGPFEQNSWSAVLNRSMAAIQSKNSSNILYTNEKEERNNEIQELLKKDILSAAS- IL GDFRRGEFNRSVVSKNHLGARLNELFEIWQDLTMDDGIRKYVDLCKDKFSRRPVKALLQYIYPYFDKITAKQFL- DA ASYNTLVETNNRKKIHPTVTGPTVCNWGPKSTINGSITPPNQMVKGRPAGSHGMIWVTMTVIDNGRWIKHHLPF- YN SRYYEEHYCYREGLPTKNQPRTKQLGTQVGSTISATSLAALKSQEEQDRRNDRKNRFKAHKSIIRSQENIEYNV- AF DKSTNFDVTRKNGEFFITISSRVATPKYSYKLNIGDMIMGLDNNQTAPCTYSIWRVVEKDTEGSFFHNKIWLQL- VT DGKITSIVDNNRQVDQLSYAGIEYSNFAEWRKDRRQFLRSINEDYVKKSDNWRNMNLYQWNAEYSRLLLDVMKE- NK GKNIQNTFRAEIEELICGKFGIRLGSLFHHSLQFLTNCKSLISSYFMLNNKKEEYDQELFDSDFFRLMKSIGDK- RV RKRKEKSSRISSTVLQIARENNIKSLCVEGDLPTATKKTKPKQNQKSIDWCARAVVKKLNDGCKVLGINLQAID- PR DTSHLDPFVYYGKKSTKVGKEARYTIVEPSNIKEYMTNRFDDWHRGVTKKSKKGDVQTSTTVLLYQEALRQFAS- HY ELDFDSLPKMKFYDLAKRLGDHEKVIIPCRGGRAYLSTYPVTKDSSKITFNGRERWYNESDVVAAVNIVLRGIR- DE DEQPDDAKKQALARTK (SEQ ID NO: 11) >3300028571|Ga0247844_1000101_88|P [aquatic-freshwater] MVSDSTIRPYASKLAPNDPKLKMLNDTFNWLDHAYKVFFDVSVALFGAIEHETAQELIGEKSKFDADLICAIMW- FR LEEKSDNPGPLQTVEQRMRLFQKYSGHEPSSFTQEYIKGNIDSEKYEWVDCRLKFIDLARNINTTQESLKIDAY- TL FMNKLIPVSKDDEFNAYGLISQLFGTGKKEDRSIKAAMLEEISNILADKKPDTWEEYHDLIKKNFNVDNYKELK- EK LSAGSSGRDSSLVIDLKEEKTGLLQPNFIKNRIVKFREDADKKKTVFLLPNRMKLREFIASQIGPFEQNSWSAV- LN RSMAAIQSKNSSNILYTNEKEERNNEIQELLKKDILSAASILGDFRRGEFNRSVVSKNHLGARLNELFEIWQDL- TM DDGIRKYVDLCKDKFSRRPVKALLQYIYPYFDKITAKQFLDAASYNTLVETNNRKKIHPTVTGPTVCNWGPKST- IN GSITPPNQMVKGRPAGSHGMIWVTMTVIDNGRWIKHHLPFYNSRYYEEHYCYREGLPTKNQPRTKQLGTQVGST- IS ATSLAALKSQEEQDRRNDRKNRFKAHKSIIRSQENIEYNVAFDKSTNFDVTRKNGEFFITISSRVATPKYSYKL- NI GDMIMGLDNNQTAPCTYSIWRVVEKDTEGSFFHNKIWLQLVTDGKITSIVDNNRQVDQLSYAGIEYSNFAEWRK- DR RQFLRSINEDYVKKSDNWRNMNLYQWNAEYSRLLLDVMKENKGKNIQNTFRAEIEELICGKFGIRLGSLFHHSL- QF LTNCKSLISSYFMLNNKKEEYDQELFDSDFFRLMKSIGDKRVRKRKEKSSRISSTVLQIARENNIKSLCVEGDL- PT ATKKTKPKQNQKSIDWCARAVVKKLNDGCKVLGINLQAIDPRDTSHLDPFVYYGKKSTKVGKEARYTIVEPSNI- KE YMTNRFDDWHRGVTKKSKKGDVQTSTTVLLYQEALRQFASHYELDFDSLPKMKFYDLAKRLGDHEKVIIPCRGG- RA YLSTYPVTKDSSKITFNGRERWYNESDVVAAVNIVLRGIRDEDEQPDDAKKQALARTK (SEQ ID NO: 12) >3300009183|Ga0114974_10028552_1|M [aquatic-freshwater-freshwater lake] MMSDNIILPYNSKLAPDERKQRLLNDTFNWFDMCNEVFFDFVKNLYGGVKHEHLILVNFAEKPKKVSNSKKPKK- KD QEVNIHVEPNQAEWVDNACATFWFRLQAKSTVQLDQSVQTAEERIRRFRDYAGHEPSSFAKSYLNGNYDPEKTE- WV DCRLLYVNFCRNLNVNLDADIRTMVEHNLLPVLPGQDFKTNNVFSNIFGVGNKEDKGQKTNWLNTVSEGLQSKE- IW NWDEYRDLISRSTGCSTAAELRSESIGRPSMLAVDFASEKSGQISQEWLAERVKSFRAAASQKSKIYDMPNRLV- LK EYIASKIGPFKLERWSAAAVSAYKDVRSKNSINLLYSKERLWRCKEIAQILVDNTQVAEAQQILVNYSSGDTNS- FT VENRHMGDLTVLFKIWEKMDMDSGIEQYSEIYRDEYSRDPITELLRYLYNHRHISAKTFRAAARLNSLLLKNDR- KK IHPTISGRTSVSFGHSTIKGCITPPDHIVKNRKENAGSTGMIWVTMQLIDNGRWADHHIPFHNSRYYRDFYAYR- AD LPTISDPRRKSFGHRIGNNISDTRMINHDCKKASKMYLRTIQNMTHNVAFDQQTQFAVRRYADNNFTITIQARV- VG RKYKKEISVGDRVMGVDQNQTTSNTYSVWEVVAEGTENSYPYKGNNYRLVEDGFIRSECSGRDQLSYDGLDFQD- FA QWRRERYAFLSSVGCILNDEIEPQIPVSAEKAKKKKKFSKWRGCSLYSWNLCYAYYLKGLMHENLANNPAGFRQ- EI LNFIQGSRGVRLCSLNHTSFRLLSKAKSLIHSFFGLNNIKDPESQRDFDPEIYDIMVNLTQRKTNKRKEKANRI- TS SILQIANRLNVSRIVIENDLPNASSKNKASANQRATDWCARNVSEKLEYACKMLGISLWQIDPRDTSHLDPFVV- GK EARFMKIKVSDINEYTISNFKKWHANIATTSTTAPLYHDALKAFSSHYGIDWDNLPEMKFWELKNALKDHKEVF- IP NRGGRCYLSTLPVTSTSEKIVFNGRERWLNASDIVAGVNIVLRSV (SEQ ID NO: 4) >3300010885|Ga0133913_10053227_5|M [aquatic-freshwater-freshwater lake] MVSESTIRPYTSKLAPNDSKLKMLNDTFNWLDHAYKVFFDVSVALFGAIEHETAQELIGEKSKFDADLLCAIMW- FR LEEKSDNPGPLQTVEQRMRLFQKYSGHEPSSFTQEYIKGNIDSEKYQWVDCRLKFIDLARNINTTQESLKIDAY- TL FMNKLIPVSKDDEFNAYGLISQLFGTGKKEDRSIKASMLEEISNILADKNPNTWEEYQDLIKKTFNVDNYKELK- EK LSAGSSGRDGSLVIDLKEEKTGLLQPNFIKNRIVKFREDADKKRTVFLLPNRMKLREFIASQIGPFEQNSWSAV- LN RSMAAIQSKNSSNILYTNEKEERNNEIQELLKKDILSAASILGDFRRGEFNRSVVSKNHLGARLNELFEIWQEL- TM DDGIKKYVDLCKDKFSRRPVKALLQYIYPYFDKINAKQFLDAASYNTLVETNNRKKIHPTVTGPTVCNWGPKST- IN GSITPPNQMVKGRPAGSHGMIWVTMTVIDNGRWIKHHLPFHNSRYYEEHYCYREGLPTKNKPRTKQLGTQVGST- IS APSLAILKSQEEQDRRNDRKNRFKAHKSIIRSQENIEYNVAFDKSTNFDVTRKNGEFFITISSRVATPKYSYKL- NI GDMIMGLDNNQTAPCTYSIWRVVEKDTEGSFFHNKIWLQLVTDGKVTSIVDNNRQVDQLSYAGIEYSNFAEWRK- DR RQFLRSINEDYVKKSDNWRNMNLYQWNAEYSRLLLDVMKENKGKNIQNTFRAEIEELICGKFGIRLGSLFHHSL- QF LTNCKSLISSYFMLNNKKEEYDQELFDSDFFRLMKSIGDKRVRKRKEKSSRISSTVLQIARENNVKSLCVEGYL-

PT STKKTKPKQNQKSIDWCARAVVKKLNDGCKVLGIYLQAIDPRDTSHLDPFVYYGKKSTKVGKEARHTIVEPSNI- KE YMTNRFDDWHRGVTKKSKKGDVQTSTTVLLYQEALRQFASHYKLDFDSLPKMKFYELAKILGDHEKVIIPCRGG- RA YLSTYPVTKDSSKITFNGRERWYNESDVVAAVNIVLRGIIDEDEQPDGAKKQATTRRT (SEQ ID NO: 13) >3300020193|Ga0194131_10013618_4|P [aquatic-freshwater-freshwater lake] MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWF- RP VSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILC- RE KCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPKNVAPIQEIILNVAKATKETFRQVY- AG NLGAPSTLEKFIAKDGQKEFDLKKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTA- LK IKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGEDLSKLYKAWEDDPADPE- NA IVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPE- KA QRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIR- VN KKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKEDVGRQKGTLQIGDRFCGYDQNQTASH- AY SLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKK- EI VTVEAKEKFDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVK- AV KGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEG- DL STTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIG- DW VLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIP- VR GGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS (SEQ ID NO: 5) >3300020214|Ga0194132_10015959_3|M [aquatic-freshwater-freshwater lake] MSSAIKSYKSVLRPNERKNQLLKSTIQCLEDGSAFFFKMLQGLFGGITPEIVRFSTEQEKQQQDIALWCAVNWF- RP VSQDSLTHTIASDNLVEKFEEYYGGTASDAIKQYFSASIGESYYWNDCRQQYYDLCRELGVEVSDLTHDLEILC- RE KCLAVATESNQNNSIISVLFGTGEKEDRSVKLRITKKILEAISNLKEIPENVAPIQEIILNVAKATKETFRQVY- AG NLGAPSTLEKFIAKDGQKEFDLEKLQTDLKKVIRGKSKERDWCCQEELRSYVEQNTIQYDLWAWGEMFNKAHTA- LK IKSTRNYNFAKQRLEQFKEIQSLNNLLVVKKLNDFFDSEFFSGEETYTICVHHLGGKDLSKLYKAWEDDPADPE- NA IVVLCDDLKNNFKKEPIRNILRYIFTIRQECSAQDILAAAKYNQQLDRYKSQKANPSVLGNQGFTWTNAVILPE- KA QRNDRPNSLDLRIWLYLKLRHPDGRWKKHHIPFYDTRFFQEIYAAGNSPVDTCQFRTPRFGYHLPKLTDQTAIR- VN KKHVKAAKTEARIRLAIQQGTLPVSNLKITEISATINSKGQVRIPVKFDVGRQKGTLQIGDRECGYDQNQTASH- AY SLWEVVKEGQYHKELGCFVRFISSGDIVSITENRGNQFDQLSYEGLAYPQYADWRKKASKFVSLWQITKKNKKK- EI VTVEAKEKEDAICKYQPRLYKFNKEYAYLLRDIVRGKSLVELQQIRQEIFRFIEQDCGVTRLGSLSLSTLETVK- AV KGIIYSYFSTALNASKNNPISDEQRKEFDPELFALLEKLELIRTRKKKQKVERIANSLIQTCLENNIKFIRGEG- DL STTNNATKKKANSRSMDWLARGVFNKIRQLAPMHNITLFGCGSLYTSHQDPLVHRNPDKAMKCRWAAIPVKDIG- DW VLRKLSQNLRAKNIGTGEYYHQGVKEFLSHYELQDLEEELLKWRSDRKSNIPCWVLQNRLAEKLGNKEAVVYIP- VR GGRIYFATHKVATGAVSIVFDQKQVWVCNADHVAAANIALTVKGIGEQSSDEENPDGSRIKLQLTS (SEQ ID NO: 5)

TABLE-US-00005 TABLE 5A Representative CLUST.029130 (Type V-I) Effector Proteins and Direct Repeats CLUST.201934 Effector Protein Accession Direct Repeat Nucleotide Sequence SRR1522973_megahit_k177_1081830_2|M CTAGCAATGACCTAATAGTGTGTCCTTAGTTGACAT (SEQ ID NO: 11) (SEQ ID NO: 19) SRR1522973_megahit_k177_427371_1|M CTAGCAATGACCTAATAGTGTGTCCTTAGTTGACAT (SEQ ID NO: 12) (SEQ ID NO: 19) SRR2179954_megahit_k177_1417524_4|M TCTCAACGATAGTCAGACATGTGTCCTCAGTGACAC (SEQ ID NO: 13) (SEQ ID NO: 20) SRR6475631_megahit_k177_2773783_7|M CCTACAATACCTAAGAAATCCGTCCTAAGTTGACGG (SEQ ID NO: 14) (SEQ ID NO: 21) SRR6837575_megahit_k177_919599_7|M GTAGCAATCAGTACATATTGTGCCTTTCATTGGCACA (SEQ ID NO: 15) (SEQ ID NO: 22) SRR6837577_megahit_k177_410843_33|P GTAGCAATCAGTACATATTGTGCCTTTCATTGGCAC (SEQ ID NO: 15) (SEQ ID NO: 23) 3300020508|Ga0208225_1000010_34|M GTTGGAATGACTAATTTTTGTGCCCACCGTTGGCAC (SEQ ID NO: 3) (SEQ ID NO: 24) 3300002408|release|scaffold05697_22|M CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 2) (SEQ ID NO: 6) 3300002408|release|scaffold05697_22|P CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 1) (SEQ ID NO: 6) 3300002408|release|scaffold08426_1|P AATTTTTGTGCCCATCGTTGGCAC (SEQ ID NO: 3) (SEQ ID NO: 7) 3300028569|Ga0247843_1000055_230|M CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 16) (SEQ ID NO: 6) 3300028569|Ga0247843_1000055_232|P CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 17) (SEQ ID NO: 6) 3300028571|Ga0247844_1000101_90|M CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 16) (SEQ ID NO: 6) 3300028571|Ga0247844_1000101_88|P CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 17) (SEQ ID NO: 6) 3300009183|Ga0114974_10028552_1|M CTCTCAATGCCTTAGAAATCCGTCCTTGGTTGACGG (SEQ ID NO: 4) (SEQ ID NO: 8) 3300010885|Ga0133913_10053227_5|M CCCACAATACCTGAGAAATCCGTCCTACGTTGACGG (SEQ ID NO: 18) (SEQ ID NO: 6) 3300020193|Ga0194131_10013618_4|P GCAACCTAAGAAATCCGTCTTTCATTGACGGG (SEQ ID NO: 5) (SEQ ID NO: 9) 3300020214|Ga0194132_10015959_3|M GTTGCAAAACCCAAGAAATCCGTCTTTCATTGACGG (SEQ ID NO: 5) (SEQ ID NO: 10)

TABLE-US-00006 TABLE 5B Example CLUST.029130 (TypeV-I) pre-crRNA sequences Spacer Spacer Spacer Effector Lens Lens Lens Accession Example pre-crRNA sequence 1 2 3 SRR1522973_megahit_k177_1081830_2|M CUAGCAAUGACCUAAUAGUGUGUCCUUAGUUGACAUNNNN 34-36 33-37 20-41 (SEQ ID NO: 11) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCUAGCAAUGA CCUAAUAGUGUGUCCUUAGUUGACAU (SEQ ID NO: 150) SRR1522973_megahit_k177_427371_1|M CUAGCAAUGACCUAAUAGUGUGUCCUUAGUUGACAUNNNN 35-36 33-37 23-38 (SEQ ID NO: 12) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCUAGCAAUG ACCUAAUAGUGUGUCCUUAGUUGACAU (SEQ ID NO: 151) SRR2179954_megahit_k177_1417524_4|M UCUCAACGAUAGUCAGACAUGUGUCCUCAGUGACACNNNN 36-45 36-51 36-59 (SEQ ID NO: 13) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNUCUCAACG AUAGUCAGACAUGUGUCCUCAGUGACAC (SEQ ID NO: 152) SRR6475631_megahit_k177_2773783_7|M CCUACAAUACCUAAGAAAUCCGUCCUAAGUUGACGGNNNN 35-38 27-44 21-47 (SEQ ID NO: 14) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCUACAAUA CCUAAGAAAUCCGUCCUAAGUUGACGG (SEQ ID NO: 153) SRR6837575_megahit_k177_919599_7|M GUAGCAAUCAGUACAUAUUGUGCCUUUCAUUGGCACANNN 33-34 30-35 26-36 (SEQ ID NO: 15) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUAGCAAUCA GUACAUAUUGUGCCUUUCAUUGGCACA (SEQ ID NO: 154) SRR6837577_megahit_k177_410843_33|P GUAGCAAUCAGUACAUAUUGUGCCUUUCAUUGGCACNNNN 34-37 27-38 20-42 (SEQ ID NO: 15) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUAGCAAUCA GUACAUAUUGUGCCUUUCAUUGGCAC (SEQ ID NO: 155) 3300020508|Ga0208225_1000010_34|M GUUGGAAUGACUAAUUUUUGUGCCCACCGUUGGCACNNNN 36-38 35-43 28-47 (SEQ ID NO: 3) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNGUUGGAAU GACUAAUUUUUGUGCCCACCGUUGGCAC (SEQ ID NO: 156) 3300002408|release|scaffold08426_1|P AAUUUUUGUGCCCAUCGUUGGCACNNNNNNNNNNNNNNNN 36-38 36-42 28-47 (SEQ ID NO: 3) NNNNNNNNNNNNNNNNNNNNAAUUUUUGUGCCCAUCGUUG GCAC (SEQ ID NO: 157) 3300028569|Ga0247843_1000055_230|M CCCACAAUACCUGAGAAAUCCGUCCUACGUUGACGGNNNN 36-37 20-38 19-41 (SEQ ID NO: 16) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCACAAU ACCUGAGAAAUCCGUCCUACGUUGACGG (SEQ ID NO: 158) 3300028569|Ga0247843_1000055_232|P CCCACAAUACCUGAGAAAUCCGUCCUACGUUGACGGNNNN 36-37 20-38 19-41 (SEQ ID NO: 17) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCACAAU ACCUGAGAAAUCCGUCCUACGUUGACGG (SEQ ID NO: 159) 3300009183|Ga0114974_10028552_1|M CUCUCAAUGCCUUAGAAAUCCGUCCUUGGUUGACGGNNNN 36-37 36-40 36-46 (SEQ ID NO: 4) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCUCUCAAU GCCUUAGAAAUCCGUCCUUGGUUGACGG (SEQ ID NO: 160) 3300010885|Ga0133913_10053227_5|M CCCACAAUACCUGAGAAAUCCGUCCUACGUUGACGGNNNN 34-37 26-38 19-39 (SEQ ID NO: 18) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCCCACAAUAC CUGAGAAAUCCGUCCUACGUUGACGG (SEQ ID NO: 161) 3300020193|Ga0194131_10013618_4|P GCAACACCUAAGAAAUCCOUCUUUCAUUGACGGGNNNNNN 24-25 21-26 20-33 (SEQ ID NO: 5) NNNNNNNNNNNNNNNNNNGCAACACCUAAGAAAUCCGUCU UUCAUUGACGGG (SEQ ID NO: 162) 3300020214|Ga0194132_10015959_3|M GUUGCAAAACCCAAGAAAUCCGUCUUUCAUUGACGGNNNN 31-33 29-35 20-47 (SEQ ID NO: 5) NNNNNNNNNNNNNNNNNNNNNNNNNNNGUUGCAAAACCCA AGAAAUCCGUCUUUCAUUGACGG (SEQ ID NO: 163)

Example 2: In Vivo Bacterial Validation of Engineered CLUST.029130 (Type V-I) CRISPR-Cas Systems (FIGS. 4A-10B)

[0333] Having identified the minimal components of Type V-I CRISPR-Cas systems, we selected two systems for functional validation, one comprising the effector designated Cas12i1 (SEQ ID NO: 3), and the other comprising the effector designated Cas12i2 (SEQ ID NO: 5).

Methods

Gene Synthesis and Oligo Library Cloning

[0334] The E. coli codon-optimized protein sequences for CRISPR effectors, accessory proteins were cloned into pET-28a(+) (EMD-Millipore) to create the Effector Plasmid. Noncoding sequences flanking Cas genes (including 150 nt of terminal CDS coding sequence) or the CRISPR array were synthesized (Genscript) into pACYC184 (New England Biolabs) to create the Non-coding Plasmid (FIG. 4A). Effector mutants (e.g., D513A or A513D) plasmids were cloned by site directed mutagenesis using the indicated primers in the sequence table: sequence changes were first introduced into PCR fragments, which were then re-assembled into a plasmid using NEBuilder HiFi DNA Assembly Master Mix or NEB Gibson Assembly Master Mix (New England Biolabs) following the manufacturer's instructions.

[0335] For the pooled spacer library, we first computationally designed an oligonucleotide library synthesis (OLS) pool (Agilent) to express a minimal CRISPR array of "repeat-spacer-repeat" sequences. The "repeat" elements were derived from the consensus direct repeat sequence found in the CRISPR array associated with the effector, and "spacer" represents .about.8,900 sequences targeting the pACYC184 plasmid and E. coli essential genes, or negative control non-targeting sequences. The spacer length was determined by the mode of the spacer lengths found in the endogenous CRISPR array. Flanking the minimal CRISPR array were unique PCR priming sites that enabled amplification of a specific library from a larger pool of oligo synthesis.

[0336] We next cloned the minimal CRISPR array library into the Effector Plasmid to create an Effector Plasmid library. We appended flanking restriction sites, a unique molecular identifier, and a J23119 promoter for array expression onto the oligo library using PCR (NEBNext High-Fidelity 2.times.PCR Master Mix), and then used NEB Golden Gate Assembly Master Mix (New England Biolabs) to assemble the full plasmid library of effectors with their targeting arrays. This represented the "input library" for the screen.

In Vivo E. coli Screen

[0337] We performed the in vivo screen using electrocompetent E. cloni EXPRESS BL21(DE3) E. coli cells (Lucigen), unless otherwise indicated. Competent cells were co-transformed with the Effector Plasmid and/or Non-coding (FIG. 4B). The cells were electroporated with the "input library" according to the manufacturer's protocols using a Gene Pulser Xcell.RTM. (Bio-rad) with a 1.0 mm cuvette. The cells were plated onto bioassay plates containing both Chloramphenicol (Fisher) and Kanamycin (Alfa Aesar), and grown for 11 hours, after which we estimated the approximate colony count to ensure sufficient library representation and harvested the cells.

[0338] Plasmid DNA fractions were extracted from the harvested cells to create the `output library` using a QIAprep.RTM. Spin Miniprep Kit (Qiagen), while total RNA=17 nt was harvested by lysing the harvested cells in Direct-Zol.RTM. (Zymo Research), followed by extraction using the Direct-zol RNA miniprep kit (Zymo Research).

[0339] The next generation sequencing library for the DNA depletion signal was prepared by performing a PCR on both the input and output libraries, using custom primers flanking the CRISPR array cassette of the Effector Plasmid library and containing barcodes and handles compatible with Illumina sequencing chemistry. This library was then normalized, pooled, and loaded onto a Nextseq 550 (Illumina) to evaluate the activity of the effectors.

Bacterial Screen Sequencing Analysis

[0340] Next generation sequencing data for screen input and output libraries were demultiplexed using Illumina bcl2fastq. Reads in resulting fastq files for each sample contained the CRISPR array elements for the screening plasmid library. The direct repeat sequence of the CRISPR array was used to determine the array orientation, and the spacer sequence was mapped to the source (pACYC184 or E. coli essential genes) or negative control sequence (GFP) to determine the corresponding target. For each sample, the total number of reads for each unique array element (r.sub.a) in a given plasmid library was counted and normalized as follows: (r.sub.a+1)/total reads for all library array elements. The depletion score was calculated by dividing normalized output reads for a given array element by normalized input reads.

[0341] To identify specific parameters resulting in enzymatic activity and bacterial cell death, we used next generation sequencing (NGS) to quantify and compare the representation of individual CRISPR arrays (i.e., repeat-spacer-repeat) in the PCR product of the input and output plasmid libraries. We defined the fold depletion for each CRISPR array as the normalized input read count divided by the normalized output read count (with 1 added to avoid division by zero). An array was considered to be "strongly depleted" if the fold depletion was greater than 3. When calculating the array fold depletion across biological replicates, we took the maximum fold depletion value for a given CRISPR array across all experiments (i.e. a strongly depleted array must be strongly depleted in all biological replicates). We generated a matrix including array fold depletion and the following features for each spacer target: target strand, transcript targeting, ORI targeting, target sequence motifs, flanking sequence motifs, and target secondary structure. We investigated the degree to which different features in this matrix explained target depletion for Type V-I systems, thereby yielding a broad survey of functional parameters within a single screen.

Results

[0342] FIGS. 5A-D depict the location of strongly depleted targets for Cas12i1 and Cas12i2 targeting pACYC184 and E. coli E. Cloni.RTM. essential genes. Notably, the location of strongly depleted targets appears dispersed throughout the potential target space.

[0343] We found that dsDNA interference activities of the Type V-I effectors, Cas12i1 (1094aa), and Cas12i2 (1054aa), are abolished by mutation of the conserved aspartate in the RuvC I motif (FIGS. 6A, and 6B). The RuvC-dependent dsDNA interference activity of Cas12i shows no requirement for non-coding sequences flanking the CRISPR array or cas genes (FIGS. 7A and 7B), indicating that the minimal V-I interference module includes only the effector and crRNA (FIGS. 8A and 8B).

[0344] Analysis of the target-flanking sequences corresponding to strongly depleted arrays from in vivo screens show that dsDNA interference by Cas12i is PAM-dependent. Specifically, we found that Cas12i1 and Cas12i2 both showed a 5' TTN PAM preference (FIGS. 9A-B and 10A-B). These results suggest that the compact Cas12i effectors are capable of autonomous PAM-dependent dsDNA interference.

Example 3: Biochemical Mechanistic Characterization of Engineered CLUST.029130 (Type V-I) CRISPR-Cas Systems (FIGS. 11A-13, 15-17B)

Cas12i Processes Pre-crRNAs in Vivo

[0345] To investigate crRNA biogenesis for Type V-I CRISPR-Cas systems, we purified and sequenced small RNAs from E. coli expressing Cas12i and the minimal CRISPR array library from the bacterial screen. FIGS. 11A and 11B show the pile-up of RNA-sequencing reads, showing a strong consensus form of the Cas12i1 and Cas12i2 mature crRNA, respectively, as well as a distribution of spacer lengths. The most common spacer length observed was 21, with length variation between 16 nt and 22 nt.

[0346] For the Type V-I CRISPR-Cas system containing Cas12i1, the mature crRNA can take the form 5'-AUUUUUGUGCCCAUCGUUGGCAC[spacer]-3' (SEQ ID NO: 100).

[0347] For the Type V-I CRISPR-Cas system containing Cas12i2, the mature crRNA can take the form 5'-AGAAAUCCGUCUUUCAUUGACGG[spacer]-3' (SEQ ID NO: 101).

[0348] Sequencing the small RNA from the in vivo bacterial screen was performed by extracting total RNA from harvested bacteria using the Direct-zol RNA MiniPrep Plus with TRI Reagent (Zymo Research). Ribosomal RNA was removed using a Ribo-Zero rRNA Removal Kit for Bacteria, followed by cleanup using a RNA Clean and Concentrator-5 kit. The resultant ribosomal RNA depleted-total RNA was treated with T4 PNK for 3 hours without ATP to enrich for 3'-P ends, after which ATP was added and the reaction incubated for another hour to enrich for 5'-OH ends. The samples were then column purified, incubated with RNA 5' polyphosphatase (Lucigen) and column purified again prior to preparation for next-generation sequencing using the NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs). The library was paired-end sequenced on a Nextseq 550 (Illumina), and the resulting paired end alignments were analyzed using Geneious 11.0.2 (Biomatters).

Cas12i Effector Purification

[0349] Effector vectors were transformed into E. coli NiCo21 (DE3) (New England BioLabs) and expressed under a T7 promoter. Transformed cells were initially grown overnight in 3 mL Luria Broth (Sigma)+50 ug/mL kanamycin, followed by inoculation of 1 L of Terrific Broth media (Sigma)+50 ug/mL kanamycin with 1 mL of overnight culture. Cells were grown at 37.degree. C. until an OD600 of 1-1.5, then protein expression was induced with 0.2 mM IPTG. Cultures were then grown at 20.degree. C. for an additional 14-18 hours. Cultures were harvested and pelleted via centrifugation, then resuspended in 80 mL of lysis buffer (50 mM HEPES pH 7.6, 0.5M NaCl, 10 mM imidazole, 14 mM 2-mercaptoethanol, and 5% glycerol)+protease inhibitors (Sigma). Cells were lysed via cell disruptor (Constant System Limited), then centrifuged twice at 28,000.times.g for 20 minutes at 4.degree. C. to clarify the lysate. The lysate was loaded onto a 5 mL HisTrap FF column (GE Life Sciences), then purified via FPLC (AKTA Pure, GE Life Sciences) over an imidazole gradient from 10 mM to 250 mM. Cas12i1 was purified in low salt buffer (50 mM HEPES-KOH pH 7.8, 500 mM KCl, 10 mM MgCl2, 14 mM 2-mercaptoethanol, and 5% glycerol). After purification, fractions were run on SDS-PAGE gels and fractions containing protein of the appropriate size were pooled and concentrated using 10kD Amicon Ultra-15 Centrifugal Units. Protein concentration was determined by Qubit protein assay (Thermo Fisher).

Cas12i Processes Pre-crRNAs in Vitro

[0350] To determine whether Cas12i1 is capable of autonomous crRNA biogenesis, we incubated the effector protein purified from E. coli with a pre-crRNA expressed from a minimal CRISPR array (repeat-spacer-repeat-spacer-repeat). We observed that purified Cas12i1 processes the pre-crRNA into fragments matching the mature crRNAs identified from the in vivo small RNAseq, suggesting Cas12i1 is capable of autonomous pre-crRNA processing (FIG. 12).

[0351] Pre-crRNA processing assays for Cas12i1 were performed at 37.degree. C. for 30 minutes in cleavage buffer at a final pre-cr-RNA concentration of 100 nM. The reaction was performed in optimized cleavage buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 10 mM MgCl.sub.2, 50 ug/ml BSA) for Cas12i. Reactions were quenched with the addition of 1 ug/uL of proteinase K (Ambion) and incubated at 37.degree. C. for 15 minutes. 50 mM EDTA was added to the reactions before mixing with equal volume of 2.times.TBE-Urea sample buffer (Invitrogen) and denaturing at 65.degree. C. for 3 minutes. Samples were analyzed on 15% TBE-Urea gels (Invitrogen). Gels were stained for 5 minutes with SYBR Gold nucleic acid stain (Invitrogen) and imaged on Gel Doc EZ (Biorad). Gels containing labeled pre-crRNA were first imaged on Odyssey CLx scanner (LI-COR Biosciences) prior to SYBR staining.

Cas12i1 DNA Manipulation using Strongly Depleted Arrays

[0352] To explore the mechanism of the interference activity of Cas12i1, we selected strongly depleted CRISPR array sequences from the in vivo negative selection screen and generated pre-crRNAs with the DR-spacer-DR-spacer-DR arrangement. The pre-crRNAs were designed to target Cas12i1 to 128 nt ssDNA and dsDNA substrates containing target sequences complementary to the second spacer of the pre-crRNA. We observed that Cas12i1 binary complex consisting of the effector protein and pre-crRNA cleaved 100 nM of target ssDNA to saturation at a 62.5 nM complex concentration (FIG. 13). Additional degradation of cleaved ssDNA to short fragments or single nucleotides was observed at increasing concentrations of the complex, suggestive of collateral ssDNA cleavage activated by binding of the binary complex to an ssDNA target (FIG. 13).

[0353] To explore the dsDNA interference activity of Cas12i, we targeted the Cas12i1 binary complex to target dsDNA substrates containing a 5' end label on the non-spacer-complementary strand. To assess both dsDNA cleavage and nicking activity comprehensively, the resulting dsDNA cleavage reactions were split into three fractions for different analyses. The first two fractions were quenched and analyzed by denaturing or nondenaturing gel electrophoresis conditions, respectively. The third fraction was treated with 0.1 U of S1 nuclease to convert any dsDNA nicks to double-stranded breaks, quenched, and analyzed by nondenaturing gel electrophoresis.

[0354] We observed dose-dependent cleavage under denaturing conditions, suggestive of either target nicking or dsDNA cleavage (FIG. 15). Under non-denaturing conditions with no S1 nuclease treatment, we observed a dose-dependent increase in a primary product that migrated with slightly lower electrophoretic mobility than the input dsDNA, suggestive of a nicked dsDNA product (FIG. 16). When these products were incubated with S1 nuclease, the upward shifted band was converted to a smaller dsDNA product indicative of the S1-mediated conversion of nicked dsDNA to double-stranded breaks (FIG. 16). We also observed minor dsDNA cleavage products at high concentrations and incubation times, indicating that Cas12i1 is a dsDNA nuclease that cleaves the spacer complementary ("SC") and non-spacer complementary ("NSC") strands of target dsDNA with substantially different efficiencies (FIG. 17A).

[0355] The observation of nicking activity accompanying 5' labeling of the spacer complementary strand of dsDNA substrates suggests that Cas12i1 preferentially nicks the DNA strand opposing the crRNA-target DNA hybrid. To validate this bias in DNA strand cleavage by Cas12i1, we generated dsDNA substrates that were IR800 dye-labeled at either the 5' end of the spacer complementary or at the 5' end of the non-spacer complementary strand. At lower concentrations of the effector complex, we observed only cleavage of the NSC strand of the DNA duplex, whereas at higher concentrations of the effector complex, cleavage of both the NSC and the SC strand was observed (FIG. 17A-B). Comparing the SYBR stain labeling all nucleic acid products versus the strand-specific labeling using IR800 dye reveals a difference in the rate of stranded product formation versus the overall accumulation of cleavage products. These results suggest an ordered series of events leading to dsDNA interference, whereby the Cas12i1 binary complex first nicks the NSC strand and then cleaves the SC strand with a lower efficiency, resulting in dsDNA cleavage. Taken together, these findings indicate that Cas12i is an effector capable of autonomous pre-crRNA processing, ssDNA target and collateral cleavage, and dsDNA cleavage. This spectrum of catalytic activities closely parallels those of Cas12a and Cas12b except for the notable bias towards non-spacer complementary strand cleavage, resulting in preferential dsDNA nicking.

crRNA and Substrate RNA Preparation

[0356] Single stranded DNA oligo templates for crRNA and substrate RNA were ordered from IDT. Substrate RNA and pre-crRNA templates were PCR amplified to generate a double stranded in vitro transcription (IVT) template DNA using NEBNEXT Hifi 2.times. master mix (New England Biolabs). Double stranded DNA templates for mature cr-RNA was generated by annealing T7 primer with templates followed by extension using DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs). Annealing was performed by incubating for 5 min at 95.degree. C. followed by a -5.degree. C./min ramp down to 4.degree. C. In vitro transcription was performed by incubating the dsDNA templates with T7 RNA polymerase at 37.degree. C. for 3 hours using HiScribe T7 Quick High Yield RNA kit (New England Biolabs). After incubation, IVT samples were treated with Turbo DNase.RTM. (Thermo Scientific) and then purified using RNA Clean & Concentrator kit (Zymo Research). Mature cr-RNA generated from IVT was treated with Calf Intestinal Alkaline Phosphatase (Thermo Fisher) or RNA 5'-polyphosphatase (Lucigen) for 2 hours at 37.degree. C. to generate 5'-hydroxyl or 5'-monophosphate, respectively, followed by clean up with RNA Clean & Concentrator kit (Zymo Research). Concentrations were measured via Nanodrop 2000 (Thermo Fisher).

[0357] Pre-crRNA sequences used in biochemical characterization Cas12i are included in Table 6. Oligonucleotide templates and primers for preparation of crRNAs are included in Table 9.

Preparation of IR-800 Labeled Substrate RNA and DNA

[0358] RNA substrates from IVT were treated with Calf Intestinal Alkaline Phosphatase (Thermo Fisher) for 30 minutes at 37.degree. C. to convert the 5'-triphosphate to 5' terminal hydroxyl group and purified using RNA Clean & Concentrator kit (Zymo Research). A thiol end group was added to the 5' terminal hydroxyl group of the DNA and RNA substrates via 5' EndTag Labeling Kit (Vector Labs), then substrates were labeled with IRDye 800CW Maleimide (LI-COR Biosciences). Substrates were purified using DNA Clean & Concentrator kit or RNA Clean & Concentrator kit (Zymo Research). Labeled dsDNA substrates were generated by labeling the non-target (non-spacer complementary) ssDNA strand, annealing with a primer, then extending with DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs) for 15 minutes at 25.degree. C. These substrates were purified with DNA Clean & Concentrator kit (Zymo Research). Concentrations were measured via Nanodrop 2000 (Thermo Fisher).

[0359] RNA and DNA substrate sequences used in the biochemical characterization of Cas12i are included in Tables 7 and 8.

Target Cleavage Assays with Cas12i

[0360] ssDNA: Cas12i target cleavage assays with ssDNA were performed in optimized cleavage buffer (50 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM DTT, 10 mM MgCl2, 50 ug/ml BSA). Binary complex was formed by incubating a 1:2 molar ratio of Cas12i:pre-crRNA for 10 minutes at 37.degree. C., followed by transfer to ice. All further complex dilutions were done on ice keeping the protein:RNA ratio fixed. The complex was added to 100 nM IR800 labeled substrates and incubated at 37.degree. C. for 30 minutes. Reactions were treated with RNAse cocktail and proteinase K and analyzed as above.

[0361] dsDNA: dsDNA target cleavage assays were set up in the optimized cleavage buffer at 37.degree. C. for 1 hour. Binary complex was formed as described above and added to 100 nM dsDNA substrate. Reactions were first treated with RNAse cocktail with incubation at 37.degree. C. for 15 minutes. Next, they were treated with proteinase K with incubation at 37.degree. C. for 15 minutes. To detect dsDNA cleavage products the reactions were analyzed with 15% TBE-Urea gel as described before. To detect nicking activity of Cas12i, reactions were SPRI purified after proteinase K treatment and split into three fractions. One fraction was analyzed on a 15% TBE-Urea gel as described above. Another fraction was mixed with 5.times. hi-density TBE sample buffer and analyzed on a non-denaturing 4-20% TBE gel to detect nicked dsDNA products. The last fraction was incubated with 0.01 U/uL of S1 Nuclease (Thermo Scientific) at 50.degree. C. for 1 hour to convert nicks into double stranded breaks followed by mixing with 5.times. hi-density TBE sample buffer and analyzed on a non-denaturing 4-20% TBE gel. All gels were imaged on Odyssey CLx scanner followed by a 5 minute SYBR stain and image on Gel Doc imager.

[0362] To identify the nicked strand, dsDNA was prepared by labeling either the target strand (complementary to crRNA) or the non-target strand (non-spacer complementary, same sequence as the crRNA). The cleavage reaction was performed as described. The labeled strands were then annealed with the corresponding primers and extended with DNA Polymerase I, Large (Klenow) Fragment (New England Biolabs) for 15 minutes at 25.degree. C. The dsDNA substrates were then purified using SPRI purification.

TABLE-US-00007 TABLE 6 Pre-crRNAs used for CLUST.029130 (Type V-I) in vitro Name Sequence DR Spacer1 Spacer2 Target FIG. Cas12i1 gggAAUUUUUGUGCCC AAUUUUU CCUAA UCCGC Cas12i1 FIG. pre- AUCGUUGGCACCCUA GUGCCCAU UGCGG AAGAA Target 1 12 crRNA 1 AUGCGGAAGUAGUGG CGUUGGC AAGUA UUGAU GUAACCCGGAAUUUU AC (SEQ ID GUGGG UGGCU UGUGCCCAUCGUUGG NO: 401) UAACC CCAAU CACUCCGCAAGAAUU CGG UCU GAUUGGCUCCAAUUC (SEQ ID (SEQ ID UAAUUUUUGUGCCCA NO: 402) NO: 403) UCGUUGGCAC (SEQ ID NO: 400) Cas12i1 gggAAUUUUUGUGCCC AAUUUUU AGGCA GCGUG Cas12i1 FIGs. pre- AUCGUUGGCACAGGC GUGCCCAU UCAUC CUGGA Target 2 13-17B crRNA 2 AUCAUCAGCAUUAAC CGUUGGC AGCAU UUGCU CACGCAAACAAUUUU AC (SEQ ID UAACC UCGAU UGUGCCCAUCGUUGG NO: 405) ACGCA GGUCU CACGCGUGCUGGAUU AAC GCG GCUUCGAUGGUCUGC (SEQ ID (SEQ ID GAAUUUUUGUGCCCA NO: 406) NO: 407) UCGUUGGCAC (SEQ ID NO: 404)

TABLE-US-00008 TABLE 7 Substrates used for CLUST.029130 (Type V-I) in vitro biochemistry Nucleic Name Sequence acid FIG. Cas12i1 CATGTGGACCACATTAGGCTGCAAAACTGCGCA DNA FIG. 12 ssDNA1, TTTACGAAAACGCGAAAGTTTGCGTGGTTAATG dsDNA1 CTGATGATGCCTTAACAATGCCGATTCGCGGTG CGGATGAACGTAATTTCTCGAGGCGTATT (SEQ ID NO: 408) Cas12i1 CATGTGGACCACATTAGGCTTGGTTGTTGCTGC DNA FIGs. 13-17B ssDNA2, CGACGACGGTGTGATGCCGCAGACCATCGAAGC dsDNA2 AATCCAGCACGCGAAAGCGGCGCAGGTACCGG TGGTGGTTGCGTAATTTCTCGAGGCGTATT (SEQ ID NO: 409)

TABLE-US-00009 TABLE 8 Collateral nucleic acids used for CLUST.029130 (Type V-I) in vitro Biochemistry Nucleic Name Sequence acid FIG. Cas12i1 AATACGCCTCGAGAAATTACAAAGTGATGCAGGCGTTTCCAGGTG DNA FIG. 14 ssDNA6_RC CTTTCCCTAATGCGGAAGTAGTGGGTAACCCGGTGCGTACCGATG TGTTGGCGCTGCCGTTGCAGCCTAATGTGGTCCACATG (SEQ ID NO: 410)

TABLE-US-00010 TABLE 9 IDT Template oligos and primers for crRNAs used for CLUST.029130 (Type V-I) in vitro biochemistry T7 Fwd Name Template Sequence primer Rev primer Cas12i1 GTGCCAACGATGGGCACAAAAATTAGAA TAATACGA GTGCCAACGAT pre- TTGGAGCCAATCAATTCTTGCGGAGTGC CTCACTAT GGGCACAAAAA crRNA 1 CAACGATGGGCACAAAAATTAGAATTGG AG (SEQ ID TTAGAATTGGA AGCCAATCAATTCTTGCGGAGTGCCAAC NO: 412) GCCAATCAATTC GATGGGCACAAAAATTccctatagtgagtcgtattac TTGCGGA (SEQ tcgagggatccTTATTACATTT (SEQ ID NO: ID NO: 413) 411) Cas12i1 GTGCCAACGATGGGCACAAAAATTCGCA TAATACGA GTGCCAACGAT pre- GACCATCGAAGCAATCCAGCACGCGTGC CTCACTAT GGGCACAAAAA crRNA 2 CAACGATGGGCACAAAAATTGTTTGCGT AG (SEQ ID TTCGCAGACCAT GGTTAATGCTGATGATGCCTGTGCCAAC NO: 415) CGAAGCAATCC GATGGGCACAAAAATTccctatagtgagtcgtattac AGCACGC (SEQ tcgagggatccTTATTACATTT (SEQ ID NO: ID NO: 416) 414)

Example 4: In Vitro Pooled Screening for Rapid Evaluation of CRISPR-Cas Systems (FIGS. 20-25)

[0363] As described herein, in vitro pooled screening serves as an efficient and high throughput method to perform biochemical evaluation. As an overview, we begin by in vitro reconstitution of the CRISPR-Cas system (FIG. 20). In one embodiment, the effector protein is produced using an in vitro transcription and translation reagent that uses dsDNA template containing a T7-RNA polymerase promoter driving the expression of the effector protein(s), and produces proteins for the reaction. In another embodiment, the minimal CRISPR arrays and the tracrRNAs include T7 promoter sequences appended onto either the top strand or bottom strand transcription directions using PCR in order to interrogate all possible RNA orientations. As shown in FIG. 20, the Apo form contains the effector only, the Binary form contains the effector protein and T7 transcript minimal CRISPR array, and the Binary+tracrRNA form adds any T7 transcribed tracrRNA elements to the complex for incubation.

[0364] In one embodiment, the endonucleolytic activity of the CRISPR-Cas systems is the primary biochemical activity assayed. FIG. 21 shows one form of the ssDNA and dsDNA substrates, in which a target sequence is flanked on both sides by 6 degenerate bases to create a pool of possible PAM sequences that may gate ssDNA and dsDNA cleavage activity. Apart from the PAM sequence, the substrates include 5' and 3' fiducial marks designed to facilitate downstream next generation sequencing library preparation protocols that selectively enrich for the substrate ssDNA or dsDNA, as well as provide unique sequences that facilitate mapping of the cleavage products. In one embodiment, the dsDNA substrate is generated by second strand synthesis in the 5'-to-3' direction using a short DNA primer and DNA polymerase I. Similar reactions can be performed using pools of different targets in the minimal CRISPR array, as well as libraries of different ssDNA and dsDNA sequences.

[0365] The CRISPR-Cas cleavage reaction is performed by mixing and incubating the preformed Apo/Binary/Binary-tracrRNA complexes with either targeting or non-targeting substrates. While other methods such as gel electrophoresis are possible, a useful embodiment for maximum sensitivity and base-pair resolution capture of the cleavage is next generation sequencing of the ssDNA or dsDNA substrate after incubation with the effector complex. FIG. 22 is a schematic that describes the library preparation for enrichment of the ssDNA substrates. By annealing a primer to well-defined sequences within the fiducial marks, the second strand synthesis and end repair occurs to produce fragments of dsDNA that represent both cut and uncut ssDNA. Afterwards, the newly-formed dsDNA molecules are a substrate for adaptor ligation, after which a selective PCR is performed using one primer (I5/P5) complementary to the ligation adaptor and another (I7/P7) that is complementary to the 3' fiducial of the original ssDNA substrate. This ultimately produces a sequencing library that contains both the full length, as well as cleaved and degraded ssDNA products, as demonstrated in FIG. 24A. The dsDNA readout NGS library prep begins without requiring the primer annealing and second strand synthesis, so the end repair and subsequent adaptor ligation can be directly performed. FIG. 23 describes the general overview of the library preparation that, similar to the ssDNA prep, labels both the cleaved/degraded as well as uncleaved fragments. Of note, either end of the dsDNA cleavage fragment can be enriched based on the PCR primer choice. In one embodiment, illustrated in FIG. 24A, dsDNA manipulation next generation sequencing libraries for readout can be prepared with a first primer complementary to a handle ligated to the 5' end of the full length or cleaved substrate (and containing I5/P5 sequences) and a second primer complementary to the 3' fiducial sequence of thes substrate (and containing I7/P7 sequences). In one embodiment, illustrated in FIG. 24B, DNA manipulation next generation sequencing libraries for readout can be prepared with a first primer complementary to the 5' fiducial sequence of the substrate (and containing I5/P5 sequences) and a second primer complementary to a handle ligated to the 3' end of the full length or cleaved substrate (and containing I7/P7 sequences). Target length and substrate length can be extracted from resulting NGS reads from RNA/ssDNA/dsDNA manipulation experiments as depicted in FIGS. 25A-B, respectively. Target length and substrate lengths extracted can be used to investigate the presence of RNA/ssDNA/dsDNA nicking or cleavage.

Example 5: Characterization of dsDNA Cleavage Activity for the Type V-I1 CRISPR-Cas System (FIGS. 26-32)

[0366] Having computationally identified the minimal components of Type V-I CRISPR-Cas systems, we investigated double stranded DNA (dsDNA) cleavage activity from the Type V-I1 system containing effector Cas12i1.

[0367] IVTT-expressed Cas12i1 in complex with a top-strand expressed crRNA targeting dsDNA resulted in a population of truncated target lengths not present in the apo (effector-only) controls as shown in FIG. 26A-B. Libraries prepared using a 5' ligation adapter and selecting for the 3' fiducial (as depicted in FIG. 24A) showed a cleavage product not present in the Apo control at the +24 position within the target sequence. This result indicates either nicking of the non-target dsDNA strand or both strands of the dsDNA between the +24 and +25 nucleotides relative to the PAM. Target length analysis shows a peak at +24 indicating truncation of the target between nucleotides +24 and +25 (FIG. 27A). This population of truncated target sequences coincides with substrate lengths indicating cleavage of the non-target dsDNA strand between between nucleotides +24 and +25 of the target sequence (FIG. 28A).

[0368] Libraries prepared using a 3' ligation adapter and selecting for the 5' fiducial (as depicted in FIG. 24B) showed a cleavage product not present in the Apo control at the -9 position. (+19 given a 28 nt target) within the target sequence. This result indicates either nicking of the target dsDNA strand or both strands of the dsDNA between the +19 and +20 nucleotides relative to the PAM. Target length analysis shows a peak at -9 nucleotides from the PAM (28 nt full length target) indicating truncation of the target between nucleotides +19 and +20 (FIG. 27B). This population of truncated target sequences coincides with substrate lengths indicating cleavage of the target dsDNA strand between nucleotides +19 and +20 of the target sequence (FIG. 28B).

[0369] Sequence motif analysis for substrates showing non-target strand cleavage between the +24/+25 nucleotides relative to the PAM revealed a 5' TTN PAM motif to the left of the target sequence for Cas12i1 (FIG. 29). No PAM sequence requirement was observed on the right side of the Cas12i1 target. Taken together, in vitro screening of Cas12i1 indicates predominant nicking between the +24/+25 nucleotides of the non-target strand relative to a TTN PAM with a significant fraction of these products converted to double strand breaks with a 5 nt 3' overhang by cleavage of the target strand between the +19/+20 nucleotides relative to the PAM (FIG. 30).

[0370] Targeting of Cas12i1 in complex with a top-strand expressed non-target crRNA resulted in no manipulation of dsDNA relative, indicating that Cas12i1 cleavage specificity is conferred by the crRNA spacer (FIG. 31A-B). Cas12i1 showed no cleavage cleavage activity in the presence of a bottom strand-expressed crRNA targeting the dsDNA substrate indicating that the top-strand oriented crRN A is required for formation of the active Cas12i1 complex (FIG. 32A-B).

Example 6: Characterization of dsDNA Cleavage Activity for the Type V-2 CRISPR-Cas System (FIGS. 33-39)

[0371] Having computationally identified the minimal components of Type V-I CRISPR-Cas systems, we investigated double stranded DNA (dsDNA) cleavage activity from the Type V-I2 system containing effector Cas12i2.

[0372] IVTT-expressed Cas12i2 in complex with a top-strand expressed crRNA targeting dsDNA resulted in a population of truncated target lengths not present in the apo (effector-only) controls as shown in FIG. 33A-B. Libraries prepared using a 5' ligation adapter and selecting for the 3' fiducial (as depicted in FIG. 24A) showed a cleavage product not present in the Apo control at the +24 position within the target sequence. This result indicates either nicking of the non-target dsDNA strand or both strands of the dsDNA between the +24 and +25 nucleotides relative to the PAM. Target length analysis shows a peak at +24 indicating truncation of the target between nucleotides +24 and +25 (FIG. 34A). This population of truncated target sequences coincides with substrate lengths indicating cleavage of the non-target dsDNA strand between nucleotides +24 and +25 of the target sequence (FIG. 35A).

[0373] Libraries prepared using a 3' ligation adapter and selecting for the 5' fiducial (as depicted in FIG. 33B) showed a cleavage product not present in the Apo control at the -7 position (+24 given 31 nt target) within the target sequence. This result indicates either nicking of the target dsDNA strand or both strands of the dsDNA between the +24 and +25 nucleotides relative to the PAM. Target length analysis shows a peak at -7 nucleotides from the PAM (28 nt full length target) indicating truncation of the target between nucleotides +24 and +25 (FIG. 34B). This population of truncated target sequences coincides with substrate lengths indicating cleavage of the target dsDNA strand between nucleotides +24 and +25 of the target sequence (FIG. 35B).

[0374] Sequence motif analysis for substrates showing non-target strand cleavage between the +24/+25 nucleotides relative to the PAM revealed a 5' TTN PAM motif to the left of the target sequence for Cas12i2 (FIG. 36). No PAM sequence requirement was observed on the right side of the Cas12i2 target. Taken together, in vitro screening of Cas12i2 indicates predominant nicking between the +24/+25 nucleotides of the non-target strand relative to a TTN PAM with a significant fraction of these products converted to double strand breaks with a blunt cut by cleavage of the target strand between the +24/+25 nucleotides relative to the PAM (FIG. 37).

[0375] Targeting of Cas12i2 in complex with a top-strand expressed non-target crRNA resulted in no manipulation of dsDNA relative, indicating that Cas12i2 cleavage specificity is conferred by the crRNA spacer (FIG. 38A-B). Cas12i2 showed no cleavage cleavage activity in the presence of a bottom strand-expressed crRNA targeting the dsDNA substrate indicating that the top-strand oriented crRN A is required for formation of the active Cas12i2 complex (FIG. 39A-B).

Example 7: CLUST.029130 (Type V-I) CRISPR Cas Systems can be Used for Gene Silencing In Vitro

[0376] An in vitro gene-silencing assay (FIGS. 18A and 18B) was developed to mimic in vivo gene silencing activity for rapid validation of the activity of a novel CRISPR-Cas system. This assay can simultaneously evaluate in an unbiased manner different activity mechanisms and functional parameters outside the natural cellular environment.

[0377] First, a reconstituted IVTT (in vitro transcription and translation) system was supplemented with E. coli RNA polymerase core enzyme to allow gene expression (protein synthesis) to occur from not only T7 promoter but also any E. coli promoter, as long as the corresponding E. coli sigma factor is present.

[0378] Second, to facilitate rapid and high throughput experimentation, linear DNA templates generated from PCR reactions were directly used. These linear DNA templates included those encoding the Type V-1 effector, a RNA guide, and E. coli sigma factor 28. Incubation of these DNA templates with the reconstituted IVTT reagent results in co-expression of the Type V-I effector and a RNA guide, and the formation of the RNP (ribonucleoprotein complex). E. coli sigma factor 28 was also expressed for subsequent expression of GFP and RFP as described below.

[0379] Third, as the target substrate, a linear or plasmid DNA encoding GFP expressed from the sigma factor 28 promoter was included in the above incubation reaction such that the newly synthesized RNP has the immediate access to the target substrate. As an internal control, a non-target linear DNA encoding RFP expressed from the sigma factor 28 promoter was also included. The RNA polymerase core enzyme alone does not recognize the sigma factor 28 promoter until sufficient sigma factor 28 protein is synthesized. This delay in the GFP and RFP expression allows the newly synthesized RNP to interfere with the GFP target substrate, which could result in a decrease in the GFP expression and a depletion of the GFP fluorescence. The RFP expression, on the other hand, was not negatively affected, which serves as the internal control for protein synthesis and fluorescence measurement.

[0380] Certain important advantages of the in vitro gene-silencing assay described herein include:

[0381] (1) Modularity--The reconstituted IVTT is a synthetic system consisting of individually purified components, which allows the assay to be custom designed for a variety of controls and activities. Each component of the CRISPR-Cas system is encoded in a separate linear DNA template, allowing rapid assays of a combination of different effectors, effector variants, and RNA guides;

[0382] (2) Complexity--The assay contains all essential components for RNA transcription and protein synthesis, allowing diverse mechanisms of interference to be tested in a single one-pot reaction, such as DNA and RNA cleavage, and transcription-dependent interference. The kinetic fluorescence readouts of the assay provide significantly more data points than endpoint activity assays;

[0383] (3) Sensitivity--The assay couples effector and RNA guide synthesis with substrate interference, allowing newly synthesized RNPs (ribonucleoprotein complexes of effector protein and RNA guide) to immediately interact with the substrate in the same reaction. There are no separate purification steps, thus potentially allowing small amounts of RNPs to be sufficient to generate signal. Furthermore, the interference of the GFP expression is amplified due to the coupled transcription and translation of GFP that can generate >100 GFP protein per DNA template.

[0384] (4) Efficiency--The assay is designed to be highly compatible to high throughput platforms. Due to its modularity, all components of the assay can be added in 96-, 384- and 1536-well formats by commonly available liquid handling instruments, and fluorescence can be measured by commonly available plate fluorometers.

[0385] (5) Relevance--The assay tests the ability of a CRISPR-Cas effector protein to interfere with the gene expression during transcription and translation in an in vitro engineered system outside of its natural cellular environment. It may be possible that a highly active CRISPR-Cas effector measured by this gene-silencing assay is also highly efficient for gene editing in mammalian cells.

[0386] This assay has been used to measure the gene-silencing effect of a Cas12i effector complex as illustrated here when targeting GFP encoded in plasmid DNA. Multiple Type V-I RNA guides are designed--one with a spacer sequence complementary to the template strand of the GFP sequence, and another with a spacer sequence complementary to the coding strand of the GFP sequence. The degree of gene-silencing by the Cas12i1 effector protein was then compared with that of the mutants Cas12i1 D647A, Cas12i1 E894A, and Cas12i1 D948A.

[0387] FIG. 19A depicts the fold-depletion of each of the four tested Cas12i effectors when complexed with an RNA guide complementary to the template strand. In this case, the non-target strand, preferentially being nicked, is the coding strand. While Cas12i1 shows approximately 2-fold depletion of GFP expression after 400 minutes, each of the three mutant forms shows smaller degrees of depletion.

[0388] FIG. 19B depicts the fold-depletion of each of the four tested Cas12i1 effectors when complexed with an RNA guide complementary to the coding strand. In this case, the non-target strand, preferentially being nicked, is the template strand. The ability for RNA polymerase to produce a functional RNA transcript appears to be significantly impaired by Cas12i1 in this configuration, with greater than 4-fold depletion in the case of Cas12i. The gene-silencing ability of the three mutant forms appears significantly diminished.

[0389] Taken together, the data shown in FIG. 19A and FIG. 19B indicate that the assay is effective in detecting the gene silencing activity of Cas12i1 when using RNA guides targeting both the coding and template strands. The significant higher depletion when targeting the coding strand than targeting the template strand suggests Cas12i1 interferes with the GFP expression by preferentially nicking the non-target strand. All three Cas12i1 mutants substitute the postulated catalytic residues (aspartic acid (D) and glutamic acid (E)) with alanine (A). The diminishing silencing activities of these Cas12i1 mutants further support that DNA stand cleavage, rather than just binding, underlies the mechanism of the gene silencing by Cas12i1

Example 8: CLUST.029130 (Type V-I) CRISPR-Cas Systems can be Used with a Fluorescent Reporter for the Specific Detection of Nucleic Acid Species

[0390] The nuclease activities of Cas12i proteins (i.e., non-specific collateral DNase activities activated by a target ssDNA substrate complementary to the crRNA spacer) make these effectors promising candidates for use in the detection of nucleic acid species. Some of these methods have been previously described (see, e.g., East-Seletsky et al. "Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection," Nature. 2016 Oct. 13; 538(7624):270-273), Gootenberg et al. (2017), Chen et al. 2018, and Gootenberg et al. (2018) "Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6" Science 15 Feb. 2018: eaaq0179), describing the general principle of RNA detection using Cas13a (East-Seletsky et al. (2016)), supplemented by amplification to increase the detection sensitivity and optimization of additional Cas13a enzymes (Gootenberg et al. (2017)), and most recently, the inclusion of additional RNA targets, orthologous and paralogous enzymes, and Csm6 activator to enable multiplexed detection of nucleic acids along with an increase in detection sensitivity (Gootenberg et al. (2018)). The addition of Cas12i to this toolkit provides an additional channel of orthogonal activity for nucleic acid detection.

[0391] The in vitro biochemical activity of Cas12i1 suggests that it may have promise in applications for sensitive nucleic acid detection, given that a dye-labeled, collateral DNA was efficiently cleaved at low target ssDNA concentrations and background nuclease activity was limited with a non-targeting substrate (FIG. 14). Adapting Cas12i1 towards sensitive nucleic acid detection application requires several steps, including, but not limited to, optimizing the substrate for sensitive readout of the collateral activity and identifying per-base mismatch tolerance between the spacer and the target substrate.

[0392] Identification of the optimal substrate for nucleic acid detection can be informed by performing next generation sequencing (NGS) on the cleavage products of Cas12i collateral activity on both DNA substrates. The enzyme concentration may have to be titrated or incubation time adjusted in order to yield cleavage fragments that are still of a sufficient size to be prepared into a next generation sequencing library. The NGS data reveal the enzyme cleavage sites and the adjacent base preferences. It has been demonstrated that the individual effectors within the Cas13a and b families have different dinucleotide base preferences for RNA cleavage, yielding markedly different cleavage magnitudes and signal to noise ratios (Gootenberg et al. (2018)). The collateral NGS data thus enable better insight into the preferences for Cas12i. A separate experimental approach to identifying the dinucleotide preference of Cas12i collateral cleavage is to create a collateral DNA substrate with degenerate N's in consecutive positions so as to have a broader sequence space than a defined sequence. The library prep and analysis of the NGS data would proceed similarly to identify base preferences for cleavage. To verify the preference, collateral substrates containing synthesized short DNAs with a fluorophore/quencher pair on the 5' and 3' ends can be introduced into a cleavage reaction to assess the signal to noise ratio. Further optimization can be done on the length of the collateral DNA substrate to determine whether Cas12i1 has a length preference.

[0393] Having identified the preferred substrate, another important parameter to determine is the mismatch tolerance of the Cas12i system, as it has implications for guide design that affects the ability of the enzyme to distinguish single base pair mismatches. The mismatch tolerance can be determined by designing a panel of targets bearing different positions and types of mismatches (for example, insertion/deletions, single base pair mismatches, adjacent double mismatches, separated double mismatches, triple mismatches, and more). Mismatch tolerance can be measured by assessing the amount of cleavage of collateral DNA for targets containing varying amounts of mismatches. As an example, the collateral DNA substrate could be a short ssDNA probe containing a fluorophore and quencher on opposite sides. For reactions containing the Cas12i effector, an RNA guide, and a target substrate containing different numbers of mismatches, insertions and deletions in the target sequence, successful activation of the Cas12i system by targeting of altered target DNA sequence will result in collateral cleavage of the fluorescent probe. Hence resulting fluorescent measurements denoting cleaved collateral substrate can be background subtracted using negative control samples and normalized to the signal from perfectly matching targets to estimate the impact of target alterations on the efficiency of collateral cleavage by Cas12i. Resulting maps of mismatch, insertion, and deletion tolerance by the Cas12i enzyme over the target length relative to the PAM can be used to design optimal RNA guides to distinguish between different DNA sequences or genotypes for specific detection or distinction between different Nucleic Acid Species. Using the fluorometric cleavage readout and the preferred collateral substrate, the fluorescence activity would be compared against the fully matched sequence to determine the position and types of mismatch to which the enzyme is most sensitive.

[0394] The optimization process can be furthermore applied to other Cas12i orthologs to yield other systems that may have different properties. For example, orthogonal dinucleotide preferences of collateral cleavage would be helpful in generating separate channels of detection.

Example 9. CLUST.029130 (Type V-I) CRISPR Cas Systems can be Used for Paired Nicking to Enable Highly Specific dsDNA Manipulation

[0395] The CLUST.029130 effector Cas12i is capable of manipulating dsDNA via nicking of the non-target strand (FIGS. 15, 16, 17A-B). Catalytically inactivated Cas12i can also be fused with a FokI nuclease domain to create a fusion protein capable of binding and nicking dsDNA. Some of these methods have been previously described. Ran et al. (2013) "Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity" Science 29 Aug. 2013 describes the general principle and optimization of double nicking using Cas9; Guilinger et al. (2014) "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification" Science 25 Apr. 2014 described the principle of double nicking using a FokI-dCas9 fusion.

[0396] The use of paired Cas12i nickases enables highly specific dsDNA manipulation as follows. A first Cas12i complex with a crRNA targeting one strand of a dsDNA target region and second Cas12i-crRNA complex targeting the opposing strand of the dsDNA are introduced together to enable a dsDNA cleavage reaction. By targeting the Cas12i complexes to different dsDNA strands, the first and second Cas12i complexes cleave opposing dsDNA strands resulting in a double strand break.

[0397] To optimize the efficiency of dsDNA double strand break formation by double nicking, pairs of crRNA spacer sequences are chosen with different lengths separating their expected nuclease cleavage sites. Cleavage of the top and bottom strand of dsDNA by Cas12i paired nickases with different target displacements produces different length sequence overhangs, resulting in different efficiencies of double strand break formation. Paired nickase targets can be selected with specific orientations to generate either 3' or 5' overhangs, or a blunt (overhang length of 0) double strand breaks.

[0398] For nicking applications with the Cas12i1 and Cas12i2-WT enzymes containing 5' TTN PAMs, orientation of the paired nickase targets with PAMs `out` (PAMs at the outside of the paired targets) results in a 5' overhang, whereas pairing of nickase targets with PAMs at the inside of the target pair results in a 3' overhang. In some instances 3' and 5' overhangs range from 1-200 nt. In some instances, 3' and 5' overhangs are between 20 and 100 nt.

[0399] Autonomous pre-crRNA processing facilitates Cas12i delivery for double nicking applications (FIG. 12), as two separate genomic loci can be targeted from a single crRNA transcript. Therein, Cas12i and a CRISPR array containing two spacer sequences targeting the Cas12i to nick opposing strands of dsDNA can be expressed from a single viral vector or plasmid. Cas12i and the CRISPR array can also be delivered on separate plasmids or viral vectors. The Cas12i protein then processes the CRISPR array into two cognate crRNAs that result in the formation of paired nicking complexes. Viral vectors can include phage or adeno-associated virus for delivery to bacteria or mammalian cells, respectively.

[0400] Apart from viral or plasmid delivery methods, paired nicking complexes can be delivered directly using nanoparticle or other direct protein delivery methods, such that complexes containing both paired crRNA elements are co-delivered. Furthermore, protein can be delivered to cells by viral vector or directly, followed by the direct delivery of a CRISPR array containing two paired spacers for double nicking. In some instances, for direct RNA delivery the RNA may be conjugated to at least one sugar moiety, such as N-acetyl galactosamine (GalNAc) (particularly, triantennary GalNAc).

Example 10: Adaptation of CLUST.029130 (Type V-I) CRISPR Cas System Effectors for Eukaryotic and Mammalian Activity

[0401] To develop CLUST.029130 (Type V-I) CRISPR Cas systems for eukaryotic applications, the constructs encoding the protein effectors were first codon-optimized for expression in mammalian cells, and specific localization tags were optionally appended to either or both the N-terminus or C-terminus of the effector protein. These localization tags can include sequences such as nuclear localization signal (NLS) sequences, which localize the effector to the nucleus for modification of genomic DNA. These sequences are described above in the "Functional Mutations" section. Some examples of non-naturally occurring, engineered nucleotide sequences to encode mammalian codon-optimized Cas12i effectors with a localization tag are provided in TABLE 10. Other accessory proteins, such as fluorescent proteins, may be further appended. It has been demonstrated that the addition of robust, "superfolding" proteins such as superfolding green fluorescent protein (GFP) can increase the activity of CRISPR enzymes in mammalian cells when appended to the effector (Abudayyeh et al. (2017) Nature 550(7675): 280-4, and Cox et al. (2017) Science 358(6366): 1019-27).

[0402] The codon-optimized sequence coding for the Cas12i and appended accessory proteins and localization signals was then cloned into a eukaryotic expression vector with the appropriate 5' Kozak eukaryotic translation initiation sequence, eukaryotic promoters, and polyadenylation signals. In mammalian expression vectors, these promoters can include, e.g., general promoters such as CMV, EFla, EFS, CAG, SV40, and cell-type specific RNA polymerase II promoters such as Syn and CamKIIa for neuronal expression, and thyroxine binding globulin (TBG) for hepatocyte expression to name a few. Similarly, useful polyadenylation signals include, but are not limited to, SV40, hGH, and BGH. Additional transcript stabilization or transcript nuclear export elements such as WPRE can be used for increasing the expression of such constructs. For expression of the pre-crRNA or mature crRNA, RNA polymerase III promoters such as H1 or U6 can be used.

[0403] Depending on the application and mode of packaging, the eukaryotic expression vector can be a lentiviral plasmid backbone, adeno-associated viral (AAV) plasmid backbone, or similar plasmid backbone capable of use in recombinant viral vector production. Notably, the small size of CLUST.029130 (Type V-I) CRISPR Cas effector proteins, e.g., Cas12i proteins, make them ideally suited for packaging along with its crRNA and appropriate control sequences into a single adeno-associated virus particle; the packaging size limit of 4.7 kb for AAV may preclude the use of larger effectors, particularly if large cell-type specific promoters are used for expression control.

[0404] After adapting the sequences, delivery vectors, and methods for eukaryotic and mammalian use, different Cas12i constructs as described herein were characterized for performance. An initial characterization was performed by lipofection of DNA constructs expressing the minimal components of the Cas12i system with the adaptations for eukaryotic use as described above. In one embodiment, the Cas12i effector is mammalian codon optimized and a nucleoplasmin nuclear localization sequence (npNLS) is appended to the C-terminus of the protein. The expression of the effector is driven by the elongation factor 1alpha short (EFS) promoter, and terminated using a bGH poly(A) signal (TABLE 10). A double-stranded, linear PCR product containing a U6 promoter was used to express the cognate RNA guides for the Cas12i system, as adapted from (Ran et al. "Genome engineering using the CRISPR-Cas9 system," Nat Protoc. 2013 November; 8(11):2281-2308.). This approach is well suited to testing a larger number of sgRNAs over plasmid cloning and sequence verification. (FIG. 40) The effector plasmid and U6-guide PCR fragment were co-transfected into 293T cells at an approximately 1:2 molar ratio of plasmid to PCR product with 400 ng of effector plasmid and 30 ng of U6-guide PCR product for a 24 well plate format. The resulting gene editing event was evaluated using next generation sequencing of a targeted PCR amplicon surrounding the target site (Hsu et al., "DNA targeting specificity of RNA-guided Cas9 nucleases," Nat Biotechnol. 2013 September; 31(9):827-32.).

[0405] Initial evaluation of Cas12i2 yielded indel activity of 13% at the VEGFA locus at a target site with a TTC PAM. We tested different RNA guide designs as described in FIG. 41, with the strongest indel efficiency achieved using pre-crRNA, and with indel rates decreasing with shorter spacer lengths. Examining the indels created by Cas12i2 reveals that the predominant location of the indels are centered around +20 relative to the PAM sequence.

[0406] Multiplexing of Type V-I effectors is accomplished using the pre-crRNA processing capability of the effectors, where multiple targets with different sequences can be programmed on a single RNA guide. As such, multiple genes or DNA targets can be manipulated simultaneously for therapeutic applications. One embodiment of a RNA guide design is a pre-crRNA expressed from a CRISPR array consisting of target sequences interleaved by unprocessed DR sequences, repeated to enable targeting of one, two, or more loci simultaneously by the intrinsic pre-crRNA processing of the effector.

[0407] In addition to testing various construct configurations and accessory sequences on individual targets, pooled library-based approaches are used to determine 1) any targeting dependency of specific Cas12i proteins in mammalian cells as well as 2) the effect of mismatch locations and combinations along the length of the targeting crRNA. Briefly, the pooled library includes a plasmid that expresses a target DNA containing different flanking sequences as well as mismatches to the guide or guides used in the screening experiment, such that the successful target recognition and cleavage results in depletion of the sequence from the library. Furthermore, targeted indel sequencing or unbiased genome-wide cleavage assays can be used to evaluate the specificity of the CLUST.029130 (Type V-I) CRISPR-Cas system (Hsu et al. (2013), Tsai et al. "GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases." Nat Biotechnol. 2015 February; 33(2):187-197, Kim et al. "Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells," Nat Methods. 2015 March; 12(3):237-43, Tsai et al., "CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets," Nat Methods. 2017 June; 14(6):607-614).

[0408] Mutations are additionally created to extend the functional range of Cas12i proteins. In some embodiment, catalytically-inactive Cas12i proteins can be made in which the conserved residues of the RuvC domain are mutated to alanine (such as the D647A mutation for Cas12i1 and D599A mutation for Cas12i2). Catalytically inactive Cas12i versions (referred to as dCas12i) retains its programmable DNA binding activity, though it will no longer be able to cleave target or collateral ssDNA or dsDNA. Direct uses of dCas12i include immunoprecipitation and transcriptional repression. Further functionality is provided by appending other domains onto the dCas12i protein

[0409] Activities of these domains include, but are not limited to, DNA base modification (ex: ecTAD and its evolved forms, APOBEC), DNA methylation (m.sup.6A methyltransferases and demethylases), localization factors (KDEL retention sequence, mitochondrial targeting signal), transcription modification factors (ex: KRAB, VP64). Additionally, domains can be appended to provide additional control, such as light-gated control (cryptochromes) and chemically inducible components (FKBP-FRB chemically inducible dimerization).

[0410] Optimizing the activity of such fusion proteins requires a systematic way of comparing linkers that connect the dCas12i with the appended domain. These linkers may include, but are not limited to, flexible glycine-serine (GS) linkers in various combinations and lengths, rigid linkers such as the alpha-helix forming EAAAK sequence, XTEN linker (Schellenberger V, et al. Nat. Biotechnol. 2009; 27:1186-1190), as well as different combinations thereof (see TABLE 11). The various designs are then assayed in parallel over the same crRNA target complex and functional readout to determine which one yields the desired properties.

[0411] For adapting Cas12i for use in targeted DNA base modification (see, e.g., Gaudelli et al. (2017) "Programmable base editing of A-T to G C in genomic DNA without DNA cleavage" Science 25 Oct. 2017), we begin with the Cas12i ortholog and NLS combination that yielded the highest endogenous mammalian DNA cleavage activity and mutate the conserved residues of the RuvC domain to create a catalytically inactive enzyme (dCas12i). Next, a linker is used to create the fusion protein between dCas12i-NLS and the base editing domain. Initially, this domain will consist of the ecTadA(wt)/ecTadA*(7.10)heterodimer (hereafter referred to as the dCas12i-TadA heterodimer) engineered previously for hyperactivity and modification of dsDNA A-T dinucleotides to G C (TABLE 11). Given the likely structural differences between the smaller Cas12i versus the previously characterized Cas9 effectors, alternate linker designs and lengths may yield the optimal design of the base editing fusion protein.

[0412] To evaluate the activity of the dCas12i-derived base editors, the HEK 293T cells are transiently transfected with the dCas12i-TadA heterodimer construct, a plasmid expressing the crRNA, and optionally, a reporter plasmid if targeting the reporter and not an endogenous locus. The cells are harvested 48 hours after transient transfection, the DNA is extracted and prepared for next generation sequencing. Analysis of the base composition of loci of samples containing the targeting vs. negative control non-targeting crRNAs provide information about the editing efficiency, and analysis of broader changes to the transcriptome will yield information about the off-target activity.

[0413] One particular advantage of developing a DNA base editing system using Cas12i is that the small size, smaller than the existing Cas9 and Cas12a effectors, enables more ready packaging in AAV of dCas12i-TadA heterodimer along with its crRNA and control elements without the need for protein truncations. This all-in-one AAV vector enables greater efficacy of in vivo base editing in tissues, which is particularly relevant as a path towards therapeutic applications of Cas12i.

[0414] In additional to editing using Cas12i and an RNA guide, additional template DNA sequences can be co-delivered either in a vector, such as an AAV viral vector, or as linear single stranded or double stranded DNA fragments. For insertion of template DNA by homology directed repair (HDR), template sequences are designed containing a payload sequence to be inserted into the locus of interest as well as flanking sequences that are homologous to endogenous sequences flanking the desired insertion site. In some instances, for insertion of short DNA payloads less than (for example: less than lkb in length), flanking homologous sequences can be short (for example: ranging from 15 to 200 nt in length). In other instances, for the insertion of long DNA payloads (for example: lkb or greater in length), long homologous flanking sequences are required to facilitate efficient HDR (for example: greater than 200 nt in length). Cleavage of target genomic loci for HDR between sequences homologous to template DNA flanking regions can significantly increase the frequency of HDR. Cas12i cleavage events facilitating HDR include, but are not limited to dsDNA cleavage, double nicking, and single strand nicking activity.

[0415] DsDNA fragments may contain overhang sequences complementary to the overhangs resulting from double nicking using Cas12i. Pairing of the insert and double-nicking overhangs and subsequent ligation by endogenous DNA repair machinery result in the seamless insertion of the template DNA at the site of double-nicking.

TABLE-US-00011 TABLE 10 Sequences enabling mammalian expression of Cas12i effectors with included N-terminal mH6 tag and C-terminal nucleoplasmin NLS sequence (bolded) >EF1alpha short (EFS) promoter GGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGATCCGGTGCCTAGAGA- AGGTGGCGCG GGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGT- GCAGTAGTCG CCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAG (SEQ ID NO: 500) >Cas12i1_mammallan_effector atgAAAATCGAAGAAGGTAAAGGTCACCATCACCATCACCACATGTCTAACAAGGAGAAGAATGCCAGCGAGAC- CCGGAAGGCC TACACCACAAAGATGATCCCCAGGAGCCACGACCGCATGAAGCTGCTGGGCAACTTTATGGACTATCTGATGGA- TGGCACCCCT ATCTTCTTTGAGCTGTGGAATCAGTTCGGCGGCGGCATCGACAGAGATATCATCAGCGGCACAGCCAACAAGGA- TAAGATCTCC GACGATCTGCTGCTGGCCGTGAACTGGTTTAAAGTGATGCCAATCAATTCTAAGCCCCAGGGCGTGTCCCCTTC- TAACCTGGCC AATCTGTTCCAGCAGTACAGCGGATCCGAGCCTGACATCCAGGCACAGGAGTATTTCGCCTCCAACTTTGACAC- CGAGAAGCAC CAGTGGAAGGATATGCGGGTGGAGTACGAGAGACTGCTGGCCGAGCTGCAGCTGTCTAGGAGCGACATGCATCA- CGATCTGAAG CTGATGTACAAGGAGAAGTGCATCGGCCTGTCCCTGTCTACCGCCCACTATATCACAAGCGTGATGTTTGGCAC- CGGCGCCAAG AACAATCGCCAGACAAAGCACCAGTTCTATTCCAAAGTGATCCAGCTGCTGGAGGAGAGCACCCAGATCAATTC- CGTGGAGCAG CTGGCCTCCATCATCCTGAAGGCCGGCGACTGCGATTCTTACAGGAAGCTGAGGATCAGGTGTTCCCGCAAGGG- AGCAACCCCA TCTATCCTGAAGATCGTGCAGGACTATGAGCTGGGCACAAACCACGACGATGAAGTGAATGTGCCCTCCCTGAT- CGCCAACCTG AAGGAGAAGCTGGGCAGGTTTGAGTACGAGTGCGAGTGGAAGTGTATGGAGAAGATCAAGGCCTTCCTGGCCTC- TAAAGTGGGC CCTTACTATCTGGGCAGCTATTCCGCCATGCTGGAGAATGCCCTGAGCCCAATCAAGGGCATGACCACAAAGAA- CTGTAAGTTC GTGCTGAAGCAGATCGACGCCAAGAACGATATCAAGTACGAGAATGAGCCCTTTGGCAAGATCGTGGAGGGCTT- CTTTGACTCT CCTTATTTCGAGAGCGATACCAATGTGAAGTGGGTGCTGCACCCTCACCACATCGGCGAGTCTAACATCAAGAC- ACTGTGGGAG GACCTGAATGCCATCCACAGCAAGTACGAGGAGGACATCGCCTCTCTGAGCGAGGATAAGAAGGAGAAGCGGAT- CAAGGTGTAC CAGGGCGATGTGTGCCAGACCATCAACACATATTGTGAGGAAGTGGGCAAGGAGGCCAAGACCCCACTGGTGCA- GCTGCTGAGG TACCTGTATTCCCGCAAGGACGATATCGCCGTGGACAAGATCATCGATGGCATCACATTCCTGTCTAAGAAGCA- CAAGGTGGAG AAGCAGAAGATCAACCCAGTGATCCAGAAGTACCCCAGCTTCAATTTTGGCAACAATTCCAAGCTGCTGGGCAA- GATCATCAGC CCAAAGGACAAGCTGAAGCACAACCTGAAGTGCAACAGAAATCAGGTGGATAATTACATCTGGATCGAGATCAA- GGTGCTGAAC ACCAAGACAATGCGGTGGGAGAAGCACCACTATGCCCTGAGCTCCACCAGATTTCTGGAGGAGGTGTACTATCC- CGCCACATCC GAGAATCCACCTGACGCACTGGCAGCACGGTTCAGAACCAAGACAAACGGCTACGAGGGCAAGCCAGCCCTGTC- TGCCGAGCAG ATCGAGCAGATCAGGAGCGCACCAGTGGGACTGAGAAAGGTGAAGAAGCGGCAGATGAGACTGGAGGCAGCAAG- GCAGCAGAAT CTGCTGCCACGCTATACCTGGGGCAAGGATTTTAACATCAATATCTGTAAGAGGGGCAACAATTTCGAGGTGAC- CCTGGCCACA AAGGTGAAGAAGAAGAAGGAGAAGAACTACAAGGTGGTGCTGGGCTATGACGCCAACATCGTGCGCAAGAATAC- CTACGCAGCA ATCGAGGCACACGCAAACGGCGATGGCGTGATCGACTATAATGATCTGCCTGTGAAGCCAATCGAGTCTGGCTT- TGTGACAGTG GAGAGCCAGGTGAGGGACAAGTCCTACGATCAGCTGTCTTATAACGGCGTGAAGCTGCTGTACTGCAAGCCTCA- CGTGGAGAGC CGGAGATCCTTCCTGGAGAAGTATCGGAACGGCACCATGAAGGACAATAGAGGCAACAATATCCAGATCGACTT- CATGAAGGAT TTTGAGGCCATCGCCGACGATGAGACAAGCCTGTACTACTTCAACATGAAGTACTGTAAGCTGCTGCAGTCTAG- CATCCGCAAC CACTCCTCTCAGGCCAAGGAGTATAGGGAGGAGATCTTCGAGCTGCTGCGCGATGGCAAGCTGTCCGTGCTGAA- GCTGAGCTCC CTGTCTAATCTGAGCTTCGTGATGTTTAAGGTGGCCAAGTCTCTGATCGGCACCTACTTTGGCCACCTGCTGAA- GAAGCCTAAG AACTCCAAGTCTGACGTGAAGGCCCCACCCATCACAGACGAGGATAAGCAGAAGGCCGATCCAGAGATGTTCGC- ACTGCGGCTG GCACTGGAGGAGAAGAGACTGAATAAGGTGAAGAGCAAGAAGGAAGTGATCGCCAACAAGATCGTGGCCAAGGC- ACTGGAGCTG AGGGACAAGTACGGACCAGTGCTGATCAAGGGCGAGAATATCAGCGATACCACAAAGAAGGGCAAGAAGTCTAG- CACCAATTCC TTCCTGATGGACTGGCTGGCCAGAGGCGTGGCCAACAAGGTGAAGGAGATGGTCATGATGCACCAGGGCCTGGA- GTTCGTGGAG GTGAACCCCAATTTTACCTCCCACCAGGATCCTTTCGTGCACAAGAACCCAGAGAATACCTTCCGGGCAAGGTA- CAGCAGGTGC ACCCCTTCCGAGCTGACAGAGAAGAACCGCAAGGAGATCCTGTCCTTCCTGTCTGACAAGCCCAGCAAGCGGCC- TACTAACGCC TACTATAATGAGGGCGCCATGGCCTTTCTGGCCACATATGGCCTGAAGAAGAATGACGTGCTGGGCGTGTCCCT- GGAGAAGTTC AAGCAGATCATGGCCAACATCCTGCACCAGCGGTCCGAGGATCAGCTGCTGTTTCCCTCTAGAGGCGGCATGTT- CTACCTGGCC ACCTATAAGCTGGACGCCGATGCCACAAGCGTGAACTGGAATGGCAAGCAGTTTTGGGTGTGTAACGCCGACCT- GGTGGCCGCC TACAATGTGGGCCTGGTGGACATCCAGAAGGATTTCAAGAAGAAGAAAAGGCCGGCGGCCACGAAAAAGGCCGG- CCAGGCAAAA AAGAAAAAGTAATAA (SEQ ID NO: 501) >Cas12i2_mammalian_effector atgAAAATCGAAGAAGGTAAAGGTCACCATCACCATCACCACATGAGCTCCGCCATCAAGTCCTACAAGTCTGT- GCTGCGGCCA AACGAGAGAAAGAATCAGCTGCTGAAGAGCACCATCCAGTGCCTGGAGGACGGCTCCGCCTTCTTTTTCAAGAT- GCTGCAGGGC CTGTTTGGCGGCATCACCCCCGAGATCGTGAGATTCAGCACAGAGCAGGAGAAGCAGCAGCAGGATATCGCCCT- GTGGTGTGCC GTGAATTGGTTCAGGCCTGTGAGCCAGGACTCCCTGACCCACACAATCGCCTCCGATAACCTGGTGGAGAAGTT- TGAGGAGTAC TATGGCGGCACAGCCAGCGACGCCATCAAGCAGTACTTCAGCGCCTCCATCGGCGAGTCCTACTATTGGAATGA- CTGCCGCCAG CAGTACTATGATCTGTGTCGGGAGCTGGGCGTGGAGGTGTCTGACCTGACCCACGATCTGGAGATCCTGTGCCG- GGAGAAGTGT CTGGCCGTGGCCACAGAGAGCAACCAGAACAATTCTATCATCAGCGTGCTGTTTGGCACCGGCGAGAAGGAGGA- TAGGTCTGTG AAGCTGCGCATCACAAAGAAGATCCTGGAGGCCATCAGCAACCTGAAGGAGATCCCAAAGAATGTGGCCCCCAT- CCAGGAGATC ATCCTGAATGTGGCCAAGGCCACCAAGGAGACATTCAGACAGGTGTACGCAGGAAACCTGGGAGCACCATCCAC- CCTGGAGAAG TTTATCGCCAAGGACGGCCAGAAGGAGTTCGATCTGAAGAAGCTGCAGACAGACCTGAAGAAAGTGATCCGGGG- CAAGTCTAAG GAGAGAGATTGGTGCTGTCAGGAGGAGCTGAGGAGCTACGTGGAGCAGAATACCATCCAGTATGACCTGTGGGC- CTGGGGCGAG ATGTTCAACAAGGCCCACACCGCCCTGAAGATCAAGTCCACAAGAAACTACAATTTTGCCAAGCAGAGGCTGGA- GCAGTTCAAG GAGATCCAGTCTCTGAACAATCTGCTGGTGGTGAAGAAGCTGAACGACTTTTTCGATAGCGAGTTTTTCTCCGG- CGAGGAGACC TACACAATCTGCGTGCACCACCTGGGCGGCAAGGACCTGTCCAAGCTGTATAAGGCCTGGGAGGACGATCCCGC- CGATCCTGAG AATGCCATCGTGGTGCTGTGCGACGATCTGAAGAACAATTTTAAGAAGGAGCCTATCAGGAACATCCTGCGCTA- CATCTTCACC ATCCGCCAGGAGTGTAGCGCACAGGACATCCTGGCAGCAGCAAAGTACAATCAGCAGCTGGATCGGTATAAGAG- CCAGAAGGCC AACCCATCCGTGCTGGGCAATCAGGGCTTTACCTGGACAAACGCCGTGATCCTGCCAGAGAAGGCCCAGCGGAA- CGACAGACCC AATTCTCTGGATCTGCGCATCTGGCTGTACCTGAAGCTGCGGCACCCTGACGGCAGATGGAAGAAGCACCACAT- CCCATTCTAC GATACCCGGTTTTTCCAGGAGATCTATGCCGCCGGCAATAGCCCTGTGGACACCTGTCAGTTTAGGACACCCCG- CTTCGGCTAT CACCTGCCTAAGCTGACCGATCAGACAGCCATCCGCGTGAACAAGAAGCACGTGAAGGCAGCAAAGACCGAGGC- ACGGATCAGA CTGGCCATCCAGCAGGGCACACTGCCAGTGTCCAATCTGAAGATCACCGAGATCTCCGCCACAATCAACTCTAA- GGGCCAGGTG CGCATCCCCGTGAAGTTTGACGTGGGAAGGCAGAAGGGAACCCTGCAGATCGGCGACCGGTTCTGCGGCTACGA- TCAGAACCAG ACAGCCTCTCACGCCTATAGCCTGTGGGAGGTGGTGAAGGAGGGCCAGTACCACAAGGAGCTGGGCTGTTTTGT- GCGCTTCATC TCTAGCGGCGACATCGTGTCCATCACCGAGAACCGGGGCAATCAGTTTGATCAGCTGTCTTATGAGGGCCTGGC- CTACCCCCAG TATGCCGACTGGAGAAAGAAGGCCTCCAAGTTCGTGTCTCTGTGGCAGATCACCAAGAAGAACAAGAAGAAGGA- GATCGTGACA GTGGAGGCCAAGGAGAAGTTTGACGCCATCTGCAAGTACCAGCCTAGGCTGTATAAGTTCAACAAGGAGTACGC- CTATCTGCTG CGGGATATCGTGAGAGGCAAGAGCCTGGTGGAGCTGCAGCAGATCAGGCAGGAGATCTTTCGCTTCATCGAGCA- GGACTGTGGA GTGACCCGCCTGGGATCTCTGAGCCTGTCCACCCTGGAGACAGTGAAGGCCGTGAAGGGCATCATCTACTCCTA- TTTTTCTACA GCCCTGAATGCCTCTAAGAACAATCCCATCAGCGACGAGCAGCGGAAGGAGTTTGATCCTGAGCTGTTCGCCCT- GCTGGAGAAG CTGGAGCTGATCAGGACTCGGAAGAAGAAGCAGAAGGTGGAGAGAATCGCCAATAGCCTGATCCAGACATGCCT- GGAGAACAAT ATCAAGTTCATCAGGGGCGAGGGCGACCTGTCCACCACAAACAATGCCACCAAGAAGAAGGCCAACTCTAGGAG- CATGGATTGG CTGGCCAGAGGCGTGTTTAATAAGATCCGGCAGCTGGCCCCAATGCACAACATCACCCTGTTCGGCTGCGGCAG- CCTGTACACA TCCCACCAGGACCCTCTGGTGCACAGAAACCCAGATAAGGCCATGAAGTGTAGATGGGCAGCAATCCCAGTGAA- GGACATCGGC GATTGGGTGCTGAGAAAGCTGTCCCAGAACCTGAGGGCCAAGAATATCGGCACCGGCGAGTACTATCACCAGGG- CGTGAAGGAG TTCCTGTCTCACTATGAGCTGCAGGACCTGGAGGAGGAGCTGCTGAAGTGGCGGTCTGATAGAAAGAGCAACAT- CCCTTGCTGG GTGCTGCAGAATAGACTGGCCGAGAAGCTGGGCAACAAGGAGGCCGTGGTGTACATCCCAGTGAGGGGCGGCCG- CATCTATTTT GCAACCCACAAGGTGGCAACAGGAGCCGTGAGCATCGTGTTCGACCAGAAGCAAGTGTGGGTGTGTAATGCAGA- TCACGTGGCA GCAGCAAACATCGCACTGACCGTGAAGGGCATCGGCGAGCAGTCCTCTGACGAGGAGAACCCCGATGGCTCCAG- GATCAAGCTG CAGCTGACATCTAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGTAATAA (SEQ ID NO: 502) >bGH polyA Tail CGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC- ACTCCCACTG TCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTG- GGGCAGGACA GCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGG (SEQ ID NO: 503)

TABLE-US-00012 TABLE 11 Amino Acid Sequences of Motifs and Functional Domains in Engineered Variants of CLUST.029130 (Type V-I) CRISPR-Cas Effector Proteins >LINKER_1 GS (SEQ ID NO: 600) >LINKER_2 GSGGGGS (SEQ ID NO: 601) >LINKER_3 GGGGSGGGGSGGGGS (SEQ ID NO: 602) >LINKER_4 GGSGGSGGSGGSGGSGGS (SEQ ID NO: 603) >LINKER 5 (Gaudelli et al., 2017) SGGSSGGSSGSETPGTSESATPESSGGSSGGS (SEQ ID NO: 604) >ecTadA(wt) (Gaudelli et al., 2017) [N-term fusion to ecTadA* (7.10)] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYR- LIDAT LYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRR- QEIKAQK KAQSSTD (SEQ ID NO: 605) >ecTadA* (7.10) (Gaudelli et al., 2017) [N-term fusion to CRISPR nuclease] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYR- LIDAT LYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRR- QEIKAQK KAQSSTD (SEQ ID NO: 606) [Cytidine deaminase, AID, APOBEC1: N-term fusion (or optionally C-term)] >AID-APOBEC1 (Dickerson et al., 2003, Komor et al., 2017) MDSLLMNRRKFLYQFKNVRWAKGRRETYLCYVVKRRDSATSFSLDFGYLRNKNGCHVELLFLRYISDWDLDPGR- CYRVTWFTSW SPCYDCARHVADFLRGNPNLSLRIFTARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENHERT- FKAWEGLHEN SVRLSRQLRRILLPLYEVDDLRDAFRTLGL (SEQ ID NO: 607) >Lamprey_AID-APOBEC1 (Rogozin et al., 2007, Komor et al., 2017) MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGERRACFWGYAVNKPQSGTERGIHAEIFSIRK- VEEYLRDNPG QFTINWYSSWSPCADCAEKILEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQC- CRKIFIQSSH NQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV (SEQ ID NO: 608) >APOBEC1_BE1 (Komor et al., 2016) MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGGRHSIWRHTSQNTNKHVEVNFIEKFTTER- YFCPNTRCSI TWFLSWSPCGECSRAITEFLSRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVN- YSPSNEAHWP RYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHILWATGLK (SEQ ID NO: 609)

[0416] These results suggest that members of the compact Type V-I CRISPR family can be engineered for activity in eukaryotic cells, and specifically, for genome editing in mammalian cells. A mammalian functional Type V-I effector enables the development of additional technologies based on further engineering on top of a DNA binding chassis.

Example 11. Type V-I CRISPR-Cas Systems can be Used to Provide Genotype-Gated Control of Genome Replication, Viral Propagation, Plasmid Propagation, Cell Death, or Cell Dormancy

[0417] Hybridization of the Type V-I CRISPR-Cas effector protein and crRNA with a specific ssDNA or dsDNA target results in nicking or cleavage of the substrate. The dependence of such activity on the presence of a specific DNA target in a cell is valuable since it enables targeting of specific genomic material or cell populations based on specific underlying genotypes. Numerous applications exist in both eukaryotic, prokaryotic, and viral/plasmid settings for such control of genome replication, cell death, or cell dormancy.

[0418] For prokaryotic, viral, and plasmid applications, a Type V-I CRISPR-Cas system (e.g., including a Type V-I effector and a RNA guide) can be delivered (e.g., in vitro or in vivo) in order to stop genome replication and/or induce cell death or dormancy of specific prokaryote populations (e.g., bacterial populations) in a genotype-specific way. For instance, the Type V-I CRISPR-Cas system can include one or more RNA guides that specifically target a particular virus, plasmid, or prokaryotic genus, species, or strain. As shown in FIG. 5A-D cleavage, nicking, or interference with the E. coli genome or plasmid DNA conferring antibiotic resistance in E. coli by a Type V-I system results in specific depletion of the E. coli containing these sequences. Specific targeting of viruses, plasmids, or prokaryotes has many therapeutic benefits as it may be used to induce death or dormancy of undesirable bacteria (e.g., pathogenic bacteria such as Clostridium difficile). In addition, the Type V-I systems provided herein may be used to target prokaryotic cells having specific genotypes. Within the microbial diversity that colonizes humans, only a small number of bacterial strains can induce pathogenesis. Further, even within pathogenic strains such as Clostridium difficile, not all members of the bacterial population exist continuously in active, disease-causing states. Thus, targeting the Type V-I system based on the genotype of a virus, plasmid, or prokaryotic cell allows for specific control of which genomes or cell populations are targeted without disrupting the entire microbiome.

[0419] Additionally, bacterial strains can be readily engineered with genetic circuits or environmentally-controlled expression elements to generate genetic kill switches that limit the growth, colonization, and/or shedding of the engineered bacterial strains. For example, the expression of a TypeV-I effector and specific crRNA, can be controlled using promoters derived from the regulatory regions of genes encoding proteins expressed in response to external stimuli, such as cold sensitive proteins (PcspA), heat shock proteins (Hsp), chemically inducible systems (Tet, Lac, AraC). The controlled expression of one or more elements of the Type V-I system allows for the full functional system to be expressed only upon exposure to an environmental stimulus, which results in genotype-specific DNA interference activity of the system and thereby induces cell death or dormancy. Kill switches including Cas12i effectors as those described herein may be advantageous over traditional kill switch designs such as toxin/antitoxin systems (e.g., CcdB/CcdA Type II toxin/antitoxin systems), since they are not dependent on relative protein expression ratios which may be affected by leaky expression from a promoter (e.g., an environmental-stimulus dependent promoter), and thus allow for more precise control of the kill-switch.

Other Embodiments

[0420] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Sequence CWU 1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 621 <210> SEQ ID NO 1 <211> LENGTH: 1046 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 1 Met Val Ser Glu Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala 35 40 45 Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe 50 55 60 Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Gln Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser 130 135 140 Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ser Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Ile Glu Asp Lys Lys Pro Asn Thr Trp Glu Glu 195 200 205 Tyr His Asp Leu Ile Lys Lys Thr Phe Asn Val Asp Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg 260 265 270 Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu 355 360 365 Asn Glu Leu Phe Glu Ile Trp Gln Glu Leu Thr Met Asp Asp Gly Ile 370 375 380 Lys Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val 385 390 395 400 Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Asn Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe 485 490 495 His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Lys Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Thr Ile Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile 595 600 605 Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Val Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp 705 710 715 720 Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu 770 775 780 Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys 785 790 795 800 Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly 820 825 830 Tyr Leu Pro Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln 930 935 940 Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Lys Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Gly Asp His 965 970 975 Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Ile Asp Glu Asp Glu Gln Pro Asp Gly Ala Lys 1025 1030 1035 Lys Gln Ala Leu Ala Arg Thr Lys 1040 1045 <210> SEQ ID NO 2 <211> LENGTH: 1091 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 2 Met Phe Thr Leu Leu Leu Ser Asp Ile Ser Gln Gln Asn Phe Asn Lys 1 5 10 15 Phe Leu Lys Asn Phe Phe Phe Thr Arg Asn Lys Thr Val Val His Cys 20 25 30 Ser Ser Glu Ile Arg His Lys Gly Tyr Arg Ser Asn Val Met Val Ser 35 40 45 Glu Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro Asn Asp Pro 50 55 60 Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp His Ala Tyr 65 70 75 80 Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala Ile Glu His 85 90 95 Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe Asp Ala Asp 100 105 110 Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys Ser Asp Asn 115 120 125 Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu Phe Gln Lys 130 135 140 Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr Ile Lys Gly 145 150 155 160 Asn Ile Asp Ser Glu Lys Tyr Gln Trp Val Asp Cys Arg Leu Lys Phe 165 170 175 Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser Leu Lys Ile 180 185 190 Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val Ser Lys Asp 195 200 205 Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe Gly Thr Gly 210 215 220 Lys Lys Glu Asp Arg Ser Ile Lys Ala Ser Met Leu Glu Glu Ile Ser 225 230 235 240 Asn Ile Ile Glu Asp Lys Lys Pro Asn Thr Trp Glu Glu Tyr His Asp 245 250 255 Leu Ile Lys Lys Thr Phe Asn Val Asp Asn Tyr Lys Glu Leu Lys Glu 260 265 270 Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu Val Ile Asp 275 280 285 Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe Ile Lys Asn 290 295 300 Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg Thr Val Phe 305 310 315 320 Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala Ser Gln Ile 325 330 335 Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn Arg Ser Met 340 345 350 Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr Thr Asn Glu 355 360 365 Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys Lys Asp Ile 370 375 380 Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly Glu Phe Asn 385 390 395 400 Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu Asn Glu Leu 405 410 415 Phe Glu Ile Trp Gln Glu Leu Thr Met Asp Asp Gly Ile Lys Lys Tyr 420 425 430 Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val Lys Ala Leu 435 440 445 Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Asn Ala Lys Gln Phe 450 455 460 Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn Asn Arg Lys 465 470 475 480 Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn Trp Gly Pro 485 490 495 Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln Met Val Lys 500 505 510 Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr Met Thr Val 515 520 525 Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe His Asn Ser 530 535 540 Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu Pro Thr Lys 545 550 555 560 Asn Lys Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly Ser Thr Ile 565 570 575 Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu Gln Asp Arg 580 585 590 Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser Ile Ile Arg 595 600 605 Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys Ser Thr Asn 610 615 620 Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr Ile Ser Ser 625 630 635 640 Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile Gly Asp Met 645 650 655 Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr Tyr Ser Ile 660 665 670 Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe His Asn Lys 675 680 685 Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Val Thr Ser Ile Val Asp 690 695 700 Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile Glu Tyr Ser 705 710 715 720 Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu Arg Ser Ile 725 730 735 Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn Met Asn Leu 740 745 750 Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp Val Met Lys 755 760 765 Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala Glu Ile Glu 770 775 780 Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser Leu Phe His 785 790 795 800 His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile Ser Ser Tyr 805 810 815 Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu Leu Phe Asp 820 825 830 Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys Arg Val Arg 835 840 845 Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val Leu Gln Ile 850 855 860 Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly Tyr Leu Pro 865 870 875 880 Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys Ser Ile Asp 885 890 895 Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly Cys Lys Val 900 905 910 Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr Ser His Leu 915 920 925 Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val Gly Lys Glu 930 935 940 Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu Tyr Met Thr 945 950 955 960 Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys Ser Lys Lys 965 970 975 Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln Glu Ala Leu 980 985 990 Arg Gln Phe Ala Ser His Tyr Lys Leu Asp Phe Asp Ser Leu Pro Lys 995 1000 1005 Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Gly Asp His Glu Lys 1010 1015 1020 Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr Tyr 1025 1030 1035 Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 1040 1045 1050 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1055 1060 1065 Leu Arg Gly Ile Ile Asp Glu Asp Glu Gln Pro Asp Gly Ala Lys 1070 1075 1080 Lys Gln Ala Leu Ala Arg Thr Lys 1085 1090 <210> SEQ ID NO 3 <211> LENGTH: 1093 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 3 Met Ser Asn Lys Glu Lys Asn Ala Ser Glu Thr Arg Lys Ala Tyr Thr 1 5 10 15 Thr Lys Met Ile Pro Arg Ser His Asp Arg Met Lys Leu Leu Gly Asn 20 25 30 Phe Met Asp Tyr Leu Met Asp Gly Thr Pro Ile Phe Phe Glu Leu Trp 35 40 45 Asn Gln Phe Gly Gly Gly Ile Asp Arg Asp Ile Ile Ser Gly Thr Ala 50 55 60 Asn Lys Asp Lys Ile Ser Asp Asp Leu Leu Leu Ala Val Asn Trp Phe 65 70 75 80 Lys Val Met Pro Ile Asn Ser Lys Pro Gln Gly Val Ser Pro Ser Asn 85 90 95 Leu Ala Asn Leu Phe Gln Gln Tyr Ser Gly Ser Glu Pro Asp Ile Gln 100 105 110 Ala Gln Glu Tyr Phe Ala Ser Asn Phe Asp Thr Glu Lys His Gln Trp 115 120 125 Lys Asp Met Arg Val Glu Tyr Glu Arg Leu Leu Ala Glu Leu Gln Leu 130 135 140 Ser Arg Ser Asp Met His His Asp Leu Lys Leu Met Tyr Lys Glu Lys 145 150 155 160 Cys Ile Gly Leu Ser Leu Ser Thr Ala His Tyr Ile Thr Ser Val Met 165 170 175 Phe Gly Thr Gly Ala Lys Asn Asn Arg Gln Thr Lys His Gln Phe Tyr 180 185 190 Ser Lys Val Ile Gln Leu Leu Glu Glu Ser Thr Gln Ile Asn Ser Val 195 200 205 Glu Gln Leu Ala Ser Ile Ile Leu Lys Ala Gly Asp Cys Asp Ser Tyr 210 215 220 Arg Lys Leu Arg Ile Arg Cys Ser Arg Lys Gly Ala Thr Pro Ser Ile 225 230 235 240 Leu Lys Ile Val Gln Asp Tyr Glu Leu Gly Thr Asn His Asp Asp Glu 245 250 255 Val Asn Val Pro Ser Leu Ile Ala Asn Leu Lys Glu Lys Leu Gly Arg 260 265 270 Phe Glu Tyr Glu Cys Glu Trp Lys Cys Met Glu Lys Ile Lys Ala Phe 275 280 285 Leu Ala Ser Lys Val Gly Pro Tyr Tyr Leu Gly Ser Tyr Ser Ala Met 290 295 300 Leu Glu Asn Ala Leu Ser Pro Ile Lys Gly Met Thr Thr Lys Asn Cys 305 310 315 320 Lys Phe Val Leu Lys Gln Ile Asp Ala Lys Asn Asp Ile Lys Tyr Glu 325 330 335 Asn Glu Pro Phe Gly Lys Ile Val Glu Gly Phe Phe Asp Ser Pro Tyr 340 345 350 Phe Glu Ser Asp Thr Asn Val Lys Trp Val Leu His Pro His His Ile 355 360 365 Gly Glu Ser Asn Ile Lys Thr Leu Trp Glu Asp Leu Asn Ala Ile His 370 375 380 Ser Lys Tyr Glu Glu Asp Ile Ala Ser Leu Ser Glu Asp Lys Lys Glu 385 390 395 400 Lys Arg Ile Lys Val Tyr Gln Gly Asp Val Cys Gln Thr Ile Asn Thr 405 410 415 Tyr Cys Glu Glu Val Gly Lys Glu Ala Lys Thr Pro Leu Val Gln Leu 420 425 430 Leu Arg Tyr Leu Tyr Ser Arg Lys Asp Asp Ile Ala Val Asp Lys Ile 435 440 445 Ile Asp Gly Ile Thr Phe Leu Ser Lys Lys His Lys Val Glu Lys Gln 450 455 460 Lys Ile Asn Pro Val Ile Gln Lys Tyr Pro Ser Phe Asn Phe Gly Asn 465 470 475 480 Asn Ser Lys Leu Leu Gly Lys Ile Ile Ser Pro Lys Asp Lys Leu Lys 485 490 495 His Asn Leu Lys Cys Asn Arg Asn Gln Val Asp Asn Tyr Ile Trp Ile 500 505 510 Glu Ile Lys Val Leu Asn Thr Lys Thr Met Arg Trp Glu Lys His His 515 520 525 Tyr Ala Leu Ser Ser Thr Arg Phe Leu Glu Glu Val Tyr Tyr Pro Ala 530 535 540 Thr Ser Glu Asn Pro Pro Asp Ala Leu Ala Ala Arg Phe Arg Thr Lys 545 550 555 560 Thr Asn Gly Tyr Glu Gly Lys Pro Ala Leu Ser Ala Glu Gln Ile Glu 565 570 575 Gln Ile Arg Ser Ala Pro Val Gly Leu Arg Lys Val Lys Lys Arg Gln 580 585 590 Met Arg Leu Glu Ala Ala Arg Gln Gln Asn Leu Leu Pro Arg Tyr Thr 595 600 605 Trp Gly Lys Asp Phe Asn Ile Asn Ile Cys Lys Arg Gly Asn Asn Phe 610 615 620 Glu Val Thr Leu Ala Thr Lys Val Lys Lys Lys Lys Glu Lys Asn Tyr 625 630 635 640 Lys Val Val Leu Gly Tyr Asp Ala Asn Ile Val Arg Lys Asn Thr Tyr 645 650 655 Ala Ala Ile Glu Ala His Ala Asn Gly Asp Gly Val Ile Asp Tyr Asn 660 665 670 Asp Leu Pro Val Lys Pro Ile Glu Ser Gly Phe Val Thr Val Glu Ser 675 680 685 Gln Val Arg Asp Lys Ser Tyr Asp Gln Leu Ser Tyr Asn Gly Val Lys 690 695 700 Leu Leu Tyr Cys Lys Pro His Val Glu Ser Arg Arg Ser Phe Leu Glu 705 710 715 720 Lys Tyr Arg Asn Gly Thr Met Lys Asp Asn Arg Gly Asn Asn Ile Gln 725 730 735 Ile Asp Phe Met Lys Asp Phe Glu Ala Ile Ala Asp Asp Glu Thr Ser 740 745 750 Leu Tyr Tyr Phe Asn Met Lys Tyr Cys Lys Leu Leu Gln Ser Ser Ile 755 760 765 Arg Asn His Ser Ser Gln Ala Lys Glu Tyr Arg Glu Glu Ile Phe Glu 770 775 780 Leu Leu Arg Asp Gly Lys Leu Ser Val Leu Lys Leu Ser Ser Leu Ser 785 790 795 800 Asn Leu Ser Phe Val Met Phe Lys Val Ala Lys Ser Leu Ile Gly Thr 805 810 815 Tyr Phe Gly His Leu Leu Lys Lys Pro Lys Asn Ser Lys Ser Asp Val 820 825 830 Lys Ala Pro Pro Ile Thr Asp Glu Asp Lys Gln Lys Ala Asp Pro Glu 835 840 845 Met Phe Ala Leu Arg Leu Ala Leu Glu Glu Lys Arg Leu Asn Lys Val 850 855 860 Lys Ser Lys Lys Glu Val Ile Ala Asn Lys Ile Val Ala Lys Ala Leu 865 870 875 880 Glu Leu Arg Asp Lys Tyr Gly Pro Val Leu Ile Lys Gly Glu Asn Ile 885 890 895 Ser Asp Thr Thr Lys Lys Gly Lys Lys Ser Ser Thr Asn Ser Phe Leu 900 905 910 Met Asp Trp Leu Ala Arg Gly Val Ala Asn Lys Val Lys Glu Met Val 915 920 925 Met Met His Gln Gly Leu Glu Phe Val Glu Val Asn Pro Asn Phe Thr 930 935 940 Ser His Gln Asp Pro Phe Val His Lys Asn Pro Glu Asn Thr Phe Arg 945 950 955 960 Ala Arg Tyr Ser Arg Cys Thr Pro Ser Glu Leu Thr Glu Lys Asn Arg 965 970 975 Lys Glu Ile Leu Ser Phe Leu Ser Asp Lys Pro Ser Lys Arg Pro Thr 980 985 990 Asn Ala Tyr Tyr Asn Glu Gly Ala Met Ala Phe Leu Ala Thr Tyr Gly 995 1000 1005 Leu Lys Lys Asn Asp Val Leu Gly Val Ser Leu Glu Lys Phe Lys 1010 1015 1020 Gln Ile Met Ala Asn Ile Leu His Gln Arg Ser Glu Asp Gln Leu 1025 1030 1035 Leu Phe Pro Ser Arg Gly Gly Met Phe Tyr Leu Ala Thr Tyr Lys 1040 1045 1050 Leu Asp Ala Asp Ala Thr Ser Val Asn Trp Asn Gly Lys Gln Phe 1055 1060 1065 Trp Val Cys Asn Ala Asp Leu Val Ala Ala Tyr Asn Val Gly Leu 1070 1075 1080 Val Asp Ile Gln Lys Asp Phe Lys Lys Lys 1085 1090 <210> SEQ ID NO 4 <211> LENGTH: 1033 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 4 Met Met Ser Asp Asn Ile Ile Leu Pro Tyr Asn Ser Lys Leu Ala Pro 1 5 10 15 Asp Glu Arg Lys Gln Arg Leu Leu Asn Asp Thr Phe Asn Trp Phe Asp 20 25 30 Met Cys Asn Glu Val Phe Phe Asp Phe Val Lys Asn Leu Tyr Gly Gly 35 40 45 Val Lys His Glu His Leu Ile Leu Val Asn Phe Ala Glu Lys Pro Lys 50 55 60 Lys Val Ser Asn Ser Lys Lys Pro Lys Lys Lys Asp Gln Glu Val Asn 65 70 75 80 Ile His Val Glu Pro Asn Gln Ala Glu Trp Val Asp Asn Ala Cys Ala 85 90 95 Thr Phe Trp Phe Arg Leu Gln Ala Lys Ser Thr Val Gln Leu Asp Gln 100 105 110 Ser Val Gln Thr Ala Glu Glu Arg Ile Arg Arg Phe Arg Asp Tyr Ala 115 120 125 Gly His Glu Pro Ser Ser Phe Ala Lys Ser Tyr Leu Asn Gly Asn Tyr 130 135 140 Asp Pro Glu Lys Thr Glu Trp Val Asp Cys Arg Leu Leu Tyr Val Asn 145 150 155 160 Phe Cys Arg Asn Leu Asn Val Asn Leu Asp Ala Asp Ile Arg Thr Met 165 170 175 Val Glu His Asn Leu Leu Pro Val Leu Pro Gly Gln Asp Phe Lys Thr 180 185 190 Asn Asn Val Phe Ser Asn Ile Phe Gly Val Gly Asn Lys Glu Asp Lys 195 200 205 Gly Gln Lys Thr Asn Trp Leu Asn Thr Val Ser Glu Gly Leu Gln Ser 210 215 220 Lys Glu Ile Trp Asn Trp Asp Glu Tyr Arg Asp Leu Ile Ser Arg Ser 225 230 235 240 Thr Gly Cys Ser Thr Ala Ala Glu Leu Arg Ser Glu Ser Ile Gly Arg 245 250 255 Pro Ser Met Leu Ala Val Asp Phe Ala Ser Glu Lys Ser Gly Gln Ile 260 265 270 Ser Gln Glu Trp Leu Ala Glu Arg Val Lys Ser Phe Arg Ala Ala Ala 275 280 285 Ser Gln Lys Ser Lys Ile Tyr Asp Met Pro Asn Arg Leu Val Leu Lys 290 295 300 Glu Tyr Ile Ala Ser Lys Ile Gly Pro Phe Lys Leu Glu Arg Trp Ser 305 310 315 320 Ala Ala Ala Val Ser Ala Tyr Lys Asp Val Arg Ser Lys Asn Ser Ile 325 330 335 Asn Leu Leu Tyr Ser Lys Glu Arg Leu Trp Arg Cys Lys Glu Ile Ala 340 345 350 Gln Ile Leu Val Asp Asn Thr Gln Val Ala Glu Ala Gln Gln Ile Leu 355 360 365 Val Asn Tyr Ser Ser Gly Asp Thr Asn Ser Phe Thr Val Glu Asn Arg 370 375 380 His Met Gly Asp Leu Thr Val Leu Phe Lys Ile Trp Glu Lys Met Asp 385 390 395 400 Met Asp Ser Gly Ile Glu Gln Tyr Ser Glu Ile Tyr Arg Asp Glu Tyr 405 410 415 Ser Arg Asp Pro Ile Thr Glu Leu Leu Arg Tyr Leu Tyr Asn His Arg 420 425 430 His Ile Ser Ala Lys Thr Phe Arg Ala Ala Ala Arg Leu Asn Ser Leu 435 440 445 Leu Leu Lys Asn Asp Arg Lys Lys Ile His Pro Thr Ile Ser Gly Arg 450 455 460 Thr Ser Val Ser Phe Gly His Ser Thr Ile Lys Gly Cys Ile Thr Pro 465 470 475 480 Pro Asp His Ile Val Lys Asn Arg Lys Glu Asn Ala Gly Ser Thr Gly 485 490 495 Met Ile Trp Val Thr Met Gln Leu Ile Asp Asn Gly Arg Trp Ala Asp 500 505 510 His His Ile Pro Phe His Asn Ser Arg Tyr Tyr Arg Asp Phe Tyr Ala 515 520 525 Tyr Arg Ala Asp Leu Pro Thr Ile Ser Asp Pro Arg Arg Lys Ser Phe 530 535 540 Gly His Arg Ile Gly Asn Asn Ile Ser Asp Thr Arg Met Ile Asn His 545 550 555 560 Asp Cys Lys Lys Ala Ser Lys Met Tyr Leu Arg Thr Ile Gln Asn Met 565 570 575 Thr His Asn Val Ala Phe Asp Gln Gln Thr Gln Phe Ala Val Arg Arg 580 585 590 Tyr Ala Asp Asn Asn Phe Thr Ile Thr Ile Gln Ala Arg Val Val Gly 595 600 605 Arg Lys Tyr Lys Lys Glu Ile Ser Val Gly Asp Arg Val Met Gly Val 610 615 620 Asp Gln Asn Gln Thr Thr Ser Asn Thr Tyr Ser Val Trp Glu Val Val 625 630 635 640 Ala Glu Gly Thr Glu Asn Ser Tyr Pro Tyr Lys Gly Asn Asn Tyr Arg 645 650 655 Leu Val Glu Asp Gly Phe Ile Arg Ser Glu Cys Ser Gly Arg Asp Gln 660 665 670 Leu Ser Tyr Asp Gly Leu Asp Phe Gln Asp Phe Ala Gln Trp Arg Arg 675 680 685 Glu Arg Tyr Ala Phe Leu Ser Ser Val Gly Cys Ile Leu Asn Asp Glu 690 695 700 Ile Glu Pro Gln Ile Pro Val Ser Ala Glu Lys Ala Lys Lys Lys Lys 705 710 715 720 Lys Phe Ser Lys Trp Arg Gly Cys Ser Leu Tyr Ser Trp Asn Leu Cys 725 730 735 Tyr Ala Tyr Tyr Leu Lys Gly Leu Met His Glu Asn Leu Ala Asn Asn 740 745 750 Pro Ala Gly Phe Arg Gln Glu Ile Leu Asn Phe Ile Gln Gly Ser Arg 755 760 765 Gly Val Arg Leu Cys Ser Leu Asn His Thr Ser Phe Arg Leu Leu Ser 770 775 780 Lys Ala Lys Ser Leu Ile His Ser Phe Phe Gly Leu Asn Asn Ile Lys 785 790 795 800 Asp Pro Glu Ser Gln Arg Asp Phe Asp Pro Glu Ile Tyr Asp Ile Met 805 810 815 Val Asn Leu Thr Gln Arg Lys Thr Asn Lys Arg Lys Glu Lys Ala Asn 820 825 830 Arg Ile Thr Ser Ser Ile Leu Gln Ile Ala Asn Arg Leu Asn Val Ser 835 840 845 Arg Ile Val Ile Glu Asn Asp Leu Pro Asn Ala Ser Ser Lys Asn Lys 850 855 860 Ala Ser Ala Asn Gln Arg Ala Thr Asp Trp Cys Ala Arg Asn Val Ser 865 870 875 880 Glu Lys Leu Glu Tyr Ala Cys Lys Met Leu Gly Ile Ser Leu Trp Gln 885 890 895 Ile Asp Pro Arg Asp Thr Ser His Leu Asp Pro Phe Val Val Gly Lys 900 905 910 Glu Ala Arg Phe Met Lys Ile Lys Val Ser Asp Ile Asn Glu Tyr Thr 915 920 925 Ile Ser Asn Phe Lys Lys Trp His Ala Asn Ile Ala Thr Thr Ser Thr 930 935 940 Thr Ala Pro Leu Tyr His Asp Ala Leu Lys Ala Phe Ser Ser His Tyr 945 950 955 960 Gly Ile Asp Trp Asp Asn Leu Pro Glu Met Lys Phe Trp Glu Leu Lys 965 970 975 Asn Ala Leu Lys Asp His Lys Glu Val Phe Ile Pro Asn Arg Gly Gly 980 985 990 Arg Cys Tyr Leu Ser Thr Leu Pro Val Thr Ser Thr Ser Glu Lys Ile 995 1000 1005 Val Phe Asn Gly Arg Glu Arg Trp Leu Asn Ala Ser Asp Ile Val 1010 1015 1020 Ala Gly Val Asn Ile Val Leu Arg Ser Val 1025 1030 <210> SEQ ID NO 5 <211> LENGTH: 1054 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 5 Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu 1 5 10 15 Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly 20 25 30 Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr 35 40 45 Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp 50 55 60 Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp 65 70 75 80 Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu 85 90 95 Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser 100 105 110 Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr 115 120 125 Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His 130 135 140 Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu 145 150 155 160 Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu 165 170 175 Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu 180 185 190 Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln 195 200 205 Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln 210 215 220 Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile 225 230 235 240 Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp 245 250 255 Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys 260 265 270 Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp 275 280 285 Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys 290 295 300 Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln 305 310 315 320 Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu 325 330 335 Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr 340 345 350 Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys 355 360 365 Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu 370 375 380 Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile 385 390 395 400 Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile 405 410 415 Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln 420 425 430 Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn 435 440 445 Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser 450 455 460 Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly 465 470 475 480 Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln 485 490 495 Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg 500 505 510 Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala 515 520 525 Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg 530 535 540 Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys 545 550 555 560 Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile 565 570 575 Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly 580 585 590 Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr 595 600 605 Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly 610 615 620 Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu 625 630 635 640 Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr 645 650 655 Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu 660 665 670 Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu 675 680 685 Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr 690 695 700 Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly 705 710 715 720 Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe 725 730 735 Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser 740 745 750 Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe 755 760 765 Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln 770 775 780 Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu 785 790 795 800 Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn 805 810 815 Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly 820 825 830 Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn 835 840 845 Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg 850 855 860 Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu 865 870 875 880 Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala 885 890 895 Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp 900 905 910 Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr 915 920 925 Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu 930 935 940 Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys 945 950 955 960 Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu 965 970 975 Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile 980 985 990 Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe 995 1000 1005 Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala 1010 1015 1020 Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser 1025 1030 1035 Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr 1040 1045 1050 Ser <210> SEQ ID NO 6 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 6 cccacaatac ctgagaaatc cgtcctacgt tgacgg 36 <210> SEQ ID NO 7 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 7 aatttttgtg cccatcgttg gcac 24 <210> SEQ ID NO 8 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 8 ctctcaatgc cttagaaatc cgtccttggt tgacgg 36 <210> SEQ ID NO 9 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 9 gcaacaccta agaaatccgt ctttcattga cggg 34 <210> SEQ ID NO 10 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 10 gttgcaaaac ccaagaaatc cgtctttcat tgacgg 36 <210> SEQ ID NO 11 <211> LENGTH: 1080 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 11 Met Pro Arg Asn Tyr Phe Leu Gly Ile Phe Ser Leu Gln Lys Asn Lys 1 5 10 15 Ser Val Val His Cys Ser Val Glu Ile Arg His Lys Gly Tyr Arg Ser 20 25 30 Ser Val Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Ala Ser Lys Leu 35 40 45 Ala Pro Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp 50 55 60 Leu Asp His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe 65 70 75 80 Gly Ala Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser 85 90 95 Lys Phe Asp Ala Asp Leu Ile Cys Ala Ile Met Trp Phe Arg Leu Glu 100 105 110 Glu Lys Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met 115 120 125 Arg Leu Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln 130 135 140 Glu Tyr Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Glu Trp Val Asp 145 150 155 160 Cys Arg Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln 165 170 175 Glu Ser Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile 180 185 190 Pro Val Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln 195 200 205 Leu Phe Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ala Met 210 215 220 Leu Glu Glu Ile Ser Asn Ile Leu Ala Asp Lys Lys Pro Asp Thr Trp 225 230 235 240 Glu Glu Tyr His Asp Leu Ile Lys Lys Asn Phe Asn Val Asp Asn Tyr 245 250 255 Lys Glu Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser 260 265 270 Ser Leu Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro 275 280 285 Asn Phe Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys 290 295 300 Lys Lys Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe 305 310 315 320 Ile Ala Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val 325 330 335 Leu Asn Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile 340 345 350 Leu Tyr Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu 355 360 365 Leu Lys Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg 370 375 380 Arg Gly Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala 385 390 395 400 Arg Leu Asn Glu Leu Phe Glu Ile Trp Gln Asp Leu Thr Met Asp Asp 405 410 415 Gly Ile Arg Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg 420 425 430 Pro Val Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile 435 440 445 Thr Ala Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu 450 455 460 Thr Asn Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val 465 470 475 480 Cys Asn Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro 485 490 495 Asn Gln Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp 500 505 510 Val Thr Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu 515 520 525 Pro Phe Tyr Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu 530 535 540 Gly Leu Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln 545 550 555 560 Val Gly Ser Thr Ile Ser Ala Thr Ser Leu Ala Ala Leu Lys Ser Gln 565 570 575 Glu Glu Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His 580 585 590 Lys Ser Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe 595 600 605 Asp Lys Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe 610 615 620 Ile Thr Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu 625 630 635 640 Asn Ile Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro 645 650 655 Cys Thr Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser 660 665 670 Phe Phe His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile 675 680 685 Thr Ser Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala 690 695 700 Gly Ile Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln 705 710 715 720 Phe Leu Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp 725 730 735 Arg Asn Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu 740 745 750 Leu Asp Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe 755 760 765 Arg Ala Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu 770 775 780 Gly Ser Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser 785 790 795 800 Leu Ile Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp 805 810 815 Gln Glu Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly 820 825 830 Asp Lys Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser 835 840 845 Thr Val Leu Gln Ile Ala Arg Glu Asn Asn Ile Lys Ser Leu Cys Val 850 855 860 Glu Gly Asp Leu Pro Thr Ala Thr Lys Lys Thr Lys Pro Lys Gln Asn 865 870 875 880 Gln Lys Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn 885 890 895 Asp Gly Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg 900 905 910 Asp Thr Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr 915 920 925 Lys Val Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile 930 935 940 Lys Glu Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr 945 950 955 960 Lys Lys Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu 965 970 975 Tyr Gln Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Glu Leu Asp Phe 980 985 990 Asp Ser Leu Pro Lys Met Lys Phe Tyr Asp Leu Ala Lys Arg Leu Gly 995 1000 1005 Asp His Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr 1010 1015 1020 Leu Ser Thr Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe 1025 1030 1035 Asn Gly Arg Glu Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala 1040 1045 1050 Val Asn Ile Val Leu Arg Gly Ile Arg Asp Glu Asp Glu Gln Pro 1055 1060 1065 Asp Asp Ala Lys Lys Gln Ala Leu Ala Arg Thr Lys 1070 1075 1080 <210> SEQ ID NO 12 <211> LENGTH: 1046 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 12 Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Ala Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala 35 40 45 Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe 50 55 60 Asp Ala Asp Leu Ile Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Glu Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser 130 135 140 Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ala Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Leu Ala Asp Lys Lys Pro Asp Thr Trp Glu Glu 195 200 205 Tyr His Asp Leu Ile Lys Lys Asn Phe Asn Val Asp Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Lys 260 265 270 Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu 355 360 365 Asn Glu Leu Phe Glu Ile Trp Gln Asp Leu Thr Met Asp Asp Gly Ile 370 375 380 Arg Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val 385 390 395 400 Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Thr Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe 485 490 495 Tyr Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Thr Ile Ser Ala Thr Ser Leu Ala Ala Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile 595 600 605 Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp 705 710 715 720 Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu 770 775 780 Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys 785 790 795 800 Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Ile Lys Ser Leu Cys Val Glu Gly 820 825 830 Asp Leu Pro Thr Ala Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln 930 935 940 Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Glu Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Asp Leu Ala Lys Arg Leu Gly Asp His 965 970 975 Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Arg Asp Glu Asp Glu Gln Pro Asp Asp Ala Lys 1025 1030 1035 Lys Gln Ala Leu Ala Arg Thr Lys 1040 1045 <210> SEQ ID NO 13 <211> LENGTH: 1046 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 13 Met Val Ser Glu Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Ser Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala 35 40 45 Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe 50 55 60 Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Gln Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser 130 135 140 Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ser Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Leu Ala Asp Lys Asn Pro Asn Thr Trp Glu Glu 195 200 205 Tyr Gln Asp Leu Ile Lys Lys Thr Phe Asn Val Asp Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Gly Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg 260 265 270 Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu 355 360 365 Asn Glu Leu Phe Glu Ile Trp Gln Glu Leu Thr Met Asp Asp Gly Ile 370 375 380 Lys Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val 385 390 395 400 Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Asn Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe 485 490 495 His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Lys Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Thr Ile Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile 595 600 605 Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Val Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp 705 710 715 720 Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu 770 775 780 Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys 785 790 795 800 Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly 820 825 830 Tyr Leu Pro Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Tyr Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg His Thr Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln 930 935 940 Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Lys Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Gly Asp His 965 970 975 Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Ile Asp Glu Asp Glu Gln Pro Asp Gly Ala Lys 1025 1030 1035 Lys Gln Ala Thr Thr Arg Arg Thr 1040 1045 <210> SEQ ID NO 14 <211> LENGTH: 1098 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 14 Met Ser Ile Ser Asn Asn Asn Ile Leu Pro Tyr Asn Pro Lys Leu Leu 1 5 10 15 Pro Asp Asp Arg Lys His Lys Met Leu Val Asp Thr Phe Asn Gln Leu 20 25 30 Asp Leu Ile Arg Asn Asn Leu His Asp Met Ile Ile Ala Leu Tyr Gly 35 40 45 Ala Leu Lys Tyr Asp Asn Ile Lys Gln Phe Ala Ser Lys Glu Lys Pro 50 55 60 His Ile Ser Ala Asp Ala Leu Cys Ser Ile Asn Trp Phe Arg Leu Val 65 70 75 80 Lys Thr Asn Glu Arg Lys Pro Ala Ile Glu Ser Asn Gln Ile Ile Ser 85 90 95 Lys Phe Ile Gln Tyr Ser Gly His Thr Pro Asp Lys Tyr Ala Leu Ser 100 105 110 His Ile Thr Gly Asn His Glu Pro Ser His Lys Trp Ile Asp Cys Arg 115 120 125 Glu Tyr Ala Ile Asn Tyr Ala Arg Ile Met His Leu Ser Phe Ser Gln 130 135 140 Phe Gln Asp Leu Ala Thr Ala Cys Leu Asn Cys Lys Ile Leu Ile Leu 145 150 155 160 Asn Gly Thr Leu Thr Ser Ser Trp Ala Trp Gly Ala Asn Ser Ala Leu 165 170 175 Phe Gly Gly Ser Asp Lys Glu Asn Phe Ser Val Lys Ala Lys Ile Leu 180 185 190 Asn Ser Phe Ile Glu Asn Leu Lys Asp Glu Met Asn Thr Thr Lys Phe 195 200 205 Gln Val Val Glu Lys Val Cys Gln Gln Ile Gly Ser Ser Asp Ala Ala 210 215 220 Asp Leu Phe Asp Leu Tyr Arg Ser Thr Val Lys Asp Gly Asn Arg Gly 225 230 235 240 Pro Ala Thr Gly Arg Asn Pro Lys Val Met Asn Leu Phe Ser Gln Asp 245 250 255 Gly Glu Ile Ser Ser Glu Gln Arg Glu Asp Phe Ile Glu Ser Phe Gln 260 265 270 Lys Val Met Gln Glu Lys Asn Ser Lys Gln Ile Ile Pro His Leu Asp 275 280 285 Lys Leu Lys Tyr His Leu Val Lys Gln Ser Gly Leu Tyr Asp Ile Tyr 290 295 300 Ser Trp Ala Ala Ala Ile Lys Asn Ala Asn Ser Thr Ile Val Ala Ser 305 310 315 320 Asn Ser Ser Asn Leu Asn Thr Ile Leu Asn Lys Thr Glu Lys Gln Gln 325 330 335 Thr Phe Glu Glu Leu Arg Lys Asp Glu Lys Ile Val Ala Cys Ser Lys 340 345 350 Ile Leu Leu Ser Val Asn Asp Thr Leu Pro Glu Asp Leu His Tyr Asn 355 360 365 Pro Ser Thr Ser Asn Leu Gly Lys Asn Leu Asp Val Phe Phe Asp Leu 370 375 380 Leu Asn Glu Asn Ser Val His Thr Ile Glu Asn Lys Glu Glu Lys Asn 385 390 395 400 Lys Ile Val Lys Glu Cys Val Asn Gln Tyr Met Glu Glu Cys Lys Gly 405 410 415 Leu Asn Lys Pro Pro Met Pro Val Leu Leu Thr Phe Ile Ser Asp Tyr 420 425 430 Ala His Lys His Gln Ala Gln Asp Phe Leu Ser Ala Ala Lys Met Asn 435 440 445 Phe Ile Asp Leu Lys Ile Lys Ser Ile Lys Val Val Pro Thr Val His 450 455 460 Gly Ser Ser Pro Tyr Thr Trp Ile Ser Asn Leu Ser Lys Lys Asn Lys 465 470 475 480 Asp Gly Lys Met Ile Arg Thr Pro Asn Ser Ser Leu Ile Gly Trp Ile 485 490 495 Ile Pro Pro Glu Glu Ile His Asp Gln Lys Phe Ala Gly Gln Asn Pro 500 505 510 Ile Ile Trp Ala Val Leu Arg Val Tyr Cys Asn Asn Lys Trp Glu Met 515 520 525 His His Phe Pro Phe Ser Asp Ser Arg Phe Phe Thr Glu Val Tyr Ala 530 535 540 Tyr Lys Pro Asn Leu Pro Tyr Leu Pro Gly Gly Glu Asn Arg Ser Lys 545 550 555 560 Arg Phe Gly Tyr Arg His Ser Thr Asn Leu Ser Asn Glu Ser Arg Gln 565 570 575 Ile Leu Leu Asp Lys Ser Lys Tyr Ala Lys Ala Asn Lys Ser Val Leu 580 585 590 Arg Cys Met Glu Asn Met Thr His Asn Val Val Phe Asp Pro Lys Thr 595 600 605 Ser Leu Asn Ile Arg Ile Lys Thr Asp Lys Asn Asn Ser Pro Val Leu 610 615 620 Asp Asp Lys Gly Arg Ile Thr Phe Val Met Gln Ile Asn His Arg Ile 625 630 635 640 Leu Glu Lys Tyr Asn Asn Thr Lys Ile Glu Ile Gly Asp Arg Ile Leu 645 650 655 Ala Tyr Asp Gln Asn Gln Ser Glu Asn His Thr Tyr Ala Ile Leu Gln 660 665 670 Arg Thr Glu Glu Gly Ser His Ala His Gln Phe Asn Gly Trp Tyr Val 675 680 685 Arg Val Leu Glu Thr Gly Lys Val Thr Ser Ile Val Gln Gly Leu Ser 690 695 700 Gly Pro Ile Asp Gln Leu Asn Tyr Asp Gly Met Pro Val Thr Ser His 705 710 715 720 Lys Phe Asn Cys Trp Gln Ala Asp Arg Ser Ala Phe Val Ser Gln Phe 725 730 735 Ala Ser Leu Lys Ile Ser Glu Thr Glu Thr Phe Asp Glu Ala Tyr Gln 740 745 750 Ala Ile Asn Ala Gln Gly Ala Tyr Thr Trp Asn Leu Phe Tyr Leu Arg 755 760 765 Ile Leu Arg Lys Ala Leu Arg Val Cys His Met Glu Asn Ile Asn Gln 770 775 780 Phe Arg Glu Glu Ile Leu Ala Ile Ser Lys Asn Arg Leu Ser Pro Met 785 790 795 800 Ser Leu Gly Ser Leu Ser Gln Asn Ser Leu Lys Met Ile Arg Ala Phe 805 810 815 Lys Ser Ile Ile Asn Cys Tyr Met Ser Arg Met Ser Phe Val Asp Glu 820 825 830 Leu Gln Lys Lys Glu Gly Asp Leu Glu Leu His Thr Ile Met Arg Leu 835 840 845 Thr Asp Asn Lys Leu Asn Asp Lys Arg Val Glu Lys Ile Asn Arg Ala 850 855 860 Ser Ser Phe Leu Thr Asn Lys Ala His Ser Met Gly Cys Lys Met Ile 865 870 875 880 Val Gly Glu Ser Asp Leu Pro Val Ala Asp Ser Lys Thr Ser Lys Lys 885 890 895 Gln Asn Val Asp Arg Met Asp Trp Cys Ala Arg Ala Leu Ser His Lys 900 905 910 Val Glu Tyr Ala Cys Lys Leu Met Gly Leu Ala Tyr Arg Gly Ile Pro 915 920 925 Ala Tyr Met Ser Ser His Gln Asp Pro Leu Val His Leu Val Glu Ser 930 935 940 Lys Arg Ser Val Leu Arg Pro Arg Phe Val Val Ala Asp Lys Ser Asp 945 950 955 960 Val Lys Gln His His Leu Asp Asn Leu Arg Arg Met Leu Asn Ser Lys 965 970 975 Thr Lys Val Gly Thr Ala Val Tyr Tyr Arg Glu Ala Val Glu Leu Met 980 985 990 Cys Glu Glu Leu Gly Ile His Lys Thr Asp Met Ala Lys Gly Lys Val 995 1000 1005 Ser Leu Ser Asp Phe Val Asp Lys Phe Ile Gly Glu Lys Ala Ile 1010 1015 1020 Phe Pro Gln Arg Gly Gly Arg Phe Tyr Met Ser Thr Lys Arg Leu 1025 1030 1035 Thr Thr Gly Ala Lys Leu Ile Cys Tyr Ser Gly Ser Asp Val Trp 1040 1045 1050 Leu Ser Asp Ala Asp Glu Ile Ala Ala Ile Asn Ile Gly Met Phe 1055 1060 1065 Val Val Cys Asp Gln Thr Gly Ala Phe Lys Lys Lys Lys Lys Glu 1070 1075 1080 Lys Leu Asp Asp Glu Glu Cys Asp Ile Leu Pro Phe Arg Pro Met 1085 1090 1095 <210> SEQ ID NO 15 <211> LENGTH: 1088 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 15 Met Ser Ser Gln Val Val Arg Pro Tyr Asn Ala Lys Phe Leu Pro Asp 1 5 10 15 Asp Arg Lys His Lys Met Leu Thr Asp Thr Ile Asn Gln Leu Asp Lys 20 25 30 Ile Ser Ser Lys His Phe Asp Leu Leu Val Ala Phe Tyr Gly Ser Ile 35 40 45 Gln His Lys His Val Ser Ile Asn Asp Lys Gln Glu Glu His Ile Thr 50 55 60 Pro Asp Ser Val Cys Ala Ile Asn Trp Phe Arg Pro Met Ser Lys Asp 65 70 75 80 Tyr Ala Lys Tyr Gln Val Lys Ile Asp Ser Met Ile Thr Asn Phe Lys 85 90 95 Glu Tyr Ala Gly His Ile Pro Asp Lys Tyr Ala Ile Glu Tyr Met Gly 100 105 110 Ser Asn Ile Asp Thr Asp Arg Phe Val Trp Val Asp Cys Arg Asn Phe 115 120 125 Ala Lys Asp Tyr Val Arg Asn Met Asp Met Ser Phe Ser Glu Phe Gln 130 135 140 Asn Leu Val Asp Ala Leu Val Phe Cys Lys Ile Leu Ala Leu Asn Glu 145 150 155 160 Ser Thr Ser Thr Asn Trp Ala Trp Gly Ala Ile Ser Ala Ile Tyr Gly 165 170 175 Gly Gly Asp Lys Glu Asp Ser Gln Phe Lys Ala Lys Val Leu Asn Thr 180 185 190 Phe Val Lys Ala Leu Asn Asp Glu Asn Asn Lys Thr Lys Phe Asp Val 195 200 205 Ile Asn Lys Val Cys Ser Asp Leu Gly Tyr Asn Asp His Leu Ser Leu 210 215 220 Ile Glu Asp Phe Arg Ser Thr Ile Asp Glu Asn Gly Asn Lys Lys Ser 225 230 235 240 Ala Ser Gly Ser Pro Pro Ala Ile Ala Lys Phe Thr Glu Asp Gly Glu 245 250 255 Ile Ser Asp Asn Tyr Arg Arg Ala Cys Ile Ser Ser Phe Ser Lys Thr 260 265 270 Ala Lys Glu Lys Gln Asp Lys Lys Ser Ile Pro His Leu Asp Ile Leu 275 280 285 Lys Thr His Met Ile Ala Met Cys Gly Glu Tyr Asn Thr Tyr Ala Trp 290 295 300 Thr Glu Ala Ile Lys Asn Ala Asn Thr Asp Ile Thr Ser Arg Asn Thr 305 310 315 320 Arg Asn Met Thr Phe Ile Lys Glu Lys Ile Glu Ser Arg Asn Ser Leu 325 330 335 Lys Ile Tyr Asp Thr Glu Glu Asn Met Lys Ala Ala Lys Ile Leu Asn 340 345 350 Gly Ile Asn His Lys Leu Thr Pro Asp Leu His Tyr Thr Pro Ala Pro 355 360 365 Lys His Leu Gly Lys Asn Leu Lys Asp Leu Phe Glu Met Leu Glu Glu 370 375 380 Lys Asn Ile Leu Ala Gln Asn Glu Lys Glu Lys Lys Ala Ala Leu Asp 385 390 395 400 Glu Cys Ile Lys Gln Tyr Ile Asp Asp Cys Lys Gly Leu Asn Gln Gln 405 410 415 Pro Ile Ala Ser Leu Leu Ala His Ile Ser Asn Tyr His Lys Glu Ile 420 425 430 Thr Ala Glu Asn Phe Leu Asp Gly Ala Lys Leu Leu Val Leu Leu Gln 435 440 445 Lys Ile Asn Arg Gln Lys Ala His Pro Ser Val Phe Ser Pro Lys Ala 450 455 460 Tyr Thr Trp Gly Ser Lys Leu Glu Lys Asn Arg Arg Ala Ala Asn Ser 465 470 475 480 Ala Leu Leu Gly Trp Ile Val Pro Pro Glu Glu Lys His Lys Asp Arg 485 490 495 His Ala Gly Gln His Pro Val Met Trp Val Thr Met Thr Leu Leu Asn 500 505 510 Asn Gly Lys Trp Glu Lys His His Val Pro Phe Thr Asn Ser Arg Phe 515 520 525 Phe Ser Glu Val Tyr Ala Tyr Gln Pro Glu Leu Pro Tyr Lys Glu Gly 530 535 540 Gly Tyr Ala Arg Asn Ser Lys Thr Ala Thr Lys Pro Ser Gln Ile Met 545 550 555 560 Leu Pro Ala Tyr Ala Glu Ser Met Arg His His Ile Ala Thr Lys Gly 565 570 575 Asn Gly His Lys Lys Ser Glu Lys Ile Val Leu Arg Ala Leu Ser Asn 580 585 590 Ile Arg His Asn Val Arg Phe Asp Pro Ser Thr Ser Phe Phe Val Arg 595 600 605 Ile Met Arg Asp Lys Lys Gly Asn His Arg Leu Asp Thr Lys Gly Arg 610 615 620 Ile Thr Phe Gly Leu Gln Ile Asn His Arg Ile Thr Val Gly Lys Thr 625 630 635 640 Lys Ser Glu Ile Asn Ile Gly Asp Arg Leu Leu Ala Phe Asp Gln Asn 645 650 655 Gln Ser Glu Asn His Thr Phe Ala Ile Met Gln Arg Val Glu Glu Asn 660 665 670 Thr Pro Asn Ser His Gln Phe Asn Gly Trp Asn Ile Arg Val Leu Glu 675 680 685 Thr Gly Lys Val Val Ser Met Thr Lys Gly Ile Glu Ser Tyr Tyr Asp 690 695 700 Gln Leu Ser Tyr Asp Gly Val Pro Tyr Glu Thr Lys Lys Phe Glu Asp 705 710 715 720 Trp Arg Asn Glu Arg Lys Ala Phe Val Lys Lys Asn Lys Asp Ile Val 725 730 735 Ile Lys Glu Glu Lys Thr Phe Gly Gln Met Phe Ala Glu Ile Lys Lys 740 745 750 Ser Ser Leu Tyr Lys Trp Asn Leu Ser Tyr Leu Lys Ile Leu Arg Met 755 760 765 Ala Ile Arg Ala Lys Ser Gly Asp Thr Val Ser Leu Phe Arg Glu Glu 770 775 780 Leu Ile Ser Ile Ala Lys Asn Arg Phe Gly Pro Leu Gly Leu Gly Ser 785 790 795 800 Leu Ser Ala Ser Ser Leu Lys Met Leu Gly Ala Phe Cys Gly Val Ile 805 810 815 Gln Ser Tyr Phe Ser Val Leu Asn Cys Leu Asp Asp Lys Asp Lys Ser 820 825 830 Asn Phe Asp Ser Glu Leu Tyr Phe Tyr Leu Val Ser Ala Phe Glu Lys 835 840 845 Arg Val Phe Lys Arg Asn Glu Lys Thr Ser Arg Ala Ser Ser Phe Ile 850 855 860 Met Ala Met Ala Tyr Asn His Gly Cys Lys Met Ile Val Cys Glu Asp 865 870 875 880 Asp Leu Pro Thr Ala Gly Ala Gly Ala Asn Lys Arg Gln Asn Ser Asp 885 890 895 Arg Met Asp Trp Cys Ala Arg Ser Leu Ala Gln Lys Ile Lys Thr Gly 900 905 910 Cys Glu Ala Met Ser Ile Ala Tyr Arg Ala Ile Pro Ala Tyr Met Ser 915 920 925 Ser His Gln Asp Pro Leu Val His Leu Ala Asp Gly Lys Thr Ser Val 930 935 940 Leu Cys Pro Arg Phe Ala Leu Val Ser Lys Asp Asp Ile Lys Gln Tyr 945 950 955 960 Gln Leu Asp Gly Met Arg Arg Met Leu Asn Ser Lys Ser Lys Ile Gly 965 970 975 Thr Ala Val Tyr Tyr Arg Ala Ala Val Glu Leu Leu Cys Lys Glu Leu 980 985 990 Gly Ile Asn Lys Thr Asp Ile Ala Lys Gly Lys Leu Ser Val Ser Gln 995 1000 1005 Phe Ala Asp Ile Val Asn Gly Glu Ile Leu Leu Pro Gln Arg Gly 1010 1015 1020 Gly Arg Val Tyr Leu Ala Thr Lys Glu Leu Thr Asn Gly Ala Lys 1025 1030 1035 Leu Val Ser Tyr Asn Gly Ser Asp Val Trp Leu Ser Asn Ala Asp 1040 1045 1050 Glu Ile Ala Ala Ile Asn Ile Gly Met Phe Val Val Cys Thr Gln 1055 1060 1065 Thr Gly Val Phe Gly Lys Lys Lys Lys Lys Asp Glu Gln Asp Gly 1070 1075 1080 Asp Ile Glu Ile Ala 1085 <210> SEQ ID NO 16 <211> LENGTH: 1074 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 16 Met Ala Ser Ile Ser Arg Pro Tyr Gly Thr Lys Leu Arg Pro Asp Ala 1 5 10 15 Arg Lys Lys Glu Met Leu Asp Lys Phe Phe Asn Thr Leu Thr Lys Gly 20 25 30 Gln Arg Val Phe Ala Asp Leu Ala Leu Cys Ile Tyr Gly Ser Leu Thr 35 40 45 Leu Glu Met Ala Lys Ser Leu Glu Pro Glu Ser Asp Ser Glu Leu Val 50 55 60 Cys Ala Ile Gly Trp Phe Arg Leu Val Asp Lys Thr Ile Trp Ser Lys 65 70 75 80 Asp Gly Ile Lys Gln Glu Asn Leu Val Lys Gln Tyr Glu Ala Tyr Ser 85 90 95 Gly Lys Glu Ala Ser Glu Val Val Lys Thr Tyr Leu Asn Ser Pro Ser 100 105 110 Ser Asp Lys Tyr Val Trp Ile Asp Cys Arg Gln Lys Phe Leu Arg Phe 115 120 125 Gln Arg Glu Leu Gly Thr Arg Asn Leu Ser Glu Asp Phe Glu Cys Met 130 135 140 Leu Phe Glu Gln Tyr Ile Arg Leu Thr Lys Gly Glu Ile Glu Gly Tyr 145 150 155 160 Ala Ala Ile Ser Asn Met Phe Gly Asn Gly Glu Lys Glu Asp Arg Ser 165 170 175 Lys Lys Arg Met Tyr Ala Thr Arg Met Lys Asp Trp Leu Glu Ala Asn 180 185 190 Glu Asn Ile Thr Trp Glu Gln Tyr Arg Glu Ala Leu Lys Asn Gln Leu 195 200 205 Asn Ala Lys Asn Leu Glu Gln Val Val Ala Asn Tyr Lys Gly Asn Ala 210 215 220 Gly Gly Ala Asp Pro Phe Phe Lys Tyr Ser Phe Ser Lys Glu Gly Met 225 230 235 240 Val Ser Lys Lys Glu His Ala Gln Gln Leu Asp Lys Phe Lys Thr Val 245 250 255 Leu Lys Asn Lys Ala Arg Asp Leu Asn Phe Pro Asn Lys Glu Lys Leu 260 265 270 Lys Gln Tyr Leu Glu Ala Glu Ile Gly Ile Pro Val Asp Ala Asn Val 275 280 285 Tyr Ser Gln Met Phe Ser Asn Gly Val Ser Glu Val Gln Pro Lys Thr 290 295 300 Thr Arg Asn Met Ser Phe Ser Asn Glu Lys Leu Asp Leu Leu Thr Glu 305 310 315 320 Leu Lys Asp Leu Asn Lys Gly Asp Gly Phe Glu Tyr Ala Arg Glu Val 325 330 335 Leu Asn Gly Phe Phe Asp Ser Glu Leu His Thr Thr Glu Asp Lys Phe 340 345 350 Asn Ile Thr Ser Arg Tyr Leu Gly Gly Asp Lys Ser Asn Arg Leu Ser 355 360 365 Lys Leu Tyr Lys Ile Trp Lys Lys Glu Gly Val Asp Cys Glu Glu Gly 370 375 380 Ile Gln Gln Phe Cys Glu Ala Val Lys Asp Lys Met Gly Gln Ile Pro 385 390 395 400 Ile Arg Asn Val Leu Lys Tyr Leu Trp Gln Phe Arg Glu Thr Val Ser 405 410 415 Ala Glu Asp Phe Glu Ala Ala Ala Lys Ala Asn His Leu Glu Glu Lys 420 425 430 Ile Ser Arg Val Lys Ala His Pro Ile Val Ile Ser Asn Arg Tyr Trp 435 440 445 Ala Phe Gly Thr Ser Ala Leu Val Gly Asn Ile Met Pro Ala Asp Lys 450 455 460 Arg His Gln Gly Glu Tyr Ala Gly Gln Asn Phe Lys Met Trp Leu Glu 465 470 475 480 Ala Glu Leu His Tyr Asp Gly Lys Lys Ala Lys His His Leu Pro Phe 485 490 495 Tyr Asn Ala Arg Phe Phe Glu Glu Val Tyr Cys Tyr His Pro Ser Val 500 505 510 Ala Glu Ile Thr Pro Phe Lys Thr Lys Gln Phe Gly Cys Glu Ile Gly 515 520 525 Lys Asp Ile Pro Asp Tyr Val Ser Val Ala Leu Lys Asp Asn Pro Tyr 530 535 540 Lys Lys Ala Thr Lys Arg Ile Leu Arg Ala Ile Tyr Asn Pro Val Ala 545 550 555 560 Asn Thr Thr Gly Val Asp Lys Thr Thr Asn Cys Ser Phe Met Ile Lys 565 570 575 Arg Glu Asn Asp Glu Tyr Lys Leu Val Ile Asn Arg Lys Ile Ser Val 580 585 590 Asp Arg Pro Lys Arg Ile Glu Val Gly Arg Thr Ile Met Gly Tyr Asp 595 600 605 Arg Asn Gln Thr Ala Ser Asp Thr Tyr Trp Ile Gly Arg Leu Val Pro 610 615 620 Pro Gly Thr Arg Gly Ala Tyr Arg Ile Gly Glu Trp Ser Val Gln Tyr 625 630 635 640 Ile Lys Ser Gly Pro Val Leu Ser Ser Thr Gln Gly Val Asn Asn Ser 645 650 655 Thr Thr Asp Gln Leu Val Tyr Asn Gly Met Pro Ser Ser Ser Glu Arg 660 665 670 Phe Lys Ala Trp Lys Lys Ala Arg Met Ala Phe Ile Arg Lys Leu Ile 675 680 685 Arg Gln Leu Asn Asp Glu Gly Leu Glu Ser Lys Gly Gln Asp Tyr Ile 690 695 700 Pro Glu Asn Pro Ser Ser Phe Asp Val Arg Gly Glu Thr Leu Tyr Val 705 710 715 720 Phe Asn Ser Asn Tyr Leu Lys Ala Leu Val Ser Lys His Arg Lys Ala 725 730 735 Lys Lys Pro Val Glu Gly Ile Leu Asp Glu Ile Glu Ala Trp Thr Ser 740 745 750 Lys Asp Lys Asp Ser Cys Ser Leu Met Arg Leu Ser Ser Leu Ser Asp 755 760 765 Ala Ser Met Gln Gly Ile Ala Ser Leu Lys Ser Leu Ile Asn Ser Tyr 770 775 780 Phe Asn Lys Asn Gly Cys Lys Thr Ile Glu Asp Lys Glu Lys Phe Asn 785 790 795 800 Pro Val Leu Tyr Ala Lys Leu Val Glu Val Glu Gln Arg Arg Thr Asn 805 810 815 Lys Arg Ser Glu Lys Val Gly Arg Ile Ala Gly Ser Leu Glu Gln Leu 820 825 830 Ala Leu Leu Asn Gly Val Glu Val Val Ile Gly Glu Ala Asp Leu Gly 835 840 845 Glu Val Glu Lys Gly Lys Ser Lys Lys Gln Asn Ser Arg Asn Met Asp 850 855 860 Trp Cys Ala Lys Gln Val Ala Gln Arg Leu Glu Tyr Lys Leu Ala Phe 865 870 875 880 His Gly Ile Gly Tyr Phe Gly Val Asn Pro Met Tyr Thr Ser His Gln 885 890 895 Asp Pro Phe Glu His Arg Arg Val Ala Asp His Ile Val Met Arg Ala 900 905 910 Arg Phe Glu Glu Val Asn Val Glu Asn Ile Ala Glu Trp His Val Arg 915 920 925 Asn Phe Ser Asn Tyr Leu Arg Ala Asp Ser Gly Thr Gly Leu Tyr Tyr 930 935 940 Lys Gln Ala Thr Met Asp Phe Leu Lys His Tyr Gly Leu Glu Glu His 945 950 955 960 Ala Glu Gly Leu Glu Asn Lys Lys Ile Lys Phe Tyr Asp Phe Arg Lys 965 970 975 Ile Leu Glu Asp Lys Asn Leu Thr Ser Val Ile Ile Pro Lys Arg Gly 980 985 990 Gly Arg Ile Tyr Met Ala Thr Asn Pro Val Thr Ser Asp Ser Thr Pro 995 1000 1005 Ile Thr Tyr Ala Gly Lys Thr Tyr Asn Arg Cys Asn Ala Asp Glu 1010 1015 1020 Val Ala Ala Ala Asn Ile Val Ile Ser Val Leu Ala Pro Arg Ser 1025 1030 1035 Lys Lys Asn Glu Glu Gln Asp Asp Ile Pro Leu Ile Thr Lys Lys 1040 1045 1050 Ala Glu Ser Lys Ser Pro Pro Lys Asp Arg Lys Arg Ser Lys Thr 1055 1060 1065 Ser Gln Leu Pro Gln Lys 1070 <210> SEQ ID NO 17 <211> LENGTH: 1031 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 17 Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Pro Lys Arg Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Gly 35 40 45 Ile Asp Tyr Glu Ala Ala Glu Glu Leu Ile Asp Glu Lys Ser Thr Phe 50 55 60 Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asn Asn Pro Gly Pro Leu Gln Thr Thr Glu Gln Arg Thr Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Ala Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Thr Asp Thr Glu Lys Tyr Glu Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ala Asp Leu Ala Arg Asn Ile His Thr Thr Gln Glu Ser 130 135 140 Leu Lys Thr Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Phe Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Val Lys Ala Ser Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Ile Glu Asp Lys Lys Pro Asn Thr Trp Glu Glu 195 200 205 Tyr Gln Asp Leu Ile Lys Lys Thr Phe Asn Val Ser Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Gly Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg 260 265 270 Thr Val Phe Ser Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ser 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Gln Lys Gln Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Glu Asp Ile Leu Ser Ala Ala Ser Ile Leu Asn Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Ser Ser Val Val Ser Lys Asn His Leu Gly Ser Arg Leu 355 360 365 Asn Glu Leu Phe Glu Met Trp Gln Ala Leu Lys Met Asn Asp Gly Ile 370 375 380 Glu Lys Tyr Thr Asp Leu Cys Lys Asp Asn Phe Ser Arg Arg Pro Val 385 390 395 400 Ser Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Thr Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Asp Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Arg Asp Asn Gly Arg Trp Val Lys His His Leu Pro Phe 485 490 495 His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Ile Ile Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Ser Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Lys Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Thr Thr Pro Lys Tyr Ser His Lys Leu Asn Val 595 600 605 Gly Asp Ile Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Ile Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Val 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Leu Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Gly 705 710 715 720 Val Met Lys Asp Asn Lys Asp Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Ser His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu His Asp Gln Glu 770 775 780 Ser Phe Asp Ser Asp Phe Phe Arg Leu Met Arg Ser Ile Asp Asp Lys 785 790 795 800 Arg Ile Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Ser Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly 820 825 830 Asp Leu Pro Thr Ala Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg Tyr Val Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Lys Lys Phe Thr Asp Trp His Arg Gly Val Ser Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Ala Pro Leu Tyr Gln 930 935 940 Glu Ala Leu Lys Gln Phe Ala Asp His Tyr Lys Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Glu Asp His 965 970 975 Lys Gln Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Ile Thr Lys Asp Ser Ser Lys Ile Asn Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Gln Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Arg Asp Glu Asn 1025 1030 <210> SEQ ID NO 18 <211> LENGTH: 1066 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 18 Met Pro Asp Pro Ile Lys Ser Tyr Lys Ser Pro Ile Ile Ile Asp Pro 1 5 10 15 Asn Asn Ala His Asp Val Glu Lys Leu Asp Phe Leu Arg Glu Thr Phe 20 25 30 Val Tyr Leu Ser Asn Gly Thr Lys Cys Phe Met His Val Phe Leu Ser 35 40 45 Leu Leu Gly Gly Met Asn Glu Thr Leu Ala Lys Lys Ile Val Ser Leu 50 55 60 Glu Thr Pro Lys Lys Glu Lys Lys Lys Lys Ser Asn Lys Pro Ser His 65 70 75 80 Lys Ile Glu Leu Phe Leu Ala Ile Cys Trp Phe Arg Leu Val Lys Ile 85 90 95 Ser Lys Asn Glu Ser Ser Val Leu Pro Ala Leu Leu Gly Asn Arg Phe 100 105 110 Glu Lys Tyr Phe Gly Ala Lys Ala Thr Pro Glu Val Met Glu Tyr Phe 115 120 125 Ser Ala Asn Tyr Asp Glu Ala Thr Tyr Ala Trp Lys Asp Met Arg Glu 130 135 140 Glu Phe Val Ser Leu Lys Ser Lys Leu Lys Val Ser Glu Lys Asp Leu 145 150 155 160 Ile Ser Asp Ile Gly Ser Met Ile Asn Glu Arg Tyr Ile Gly Leu Lys 165 170 175 Phe Gly Lys Pro Trp Gly Ile Ile Ser Gly Leu Phe Gly Glu Gly Lys 180 185 190 Lys Val Asp Arg Ser Leu Lys Val Glu Leu Leu Lys Asn Val Leu Glu 195 200 205 Glu Ile Glu Lys Asn Pro Pro Lys Thr Lys Asp Gln Leu Ala Lys Met 210 215 220 Ile Leu Lys Cys Ala Asp Cys Lys Asn Gly Gln Glu Ile His Ala Lys 225 230 235 240 Cys Gly Lys Ile Gly Arg Met Ser Ser Val Ser Asn Trp Ala Asp Glu 245 250 255 Val Gly Ser Glu Lys Glu Ile Val Leu Ser Phe Val Lys Ser Lys Ile 260 265 270 Ser Gln Asp Leu Ala Lys Gln Ser Asn Glu Arg Asn Trp Lys Cys Val 275 280 285 Asn Ala Leu Lys Ser Tyr Ile Leu Ser Glu Ile Gly Asn Cys Phe Asp 290 295 300 Gln Ser Ser Trp Ser Glu Met Leu Asn Asn Ser Leu Ser Val Ile Gln 305 310 315 320 Ser Lys Thr Thr Arg Asn Tyr Asn Phe Cys Ile Glu Gln Leu Glu Glu 325 330 335 Lys Lys Asn Leu Asn Gln Asn His Arg Lys Phe Gly Thr Met Ile Glu 340 345 350 Asp Tyr Phe Ser Ser Arg Phe Phe Thr Gly Glu Asn Lys Phe Ile Ile 355 360 365 Cys Asn Phe His Val Gly Asp Lys Asp Lys Val Ser Ala Leu Leu Ala 370 375 380 Ser Cys Glu Gly Leu Ser Glu Glu Glu Leu Glu Glu Lys Ile Gln Asn 385 390 395 400 Phe Cys Glu Ser Gln Lys Gln Glu Ser Lys Met Pro Ile Pro Ala Leu 405 410 415 Leu Met Tyr Leu Asn Ser Leu Lys Asp Ser Ile Thr Val Asp Gln Met 420 425 430 Phe Gln Gly Ile Leu Tyr Asn Lys Ile Arg Asp Lys Ile Glu Arg Gln 435 440 445 Lys Leu His Pro Ile Val Pro Asn Asn Asp Ser Phe Asp Trp Gly Met 450 455 460 Ser Ser Lys Ile Asn Gly Arg Ile Ile Ser Pro Lys Glu Lys Ala Lys 465 470 475 480 His Asn Ala Gln Asn Asn Arg Ser Leu Tyr Asp Ser Gly Ile Trp Ile 485 490 495 Glu Ile Ser Val Leu Lys Asn Lys Glu Trp Ala Lys His His Tyr Lys 500 505 510 Ile Ser Asn Thr Arg Phe Val Glu Glu Phe Tyr Tyr Pro Ser Ser Asn 515 520 525 Asp Glu Asn Ser Leu Asp Gln Val Phe Arg Thr Gly Arg Asn Gly Phe 530 535 540 Asn Asn Pro Ala Lys Asn Asn Leu Ser Leu Glu Gln Val Ser Asn Ile 545 550 555 560 Lys Asn Ala Pro Lys Asn Arg Arg Arg Ala Ile Lys Arg Gln Met Arg 565 570 575 Val Glu Ala Ala His Gln Gln Asn Val Leu Pro His Val Lys Trp Asp 580 585 590 Asp Asn Tyr Cys Ile Thr Ile Ser Lys Tyr Gly Asp Lys Phe Val Thr 595 600 605 Phe Ile Ser Lys Lys Phe Lys Ser Lys Lys Ser Lys Glu Tyr Val Val 610 615 620 Phe Leu Gly Phe Asp Gln Asn Gln Thr Ala Ser His Thr Phe Ala Ala 625 630 635 640 Val Gln Ile Cys Asp Ser Lys Asp Glu Asn Val Ile Pro Tyr Cys Gly 645 650 655 Leu Phe Val Lys Pro Leu Glu Cys Gly His Ile Thr Ser Val Gln Lys 660 665 670 Val Lys Asp Arg Ser Ile Asp Gln Leu Ser Tyr Ser Gly Leu Pro Trp 675 680 685 Lys Asp Phe Ile Ser Trp Ser Gln Glu Arg Lys Glu Phe Val Ser Lys 690 695 700 Trp Arg Met Val Glu Val Lys Thr Arg Asn Gly Glu Lys Leu Asp Asp 705 710 715 720 Leu Thr Val Lys Ile Asn Lys Leu Asp Glu Asn Lys His Gly Leu Tyr 725 730 735 Ala Tyr Asn Ser Lys Tyr Phe Trp Tyr Leu Lys Ser Ile Met Arg Lys 740 745 750 Lys Thr Lys Asp Glu Leu Phe Glu Ile Arg Lys Glu Leu Leu Thr Val 755 760 765 Ile Lys Thr Gly Arg Leu Cys Val Leu Arg Leu Ser Ser Leu Asn His 770 775 780 Ser Ser Phe Leu Met Leu Lys Asn Ala Lys Ser Ala Ile Ser Cys Tyr 785 790 795 800 Phe Asn Asn Leu Leu Lys Gly Val Ser Asn Asp Gln Glu Lys Tyr Glu 805 810 815 Ala Asp Pro Glu Met Phe Glu Leu Arg Arg Glu Val Glu Ala Lys Arg 820 825 830 Gln Asn Lys Cys Met Ser Lys Lys Asn Leu Ile Ser Ser Gln Ile Val 835 840 845 Ser Lys Ala Ile Glu Leu Arg Gly Asn Tyr Gly Ser Val Ala Ile Ile 850 855 860 Gly Glu Asp Leu Ser Asp Tyr Val Pro Asp Lys Gly Lys Lys Ser Thr 865 870 875 880 Gln Asn Ala Asn Leu Leu Asp Trp Leu Ser Arg Gly Val Ala Asn Lys 885 890 895 Val Lys Gln Ile Ala Asn Met His Asp Asn Ile Ser Phe Lys Asp Val 900 905 910 Ser Pro Gln Trp Thr Ser His Gln Asp Ser Phe Val Asp Arg Asn Pro 915 920 925 Asn Ser Ala Leu Arg Val Arg Phe Gly Ser Cys Asp Pro Glu Glu Met 930 935 940 Tyr Glu Lys Asp Phe Glu Ser Leu Ile Lys Phe Leu Lys Glu Asp Cys 945 950 955 960 Gly His Tyr Thr Asn Ser Met Asn Asp Phe Leu Ser His Tyr Gly Val 965 970 975 Ser Arg Lys Asp Met Leu Glu Ile Lys Phe Ser Ala Phe Lys Ile Leu 980 985 990 Met Lys Asn Ile Leu Asn Lys Thr Gly Glu Lys Ser Leu Leu Tyr Pro 995 1000 1005 Lys Arg Gly Gly Arg Leu Tyr Leu Ala Thr His Lys Leu Gly Gln 1010 1015 1020 Cys Thr Arg Arg Thr Tyr Asn Gly Val Asp Phe Trp Glu Cys Asp 1025 1030 1035 Ala Asp Cys Val Ala Ala Phe Asn Ile Ala Leu Ser Gly Ile Arg 1040 1045 1050 Lys Tyr Tyr Gly Ile Lys Ser Glu Ala Val Ser Pro Val 1055 1060 1065 <210> SEQ ID NO 19 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 19 ctagcaatga cctaatagtg tgtccttagt tgacat 36 <210> SEQ ID NO 20 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 20 tctcaacgat agtcagacat gtgtcctcag tgacac 36 <210> SEQ ID NO 21 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 21 cctacaatac ctaagaaatc cgtcctaagt tgacgg 36 <210> SEQ ID NO 22 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 22 gtagcaatca gtacatattg tgcctttcat tggcaca 37 <210> SEQ ID NO 23 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 23 gtagcaatca gtacatattg tgcctttcat tggcac 36 <210> SEQ ID NO 24 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 24 gttggaatga ctaatttttg tgcccaccgt tggcac 36 <210> SEQ ID NO 25 <400> SEQUENCE: 25 000 <210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000 <210> SEQ ID NO 28 <400> SEQUENCE: 28 000 <210> SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30 <400> SEQUENCE: 30 000 <210> SEQ ID NO 31 <400> SEQUENCE: 31 000 <210> SEQ ID NO 32 <400> SEQUENCE: 32 000 <210> SEQ ID NO 33 <400> SEQUENCE: 33 000 <210> SEQ ID NO 34 <400> SEQUENCE: 34 000 <210> SEQ ID NO 35 <400> SEQUENCE: 35 000 <210> SEQ ID NO 36 <400> SEQUENCE: 36 000 <210> SEQ ID NO 37 <400> SEQUENCE: 37 000 <210> SEQ ID NO 38 <400> SEQUENCE: 38 000 <210> SEQ ID NO 39 <400> SEQUENCE: 39 000 <210> SEQ ID NO 40 <400> SEQUENCE: 40 000 <210> SEQ ID NO 41 <400> SEQUENCE: 41 000 <210> SEQ ID NO 42 <400> SEQUENCE: 42 000 <210> SEQ ID NO 43 <400> SEQUENCE: 43 000 <210> SEQ ID NO 44 <400> SEQUENCE: 44 000 <210> SEQ ID NO 45 <400> SEQUENCE: 45 000 <210> SEQ ID NO 46 <400> SEQUENCE: 46 000 <210> SEQ ID NO 47 <400> SEQUENCE: 47 000 <210> SEQ ID NO 48 <400> SEQUENCE: 48 000 <210> SEQ ID NO 49 <400> SEQUENCE: 49 000 <210> SEQ ID NO 50 <400> SEQUENCE: 50 000 <210> SEQ ID NO 51 <400> SEQUENCE: 51 000 <210> SEQ ID NO 52 <400> SEQUENCE: 52 000 <210> SEQ ID NO 53 <400> SEQUENCE: 53 000 <210> SEQ ID NO 54 <400> SEQUENCE: 54 000 <210> SEQ ID NO 55 <400> SEQUENCE: 55 000 <210> SEQ ID NO 56 <400> SEQUENCE: 56 000 <210> SEQ ID NO 57 <400> SEQUENCE: 57 000 <210> SEQ ID NO 58 <400> SEQUENCE: 58 000 <210> SEQ ID NO 59 <400> SEQUENCE: 59 000 <210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210> SEQ ID NO 61 <400> SEQUENCE: 61 000 <210> SEQ ID NO 62 <400> SEQUENCE: 62 000 <210> SEQ ID NO 63 <400> SEQUENCE: 63 000 <210> SEQ ID NO 64 <400> SEQUENCE: 64 000 <210> SEQ ID NO 65 <400> SEQUENCE: 65 000 <210> SEQ ID NO 66 <400> SEQUENCE: 66 000 <210> SEQ ID NO 67 <400> SEQUENCE: 67 000 <210> SEQ ID NO 68 <400> SEQUENCE: 68 000 <210> SEQ ID NO 69 <400> SEQUENCE: 69 000 <210> SEQ ID NO 70 <400> SEQUENCE: 70 000 <210> SEQ ID NO 71 <400> SEQUENCE: 71 000 <210> SEQ ID NO 72 <400> SEQUENCE: 72 000 <210> SEQ ID NO 73 <400> SEQUENCE: 73 000 <210> SEQ ID NO 74 <400> SEQUENCE: 74 000 <210> SEQ ID NO 75 <400> SEQUENCE: 75 000 <210> SEQ ID NO 76 <400> SEQUENCE: 76 000 <210> SEQ ID NO 77 <400> SEQUENCE: 77 000 <210> SEQ ID NO 78 <400> SEQUENCE: 78 000 <210> SEQ ID NO 79 <400> SEQUENCE: 79 000 <210> SEQ ID NO 80 <400> SEQUENCE: 80 000 <210> SEQ ID NO 81 <400> SEQUENCE: 81 000 <210> SEQ ID NO 82 <400> SEQUENCE: 82 000 <210> SEQ ID NO 83 <400> SEQUENCE: 83 000 <210> SEQ ID NO 84 <400> SEQUENCE: 84 000 <210> SEQ ID NO 85 <400> SEQUENCE: 85 000 <210> SEQ ID NO 86 <400> SEQUENCE: 86 000 <210> SEQ ID NO 87 <400> SEQUENCE: 87 000 <210> SEQ ID NO 88 <400> SEQUENCE: 88 000 <210> SEQ ID NO 89 <400> SEQUENCE: 89 000 <210> SEQ ID NO 90 <400> SEQUENCE: 90 000 <210> SEQ ID NO 91 <400> SEQUENCE: 91 000 <210> SEQ ID NO 92 <400> SEQUENCE: 92 000 <210> SEQ ID NO 93 <400> SEQUENCE: 93 000 <210> SEQ ID NO 94 <400> SEQUENCE: 94 000 <210> SEQ ID NO 95 <400> SEQUENCE: 95 000 <210> SEQ ID NO 96 <400> SEQUENCE: 96 000 <210> SEQ ID NO 97 <400> SEQUENCE: 97 000 <210> SEQ ID NO 98 <400> SEQUENCE: 98 000 <210> SEQ ID NO 99 <400> SEQUENCE: 99 000 <210> SEQ ID NO 100 <211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 100 auuuuugugc ccaucguugg cac 23 <210> SEQ ID NO 101 <211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 101 agaaauccgu cuuucauuga cgg 23 <210> SEQ ID NO 102 <400> SEQUENCE: 102 000 <210> SEQ ID NO 103 <400> SEQUENCE: 103 000 <210> SEQ ID NO 104 <400> SEQUENCE: 104 000 <210> SEQ ID NO 105 <400> SEQUENCE: 105 000 <210> SEQ ID NO 106 <400> SEQUENCE: 106 000 <210> SEQ ID NO 107 <400> SEQUENCE: 107 000 <210> SEQ ID NO 108 <400> SEQUENCE: 108 000 <210> SEQ ID NO 109 <400> SEQUENCE: 109 000 <210> SEQ ID NO 110 <400> SEQUENCE: 110 000 <210> SEQ ID NO 111 <400> SEQUENCE: 111 000 <210> SEQ ID NO 112 <400> SEQUENCE: 112 000 <210> SEQ ID NO 113 <400> SEQUENCE: 113 000 <210> SEQ ID NO 114 <400> SEQUENCE: 114 000 <210> SEQ ID NO 115 <400> SEQUENCE: 115 000 <210> SEQ ID NO 116 <400> SEQUENCE: 116 000 <210> SEQ ID NO 117 <400> SEQUENCE: 117 000 <210> SEQ ID NO 118 <400> SEQUENCE: 118 000 <210> SEQ ID NO 119 <400> SEQUENCE: 119 000 <210> SEQ ID NO 120 <400> SEQUENCE: 120 000 <210> SEQ ID NO 121 <400> SEQUENCE: 121 000 <210> SEQ ID NO 122 <400> SEQUENCE: 122 000 <210> SEQ ID NO 123 <400> SEQUENCE: 123 000 <210> SEQ ID NO 124 <400> SEQUENCE: 124 000 <210> SEQ ID NO 125 <400> SEQUENCE: 125 000 <210> SEQ ID NO 126 <400> SEQUENCE: 126 000 <210> SEQ ID NO 127 <400> SEQUENCE: 127 000 <210> SEQ ID NO 128 <400> SEQUENCE: 128 000 <210> SEQ ID NO 129 <400> SEQUENCE: 129 000 <210> SEQ ID NO 130 <400> SEQUENCE: 130 000 <210> SEQ ID NO 131 <400> SEQUENCE: 131 000 <210> SEQ ID NO 132 <400> SEQUENCE: 132 000 <210> SEQ ID NO 133 <400> SEQUENCE: 133 000 <210> SEQ ID NO 134 <400> SEQUENCE: 134 000 <210> SEQ ID NO 135 <400> SEQUENCE: 135 000 <210> SEQ ID NO 136 <400> SEQUENCE: 136 000 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000 <210> SEQ ID NO 138 <400> SEQUENCE: 138 000 <210> SEQ ID NO 139 <400> SEQUENCE: 139 000 <210> SEQ ID NO 140 <400> SEQUENCE: 140 000 <210> SEQ ID NO 141 <400> SEQUENCE: 141 000 <210> SEQ ID NO 142 <400> SEQUENCE: 142 000 <210> SEQ ID NO 143 <400> SEQUENCE: 143 000 <210> SEQ ID NO 144 <400> SEQUENCE: 144 000 <210> SEQ ID NO 145 <400> SEQUENCE: 145 000 <210> SEQ ID NO 146 <400> SEQUENCE: 146 000 <210> SEQ ID NO 147 <400> SEQUENCE: 147 000 <210> SEQ ID NO 148 <400> SEQUENCE: 148 000 <210> SEQ ID NO 149 <400> SEQUENCE: 149 000 <210> SEQ ID NO 150 <211> LENGTH: 106 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 150 cuagcaauga ccuaauagug uguccuuagu ugacaunnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn cuagcaauga ccuaauagug uguccuuagu ugacau 106 <210> SEQ ID NO 151 <211> LENGTH: 107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(71) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 151 cuagcaauga ccuaauagug uguccuuagu ugacaunnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn ncuagcaaug accuaauagu guguccuuag uugacau 107 <210> SEQ ID NO 152 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 152 ucucaacgau agucagacau guguccucag ugacacnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnucucaacg auagucagac auguguccuc agugacac 108 <210> SEQ ID NO 153 <211> LENGTH: 107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(71) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 153 ccuacaauac cuaagaaauc cguccuaagu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nccuacaaua ccuaagaaau ccguccuaag uugacgg 107 <210> SEQ ID NO 154 <211> LENGTH: 107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (38)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 154 guagcaauca guacauauug ugccuuucau uggcacannn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn guagcaauca guacauauug ugccuuucau uggcaca 107 <210> SEQ ID NO 155 <211> LENGTH: 106 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 155 guagcaauca guacauauug ugccuuucau uggcacnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn guagcaauca guacauauug ugccuuucau uggcac 106 <210> SEQ ID NO 156 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 156 guuggaauga cuaauuuuug ugcccaccgu uggcacnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnguuggaau gacuaauuuu ugugcccacc guuggcac 108 <210> SEQ ID NO 157 <211> LENGTH: 84 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (25)..(60) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 157 aauuuuugug cccaucguug gcacnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 aauuuuugug cccaucguug gcac 84 <210> SEQ ID NO 158 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 158 cccacaauac cugagaaauc cguccuacgu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nncccacaau accugagaaa uccguccuac guugacgg 108 <210> SEQ ID NO 159 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 159 cccacaauac cugagaaauc cguccuacgu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nncccacaau accugagaaa uccguccuac guugacgg 108 <210> SEQ ID NO 160 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 160 cucucaaugc cuuagaaauc cguccuuggu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nncucucaau gccuuagaaa uccguccuug guugacgg 108 <210> SEQ ID NO 161 <211> LENGTH: 106 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 161 cccacaauac cugagaaauc cguccuacgu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn cccacaauac cugagaaauc cguccuacgu ugacgg 106 <210> SEQ ID NO 162 <211> LENGTH: 92 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (35)..(58) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 162 gcaacaccua agaaauccgu cuuucauuga cgggnnnnnn nnnnnnnnnn nnnnnnnngc 60 aacaccuaag aaauccgucu uucauugacg gg 92 <210> SEQ ID NO 163 <211> LENGTH: 103 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(67) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 163 guugcaaaac ccaagaaauc cgucuuucau ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnguu gcaaaaccca agaaauccgu cuuucauuga cgg 103 <210> SEQ ID NO 164 <400> SEQUENCE: 164 000 <210> SEQ ID NO 165 <400> SEQUENCE: 165 000 <210> SEQ ID NO 166 <400> SEQUENCE: 166 000 <210> SEQ ID NO 167 <400> SEQUENCE: 167 000 <210> SEQ ID NO 168 <400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <400> SEQUENCE: 169 000 <210> SEQ ID NO 170 <400> SEQUENCE: 170 000 <210> SEQ ID NO 171 <400> SEQUENCE: 171 000 <210> SEQ ID NO 172 <400> SEQUENCE: 172 000 <210> SEQ ID NO 173 <400> SEQUENCE: 173 000 <210> SEQ ID NO 174 <400> SEQUENCE: 174 000 <210> SEQ ID NO 175 <400> SEQUENCE: 175 000 <210> SEQ ID NO 176 <400> SEQUENCE: 176 000 <210> SEQ ID NO 177 <400> SEQUENCE: 177 000 <210> SEQ ID NO 178 <400> SEQUENCE: 178 000 <210> SEQ ID NO 179 <400> SEQUENCE: 179 000 <210> SEQ ID NO 180 <400> SEQUENCE: 180 000 <210> SEQ ID NO 181 <400> SEQUENCE: 181 000 <210> SEQ ID NO 182 <400> SEQUENCE: 182 000 <210> SEQ ID NO 183 <400> SEQUENCE: 183 000 <210> SEQ ID NO 184 <400> SEQUENCE: 184 000 <210> SEQ ID NO 185 <400> SEQUENCE: 185 000 <210> SEQ ID NO 186 <400> SEQUENCE: 186 000 <210> SEQ ID NO 187 <400> SEQUENCE: 187 000 <210> SEQ ID NO 188 <400> SEQUENCE: 188 000 <210> SEQ ID NO 189 <400> SEQUENCE: 189 000 <210> SEQ ID NO 190 <400> SEQUENCE: 190 000 <210> SEQ ID NO 191 <400> SEQUENCE: 191 000 <210> SEQ ID NO 192 <400> SEQUENCE: 192 000 <210> SEQ ID NO 193 <400> SEQUENCE: 193 000 <210> SEQ ID NO 194 <400> SEQUENCE: 194 000 <210> SEQ ID NO 195 <400> SEQUENCE: 195 000 <210> SEQ ID NO 196 <400> SEQUENCE: 196 000 <210> SEQ ID NO 197 <400> SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE: 198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000 <210> SEQ ID NO 200 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: /replace="Thr" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: /replace="Leu" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: /replace="Ser" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (7)..(7) <223> OTHER INFORMATION: /replace="Leu" <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)..(7) <223> OTHER INFORMATION: /note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> SEQUENCE: 200 Ser Ser His Gln Asp Pro Phe 1 5 <210> SEQ ID NO 201 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: /replace="Gly" or "Ser" <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2)..(2) <223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: /replace="Ile" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (7)..(7) <223> OTHER INFORMATION: /replace="Ser" or "Val" <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (8)..(10) <223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: /replace="Ala" <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)..(11) <223> OTHER INFORMATION: /note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> SEQUENCE: 201 Ala Xaa Asp Xaa Asn Gln Thr Xaa Xaa Xaa Thr 1 5 10 <210> SEQ ID NO 202 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(11) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 202 ccgucnnnnn nugacgg 17 <210> SEQ ID NO 203 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(11) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 203 gugccnnnnn nuggcac 17 <210> SEQ ID NO 204 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(11) <223> OTHER INFORMATION: a, c, u, g, unknown or other <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: May or may not be present <400> SEQUENCE: 204 gugucnnnnn nugacay 17 <210> SEQ ID NO 205 <211> LENGTH: 14 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 205 ucyuwvruug acgg 14 <210> SEQ ID NO 206 <211> LENGTH: 14 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 206 ccywycruug gcac 14 <210> SEQ ID NO 207 <400> SEQUENCE: 207 000 <210> SEQ ID NO 208 <400> SEQUENCE: 208 000 <210> SEQ ID NO 209 <400> SEQUENCE: 209 000 <210> SEQ ID NO 210 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: /replace="Phe" or "Ile" or "Leu" or "Met" or "Pro" or "Val" or "Trp" or "Tyr" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (2)..(2) <223> OTHER INFORMATION: /replace="Phe" or "Ile" or "Leu" or "Met" or "Pro" or "Arg" or "Val" or "Trp" or "Tyr" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (3)..(3) <223> OTHER INFORMATION: /replace="Phe" or "Gly" or "Ile" or "Leu" or "Met" or "Pro" or "Val" or "Trp" or "Tyr" <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)..(4) <223> OTHER INFORMATION: /note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> SEQUENCE: 210 Cys Cys Cys Glu 1 <210> SEQ ID NO 211 <400> SEQUENCE: 211 000 <210> SEQ ID NO 212 <400> SEQUENCE: 212 000 <210> SEQ ID NO 213 <400> SEQUENCE: 213 000 <210> SEQ ID NO 214 <400> SEQUENCE: 214 000 <210> SEQ ID NO 215 <400> SEQUENCE: 215 000 <210> SEQ ID NO 216 <400> SEQUENCE: 216 000 <210> SEQ ID NO 217 <400> SEQUENCE: 217 000 <210> SEQ ID NO 218 <400> SEQUENCE: 218 000 <210> SEQ ID NO 219 <400> SEQUENCE: 219 000 <210> SEQ ID NO 220 <400> SEQUENCE: 220 000 <210> SEQ ID NO 221 <400> SEQUENCE: 221 000 <210> SEQ ID NO 222 <400> SEQUENCE: 222 000 <210> SEQ ID NO 223 <400> SEQUENCE: 223 000 <210> SEQ ID NO 224 <400> SEQUENCE: 224 000 <210> SEQ ID NO 225 <400> SEQUENCE: 225 000 <210> SEQ ID NO 226 <400> SEQUENCE: 226 000 <210> SEQ ID NO 227 <400> SEQUENCE: 227 000 <210> SEQ ID NO 228 <400> SEQUENCE: 228 000 <210> SEQ ID NO 229 <400> SEQUENCE: 229 000 <210> SEQ ID NO 230 <400> SEQUENCE: 230 000 <210> SEQ ID NO 231 <400> SEQUENCE: 231 000 <210> SEQ ID NO 232 <400> SEQUENCE: 232 000 <210> SEQ ID NO 233 <400> SEQUENCE: 233 000 <210> SEQ ID NO 234 <400> SEQUENCE: 234 000 <210> SEQ ID NO 235 <400> SEQUENCE: 235 000 <210> SEQ ID NO 236 <400> SEQUENCE: 236 000 <210> SEQ ID NO 237 <400> SEQUENCE: 237 000 <210> SEQ ID NO 238 <400> SEQUENCE: 238 000 <210> SEQ ID NO 239 <400> SEQUENCE: 239 000 <210> SEQ ID NO 240 <400> SEQUENCE: 240 000 <210> SEQ ID NO 241 <400> SEQUENCE: 241 000 <210> SEQ ID NO 242 <400> SEQUENCE: 242 000 <210> SEQ ID NO 243 <400> SEQUENCE: 243 000 <210> SEQ ID NO 244 <400> SEQUENCE: 244 000 <210> SEQ ID NO 245 <400> SEQUENCE: 245 000 <210> SEQ ID NO 246 <400> SEQUENCE: 246 000 <210> SEQ ID NO 247 <400> SEQUENCE: 247 000 <210> SEQ ID NO 248 <400> SEQUENCE: 248 000 <210> SEQ ID NO 249 <400> SEQUENCE: 249 000 <210> SEQ ID NO 250 <400> SEQUENCE: 250 000 <210> SEQ ID NO 251 <400> SEQUENCE: 251 000 <210> SEQ ID NO 252 <400> SEQUENCE: 252 000 <210> SEQ ID NO 253 <400> SEQUENCE: 253 000 <210> SEQ ID NO 254 <400> SEQUENCE: 254 000 <210> SEQ ID NO 255 <400> SEQUENCE: 255 000 <210> SEQ ID NO 256 <400> SEQUENCE: 256 000 <210> SEQ ID NO 257 <400> SEQUENCE: 257 000 <210> SEQ ID NO 258 <400> SEQUENCE: 258 000 <210> SEQ ID NO 259 <400> SEQUENCE: 259 000 <210> SEQ ID NO 260 <400> SEQUENCE: 260 000 <210> SEQ ID NO 261 <400> SEQUENCE: 261 000 <210> SEQ ID NO 262 <400> SEQUENCE: 262 000 <210> SEQ ID NO 263 <400> SEQUENCE: 263 000 <210> SEQ ID NO 264 <400> SEQUENCE: 264 000 <210> SEQ ID NO 265 <400> SEQUENCE: 265 000 <210> SEQ ID NO 266 <400> SEQUENCE: 266 000 <210> SEQ ID NO 267 <400> SEQUENCE: 267 000 <210> SEQ ID NO 268 <400> SEQUENCE: 268 000 <210> SEQ ID NO 269 <400> SEQUENCE: 269 000 <210> SEQ ID NO 270 <400> SEQUENCE: 270 000 <210> SEQ ID NO 271 <400> SEQUENCE: 271 000 <210> SEQ ID NO 272 <400> SEQUENCE: 272 000 <210> SEQ ID NO 273 <400> SEQUENCE: 273 000 <210> SEQ ID NO 274 <400> SEQUENCE: 274 000 <210> SEQ ID NO 275 <400> SEQUENCE: 275 000 <210> SEQ ID NO 276 <400> SEQUENCE: 276 000 <210> SEQ ID NO 277 <400> SEQUENCE: 277 000 <210> SEQ ID NO 278 <400> SEQUENCE: 278 000 <210> SEQ ID NO 279 <400> SEQUENCE: 279 000 <210> SEQ ID NO 280 <400> SEQUENCE: 280 000 <210> SEQ ID NO 281 <400> SEQUENCE: 281 000 <210> SEQ ID NO 282 <400> SEQUENCE: 282 000 <210> SEQ ID NO 283 <400> SEQUENCE: 283 000 <210> SEQ ID NO 284 <400> SEQUENCE: 284 000 <210> SEQ ID NO 285 <400> SEQUENCE: 285 000 <210> SEQ ID NO 286 <400> SEQUENCE: 286 000 <210> SEQ ID NO 287 <400> SEQUENCE: 287 000 <210> SEQ ID NO 288 <400> SEQUENCE: 288 000 <210> SEQ ID NO 289 <400> SEQUENCE: 289 000 <210> SEQ ID NO 290 <400> SEQUENCE: 290 000 <210> SEQ ID NO 291 <400> SEQUENCE: 291 000 <210> SEQ ID NO 292 <400> SEQUENCE: 292 000 <210> SEQ ID NO 293 <400> SEQUENCE: 293 000 <210> SEQ ID NO 294 <400> SEQUENCE: 294 000 <210> SEQ ID NO 295 <400> SEQUENCE: 295 000 <210> SEQ ID NO 296 <400> SEQUENCE: 296 000 <210> SEQ ID NO 297 <400> SEQUENCE: 297 000 <210> SEQ ID NO 298 <400> SEQUENCE: 298 000 <210> SEQ ID NO 299 <400> SEQUENCE: 299 000 <210> SEQ ID NO 300 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Simian virus 40 <400> SEQUENCE: 300 Pro Lys Lys Lys Arg Lys Val 1 5 <210> SEQ ID NO 301 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: Nucleoplasmin bipartite NLS sequence" <400> SEQUENCE: 301 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> SEQ ID NO 302 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: c-Myc NLS sequence" <400> SEQUENCE: 302 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> SEQ ID NO 303 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: c-Myc NLS sequence" <400> SEQUENCE: 303 Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10 <210> SEQ ID NO 304 <211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 304 Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15 Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro 20 25 30 Arg Asn Gln Gly Gly Tyr 35 <210> SEQ ID NO 305 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: IBB domain from importin-alpha sequence" <400> SEQUENCE: 305 Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu 1 5 10 15 Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys 20 25 30 Asp Glu Gln Ile Leu Lys Arg Arg Asn Val 35 40 <210> SEQ ID NO 306 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: Myoma T protein sequence" <400> SEQUENCE: 306 Val Ser Arg Lys Arg Pro Arg Pro 1 5 <210> SEQ ID NO 307 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: Myoma T protein sequence" <400> SEQUENCE: 307 Pro Pro Lys Lys Ala Arg Glu Asp 1 5 <210> SEQ ID NO 308 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 308 Pro Gln Pro Lys Lys Lys Pro Leu 1 5 <210> SEQ ID NO 309 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 309 Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 <210> SEQ ID NO 310 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Influenza virus <400> SEQUENCE: 310 Asp Arg Leu Arg Arg 1 5 <210> SEQ ID NO 311 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Influenza virus <400> SEQUENCE: 311 Pro Lys Gln Lys Lys Arg Lys 1 5 <210> SEQ ID NO 312 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Hepatitis virus <400> SEQUENCE: 312 Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1 5 10 <210> SEQ ID NO 313 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 313 Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 <210> SEQ ID NO 314 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 314 Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15 Lys Ser Lys Lys 20 <210> SEQ ID NO 315 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 315 Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys <210> SEQ ID NO 316 <400> SEQUENCE: 316 000 <210> SEQ ID NO 317 <400> SEQUENCE: 317 000 <210> SEQ ID NO 318 <400> SEQUENCE: 318 000 <210> SEQ ID NO 319 <400> SEQUENCE: 319 000 <210> SEQ ID NO 320 <400> SEQUENCE: 320 000 <210> SEQ ID NO 321 <400> SEQUENCE: 321 000 <210> SEQ ID NO 322 <400> SEQUENCE: 322 000 <210> SEQ ID NO 323 <400> SEQUENCE: 323 000 <210> SEQ ID NO 324 <400> SEQUENCE: 324 000 <210> SEQ ID NO 325 <400> SEQUENCE: 325 000 <210> SEQ ID NO 326 <400> SEQUENCE: 326 000 <210> SEQ ID NO 327 <400> SEQUENCE: 327 000 <210> SEQ ID NO 328 <400> SEQUENCE: 328 000 <210> SEQ ID NO 329 <400> SEQUENCE: 329 000 <210> SEQ ID NO 330 <400> SEQUENCE: 330 000 <210> SEQ ID NO 331 <400> SEQUENCE: 331 000 <210> SEQ ID NO 332 <400> SEQUENCE: 332 000 <210> SEQ ID NO 333 <400> SEQUENCE: 333 000 <210> SEQ ID NO 334 <400> SEQUENCE: 334 000 <210> SEQ ID NO 335 <400> SEQUENCE: 335 000 <210> SEQ ID NO 336 <400> SEQUENCE: 336 000 <210> SEQ ID NO 337 <400> SEQUENCE: 337 000 <210> SEQ ID NO 338 <400> SEQUENCE: 338 000 <210> SEQ ID NO 339 <400> SEQUENCE: 339 000 <210> SEQ ID NO 340 <400> SEQUENCE: 340 000 <210> SEQ ID NO 341 <400> SEQUENCE: 341 000 <210> SEQ ID NO 342 <400> SEQUENCE: 342 000 <210> SEQ ID NO 343 <400> SEQUENCE: 343 000 <210> SEQ ID NO 344 <400> SEQUENCE: 344 000 <210> SEQ ID NO 345 <400> SEQUENCE: 345 000 <210> SEQ ID NO 346 <400> SEQUENCE: 346 000 <210> SEQ ID NO 347 <400> SEQUENCE: 347 000 <210> SEQ ID NO 348 <400> SEQUENCE: 348 000 <210> SEQ ID NO 349 <400> SEQUENCE: 349 000 <210> SEQ ID NO 350 <400> SEQUENCE: 350 000 <210> SEQ ID NO 351 <400> SEQUENCE: 351 000 <210> SEQ ID NO 352 <400> SEQUENCE: 352 000 <210> SEQ ID NO 353 <400> SEQUENCE: 353 000 <210> SEQ ID NO 354 <400> SEQUENCE: 354 000 <210> SEQ ID NO 355 <400> SEQUENCE: 355 000 <210> SEQ ID NO 356 <400> SEQUENCE: 356 000 <210> SEQ ID NO 357 <400> SEQUENCE: 357 000 <210> SEQ ID NO 358 <400> SEQUENCE: 358 000 <210> SEQ ID NO 359 <400> SEQUENCE: 359 000 <210> SEQ ID NO 360 <400> SEQUENCE: 360 000 <210> SEQ ID NO 361 <400> SEQUENCE: 361 000 <210> SEQ ID NO 362 <400> SEQUENCE: 362 000 <210> SEQ ID NO 363 <400> SEQUENCE: 363 000 <210> SEQ ID NO 364 <400> SEQUENCE: 364 000 <210> SEQ ID NO 365 <400> SEQUENCE: 365 000 <210> SEQ ID NO 366 <400> SEQUENCE: 366 000 <210> SEQ ID NO 367 <400> SEQUENCE: 367 000 <210> SEQ ID NO 368 <400> SEQUENCE: 368 000 <210> SEQ ID NO 369 <400> SEQUENCE: 369 000 <210> SEQ ID NO 370 <400> SEQUENCE: 370 000 <210> SEQ ID NO 371 <400> SEQUENCE: 371 000 <210> SEQ ID NO 372 <400> SEQUENCE: 372 000 <210> SEQ ID NO 373 <400> SEQUENCE: 373 000 <210> SEQ ID NO 374 <400> SEQUENCE: 374 000 <210> SEQ ID NO 375 <400> SEQUENCE: 375 000 <210> SEQ ID NO 376 <400> SEQUENCE: 376 000 <210> SEQ ID NO 377 <400> SEQUENCE: 377 000 <210> SEQ ID NO 378 <400> SEQUENCE: 378 000 <210> SEQ ID NO 379 <400> SEQUENCE: 379 000 <210> SEQ ID NO 380 <400> SEQUENCE: 380 000 <210> SEQ ID NO 381 <400> SEQUENCE: 381 000 <210> SEQ ID NO 382 <400> SEQUENCE: 382 000 <210> SEQ ID NO 383 <400> SEQUENCE: 383 000 <210> SEQ ID NO 384 <400> SEQUENCE: 384 000 <210> SEQ ID NO 385 <400> SEQUENCE: 385 000 <210> SEQ ID NO 386 <400> SEQUENCE: 386 000 <210> SEQ ID NO 387 <400> SEQUENCE: 387 000 <210> SEQ ID NO 388 <400> SEQUENCE: 388 000 <210> SEQ ID NO 389 <400> SEQUENCE: 389 000 <210> SEQ ID NO 390 <400> SEQUENCE: 390 000 <210> SEQ ID NO 391 <400> SEQUENCE: 391 000 <210> SEQ ID NO 392 <400> SEQUENCE: 392 000 <210> SEQ ID NO 393 <400> SEQUENCE: 393 000 <210> SEQ ID NO 394 <400> SEQUENCE: 394 000 <210> SEQ ID NO 395 <400> SEQUENCE: 395 000 <210> SEQ ID NO 396 <400> SEQUENCE: 396 000 <210> SEQ ID NO 397 <400> SEQUENCE: 397 000 <210> SEQ ID NO 398 <400> SEQUENCE: 398 000 <210> SEQ ID NO 399 <400> SEQUENCE: 399 000 <210> SEQ ID NO 400 <211> LENGTH: 131 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 400 gggaauuuuu gugcccaucg uuggcacccu aaugcggaag uaguggguaa cccggaauuu 60 uugugcccau cguuggcacu ccgcaagaau ugauuggcuc caauucuaau uuuugugccc 120 aucguuggca c 131 <210> SEQ ID NO 401 <211> LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 401 aauuuuugug cccaucguug gcac 24 <210> SEQ ID NO 402 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 402 ccuaaugcgg aaguaguggg uaacccgg 28 <210> SEQ ID NO 403 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 403 uccgcaagaa uugauuggcu ccaauucu 28 <210> SEQ ID NO 404 <211> LENGTH: 131 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 404 gggaauuuuu gugcccaucg uuggcacagg caucaucagc auuaaccacg caaacaauuu 60 uugugcccau cguuggcacg cgugcuggau ugcuucgaug gucugcgaau uuuugugccc 120 aucguuggca c 131 <210> SEQ ID NO 405 <211> LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 405 aauuuuugug cccaucguug gcac 24 <210> SEQ ID NO 406 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 406 aggcaucauc agcauuaacc acgcaaac 28 <210> SEQ ID NO 407 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 407 gcgugcugga uugcuucgau ggucugcg 28 <210> SEQ ID NO 408 <211> LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 408 catgtggacc acattaggct gcaaaactgc gcatttacga aaacgcgaaa gtttgcgtgg 60 ttaatgctga tgatgcctta acaatgccga ttcgcggtgc ggatgaacgt aatttctcga 120 ggcgtatt 128 <210> SEQ ID NO 409 <211> LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 409 catgtggacc acattaggct tggttgttgc tgccgacgac ggtgtgatgc cgcagaccat 60 cgaagcaatc cagcacgcga aagcggcgca ggtaccggtg gtggttgcgt aatttctcga 120 ggcgtatt 128 <210> SEQ ID NO 410 <211> LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 410 aatacgcctc gagaaattac aaagtgatgc aggcgtttcc aggtgctttc cctaatgcgg 60 aagtagtggg taacccggtg cgtaccgatg tgttggcgct gccgttgcag cctaatgtgg 120 tccacatg 128 <210> SEQ ID NO 411 <211> LENGTH: 171 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 411 gtgccaacga tgggcacaaa aattagaatt ggagccaatc aattcttgcg gagtgccaac 60 gatgggcaca aaaattagaa ttggagccaa tcaattcttg cggagtgcca acgatgggca 120 caaaaattcc ctatagtgag tcgtattact cgagggatcc ttattacatt t 171 <210> SEQ ID NO 412 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 412 taatacgact cactatag 18 <210> SEQ ID NO 413 <211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 413 gtgccaacga tgggcacaaa aattagaatt ggagccaatc aattcttgcg ga 52 <210> SEQ ID NO 414 <211> LENGTH: 171 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 414 gtgccaacga tgggcacaaa aattcgcaga ccatcgaagc aatccagcac gcgtgccaac 60 gatgggcaca aaaattgttt gcgtggttaa tgctgatgat gcctgtgcca acgatgggca 120 caaaaattcc ctatagtgag tcgtattact cgagggatcc ttattacatt t 171 <210> SEQ ID NO 415 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 415 taatacgact cactatag 18 <210> SEQ ID NO 416 <211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 416 gtgccaacga tgggcacaaa aattcgcaga ccatcgaagc aatccagcac gc 52 <210> SEQ ID NO 417 <400> SEQUENCE: 417 000 <210> SEQ ID NO 418 <400> SEQUENCE: 418 000 <210> SEQ ID NO 419 <400> SEQUENCE: 419 000 <210> SEQ ID NO 420 <400> SEQUENCE: 420 000 <210> SEQ ID NO 421 <400> SEQUENCE: 421 000 <210> SEQ ID NO 422 <400> SEQUENCE: 422 000 <210> SEQ ID NO 423 <400> SEQUENCE: 423 000 <210> SEQ ID NO 424 <400> SEQUENCE: 424 000 <210> SEQ ID NO 425 <400> SEQUENCE: 425 000 <210> SEQ ID NO 426 <400> SEQUENCE: 426 000 <210> SEQ ID NO 427 <400> SEQUENCE: 427 000 <210> SEQ ID NO 428 <400> SEQUENCE: 428 000 <210> SEQ ID NO 429 <400> SEQUENCE: 429 000 <210> SEQ ID NO 430 <400> SEQUENCE: 430 000 <210> SEQ ID NO 431 <400> SEQUENCE: 431 000 <210> SEQ ID NO 432 <400> SEQUENCE: 432 000 <210> SEQ ID NO 433 <400> SEQUENCE: 433 000 <210> SEQ ID NO 434 <400> SEQUENCE: 434 000 <210> SEQ ID NO 435 <400> SEQUENCE: 435 000 <210> SEQ ID NO 436 <400> SEQUENCE: 436 000 <210> SEQ ID NO 437 <400> SEQUENCE: 437 000 <210> SEQ ID NO 438 <400> SEQUENCE: 438 000 <210> SEQ ID NO 439 <400> SEQUENCE: 439 000 <210> SEQ ID NO 440 <400> SEQUENCE: 440 000 <210> SEQ ID NO 441 <400> SEQUENCE: 441 000 <210> SEQ ID NO 442 <400> SEQUENCE: 442 000 <210> SEQ ID NO 443 <400> SEQUENCE: 443 000 <210> SEQ ID NO 444 <400> SEQUENCE: 444 000 <210> SEQ ID NO 445 <400> SEQUENCE: 445 000 <210> SEQ ID NO 446 <400> SEQUENCE: 446 000 <210> SEQ ID NO 447 <400> SEQUENCE: 447 000 <210> SEQ ID NO 448 <400> SEQUENCE: 448 000 <210> SEQ ID NO 449 <400> SEQUENCE: 449 000 <210> SEQ ID NO 450 <400> SEQUENCE: 450 000 <210> SEQ ID NO 451 <400> SEQUENCE: 451 000 <210> SEQ ID NO 452 <400> SEQUENCE: 452 000 <210> SEQ ID NO 453 <400> SEQUENCE: 453 000 <210> SEQ ID NO 454 <400> SEQUENCE: 454 000 <210> SEQ ID NO 455 <400> SEQUENCE: 455 000 <210> SEQ ID NO 456 <400> SEQUENCE: 456 000 <210> SEQ ID NO 457 <400> SEQUENCE: 457 000 <210> SEQ ID NO 458 <400> SEQUENCE: 458 000 <210> SEQ ID NO 459 <400> SEQUENCE: 459 000 <210> SEQ ID NO 460 <400> SEQUENCE: 460 000 <210> SEQ ID NO 461 <400> SEQUENCE: 461 000 <210> SEQ ID NO 462 <400> SEQUENCE: 462 000 <210> SEQ ID NO 463 <400> SEQUENCE: 463 000 <210> SEQ ID NO 464 <400> SEQUENCE: 464 000 <210> SEQ ID NO 465 <400> SEQUENCE: 465 000 <210> SEQ ID NO 466 <400> SEQUENCE: 466 000 <210> SEQ ID NO 467 <400> SEQUENCE: 467 000 <210> SEQ ID NO 468 <400> SEQUENCE: 468 000 <210> SEQ ID NO 469 <400> SEQUENCE: 469 000 <210> SEQ ID NO 470 <400> SEQUENCE: 470 000 <210> SEQ ID NO 471 <400> SEQUENCE: 471 000 <210> SEQ ID NO 472 <400> SEQUENCE: 472 000 <210> SEQ ID NO 473 <400> SEQUENCE: 473 000 <210> SEQ ID NO 474 <400> SEQUENCE: 474 000 <210> SEQ ID NO 475 <400> SEQUENCE: 475 000 <210> SEQ ID NO 476 <400> SEQUENCE: 476 000 <210> SEQ ID NO 477 <400> SEQUENCE: 477 000 <210> SEQ ID NO 478 <400> SEQUENCE: 478 000 <210> SEQ ID NO 479 <400> SEQUENCE: 479 000 <210> SEQ ID NO 480 <400> SEQUENCE: 480 000 <210> SEQ ID NO 481 <400> SEQUENCE: 481 000 <210> SEQ ID NO 482 <400> SEQUENCE: 482 000 <210> SEQ ID NO 483 <400> SEQUENCE: 483 000 <210> SEQ ID NO 484 <400> SEQUENCE: 484 000 <210> SEQ ID NO 485 <400> SEQUENCE: 485 000 <210> SEQ ID NO 486 <400> SEQUENCE: 486 000 <210> SEQ ID NO 487 <400> SEQUENCE: 487 000 <210> SEQ ID NO 488 <400> SEQUENCE: 488 000 <210> SEQ ID NO 489 <400> SEQUENCE: 489 000 <210> SEQ ID NO 490 <400> SEQUENCE: 490 000 <210> SEQ ID NO 491 <400> SEQUENCE: 491 000 <210> SEQ ID NO 492 <400> SEQUENCE: 492 000 <210> SEQ ID NO 493 <400> SEQUENCE: 493 000 <210> SEQ ID NO 494 <400> SEQUENCE: 494 000 <210> SEQ ID NO 495 <400> SEQUENCE: 495 000 <210> SEQ ID NO 496 <400> SEQUENCE: 496 000 <210> SEQ ID NO 497 <400> SEQUENCE: 497 000 <210> SEQ ID NO 498 <400> SEQUENCE: 498 000 <210> SEQ ID NO 499 <400> SEQUENCE: 499 000 <210> SEQ ID NO 500 <211> LENGTH: 212 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 500 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat 60 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac ag 212 <210> SEQ ID NO 501 <211> LENGTH: 3375 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 501 atgaaaatcg aagaaggtaa aggtcaccat caccatcacc acatgtctaa caaggagaag 60 aatgccagcg agacccggaa ggcctacacc acaaagatga tccccaggag ccacgaccgc 120 atgaagctgc tgggcaactt tatggactat ctgatggatg gcacccctat cttctttgag 180 ctgtggaatc agttcggcgg cggcatcgac agagatatca tcagcggcac agccaacaag 240 gataagatct ccgacgatct gctgctggcc gtgaactggt ttaaagtgat gccaatcaat 300 tctaagcccc agggcgtgtc cccttctaac ctggccaatc tgttccagca gtacagcgga 360 tccgagcctg acatccaggc acaggagtat ttcgcctcca actttgacac cgagaagcac 420 cagtggaagg atatgcgggt ggagtacgag agactgctgg ccgagctgca gctgtctagg 480 agcgacatgc atcacgatct gaagctgatg tacaaggaga agtgcatcgg cctgtccctg 540 tctaccgccc actatatcac aagcgtgatg tttggcaccg gcgccaagaa caatcgccag 600 acaaagcacc agttctattc caaagtgatc cagctgctgg aggagagcac ccagatcaat 660 tccgtggagc agctggcctc catcatcctg aaggccggcg actgcgattc ttacaggaag 720 ctgaggatca ggtgttcccg caagggagca accccatcta tcctgaagat cgtgcaggac 780 tatgagctgg gcacaaacca cgacgatgaa gtgaatgtgc cctccctgat cgccaacctg 840 aaggagaagc tgggcaggtt tgagtacgag tgcgagtgga agtgtatgga gaagatcaag 900 gccttcctgg cctctaaagt gggcccttac tatctgggca gctattccgc catgctggag 960 aatgccctga gcccaatcaa gggcatgacc acaaagaact gtaagttcgt gctgaagcag 1020 atcgacgcca agaacgatat caagtacgag aatgagccct ttggcaagat cgtggagggc 1080 ttctttgact ctccttattt cgagagcgat accaatgtga agtgggtgct gcaccctcac 1140 cacatcggcg agtctaacat caagacactg tgggaggacc tgaatgccat ccacagcaag 1200 tacgaggagg acatcgcctc tctgagcgag gataagaagg agaagcggat caaggtgtac 1260 cagggcgatg tgtgccagac catcaacaca tattgtgagg aagtgggcaa ggaggccaag 1320 accccactgg tgcagctgct gaggtacctg tattcccgca aggacgatat cgccgtggac 1380 aagatcatcg atggcatcac attcctgtct aagaagcaca aggtggagaa gcagaagatc 1440 aacccagtga tccagaagta ccccagcttc aattttggca acaattccaa gctgctgggc 1500 aagatcatca gcccaaagga caagctgaag cacaacctga agtgcaacag aaatcaggtg 1560 gataattaca tctggatcga gatcaaggtg ctgaacacca agacaatgcg gtgggagaag 1620 caccactatg ccctgagctc caccagattt ctggaggagg tgtactatcc cgccacatcc 1680 gagaatccac ctgacgcact ggcagcacgg ttcagaacca agacaaacgg ctacgagggc 1740 aagccagccc tgtctgccga gcagatcgag cagatcagga gcgcaccagt gggactgaga 1800 aaggtgaaga agcggcagat gagactggag gcagcaaggc agcagaatct gctgccacgc 1860 tatacctggg gcaaggattt taacatcaat atctgtaaga ggggcaacaa tttcgaggtg 1920 accctggcca caaaggtgaa gaagaagaag gagaagaact acaaggtggt gctgggctat 1980 gacgccaaca tcgtgcgcaa gaatacctac gcagcaatcg aggcacacgc aaacggcgat 2040 ggcgtgatcg actataatga tctgcctgtg aagccaatcg agtctggctt tgtgacagtg 2100 gagagccagg tgagggacaa gtcctacgat cagctgtctt ataacggcgt gaagctgctg 2160 tactgcaagc ctcacgtgga gagccggaga tccttcctgg agaagtatcg gaacggcacc 2220 atgaaggaca atagaggcaa caatatccag atcgacttca tgaaggattt tgaggccatc 2280 gccgacgatg agacaagcct gtactacttc aacatgaagt actgtaagct gctgcagtct 2340 agcatccgca accactcctc tcaggccaag gagtataggg aggagatctt cgagctgctg 2400 cgcgatggca agctgtccgt gctgaagctg agctccctgt ctaatctgag cttcgtgatg 2460 tttaaggtgg ccaagtctct gatcggcacc tactttggcc acctgctgaa gaagcctaag 2520 aactccaagt ctgacgtgaa ggccccaccc atcacagacg aggataagca gaaggccgat 2580 ccagagatgt tcgcactgcg gctggcactg gaggagaaga gactgaataa ggtgaagagc 2640 aagaaggaag tgatcgccaa caagatcgtg gccaaggcac tggagctgag ggacaagtac 2700 ggaccagtgc tgatcaaggg cgagaatatc agcgatacca caaagaaggg caagaagtct 2760 agcaccaatt ccttcctgat ggactggctg gccagaggcg tggccaacaa ggtgaaggag 2820 atggtcatga tgcaccaggg cctggagttc gtggaggtga accccaattt tacctcccac 2880 caggatcctt tcgtgcacaa gaacccagag aataccttcc gggcaaggta cagcaggtgc 2940 accccttccg agctgacaga gaagaaccgc aaggagatcc tgtccttcct gtctgacaag 3000 cccagcaagc ggcctactaa cgcctactat aatgagggcg ccatggcctt tctggccaca 3060 tatggcctga agaagaatga cgtgctgggc gtgtccctgg agaagttcaa gcagatcatg 3120 gccaacatcc tgcaccagcg gtccgaggat cagctgctgt ttccctctag aggcggcatg 3180 ttctacctgg ccacctataa gctggacgcc gatgccacaa gcgtgaactg gaatggcaag 3240 cagttttggg tgtgtaacgc cgacctggtg gccgcctaca atgtgggcct ggtggacatc 3300 cagaaggatt tcaagaagaa gaaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 3360 aagaaaaagt aataa 3375 <210> SEQ ID NO 502 <211> LENGTH: 3258 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 502 atgaaaatcg aagaaggtaa aggtcaccat caccatcacc acatgagctc cgccatcaag 60 tcctacaagt ctgtgctgcg gccaaacgag agaaagaatc agctgctgaa gagcaccatc 120 cagtgcctgg aggacggctc cgccttcttt ttcaagatgc tgcagggcct gtttggcggc 180 atcacccccg agatcgtgag attcagcaca gagcaggaga agcagcagca ggatatcgcc 240 ctgtggtgtg ccgtgaattg gttcaggcct gtgagccagg actccctgac ccacacaatc 300 gcctccgata acctggtgga gaagtttgag gagtactatg gcggcacagc cagcgacgcc 360 atcaagcagt acttcagcgc ctccatcggc gagtcctact attggaatga ctgccgccag 420 cagtactatg atctgtgtcg ggagctgggc gtggaggtgt ctgacctgac ccacgatctg 480 gagatcctgt gccgggagaa gtgtctggcc gtggccacag agagcaacca gaacaattct 540 atcatcagcg tgctgtttgg caccggcgag aaggaggata ggtctgtgaa gctgcgcatc 600 acaaagaaga tcctggaggc catcagcaac ctgaaggaga tcccaaagaa tgtggccccc 660 atccaggaga tcatcctgaa tgtggccaag gccaccaagg agacattcag acaggtgtac 720 gcaggaaacc tgggagcacc atccaccctg gagaagttta tcgccaagga cggccagaag 780 gagttcgatc tgaagaagct gcagacagac ctgaagaaag tgatccgggg caagtctaag 840 gagagagatt ggtgctgtca ggaggagctg aggagctacg tggagcagaa taccatccag 900 tatgacctgt gggcctgggg cgagatgttc aacaaggccc acaccgccct gaagatcaag 960 tccacaagaa actacaattt tgccaagcag aggctggagc agttcaagga gatccagtct 1020 ctgaacaatc tgctggtggt gaagaagctg aacgactttt tcgatagcga gtttttctcc 1080 ggcgaggaga cctacacaat ctgcgtgcac cacctgggcg gcaaggacct gtccaagctg 1140 tataaggcct gggaggacga tcccgccgat cctgagaatg ccatcgtggt gctgtgcgac 1200 gatctgaaga acaattttaa gaaggagcct atcaggaaca tcctgcgcta catcttcacc 1260 atccgccagg agtgtagcgc acaggacatc ctggcagcag caaagtacaa tcagcagctg 1320 gatcggtata agagccagaa ggccaaccca tccgtgctgg gcaatcaggg ctttacctgg 1380 acaaacgccg tgatcctgcc agagaaggcc cagcggaacg acagacccaa ttctctggat 1440 ctgcgcatct ggctgtacct gaagctgcgg caccctgacg gcagatggaa gaagcaccac 1500 atcccattct acgatacccg gtttttccag gagatctatg ccgccggcaa tagccctgtg 1560 gacacctgtc agtttaggac accccgcttc ggctatcacc tgcctaagct gaccgatcag 1620 acagccatcc gcgtgaacaa gaagcacgtg aaggcagcaa agaccgaggc acggatcaga 1680 ctggccatcc agcagggcac actgccagtg tccaatctga agatcaccga gatctccgcc 1740 acaatcaact ctaagggcca ggtgcgcatc cccgtgaagt ttgacgtggg aaggcagaag 1800 ggaaccctgc agatcggcga ccggttctgc ggctacgatc agaaccagac agcctctcac 1860 gcctatagcc tgtgggaggt ggtgaaggag ggccagtacc acaaggagct gggctgtttt 1920 gtgcgcttca tctctagcgg cgacatcgtg tccatcaccg agaaccgggg caatcagttt 1980 gatcagctgt cttatgaggg cctggcctac ccccagtatg ccgactggag aaagaaggcc 2040 tccaagttcg tgtctctgtg gcagatcacc aagaagaaca agaagaagga gatcgtgaca 2100 gtggaggcca aggagaagtt tgacgccatc tgcaagtacc agcctaggct gtataagttc 2160 aacaaggagt acgcctatct gctgcgggat atcgtgagag gcaagagcct ggtggagctg 2220 cagcagatca ggcaggagat ctttcgcttc atcgagcagg actgtggagt gacccgcctg 2280 ggatctctga gcctgtccac cctggagaca gtgaaggccg tgaagggcat catctactcc 2340 tatttttcta cagccctgaa tgcctctaag aacaatccca tcagcgacga gcagcggaag 2400 gagtttgatc ctgagctgtt cgccctgctg gagaagctgg agctgatcag gactcggaag 2460 aagaagcaga aggtggagag aatcgccaat agcctgatcc agacatgcct ggagaacaat 2520 atcaagttca tcaggggcga gggcgacctg tccaccacaa acaatgccac caagaagaag 2580 gccaactcta ggagcatgga ttggctggcc agaggcgtgt ttaataagat ccggcagctg 2640 gccccaatgc acaacatcac cctgttcggc tgcggcagcc tgtacacatc ccaccaggac 2700 cctctggtgc acagaaaccc agataaggcc atgaagtgta gatgggcagc aatcccagtg 2760 aaggacatcg gcgattgggt gctgagaaag ctgtcccaga acctgagggc caagaatatc 2820 ggcaccggcg agtactatca ccagggcgtg aaggagttcc tgtctcacta tgagctgcag 2880 gacctggagg aggagctgct gaagtggcgg tctgatagaa agagcaacat cccttgctgg 2940 gtgctgcaga atagactggc cgagaagctg ggcaacaagg aggccgtggt gtacatccca 3000 gtgaggggcg gccgcatcta ttttgcaacc cacaaggtgg caacaggagc cgtgagcatc 3060 gtgttcgacc agaagcaagt gtgggtgtgt aatgcagatc acgtggcagc agcaaacatc 3120 gcactgaccg tgaagggcat cggcgagcag tcctctgacg aggagaaccc cgatggctcc 3180 aggatcaagc tgcagctgac atctaaaagg ccggcggcca cgaaaaaggc cggccaggca 3240 aaaaagaaaa agtaataa 3258 <210> SEQ ID NO 503 <211> LENGTH: 228 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 503 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 60 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 120 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 180 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 228 <210> SEQ ID NO 504 <400> SEQUENCE: 504 000 <210> SEQ ID NO 505 <400> SEQUENCE: 505 000 <210> SEQ ID NO 506 <400> SEQUENCE: 506 000 <210> SEQ ID NO 507 <400> SEQUENCE: 507 000 <210> SEQ ID NO 508 <400> SEQUENCE: 508 000 <210> SEQ ID NO 509 <400> SEQUENCE: 509 000 <210> SEQ ID NO 510 <400> SEQUENCE: 510 000 <210> SEQ ID NO 511 <400> SEQUENCE: 511 000 <210> SEQ ID NO 512 <400> SEQUENCE: 512 000 <210> SEQ ID NO 513 <400> SEQUENCE: 513 000 <210> SEQ ID NO 514 <400> SEQUENCE: 514 000 <210> SEQ ID NO 515 <400> SEQUENCE: 515 000 <210> SEQ ID NO 516 <400> SEQUENCE: 516 000 <210> SEQ ID NO 517 <400> SEQUENCE: 517 000 <210> SEQ ID NO 518 <400> SEQUENCE: 518 000 <210> SEQ ID NO 519 <400> SEQUENCE: 519 000 <210> SEQ ID NO 520 <400> SEQUENCE: 520 000 <210> SEQ ID NO 521 <400> SEQUENCE: 521 000 <210> SEQ ID NO 522 <400> SEQUENCE: 522 000 <210> SEQ ID NO 523 <400> SEQUENCE: 523 000 <210> SEQ ID NO 524 <400> SEQUENCE: 524 000 <210> SEQ ID NO 525 <400> SEQUENCE: 525 000 <210> SEQ ID NO 526 <400> SEQUENCE: 526 000 <210> SEQ ID NO 527 <400> SEQUENCE: 527 000 <210> SEQ ID NO 528 <400> SEQUENCE: 528 000 <210> SEQ ID NO 529 <400> SEQUENCE: 529 000 <210> SEQ ID NO 530 <400> SEQUENCE: 530 000 <210> SEQ ID NO 531 <400> SEQUENCE: 531 000 <210> SEQ ID NO 532 <400> SEQUENCE: 532 000 <210> SEQ ID NO 533 <400> SEQUENCE: 533 000 <210> SEQ ID NO 534 <400> SEQUENCE: 534 000 <210> SEQ ID NO 535 <400> SEQUENCE: 535 000 <210> SEQ ID NO 536 <400> SEQUENCE: 536 000 <210> SEQ ID NO 537 <400> SEQUENCE: 537 000 <210> SEQ ID NO 538 <400> SEQUENCE: 538 000 <210> SEQ ID NO 539 <400> SEQUENCE: 539 000 <210> SEQ ID NO 540 <400> SEQUENCE: 540 000 <210> SEQ ID NO 541 <400> SEQUENCE: 541 000 <210> SEQ ID NO 542 <400> SEQUENCE: 542 000 <210> SEQ ID NO 543 <400> SEQUENCE: 543 000 <210> SEQ ID NO 544 <400> SEQUENCE: 544 000 <210> SEQ ID NO 545 <400> SEQUENCE: 545 000 <210> SEQ ID NO 546 <400> SEQUENCE: 546 000 <210> SEQ ID NO 547 <400> SEQUENCE: 547 000 <210> SEQ ID NO 548 <400> SEQUENCE: 548 000 <210> SEQ ID NO 549 <400> SEQUENCE: 549 000 <210> SEQ ID NO 550 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 550 accccctttc caaagcccat tccctctttt cgagccgggg tgtgc 45 <210> SEQ ID NO 551 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 551 accccctttc caaagcccat tccctctttt tgagccgggg tgtgc 45 <210> SEQ ID NO 552 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 552 accccctttc caaagcccat tccctgttta tgagccgggg tgtgc 45 <210> SEQ ID NO 553 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 553 accccctttc caaagcccat tccctcttta agagccgggg tgtg 44 <210> SEQ ID NO 554 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 554 accccctttc caaagcccat tacctcttta agagccgggg tgtg 44 <210> SEQ ID NO 555 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 555 accccctttc caaagcccat tccctctgta agagccgggg tgtg 44 <210> SEQ ID NO 556 <400> SEQUENCE: 556 000 <210> SEQ ID NO 557 <400> SEQUENCE: 557 000 <210> SEQ ID NO 558 <400> SEQUENCE: 558 000 <210> SEQ ID NO 559 <400> SEQUENCE: 559 000 <210> SEQ ID NO 560 <400> SEQUENCE: 560 000 <210> SEQ ID NO 561 <400> SEQUENCE: 561 000 <210> SEQ ID NO 562 <400> SEQUENCE: 562 000 <210> SEQ ID NO 563 <400> SEQUENCE: 563 000 <210> SEQ ID NO 564 <400> SEQUENCE: 564 000 <210> SEQ ID NO 565 <400> SEQUENCE: 565 000 <210> SEQ ID NO 566 <400> SEQUENCE: 566 000 <210> SEQ ID NO 567 <400> SEQUENCE: 567 000 <210> SEQ ID NO 568 <400> SEQUENCE: 568 000 <210> SEQ ID NO 569 <400> SEQUENCE: 569 000 <210> SEQ ID NO 570 <400> SEQUENCE: 570 000 <210> SEQ ID NO 571 <400> SEQUENCE: 571 000 <210> SEQ ID NO 572 <400> SEQUENCE: 572 000 <210> SEQ ID NO 573 <400> SEQUENCE: 573 000 <210> SEQ ID NO 574 <400> SEQUENCE: 574 000 <210> SEQ ID NO 575 <400> SEQUENCE: 575 000 <210> SEQ ID NO 576 <400> SEQUENCE: 576 000 <210> SEQ ID NO 577 <400> SEQUENCE: 577 000 <210> SEQ ID NO 578 <400> SEQUENCE: 578 000 <210> SEQ ID NO 579 <400> SEQUENCE: 579 000 <210> SEQ ID NO 580 <400> SEQUENCE: 580 000 <210> SEQ ID NO 581 <400> SEQUENCE: 581 000 <210> SEQ ID NO 582 <400> SEQUENCE: 582 000 <210> SEQ ID NO 583 <400> SEQUENCE: 583 000 <210> SEQ ID NO 584 <400> SEQUENCE: 584 000 <210> SEQ ID NO 585 <400> SEQUENCE: 585 000 <210> SEQ ID NO 586 <400> SEQUENCE: 586 000 <210> SEQ ID NO 587 <400> SEQUENCE: 587 000 <210> SEQ ID NO 588 <400> SEQUENCE: 588 000 <210> SEQ ID NO 589 <400> SEQUENCE: 589 000 <210> SEQ ID NO 590 <400> SEQUENCE: 590 000 <210> SEQ ID NO 591 <400> SEQUENCE: 591 000 <210> SEQ ID NO 592 <400> SEQUENCE: 592 000 <210> SEQ ID NO 593 <400> SEQUENCE: 593 000 <210> SEQ ID NO 594 <400> SEQUENCE: 594 000 <210> SEQ ID NO 595 <400> SEQUENCE: 595 000 <210> SEQ ID NO 596 <400> SEQUENCE: 596 000 <210> SEQ ID NO 597 <400> SEQUENCE: 597 000 <210> SEQ ID NO 598 <400> SEQUENCE: 598 000 <210> SEQ ID NO 599 <400> SEQUENCE: 599 000 <210> SEQ ID NO 600 <211> LENGTH: 2 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 600 Gly Ser 1 <210> SEQ ID NO 601 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 601 Gly Ser Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 602 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 602 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 603 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 603 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser <210> SEQ ID NO 604 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 604 Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr 1 5 10 15 Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser 20 25 30 <210> SEQ ID NO 605 <211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 605 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> SEQ ID NO 606 <211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 606 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> SEQ ID NO 607 <211> LENGTH: 198 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 607 Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys 1 5 10 15 Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val 20 25 30 Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr 35 40 45 Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr 50 55 60 Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp 65 70 75 80 Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp 85 90 95 Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg 100 105 110 Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg 115 120 125 Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr 130 135 140 Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys 145 150 155 160 Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu 165 170 175 Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala 180 185 190 Phe Arg Thr Leu Gly Leu 195 <210> SEQ ID NO 608 <211> LENGTH: 208 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 608 Met Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr 1 5 10 15 Thr Phe Lys Lys Gln Phe Phe Asn Asn Lys Lys Ser Val Ser His Arg 20 25 30 Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys 35 40 45 Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly 50 55 60 Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg 65 70 75 80 Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro 85 90 95 Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu 100 105 110 Arg Gly Asn Gly His Thr Leu Lys Ile Trp Ala Cys Lys Leu Tyr Tyr 115 120 125 Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn 130 135 140 Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg 145 150 155 160 Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp 165 170 175 Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Arg Arg Ser Glu Leu Ser 180 185 190 Ile Met Ile Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val 195 200 205 <210> SEQ ID NO 609 <211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 609 Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg 1 5 10 15 Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu 20 25 30 Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His 35 40 45 Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val 50 55 60 Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr 65 70 75 80 Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys 85 90 95 Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu 100 105 110 Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg 115 120 125 Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met 130 135 140 Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser 145 150 155 160 Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg 165 170 175 Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys 180 185 190 Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile 195 200 205 Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp 210 215 220 Ala Thr Gly Leu Lys 225 <210> SEQ ID NO 610 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: KDEL motif sequence" <400> SEQUENCE: 610 Lys Asp Glu Leu 1 <210> SEQ ID NO 611 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 611 Glu Ala Ala Ala Lys 1 5 <210> SEQ ID NO 612 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(44) <223> OTHER INFORMATION: a, c, t, g, unknown or other <400> SEQUENCE: 612 atttttgtgc ccatcgttgg cacnnnnnnn nnnnnnnnnn nnnn 44 <210> SEQ ID NO 613 <211> LENGTH: 44 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(44) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 613 agaaauccgu cuuucauuga cggnnnnnnn nnnnnnnnnn nnnn 44 <210> SEQ ID NO 614 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 614 ttcgcgtgct ggattgcttc gatggtctgc ggcatc 36 <210> SEQ ID NO 615 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 615 gatgccgcag accatcgaag caatccagca cgcgaa 36 <210> SEQ ID NO 616 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 616 gcgugcugga uugcuucgau ggucugcg 28 <210> SEQ ID NO 617 <211> LENGTH: 34 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 617 gcaacaccua agaaauccgu cuuucauuga cggg 34 <210> SEQ ID NO 618 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 618 guugcaaaac ccaagaaauc cgucuuucau ugacgg 36 <210> SEQ ID NO 619 <211> LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 619 aauuuuugug cccaucguug gcac 24 <210> SEQ ID NO 620 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 620 cucucaaugc cuuagaaauc cguccuuggu ugacgg 36 <210> SEQ ID NO 621 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 621 cccacaauac cugagaaauc cguccuacgu ugacgg 36

1 SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 621 <210> SEQ ID NO 1 <211> LENGTH: 1046 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 1 Met Val Ser Glu Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala 35 40 45 Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe 50 55 60 Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Gln Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser 130 135 140 Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ser Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Ile Glu Asp Lys Lys Pro Asn Thr Trp Glu Glu 195 200 205 Tyr His Asp Leu Ile Lys Lys Thr Phe Asn Val Asp Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg 260 265 270 Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu 355 360 365 Asn Glu Leu Phe Glu Ile Trp Gln Glu Leu Thr Met Asp Asp Gly Ile 370 375 380 Lys Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val 385 390 395 400 Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Asn Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe 485 490 495 His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Lys Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Thr Ile Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile 595 600 605 Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Val Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp 705 710 715 720 Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu 770 775 780 Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys 785 790 795 800 Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly 820 825 830 Tyr Leu Pro Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln 930 935 940 Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Lys Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Gly Asp His 965 970 975 Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Ile Asp Glu Asp Glu Gln Pro Asp Gly Ala Lys 1025 1030 1035 Lys Gln Ala Leu Ala Arg Thr Lys 1040 1045 <210> SEQ ID NO 2 <211> LENGTH: 1091 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 2 Met Phe Thr Leu Leu Leu Ser Asp Ile Ser Gln Gln Asn Phe Asn Lys 1 5 10 15 Phe Leu Lys Asn Phe Phe Phe Thr Arg Asn Lys Thr Val Val His Cys 20 25 30 Ser Ser Glu Ile Arg His Lys Gly Tyr Arg Ser Asn Val Met Val Ser 35 40 45 Glu Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro Asn Asp Pro 50 55 60 Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp His Ala Tyr 65 70 75 80 Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala Ile Glu His 85 90 95 Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe Asp Ala Asp 100 105 110 Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys Ser Asp Asn 115 120 125

Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu Phe Gln Lys 130 135 140 Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr Ile Lys Gly 145 150 155 160 Asn Ile Asp Ser Glu Lys Tyr Gln Trp Val Asp Cys Arg Leu Lys Phe 165 170 175 Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser Leu Lys Ile 180 185 190 Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val Ser Lys Asp 195 200 205 Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe Gly Thr Gly 210 215 220 Lys Lys Glu Asp Arg Ser Ile Lys Ala Ser Met Leu Glu Glu Ile Ser 225 230 235 240 Asn Ile Ile Glu Asp Lys Lys Pro Asn Thr Trp Glu Glu Tyr His Asp 245 250 255 Leu Ile Lys Lys Thr Phe Asn Val Asp Asn Tyr Lys Glu Leu Lys Glu 260 265 270 Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu Val Ile Asp 275 280 285 Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe Ile Lys Asn 290 295 300 Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg Thr Val Phe 305 310 315 320 Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala Ser Gln Ile 325 330 335 Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn Arg Ser Met 340 345 350 Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr Thr Asn Glu 355 360 365 Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys Lys Asp Ile 370 375 380 Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly Glu Phe Asn 385 390 395 400 Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu Asn Glu Leu 405 410 415 Phe Glu Ile Trp Gln Glu Leu Thr Met Asp Asp Gly Ile Lys Lys Tyr 420 425 430 Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val Lys Ala Leu 435 440 445 Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Asn Ala Lys Gln Phe 450 455 460 Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn Asn Arg Lys 465 470 475 480 Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn Trp Gly Pro 485 490 495 Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln Met Val Lys 500 505 510 Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr Met Thr Val 515 520 525 Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe His Asn Ser 530 535 540 Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu Pro Thr Lys 545 550 555 560 Asn Lys Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly Ser Thr Ile 565 570 575 Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu Gln Asp Arg 580 585 590 Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser Ile Ile Arg 595 600 605 Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys Ser Thr Asn 610 615 620 Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr Ile Ser Ser 625 630 635 640 Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile Gly Asp Met 645 650 655 Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr Tyr Ser Ile 660 665 670 Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe His Asn Lys 675 680 685 Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Val Thr Ser Ile Val Asp 690 695 700 Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile Glu Tyr Ser 705 710 715 720 Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu Arg Ser Ile 725 730 735 Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn Met Asn Leu 740 745 750 Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp Val Met Lys 755 760 765 Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala Glu Ile Glu 770 775 780 Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser Leu Phe His 785 790 795 800 His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile Ser Ser Tyr 805 810 815 Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu Leu Phe Asp 820 825 830 Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys Arg Val Arg 835 840 845 Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val Leu Gln Ile 850 855 860 Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly Tyr Leu Pro 865 870 875 880 Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys Ser Ile Asp 885 890 895 Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly Cys Lys Val 900 905 910 Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr Ser His Leu 915 920 925 Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val Gly Lys Glu 930 935 940 Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu Tyr Met Thr 945 950 955 960 Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys Ser Lys Lys 965 970 975 Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln Glu Ala Leu 980 985 990 Arg Gln Phe Ala Ser His Tyr Lys Leu Asp Phe Asp Ser Leu Pro Lys 995 1000 1005 Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Gly Asp His Glu Lys 1010 1015 1020 Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr Tyr 1025 1030 1035 Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 1040 1045 1050 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1055 1060 1065 Leu Arg Gly Ile Ile Asp Glu Asp Glu Gln Pro Asp Gly Ala Lys 1070 1075 1080 Lys Gln Ala Leu Ala Arg Thr Lys 1085 1090 <210> SEQ ID NO 3 <211> LENGTH: 1093 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 3 Met Ser Asn Lys Glu Lys Asn Ala Ser Glu Thr Arg Lys Ala Tyr Thr 1 5 10 15 Thr Lys Met Ile Pro Arg Ser His Asp Arg Met Lys Leu Leu Gly Asn 20 25 30 Phe Met Asp Tyr Leu Met Asp Gly Thr Pro Ile Phe Phe Glu Leu Trp 35 40 45 Asn Gln Phe Gly Gly Gly Ile Asp Arg Asp Ile Ile Ser Gly Thr Ala 50 55 60 Asn Lys Asp Lys Ile Ser Asp Asp Leu Leu Leu Ala Val Asn Trp Phe 65 70 75 80 Lys Val Met Pro Ile Asn Ser Lys Pro Gln Gly Val Ser Pro Ser Asn 85 90 95 Leu Ala Asn Leu Phe Gln Gln Tyr Ser Gly Ser Glu Pro Asp Ile Gln 100 105 110 Ala Gln Glu Tyr Phe Ala Ser Asn Phe Asp Thr Glu Lys His Gln Trp 115 120 125 Lys Asp Met Arg Val Glu Tyr Glu Arg Leu Leu Ala Glu Leu Gln Leu 130 135 140 Ser Arg Ser Asp Met His His Asp Leu Lys Leu Met Tyr Lys Glu Lys 145 150 155 160 Cys Ile Gly Leu Ser Leu Ser Thr Ala His Tyr Ile Thr Ser Val Met 165 170 175 Phe Gly Thr Gly Ala Lys Asn Asn Arg Gln Thr Lys His Gln Phe Tyr 180 185 190 Ser Lys Val Ile Gln Leu Leu Glu Glu Ser Thr Gln Ile Asn Ser Val 195 200 205 Glu Gln Leu Ala Ser Ile Ile Leu Lys Ala Gly Asp Cys Asp Ser Tyr 210 215 220 Arg Lys Leu Arg Ile Arg Cys Ser Arg Lys Gly Ala Thr Pro Ser Ile 225 230 235 240 Leu Lys Ile Val Gln Asp Tyr Glu Leu Gly Thr Asn His Asp Asp Glu 245 250 255 Val Asn Val Pro Ser Leu Ile Ala Asn Leu Lys Glu Lys Leu Gly Arg 260 265 270 Phe Glu Tyr Glu Cys Glu Trp Lys Cys Met Glu Lys Ile Lys Ala Phe 275 280 285 Leu Ala Ser Lys Val Gly Pro Tyr Tyr Leu Gly Ser Tyr Ser Ala Met

290 295 300 Leu Glu Asn Ala Leu Ser Pro Ile Lys Gly Met Thr Thr Lys Asn Cys 305 310 315 320 Lys Phe Val Leu Lys Gln Ile Asp Ala Lys Asn Asp Ile Lys Tyr Glu 325 330 335 Asn Glu Pro Phe Gly Lys Ile Val Glu Gly Phe Phe Asp Ser Pro Tyr 340 345 350 Phe Glu Ser Asp Thr Asn Val Lys Trp Val Leu His Pro His His Ile 355 360 365 Gly Glu Ser Asn Ile Lys Thr Leu Trp Glu Asp Leu Asn Ala Ile His 370 375 380 Ser Lys Tyr Glu Glu Asp Ile Ala Ser Leu Ser Glu Asp Lys Lys Glu 385 390 395 400 Lys Arg Ile Lys Val Tyr Gln Gly Asp Val Cys Gln Thr Ile Asn Thr 405 410 415 Tyr Cys Glu Glu Val Gly Lys Glu Ala Lys Thr Pro Leu Val Gln Leu 420 425 430 Leu Arg Tyr Leu Tyr Ser Arg Lys Asp Asp Ile Ala Val Asp Lys Ile 435 440 445 Ile Asp Gly Ile Thr Phe Leu Ser Lys Lys His Lys Val Glu Lys Gln 450 455 460 Lys Ile Asn Pro Val Ile Gln Lys Tyr Pro Ser Phe Asn Phe Gly Asn 465 470 475 480 Asn Ser Lys Leu Leu Gly Lys Ile Ile Ser Pro Lys Asp Lys Leu Lys 485 490 495 His Asn Leu Lys Cys Asn Arg Asn Gln Val Asp Asn Tyr Ile Trp Ile 500 505 510 Glu Ile Lys Val Leu Asn Thr Lys Thr Met Arg Trp Glu Lys His His 515 520 525 Tyr Ala Leu Ser Ser Thr Arg Phe Leu Glu Glu Val Tyr Tyr Pro Ala 530 535 540 Thr Ser Glu Asn Pro Pro Asp Ala Leu Ala Ala Arg Phe Arg Thr Lys 545 550 555 560 Thr Asn Gly Tyr Glu Gly Lys Pro Ala Leu Ser Ala Glu Gln Ile Glu 565 570 575 Gln Ile Arg Ser Ala Pro Val Gly Leu Arg Lys Val Lys Lys Arg Gln 580 585 590 Met Arg Leu Glu Ala Ala Arg Gln Gln Asn Leu Leu Pro Arg Tyr Thr 595 600 605 Trp Gly Lys Asp Phe Asn Ile Asn Ile Cys Lys Arg Gly Asn Asn Phe 610 615 620 Glu Val Thr Leu Ala Thr Lys Val Lys Lys Lys Lys Glu Lys Asn Tyr 625 630 635 640 Lys Val Val Leu Gly Tyr Asp Ala Asn Ile Val Arg Lys Asn Thr Tyr 645 650 655 Ala Ala Ile Glu Ala His Ala Asn Gly Asp Gly Val Ile Asp Tyr Asn 660 665 670 Asp Leu Pro Val Lys Pro Ile Glu Ser Gly Phe Val Thr Val Glu Ser 675 680 685 Gln Val Arg Asp Lys Ser Tyr Asp Gln Leu Ser Tyr Asn Gly Val Lys 690 695 700 Leu Leu Tyr Cys Lys Pro His Val Glu Ser Arg Arg Ser Phe Leu Glu 705 710 715 720 Lys Tyr Arg Asn Gly Thr Met Lys Asp Asn Arg Gly Asn Asn Ile Gln 725 730 735 Ile Asp Phe Met Lys Asp Phe Glu Ala Ile Ala Asp Asp Glu Thr Ser 740 745 750 Leu Tyr Tyr Phe Asn Met Lys Tyr Cys Lys Leu Leu Gln Ser Ser Ile 755 760 765 Arg Asn His Ser Ser Gln Ala Lys Glu Tyr Arg Glu Glu Ile Phe Glu 770 775 780 Leu Leu Arg Asp Gly Lys Leu Ser Val Leu Lys Leu Ser Ser Leu Ser 785 790 795 800 Asn Leu Ser Phe Val Met Phe Lys Val Ala Lys Ser Leu Ile Gly Thr 805 810 815 Tyr Phe Gly His Leu Leu Lys Lys Pro Lys Asn Ser Lys Ser Asp Val 820 825 830 Lys Ala Pro Pro Ile Thr Asp Glu Asp Lys Gln Lys Ala Asp Pro Glu 835 840 845 Met Phe Ala Leu Arg Leu Ala Leu Glu Glu Lys Arg Leu Asn Lys Val 850 855 860 Lys Ser Lys Lys Glu Val Ile Ala Asn Lys Ile Val Ala Lys Ala Leu 865 870 875 880 Glu Leu Arg Asp Lys Tyr Gly Pro Val Leu Ile Lys Gly Glu Asn Ile 885 890 895 Ser Asp Thr Thr Lys Lys Gly Lys Lys Ser Ser Thr Asn Ser Phe Leu 900 905 910 Met Asp Trp Leu Ala Arg Gly Val Ala Asn Lys Val Lys Glu Met Val 915 920 925 Met Met His Gln Gly Leu Glu Phe Val Glu Val Asn Pro Asn Phe Thr 930 935 940 Ser His Gln Asp Pro Phe Val His Lys Asn Pro Glu Asn Thr Phe Arg 945 950 955 960 Ala Arg Tyr Ser Arg Cys Thr Pro Ser Glu Leu Thr Glu Lys Asn Arg 965 970 975 Lys Glu Ile Leu Ser Phe Leu Ser Asp Lys Pro Ser Lys Arg Pro Thr 980 985 990 Asn Ala Tyr Tyr Asn Glu Gly Ala Met Ala Phe Leu Ala Thr Tyr Gly 995 1000 1005 Leu Lys Lys Asn Asp Val Leu Gly Val Ser Leu Glu Lys Phe Lys 1010 1015 1020 Gln Ile Met Ala Asn Ile Leu His Gln Arg Ser Glu Asp Gln Leu 1025 1030 1035 Leu Phe Pro Ser Arg Gly Gly Met Phe Tyr Leu Ala Thr Tyr Lys 1040 1045 1050 Leu Asp Ala Asp Ala Thr Ser Val Asn Trp Asn Gly Lys Gln Phe 1055 1060 1065 Trp Val Cys Asn Ala Asp Leu Val Ala Ala Tyr Asn Val Gly Leu 1070 1075 1080 Val Asp Ile Gln Lys Asp Phe Lys Lys Lys 1085 1090 <210> SEQ ID NO 4 <211> LENGTH: 1033 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 4 Met Met Ser Asp Asn Ile Ile Leu Pro Tyr Asn Ser Lys Leu Ala Pro 1 5 10 15 Asp Glu Arg Lys Gln Arg Leu Leu Asn Asp Thr Phe Asn Trp Phe Asp 20 25 30 Met Cys Asn Glu Val Phe Phe Asp Phe Val Lys Asn Leu Tyr Gly Gly 35 40 45 Val Lys His Glu His Leu Ile Leu Val Asn Phe Ala Glu Lys Pro Lys 50 55 60 Lys Val Ser Asn Ser Lys Lys Pro Lys Lys Lys Asp Gln Glu Val Asn 65 70 75 80 Ile His Val Glu Pro Asn Gln Ala Glu Trp Val Asp Asn Ala Cys Ala 85 90 95 Thr Phe Trp Phe Arg Leu Gln Ala Lys Ser Thr Val Gln Leu Asp Gln 100 105 110 Ser Val Gln Thr Ala Glu Glu Arg Ile Arg Arg Phe Arg Asp Tyr Ala 115 120 125 Gly His Glu Pro Ser Ser Phe Ala Lys Ser Tyr Leu Asn Gly Asn Tyr 130 135 140 Asp Pro Glu Lys Thr Glu Trp Val Asp Cys Arg Leu Leu Tyr Val Asn 145 150 155 160 Phe Cys Arg Asn Leu Asn Val Asn Leu Asp Ala Asp Ile Arg Thr Met 165 170 175 Val Glu His Asn Leu Leu Pro Val Leu Pro Gly Gln Asp Phe Lys Thr 180 185 190 Asn Asn Val Phe Ser Asn Ile Phe Gly Val Gly Asn Lys Glu Asp Lys 195 200 205 Gly Gln Lys Thr Asn Trp Leu Asn Thr Val Ser Glu Gly Leu Gln Ser 210 215 220 Lys Glu Ile Trp Asn Trp Asp Glu Tyr Arg Asp Leu Ile Ser Arg Ser 225 230 235 240 Thr Gly Cys Ser Thr Ala Ala Glu Leu Arg Ser Glu Ser Ile Gly Arg 245 250 255 Pro Ser Met Leu Ala Val Asp Phe Ala Ser Glu Lys Ser Gly Gln Ile 260 265 270 Ser Gln Glu Trp Leu Ala Glu Arg Val Lys Ser Phe Arg Ala Ala Ala 275 280 285 Ser Gln Lys Ser Lys Ile Tyr Asp Met Pro Asn Arg Leu Val Leu Lys 290 295 300 Glu Tyr Ile Ala Ser Lys Ile Gly Pro Phe Lys Leu Glu Arg Trp Ser 305 310 315 320 Ala Ala Ala Val Ser Ala Tyr Lys Asp Val Arg Ser Lys Asn Ser Ile 325 330 335 Asn Leu Leu Tyr Ser Lys Glu Arg Leu Trp Arg Cys Lys Glu Ile Ala 340 345 350 Gln Ile Leu Val Asp Asn Thr Gln Val Ala Glu Ala Gln Gln Ile Leu 355 360 365 Val Asn Tyr Ser Ser Gly Asp Thr Asn Ser Phe Thr Val Glu Asn Arg 370 375 380 His Met Gly Asp Leu Thr Val Leu Phe Lys Ile Trp Glu Lys Met Asp 385 390 395 400 Met Asp Ser Gly Ile Glu Gln Tyr Ser Glu Ile Tyr Arg Asp Glu Tyr 405 410 415 Ser Arg Asp Pro Ile Thr Glu Leu Leu Arg Tyr Leu Tyr Asn His Arg 420 425 430 His Ile Ser Ala Lys Thr Phe Arg Ala Ala Ala Arg Leu Asn Ser Leu 435 440 445 Leu Leu Lys Asn Asp Arg Lys Lys Ile His Pro Thr Ile Ser Gly Arg 450 455 460

Thr Ser Val Ser Phe Gly His Ser Thr Ile Lys Gly Cys Ile Thr Pro 465 470 475 480 Pro Asp His Ile Val Lys Asn Arg Lys Glu Asn Ala Gly Ser Thr Gly 485 490 495 Met Ile Trp Val Thr Met Gln Leu Ile Asp Asn Gly Arg Trp Ala Asp 500 505 510 His His Ile Pro Phe His Asn Ser Arg Tyr Tyr Arg Asp Phe Tyr Ala 515 520 525 Tyr Arg Ala Asp Leu Pro Thr Ile Ser Asp Pro Arg Arg Lys Ser Phe 530 535 540 Gly His Arg Ile Gly Asn Asn Ile Ser Asp Thr Arg Met Ile Asn His 545 550 555 560 Asp Cys Lys Lys Ala Ser Lys Met Tyr Leu Arg Thr Ile Gln Asn Met 565 570 575 Thr His Asn Val Ala Phe Asp Gln Gln Thr Gln Phe Ala Val Arg Arg 580 585 590 Tyr Ala Asp Asn Asn Phe Thr Ile Thr Ile Gln Ala Arg Val Val Gly 595 600 605 Arg Lys Tyr Lys Lys Glu Ile Ser Val Gly Asp Arg Val Met Gly Val 610 615 620 Asp Gln Asn Gln Thr Thr Ser Asn Thr Tyr Ser Val Trp Glu Val Val 625 630 635 640 Ala Glu Gly Thr Glu Asn Ser Tyr Pro Tyr Lys Gly Asn Asn Tyr Arg 645 650 655 Leu Val Glu Asp Gly Phe Ile Arg Ser Glu Cys Ser Gly Arg Asp Gln 660 665 670 Leu Ser Tyr Asp Gly Leu Asp Phe Gln Asp Phe Ala Gln Trp Arg Arg 675 680 685 Glu Arg Tyr Ala Phe Leu Ser Ser Val Gly Cys Ile Leu Asn Asp Glu 690 695 700 Ile Glu Pro Gln Ile Pro Val Ser Ala Glu Lys Ala Lys Lys Lys Lys 705 710 715 720 Lys Phe Ser Lys Trp Arg Gly Cys Ser Leu Tyr Ser Trp Asn Leu Cys 725 730 735 Tyr Ala Tyr Tyr Leu Lys Gly Leu Met His Glu Asn Leu Ala Asn Asn 740 745 750 Pro Ala Gly Phe Arg Gln Glu Ile Leu Asn Phe Ile Gln Gly Ser Arg 755 760 765 Gly Val Arg Leu Cys Ser Leu Asn His Thr Ser Phe Arg Leu Leu Ser 770 775 780 Lys Ala Lys Ser Leu Ile His Ser Phe Phe Gly Leu Asn Asn Ile Lys 785 790 795 800 Asp Pro Glu Ser Gln Arg Asp Phe Asp Pro Glu Ile Tyr Asp Ile Met 805 810 815 Val Asn Leu Thr Gln Arg Lys Thr Asn Lys Arg Lys Glu Lys Ala Asn 820 825 830 Arg Ile Thr Ser Ser Ile Leu Gln Ile Ala Asn Arg Leu Asn Val Ser 835 840 845 Arg Ile Val Ile Glu Asn Asp Leu Pro Asn Ala Ser Ser Lys Asn Lys 850 855 860 Ala Ser Ala Asn Gln Arg Ala Thr Asp Trp Cys Ala Arg Asn Val Ser 865 870 875 880 Glu Lys Leu Glu Tyr Ala Cys Lys Met Leu Gly Ile Ser Leu Trp Gln 885 890 895 Ile Asp Pro Arg Asp Thr Ser His Leu Asp Pro Phe Val Val Gly Lys 900 905 910 Glu Ala Arg Phe Met Lys Ile Lys Val Ser Asp Ile Asn Glu Tyr Thr 915 920 925 Ile Ser Asn Phe Lys Lys Trp His Ala Asn Ile Ala Thr Thr Ser Thr 930 935 940 Thr Ala Pro Leu Tyr His Asp Ala Leu Lys Ala Phe Ser Ser His Tyr 945 950 955 960 Gly Ile Asp Trp Asp Asn Leu Pro Glu Met Lys Phe Trp Glu Leu Lys 965 970 975 Asn Ala Leu Lys Asp His Lys Glu Val Phe Ile Pro Asn Arg Gly Gly 980 985 990 Arg Cys Tyr Leu Ser Thr Leu Pro Val Thr Ser Thr Ser Glu Lys Ile 995 1000 1005 Val Phe Asn Gly Arg Glu Arg Trp Leu Asn Ala Ser Asp Ile Val 1010 1015 1020 Ala Gly Val Asn Ile Val Leu Arg Ser Val 1025 1030 <210> SEQ ID NO 5 <211> LENGTH: 1054 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 5 Met Ser Ser Ala Ile Lys Ser Tyr Lys Ser Val Leu Arg Pro Asn Glu 1 5 10 15 Arg Lys Asn Gln Leu Leu Lys Ser Thr Ile Gln Cys Leu Glu Asp Gly 20 25 30 Ser Ala Phe Phe Phe Lys Met Leu Gln Gly Leu Phe Gly Gly Ile Thr 35 40 45 Pro Glu Ile Val Arg Phe Ser Thr Glu Gln Glu Lys Gln Gln Gln Asp 50 55 60 Ile Ala Leu Trp Cys Ala Val Asn Trp Phe Arg Pro Val Ser Gln Asp 65 70 75 80 Ser Leu Thr His Thr Ile Ala Ser Asp Asn Leu Val Glu Lys Phe Glu 85 90 95 Glu Tyr Tyr Gly Gly Thr Ala Ser Asp Ala Ile Lys Gln Tyr Phe Ser 100 105 110 Ala Ser Ile Gly Glu Ser Tyr Tyr Trp Asn Asp Cys Arg Gln Gln Tyr 115 120 125 Tyr Asp Leu Cys Arg Glu Leu Gly Val Glu Val Ser Asp Leu Thr His 130 135 140 Asp Leu Glu Ile Leu Cys Arg Glu Lys Cys Leu Ala Val Ala Thr Glu 145 150 155 160 Ser Asn Gln Asn Asn Ser Ile Ile Ser Val Leu Phe Gly Thr Gly Glu 165 170 175 Lys Glu Asp Arg Ser Val Lys Leu Arg Ile Thr Lys Lys Ile Leu Glu 180 185 190 Ala Ile Ser Asn Leu Lys Glu Ile Pro Lys Asn Val Ala Pro Ile Gln 195 200 205 Glu Ile Ile Leu Asn Val Ala Lys Ala Thr Lys Glu Thr Phe Arg Gln 210 215 220 Val Tyr Ala Gly Asn Leu Gly Ala Pro Ser Thr Leu Glu Lys Phe Ile 225 230 235 240 Ala Lys Asp Gly Gln Lys Glu Phe Asp Leu Lys Lys Leu Gln Thr Asp 245 250 255 Leu Lys Lys Val Ile Arg Gly Lys Ser Lys Glu Arg Asp Trp Cys Cys 260 265 270 Gln Glu Glu Leu Arg Ser Tyr Val Glu Gln Asn Thr Ile Gln Tyr Asp 275 280 285 Leu Trp Ala Trp Gly Glu Met Phe Asn Lys Ala His Thr Ala Leu Lys 290 295 300 Ile Lys Ser Thr Arg Asn Tyr Asn Phe Ala Lys Gln Arg Leu Glu Gln 305 310 315 320 Phe Lys Glu Ile Gln Ser Leu Asn Asn Leu Leu Val Val Lys Lys Leu 325 330 335 Asn Asp Phe Phe Asp Ser Glu Phe Phe Ser Gly Glu Glu Thr Tyr Thr 340 345 350 Ile Cys Val His His Leu Gly Gly Lys Asp Leu Ser Lys Leu Tyr Lys 355 360 365 Ala Trp Glu Asp Asp Pro Ala Asp Pro Glu Asn Ala Ile Val Val Leu 370 375 380 Cys Asp Asp Leu Lys Asn Asn Phe Lys Lys Glu Pro Ile Arg Asn Ile 385 390 395 400 Leu Arg Tyr Ile Phe Thr Ile Arg Gln Glu Cys Ser Ala Gln Asp Ile 405 410 415 Leu Ala Ala Ala Lys Tyr Asn Gln Gln Leu Asp Arg Tyr Lys Ser Gln 420 425 430 Lys Ala Asn Pro Ser Val Leu Gly Asn Gln Gly Phe Thr Trp Thr Asn 435 440 445 Ala Val Ile Leu Pro Glu Lys Ala Gln Arg Asn Asp Arg Pro Asn Ser 450 455 460 Leu Asp Leu Arg Ile Trp Leu Tyr Leu Lys Leu Arg His Pro Asp Gly 465 470 475 480 Arg Trp Lys Lys His His Ile Pro Phe Tyr Asp Thr Arg Phe Phe Gln 485 490 495 Glu Ile Tyr Ala Ala Gly Asn Ser Pro Val Asp Thr Cys Gln Phe Arg 500 505 510 Thr Pro Arg Phe Gly Tyr His Leu Pro Lys Leu Thr Asp Gln Thr Ala 515 520 525 Ile Arg Val Asn Lys Lys His Val Lys Ala Ala Lys Thr Glu Ala Arg 530 535 540 Ile Arg Leu Ala Ile Gln Gln Gly Thr Leu Pro Val Ser Asn Leu Lys 545 550 555 560 Ile Thr Glu Ile Ser Ala Thr Ile Asn Ser Lys Gly Gln Val Arg Ile 565 570 575 Pro Val Lys Phe Asp Val Gly Arg Gln Lys Gly Thr Leu Gln Ile Gly 580 585 590 Asp Arg Phe Cys Gly Tyr Asp Gln Asn Gln Thr Ala Ser His Ala Tyr 595 600 605 Ser Leu Trp Glu Val Val Lys Glu Gly Gln Tyr His Lys Glu Leu Gly 610 615 620 Cys Phe Val Arg Phe Ile Ser Ser Gly Asp Ile Val Ser Ile Thr Glu 625 630 635 640 Asn Arg Gly Asn Gln Phe Asp Gln Leu Ser Tyr Glu Gly Leu Ala Tyr 645 650 655 Pro Gln Tyr Ala Asp Trp Arg Lys Lys Ala Ser Lys Phe Val Ser Leu 660 665 670 Trp Gln Ile Thr Lys Lys Asn Lys Lys Lys Glu Ile Val Thr Val Glu 675 680 685 Ala Lys Glu Lys Phe Asp Ala Ile Cys Lys Tyr Gln Pro Arg Leu Tyr 690 695 700

Lys Phe Asn Lys Glu Tyr Ala Tyr Leu Leu Arg Asp Ile Val Arg Gly 705 710 715 720 Lys Ser Leu Val Glu Leu Gln Gln Ile Arg Gln Glu Ile Phe Arg Phe 725 730 735 Ile Glu Gln Asp Cys Gly Val Thr Arg Leu Gly Ser Leu Ser Leu Ser 740 745 750 Thr Leu Glu Thr Val Lys Ala Val Lys Gly Ile Ile Tyr Ser Tyr Phe 755 760 765 Ser Thr Ala Leu Asn Ala Ser Lys Asn Asn Pro Ile Ser Asp Glu Gln 770 775 780 Arg Lys Glu Phe Asp Pro Glu Leu Phe Ala Leu Leu Glu Lys Leu Glu 785 790 795 800 Leu Ile Arg Thr Arg Lys Lys Lys Gln Lys Val Glu Arg Ile Ala Asn 805 810 815 Ser Leu Ile Gln Thr Cys Leu Glu Asn Asn Ile Lys Phe Ile Arg Gly 820 825 830 Glu Gly Asp Leu Ser Thr Thr Asn Asn Ala Thr Lys Lys Lys Ala Asn 835 840 845 Ser Arg Ser Met Asp Trp Leu Ala Arg Gly Val Phe Asn Lys Ile Arg 850 855 860 Gln Leu Ala Pro Met His Asn Ile Thr Leu Phe Gly Cys Gly Ser Leu 865 870 875 880 Tyr Thr Ser His Gln Asp Pro Leu Val His Arg Asn Pro Asp Lys Ala 885 890 895 Met Lys Cys Arg Trp Ala Ala Ile Pro Val Lys Asp Ile Gly Asp Trp 900 905 910 Val Leu Arg Lys Leu Ser Gln Asn Leu Arg Ala Lys Asn Ile Gly Thr 915 920 925 Gly Glu Tyr Tyr His Gln Gly Val Lys Glu Phe Leu Ser His Tyr Glu 930 935 940 Leu Gln Asp Leu Glu Glu Glu Leu Leu Lys Trp Arg Ser Asp Arg Lys 945 950 955 960 Ser Asn Ile Pro Cys Trp Val Leu Gln Asn Arg Leu Ala Glu Lys Leu 965 970 975 Gly Asn Lys Glu Ala Val Val Tyr Ile Pro Val Arg Gly Gly Arg Ile 980 985 990 Tyr Phe Ala Thr His Lys Val Ala Thr Gly Ala Val Ser Ile Val Phe 995 1000 1005 Asp Gln Lys Gln Val Trp Val Cys Asn Ala Asp His Val Ala Ala 1010 1015 1020 Ala Asn Ile Ala Leu Thr Val Lys Gly Ile Gly Glu Gln Ser Ser 1025 1030 1035 Asp Glu Glu Asn Pro Asp Gly Ser Arg Ile Lys Leu Gln Leu Thr 1040 1045 1050 Ser <210> SEQ ID NO 6 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 6 cccacaatac ctgagaaatc cgtcctacgt tgacgg 36 <210> SEQ ID NO 7 <211> LENGTH: 24 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 7 aatttttgtg cccatcgttg gcac 24 <210> SEQ ID NO 8 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 8 ctctcaatgc cttagaaatc cgtccttggt tgacgg 36 <210> SEQ ID NO 9 <211> LENGTH: 34 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 9 gcaacaccta agaaatccgt ctttcattga cggg 34 <210> SEQ ID NO 10 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 10 gttgcaaaac ccaagaaatc cgtctttcat tgacgg 36 <210> SEQ ID NO 11 <211> LENGTH: 1080 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 11 Met Pro Arg Asn Tyr Phe Leu Gly Ile Phe Ser Leu Gln Lys Asn Lys 1 5 10 15 Ser Val Val His Cys Ser Val Glu Ile Arg His Lys Gly Tyr Arg Ser 20 25 30 Ser Val Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Ala Ser Lys Leu 35 40 45 Ala Pro Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp 50 55 60 Leu Asp His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe 65 70 75 80 Gly Ala Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser 85 90 95 Lys Phe Asp Ala Asp Leu Ile Cys Ala Ile Met Trp Phe Arg Leu Glu 100 105 110 Glu Lys Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met 115 120 125 Arg Leu Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln 130 135 140 Glu Tyr Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Glu Trp Val Asp 145 150 155 160 Cys Arg Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln 165 170 175 Glu Ser Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile 180 185 190 Pro Val Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln 195 200 205 Leu Phe Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ala Met 210 215 220 Leu Glu Glu Ile Ser Asn Ile Leu Ala Asp Lys Lys Pro Asp Thr Trp 225 230 235 240 Glu Glu Tyr His Asp Leu Ile Lys Lys Asn Phe Asn Val Asp Asn Tyr 245 250 255 Lys Glu Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser 260 265 270 Ser Leu Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro 275 280 285 Asn Phe Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys 290 295 300 Lys Lys Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe 305 310 315 320 Ile Ala Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val 325 330 335 Leu Asn Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile 340 345 350 Leu Tyr Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu 355 360 365 Leu Lys Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg 370 375 380 Arg Gly Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala 385 390 395 400 Arg Leu Asn Glu Leu Phe Glu Ile Trp Gln Asp Leu Thr Met Asp Asp 405 410 415 Gly Ile Arg Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg 420 425 430 Pro Val Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile 435 440 445 Thr Ala Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu 450 455 460 Thr Asn Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val 465 470 475 480 Cys Asn Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro 485 490 495 Asn Gln Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp 500 505 510 Val Thr Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu 515 520 525 Pro Phe Tyr Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu

530 535 540 Gly Leu Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln 545 550 555 560 Val Gly Ser Thr Ile Ser Ala Thr Ser Leu Ala Ala Leu Lys Ser Gln 565 570 575 Glu Glu Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His 580 585 590 Lys Ser Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe 595 600 605 Asp Lys Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe 610 615 620 Ile Thr Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu 625 630 635 640 Asn Ile Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro 645 650 655 Cys Thr Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser 660 665 670 Phe Phe His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile 675 680 685 Thr Ser Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala 690 695 700 Gly Ile Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln 705 710 715 720 Phe Leu Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp 725 730 735 Arg Asn Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu 740 745 750 Leu Asp Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe 755 760 765 Arg Ala Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu 770 775 780 Gly Ser Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser 785 790 795 800 Leu Ile Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp 805 810 815 Gln Glu Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly 820 825 830 Asp Lys Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser 835 840 845 Thr Val Leu Gln Ile Ala Arg Glu Asn Asn Ile Lys Ser Leu Cys Val 850 855 860 Glu Gly Asp Leu Pro Thr Ala Thr Lys Lys Thr Lys Pro Lys Gln Asn 865 870 875 880 Gln Lys Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn 885 890 895 Asp Gly Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg 900 905 910 Asp Thr Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr 915 920 925 Lys Val Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile 930 935 940 Lys Glu Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr 945 950 955 960 Lys Lys Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu 965 970 975 Tyr Gln Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Glu Leu Asp Phe 980 985 990 Asp Ser Leu Pro Lys Met Lys Phe Tyr Asp Leu Ala Lys Arg Leu Gly 995 1000 1005 Asp His Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr 1010 1015 1020 Leu Ser Thr Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe 1025 1030 1035 Asn Gly Arg Glu Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala 1040 1045 1050 Val Asn Ile Val Leu Arg Gly Ile Arg Asp Glu Asp Glu Gln Pro 1055 1060 1065 Asp Asp Ala Lys Lys Gln Ala Leu Ala Arg Thr Lys 1070 1075 1080 <210> SEQ ID NO 12 <211> LENGTH: 1046 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 12 Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Ala Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Pro Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala 35 40 45 Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe 50 55 60 Asp Ala Asp Leu Ile Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Glu Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser 130 135 140 Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ala Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Leu Ala Asp Lys Lys Pro Asp Thr Trp Glu Glu 195 200 205 Tyr His Asp Leu Ile Lys Lys Asn Phe Asn Val Asp Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Ser Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Lys 260 265 270 Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu 355 360 365 Asn Glu Leu Phe Glu Ile Trp Gln Asp Leu Thr Met Asp Asp Gly Ile 370 375 380 Arg Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val 385 390 395 400 Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Thr Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe 485 490 495 Tyr Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Thr Ile Ser Ala Thr Ser Leu Ala Ala Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile 595 600 605 Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp 705 710 715 720

Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu 770 775 780 Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys 785 790 795 800 Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Ile Lys Ser Leu Cys Val Glu Gly 820 825 830 Asp Leu Pro Thr Ala Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg Tyr Thr Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln 930 935 940 Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Glu Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Asp Leu Ala Lys Arg Leu Gly Asp His 965 970 975 Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Arg Asp Glu Asp Glu Gln Pro Asp Asp Ala Lys 1025 1030 1035 Lys Gln Ala Leu Ala Arg Thr Lys 1040 1045 <210> SEQ ID NO 13 <211> LENGTH: 1046 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 13 Met Val Ser Glu Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Ser Lys Leu Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Ala 35 40 45 Ile Glu His Glu Thr Ala Gln Glu Leu Ile Gly Glu Lys Ser Lys Phe 50 55 60 Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asp Asn Pro Gly Pro Leu Gln Thr Val Glu Gln Arg Met Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Thr Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Ile Asp Ser Glu Lys Tyr Gln Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ile Asp Leu Ala Arg Asn Ile Asn Thr Thr Gln Glu Ser 130 135 140 Leu Lys Ile Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Leu Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Ile Lys Ala Ser Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Leu Ala Asp Lys Asn Pro Asn Thr Trp Glu Glu 195 200 205 Tyr Gln Asp Leu Ile Lys Lys Thr Phe Asn Val Asp Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Gly Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg 260 265 270 Thr Val Phe Leu Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ala 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Glu Lys Glu Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Lys Asp Ile Leu Ser Ala Ala Ser Ile Leu Gly Asp Phe Arg Arg Gly 340 345 350 Glu Phe Asn Arg Ser Val Val Ser Lys Asn His Leu Gly Ala Arg Leu 355 360 365 Asn Glu Leu Phe Glu Ile Trp Gln Glu Leu Thr Met Asp Asp Gly Ile 370 375 380 Lys Lys Tyr Val Asp Leu Cys Lys Asp Lys Phe Ser Arg Arg Pro Val 385 390 395 400 Lys Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Asn Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Gly Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Ile Asp Asn Gly Arg Trp Ile Lys His His Leu Pro Phe 485 490 495 His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Lys Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Thr Ile Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Asn Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Glu Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Ala Thr Pro Lys Tyr Ser Tyr Lys Leu Asn Ile 595 600 605 Gly Asp Met Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Val Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Val Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Ile 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Arg Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Asp 705 710 715 720 Val Met Lys Glu Asn Lys Gly Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Phe His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu Tyr Asp Gln Glu 770 775 780 Leu Phe Asp Ser Asp Phe Phe Arg Leu Met Lys Ser Ile Gly Asp Lys 785 790 795 800 Arg Val Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Thr Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly 820 825 830 Tyr Leu Pro Thr Ser Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Tyr Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg His Thr Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Asn Arg Phe Asp Asp Trp His Arg Gly Val Thr Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Val Leu Leu Tyr Gln 930 935 940

Glu Ala Leu Arg Gln Phe Ala Ser His Tyr Lys Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Gly Asp His 965 970 975 Glu Lys Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Val Thr Lys Asp Ser Ser Lys Ile Thr Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Glu Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Ile Asp Glu Asp Glu Gln Pro Asp Gly Ala Lys 1025 1030 1035 Lys Gln Ala Thr Thr Arg Arg Thr 1040 1045 <210> SEQ ID NO 14 <211> LENGTH: 1098 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 14 Met Ser Ile Ser Asn Asn Asn Ile Leu Pro Tyr Asn Pro Lys Leu Leu 1 5 10 15 Pro Asp Asp Arg Lys His Lys Met Leu Val Asp Thr Phe Asn Gln Leu 20 25 30 Asp Leu Ile Arg Asn Asn Leu His Asp Met Ile Ile Ala Leu Tyr Gly 35 40 45 Ala Leu Lys Tyr Asp Asn Ile Lys Gln Phe Ala Ser Lys Glu Lys Pro 50 55 60 His Ile Ser Ala Asp Ala Leu Cys Ser Ile Asn Trp Phe Arg Leu Val 65 70 75 80 Lys Thr Asn Glu Arg Lys Pro Ala Ile Glu Ser Asn Gln Ile Ile Ser 85 90 95 Lys Phe Ile Gln Tyr Ser Gly His Thr Pro Asp Lys Tyr Ala Leu Ser 100 105 110 His Ile Thr Gly Asn His Glu Pro Ser His Lys Trp Ile Asp Cys Arg 115 120 125 Glu Tyr Ala Ile Asn Tyr Ala Arg Ile Met His Leu Ser Phe Ser Gln 130 135 140 Phe Gln Asp Leu Ala Thr Ala Cys Leu Asn Cys Lys Ile Leu Ile Leu 145 150 155 160 Asn Gly Thr Leu Thr Ser Ser Trp Ala Trp Gly Ala Asn Ser Ala Leu 165 170 175 Phe Gly Gly Ser Asp Lys Glu Asn Phe Ser Val Lys Ala Lys Ile Leu 180 185 190 Asn Ser Phe Ile Glu Asn Leu Lys Asp Glu Met Asn Thr Thr Lys Phe 195 200 205 Gln Val Val Glu Lys Val Cys Gln Gln Ile Gly Ser Ser Asp Ala Ala 210 215 220 Asp Leu Phe Asp Leu Tyr Arg Ser Thr Val Lys Asp Gly Asn Arg Gly 225 230 235 240 Pro Ala Thr Gly Arg Asn Pro Lys Val Met Asn Leu Phe Ser Gln Asp 245 250 255 Gly Glu Ile Ser Ser Glu Gln Arg Glu Asp Phe Ile Glu Ser Phe Gln 260 265 270 Lys Val Met Gln Glu Lys Asn Ser Lys Gln Ile Ile Pro His Leu Asp 275 280 285 Lys Leu Lys Tyr His Leu Val Lys Gln Ser Gly Leu Tyr Asp Ile Tyr 290 295 300 Ser Trp Ala Ala Ala Ile Lys Asn Ala Asn Ser Thr Ile Val Ala Ser 305 310 315 320 Asn Ser Ser Asn Leu Asn Thr Ile Leu Asn Lys Thr Glu Lys Gln Gln 325 330 335 Thr Phe Glu Glu Leu Arg Lys Asp Glu Lys Ile Val Ala Cys Ser Lys 340 345 350 Ile Leu Leu Ser Val Asn Asp Thr Leu Pro Glu Asp Leu His Tyr Asn 355 360 365 Pro Ser Thr Ser Asn Leu Gly Lys Asn Leu Asp Val Phe Phe Asp Leu 370 375 380 Leu Asn Glu Asn Ser Val His Thr Ile Glu Asn Lys Glu Glu Lys Asn 385 390 395 400 Lys Ile Val Lys Glu Cys Val Asn Gln Tyr Met Glu Glu Cys Lys Gly 405 410 415 Leu Asn Lys Pro Pro Met Pro Val Leu Leu Thr Phe Ile Ser Asp Tyr 420 425 430 Ala His Lys His Gln Ala Gln Asp Phe Leu Ser Ala Ala Lys Met Asn 435 440 445 Phe Ile Asp Leu Lys Ile Lys Ser Ile Lys Val Val Pro Thr Val His 450 455 460 Gly Ser Ser Pro Tyr Thr Trp Ile Ser Asn Leu Ser Lys Lys Asn Lys 465 470 475 480 Asp Gly Lys Met Ile Arg Thr Pro Asn Ser Ser Leu Ile Gly Trp Ile 485 490 495 Ile Pro Pro Glu Glu Ile His Asp Gln Lys Phe Ala Gly Gln Asn Pro 500 505 510 Ile Ile Trp Ala Val Leu Arg Val Tyr Cys Asn Asn Lys Trp Glu Met 515 520 525 His His Phe Pro Phe Ser Asp Ser Arg Phe Phe Thr Glu Val Tyr Ala 530 535 540 Tyr Lys Pro Asn Leu Pro Tyr Leu Pro Gly Gly Glu Asn Arg Ser Lys 545 550 555 560 Arg Phe Gly Tyr Arg His Ser Thr Asn Leu Ser Asn Glu Ser Arg Gln 565 570 575 Ile Leu Leu Asp Lys Ser Lys Tyr Ala Lys Ala Asn Lys Ser Val Leu 580 585 590 Arg Cys Met Glu Asn Met Thr His Asn Val Val Phe Asp Pro Lys Thr 595 600 605 Ser Leu Asn Ile Arg Ile Lys Thr Asp Lys Asn Asn Ser Pro Val Leu 610 615 620 Asp Asp Lys Gly Arg Ile Thr Phe Val Met Gln Ile Asn His Arg Ile 625 630 635 640 Leu Glu Lys Tyr Asn Asn Thr Lys Ile Glu Ile Gly Asp Arg Ile Leu 645 650 655 Ala Tyr Asp Gln Asn Gln Ser Glu Asn His Thr Tyr Ala Ile Leu Gln 660 665 670 Arg Thr Glu Glu Gly Ser His Ala His Gln Phe Asn Gly Trp Tyr Val 675 680 685 Arg Val Leu Glu Thr Gly Lys Val Thr Ser Ile Val Gln Gly Leu Ser 690 695 700 Gly Pro Ile Asp Gln Leu Asn Tyr Asp Gly Met Pro Val Thr Ser His 705 710 715 720 Lys Phe Asn Cys Trp Gln Ala Asp Arg Ser Ala Phe Val Ser Gln Phe 725 730 735 Ala Ser Leu Lys Ile Ser Glu Thr Glu Thr Phe Asp Glu Ala Tyr Gln 740 745 750 Ala Ile Asn Ala Gln Gly Ala Tyr Thr Trp Asn Leu Phe Tyr Leu Arg 755 760 765 Ile Leu Arg Lys Ala Leu Arg Val Cys His Met Glu Asn Ile Asn Gln 770 775 780 Phe Arg Glu Glu Ile Leu Ala Ile Ser Lys Asn Arg Leu Ser Pro Met 785 790 795 800 Ser Leu Gly Ser Leu Ser Gln Asn Ser Leu Lys Met Ile Arg Ala Phe 805 810 815 Lys Ser Ile Ile Asn Cys Tyr Met Ser Arg Met Ser Phe Val Asp Glu 820 825 830 Leu Gln Lys Lys Glu Gly Asp Leu Glu Leu His Thr Ile Met Arg Leu 835 840 845 Thr Asp Asn Lys Leu Asn Asp Lys Arg Val Glu Lys Ile Asn Arg Ala 850 855 860 Ser Ser Phe Leu Thr Asn Lys Ala His Ser Met Gly Cys Lys Met Ile 865 870 875 880 Val Gly Glu Ser Asp Leu Pro Val Ala Asp Ser Lys Thr Ser Lys Lys 885 890 895 Gln Asn Val Asp Arg Met Asp Trp Cys Ala Arg Ala Leu Ser His Lys 900 905 910 Val Glu Tyr Ala Cys Lys Leu Met Gly Leu Ala Tyr Arg Gly Ile Pro 915 920 925 Ala Tyr Met Ser Ser His Gln Asp Pro Leu Val His Leu Val Glu Ser 930 935 940 Lys Arg Ser Val Leu Arg Pro Arg Phe Val Val Ala Asp Lys Ser Asp 945 950 955 960 Val Lys Gln His His Leu Asp Asn Leu Arg Arg Met Leu Asn Ser Lys 965 970 975 Thr Lys Val Gly Thr Ala Val Tyr Tyr Arg Glu Ala Val Glu Leu Met 980 985 990 Cys Glu Glu Leu Gly Ile His Lys Thr Asp Met Ala Lys Gly Lys Val 995 1000 1005 Ser Leu Ser Asp Phe Val Asp Lys Phe Ile Gly Glu Lys Ala Ile 1010 1015 1020 Phe Pro Gln Arg Gly Gly Arg Phe Tyr Met Ser Thr Lys Arg Leu 1025 1030 1035 Thr Thr Gly Ala Lys Leu Ile Cys Tyr Ser Gly Ser Asp Val Trp 1040 1045 1050 Leu Ser Asp Ala Asp Glu Ile Ala Ala Ile Asn Ile Gly Met Phe 1055 1060 1065 Val Val Cys Asp Gln Thr Gly Ala Phe Lys Lys Lys Lys Lys Glu 1070 1075 1080 Lys Leu Asp Asp Glu Glu Cys Asp Ile Leu Pro Phe Arg Pro Met 1085 1090 1095 <210> SEQ ID NO 15 <211> LENGTH: 1088 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide"

<400> SEQUENCE: 15 Met Ser Ser Gln Val Val Arg Pro Tyr Asn Ala Lys Phe Leu Pro Asp 1 5 10 15 Asp Arg Lys His Lys Met Leu Thr Asp Thr Ile Asn Gln Leu Asp Lys 20 25 30 Ile Ser Ser Lys His Phe Asp Leu Leu Val Ala Phe Tyr Gly Ser Ile 35 40 45 Gln His Lys His Val Ser Ile Asn Asp Lys Gln Glu Glu His Ile Thr 50 55 60 Pro Asp Ser Val Cys Ala Ile Asn Trp Phe Arg Pro Met Ser Lys Asp 65 70 75 80 Tyr Ala Lys Tyr Gln Val Lys Ile Asp Ser Met Ile Thr Asn Phe Lys 85 90 95 Glu Tyr Ala Gly His Ile Pro Asp Lys Tyr Ala Ile Glu Tyr Met Gly 100 105 110 Ser Asn Ile Asp Thr Asp Arg Phe Val Trp Val Asp Cys Arg Asn Phe 115 120 125 Ala Lys Asp Tyr Val Arg Asn Met Asp Met Ser Phe Ser Glu Phe Gln 130 135 140 Asn Leu Val Asp Ala Leu Val Phe Cys Lys Ile Leu Ala Leu Asn Glu 145 150 155 160 Ser Thr Ser Thr Asn Trp Ala Trp Gly Ala Ile Ser Ala Ile Tyr Gly 165 170 175 Gly Gly Asp Lys Glu Asp Ser Gln Phe Lys Ala Lys Val Leu Asn Thr 180 185 190 Phe Val Lys Ala Leu Asn Asp Glu Asn Asn Lys Thr Lys Phe Asp Val 195 200 205 Ile Asn Lys Val Cys Ser Asp Leu Gly Tyr Asn Asp His Leu Ser Leu 210 215 220 Ile Glu Asp Phe Arg Ser Thr Ile Asp Glu Asn Gly Asn Lys Lys Ser 225 230 235 240 Ala Ser Gly Ser Pro Pro Ala Ile Ala Lys Phe Thr Glu Asp Gly Glu 245 250 255 Ile Ser Asp Asn Tyr Arg Arg Ala Cys Ile Ser Ser Phe Ser Lys Thr 260 265 270 Ala Lys Glu Lys Gln Asp Lys Lys Ser Ile Pro His Leu Asp Ile Leu 275 280 285 Lys Thr His Met Ile Ala Met Cys Gly Glu Tyr Asn Thr Tyr Ala Trp 290 295 300 Thr Glu Ala Ile Lys Asn Ala Asn Thr Asp Ile Thr Ser Arg Asn Thr 305 310 315 320 Arg Asn Met Thr Phe Ile Lys Glu Lys Ile Glu Ser Arg Asn Ser Leu 325 330 335 Lys Ile Tyr Asp Thr Glu Glu Asn Met Lys Ala Ala Lys Ile Leu Asn 340 345 350 Gly Ile Asn His Lys Leu Thr Pro Asp Leu His Tyr Thr Pro Ala Pro 355 360 365 Lys His Leu Gly Lys Asn Leu Lys Asp Leu Phe Glu Met Leu Glu Glu 370 375 380 Lys Asn Ile Leu Ala Gln Asn Glu Lys Glu Lys Lys Ala Ala Leu Asp 385 390 395 400 Glu Cys Ile Lys Gln Tyr Ile Asp Asp Cys Lys Gly Leu Asn Gln Gln 405 410 415 Pro Ile Ala Ser Leu Leu Ala His Ile Ser Asn Tyr His Lys Glu Ile 420 425 430 Thr Ala Glu Asn Phe Leu Asp Gly Ala Lys Leu Leu Val Leu Leu Gln 435 440 445 Lys Ile Asn Arg Gln Lys Ala His Pro Ser Val Phe Ser Pro Lys Ala 450 455 460 Tyr Thr Trp Gly Ser Lys Leu Glu Lys Asn Arg Arg Ala Ala Asn Ser 465 470 475 480 Ala Leu Leu Gly Trp Ile Val Pro Pro Glu Glu Lys His Lys Asp Arg 485 490 495 His Ala Gly Gln His Pro Val Met Trp Val Thr Met Thr Leu Leu Asn 500 505 510 Asn Gly Lys Trp Glu Lys His His Val Pro Phe Thr Asn Ser Arg Phe 515 520 525 Phe Ser Glu Val Tyr Ala Tyr Gln Pro Glu Leu Pro Tyr Lys Glu Gly 530 535 540 Gly Tyr Ala Arg Asn Ser Lys Thr Ala Thr Lys Pro Ser Gln Ile Met 545 550 555 560 Leu Pro Ala Tyr Ala Glu Ser Met Arg His His Ile Ala Thr Lys Gly 565 570 575 Asn Gly His Lys Lys Ser Glu Lys Ile Val Leu Arg Ala Leu Ser Asn 580 585 590 Ile Arg His Asn Val Arg Phe Asp Pro Ser Thr Ser Phe Phe Val Arg 595 600 605 Ile Met Arg Asp Lys Lys Gly Asn His Arg Leu Asp Thr Lys Gly Arg 610 615 620 Ile Thr Phe Gly Leu Gln Ile Asn His Arg Ile Thr Val Gly Lys Thr 625 630 635 640 Lys Ser Glu Ile Asn Ile Gly Asp Arg Leu Leu Ala Phe Asp Gln Asn 645 650 655 Gln Ser Glu Asn His Thr Phe Ala Ile Met Gln Arg Val Glu Glu Asn 660 665 670 Thr Pro Asn Ser His Gln Phe Asn Gly Trp Asn Ile Arg Val Leu Glu 675 680 685 Thr Gly Lys Val Val Ser Met Thr Lys Gly Ile Glu Ser Tyr Tyr Asp 690 695 700 Gln Leu Ser Tyr Asp Gly Val Pro Tyr Glu Thr Lys Lys Phe Glu Asp 705 710 715 720 Trp Arg Asn Glu Arg Lys Ala Phe Val Lys Lys Asn Lys Asp Ile Val 725 730 735 Ile Lys Glu Glu Lys Thr Phe Gly Gln Met Phe Ala Glu Ile Lys Lys 740 745 750 Ser Ser Leu Tyr Lys Trp Asn Leu Ser Tyr Leu Lys Ile Leu Arg Met 755 760 765 Ala Ile Arg Ala Lys Ser Gly Asp Thr Val Ser Leu Phe Arg Glu Glu 770 775 780 Leu Ile Ser Ile Ala Lys Asn Arg Phe Gly Pro Leu Gly Leu Gly Ser 785 790 795 800 Leu Ser Ala Ser Ser Leu Lys Met Leu Gly Ala Phe Cys Gly Val Ile 805 810 815 Gln Ser Tyr Phe Ser Val Leu Asn Cys Leu Asp Asp Lys Asp Lys Ser 820 825 830 Asn Phe Asp Ser Glu Leu Tyr Phe Tyr Leu Val Ser Ala Phe Glu Lys 835 840 845 Arg Val Phe Lys Arg Asn Glu Lys Thr Ser Arg Ala Ser Ser Phe Ile 850 855 860 Met Ala Met Ala Tyr Asn His Gly Cys Lys Met Ile Val Cys Glu Asp 865 870 875 880 Asp Leu Pro Thr Ala Gly Ala Gly Ala Asn Lys Arg Gln Asn Ser Asp 885 890 895 Arg Met Asp Trp Cys Ala Arg Ser Leu Ala Gln Lys Ile Lys Thr Gly 900 905 910 Cys Glu Ala Met Ser Ile Ala Tyr Arg Ala Ile Pro Ala Tyr Met Ser 915 920 925 Ser His Gln Asp Pro Leu Val His Leu Ala Asp Gly Lys Thr Ser Val 930 935 940 Leu Cys Pro Arg Phe Ala Leu Val Ser Lys Asp Asp Ile Lys Gln Tyr 945 950 955 960 Gln Leu Asp Gly Met Arg Arg Met Leu Asn Ser Lys Ser Lys Ile Gly 965 970 975 Thr Ala Val Tyr Tyr Arg Ala Ala Val Glu Leu Leu Cys Lys Glu Leu 980 985 990 Gly Ile Asn Lys Thr Asp Ile Ala Lys Gly Lys Leu Ser Val Ser Gln 995 1000 1005 Phe Ala Asp Ile Val Asn Gly Glu Ile Leu Leu Pro Gln Arg Gly 1010 1015 1020 Gly Arg Val Tyr Leu Ala Thr Lys Glu Leu Thr Asn Gly Ala Lys 1025 1030 1035 Leu Val Ser Tyr Asn Gly Ser Asp Val Trp Leu Ser Asn Ala Asp 1040 1045 1050 Glu Ile Ala Ala Ile Asn Ile Gly Met Phe Val Val Cys Thr Gln 1055 1060 1065 Thr Gly Val Phe Gly Lys Lys Lys Lys Lys Asp Glu Gln Asp Gly 1070 1075 1080 Asp Ile Glu Ile Ala 1085 <210> SEQ ID NO 16 <211> LENGTH: 1074 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 16 Met Ala Ser Ile Ser Arg Pro Tyr Gly Thr Lys Leu Arg Pro Asp Ala 1 5 10 15 Arg Lys Lys Glu Met Leu Asp Lys Phe Phe Asn Thr Leu Thr Lys Gly 20 25 30 Gln Arg Val Phe Ala Asp Leu Ala Leu Cys Ile Tyr Gly Ser Leu Thr 35 40 45 Leu Glu Met Ala Lys Ser Leu Glu Pro Glu Ser Asp Ser Glu Leu Val 50 55 60 Cys Ala Ile Gly Trp Phe Arg Leu Val Asp Lys Thr Ile Trp Ser Lys 65 70 75 80 Asp Gly Ile Lys Gln Glu Asn Leu Val Lys Gln Tyr Glu Ala Tyr Ser 85 90 95 Gly Lys Glu Ala Ser Glu Val Val Lys Thr Tyr Leu Asn Ser Pro Ser 100 105 110 Ser Asp Lys Tyr Val Trp Ile Asp Cys Arg Gln Lys Phe Leu Arg Phe 115 120 125 Gln Arg Glu Leu Gly Thr Arg Asn Leu Ser Glu Asp Phe Glu Cys Met 130 135 140 Leu Phe Glu Gln Tyr Ile Arg Leu Thr Lys Gly Glu Ile Glu Gly Tyr 145 150 155 160

Ala Ala Ile Ser Asn Met Phe Gly Asn Gly Glu Lys Glu Asp Arg Ser 165 170 175 Lys Lys Arg Met Tyr Ala Thr Arg Met Lys Asp Trp Leu Glu Ala Asn 180 185 190 Glu Asn Ile Thr Trp Glu Gln Tyr Arg Glu Ala Leu Lys Asn Gln Leu 195 200 205 Asn Ala Lys Asn Leu Glu Gln Val Val Ala Asn Tyr Lys Gly Asn Ala 210 215 220 Gly Gly Ala Asp Pro Phe Phe Lys Tyr Ser Phe Ser Lys Glu Gly Met 225 230 235 240 Val Ser Lys Lys Glu His Ala Gln Gln Leu Asp Lys Phe Lys Thr Val 245 250 255 Leu Lys Asn Lys Ala Arg Asp Leu Asn Phe Pro Asn Lys Glu Lys Leu 260 265 270 Lys Gln Tyr Leu Glu Ala Glu Ile Gly Ile Pro Val Asp Ala Asn Val 275 280 285 Tyr Ser Gln Met Phe Ser Asn Gly Val Ser Glu Val Gln Pro Lys Thr 290 295 300 Thr Arg Asn Met Ser Phe Ser Asn Glu Lys Leu Asp Leu Leu Thr Glu 305 310 315 320 Leu Lys Asp Leu Asn Lys Gly Asp Gly Phe Glu Tyr Ala Arg Glu Val 325 330 335 Leu Asn Gly Phe Phe Asp Ser Glu Leu His Thr Thr Glu Asp Lys Phe 340 345 350 Asn Ile Thr Ser Arg Tyr Leu Gly Gly Asp Lys Ser Asn Arg Leu Ser 355 360 365 Lys Leu Tyr Lys Ile Trp Lys Lys Glu Gly Val Asp Cys Glu Glu Gly 370 375 380 Ile Gln Gln Phe Cys Glu Ala Val Lys Asp Lys Met Gly Gln Ile Pro 385 390 395 400 Ile Arg Asn Val Leu Lys Tyr Leu Trp Gln Phe Arg Glu Thr Val Ser 405 410 415 Ala Glu Asp Phe Glu Ala Ala Ala Lys Ala Asn His Leu Glu Glu Lys 420 425 430 Ile Ser Arg Val Lys Ala His Pro Ile Val Ile Ser Asn Arg Tyr Trp 435 440 445 Ala Phe Gly Thr Ser Ala Leu Val Gly Asn Ile Met Pro Ala Asp Lys 450 455 460 Arg His Gln Gly Glu Tyr Ala Gly Gln Asn Phe Lys Met Trp Leu Glu 465 470 475 480 Ala Glu Leu His Tyr Asp Gly Lys Lys Ala Lys His His Leu Pro Phe 485 490 495 Tyr Asn Ala Arg Phe Phe Glu Glu Val Tyr Cys Tyr His Pro Ser Val 500 505 510 Ala Glu Ile Thr Pro Phe Lys Thr Lys Gln Phe Gly Cys Glu Ile Gly 515 520 525 Lys Asp Ile Pro Asp Tyr Val Ser Val Ala Leu Lys Asp Asn Pro Tyr 530 535 540 Lys Lys Ala Thr Lys Arg Ile Leu Arg Ala Ile Tyr Asn Pro Val Ala 545 550 555 560 Asn Thr Thr Gly Val Asp Lys Thr Thr Asn Cys Ser Phe Met Ile Lys 565 570 575 Arg Glu Asn Asp Glu Tyr Lys Leu Val Ile Asn Arg Lys Ile Ser Val 580 585 590 Asp Arg Pro Lys Arg Ile Glu Val Gly Arg Thr Ile Met Gly Tyr Asp 595 600 605 Arg Asn Gln Thr Ala Ser Asp Thr Tyr Trp Ile Gly Arg Leu Val Pro 610 615 620 Pro Gly Thr Arg Gly Ala Tyr Arg Ile Gly Glu Trp Ser Val Gln Tyr 625 630 635 640 Ile Lys Ser Gly Pro Val Leu Ser Ser Thr Gln Gly Val Asn Asn Ser 645 650 655 Thr Thr Asp Gln Leu Val Tyr Asn Gly Met Pro Ser Ser Ser Glu Arg 660 665 670 Phe Lys Ala Trp Lys Lys Ala Arg Met Ala Phe Ile Arg Lys Leu Ile 675 680 685 Arg Gln Leu Asn Asp Glu Gly Leu Glu Ser Lys Gly Gln Asp Tyr Ile 690 695 700 Pro Glu Asn Pro Ser Ser Phe Asp Val Arg Gly Glu Thr Leu Tyr Val 705 710 715 720 Phe Asn Ser Asn Tyr Leu Lys Ala Leu Val Ser Lys His Arg Lys Ala 725 730 735 Lys Lys Pro Val Glu Gly Ile Leu Asp Glu Ile Glu Ala Trp Thr Ser 740 745 750 Lys Asp Lys Asp Ser Cys Ser Leu Met Arg Leu Ser Ser Leu Ser Asp 755 760 765 Ala Ser Met Gln Gly Ile Ala Ser Leu Lys Ser Leu Ile Asn Ser Tyr 770 775 780 Phe Asn Lys Asn Gly Cys Lys Thr Ile Glu Asp Lys Glu Lys Phe Asn 785 790 795 800 Pro Val Leu Tyr Ala Lys Leu Val Glu Val Glu Gln Arg Arg Thr Asn 805 810 815 Lys Arg Ser Glu Lys Val Gly Arg Ile Ala Gly Ser Leu Glu Gln Leu 820 825 830 Ala Leu Leu Asn Gly Val Glu Val Val Ile Gly Glu Ala Asp Leu Gly 835 840 845 Glu Val Glu Lys Gly Lys Ser Lys Lys Gln Asn Ser Arg Asn Met Asp 850 855 860 Trp Cys Ala Lys Gln Val Ala Gln Arg Leu Glu Tyr Lys Leu Ala Phe 865 870 875 880 His Gly Ile Gly Tyr Phe Gly Val Asn Pro Met Tyr Thr Ser His Gln 885 890 895 Asp Pro Phe Glu His Arg Arg Val Ala Asp His Ile Val Met Arg Ala 900 905 910 Arg Phe Glu Glu Val Asn Val Glu Asn Ile Ala Glu Trp His Val Arg 915 920 925 Asn Phe Ser Asn Tyr Leu Arg Ala Asp Ser Gly Thr Gly Leu Tyr Tyr 930 935 940 Lys Gln Ala Thr Met Asp Phe Leu Lys His Tyr Gly Leu Glu Glu His 945 950 955 960 Ala Glu Gly Leu Glu Asn Lys Lys Ile Lys Phe Tyr Asp Phe Arg Lys 965 970 975 Ile Leu Glu Asp Lys Asn Leu Thr Ser Val Ile Ile Pro Lys Arg Gly 980 985 990 Gly Arg Ile Tyr Met Ala Thr Asn Pro Val Thr Ser Asp Ser Thr Pro 995 1000 1005 Ile Thr Tyr Ala Gly Lys Thr Tyr Asn Arg Cys Asn Ala Asp Glu 1010 1015 1020 Val Ala Ala Ala Asn Ile Val Ile Ser Val Leu Ala Pro Arg Ser 1025 1030 1035 Lys Lys Asn Glu Glu Gln Asp Asp Ile Pro Leu Ile Thr Lys Lys 1040 1045 1050 Ala Glu Ser Lys Ser Pro Pro Lys Asp Arg Lys Arg Ser Lys Thr 1055 1060 1065 Ser Gln Leu Pro Gln Lys 1070 <210> SEQ ID NO 17 <211> LENGTH: 1031 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 17 Met Val Ser Asp Ser Thr Ile Arg Pro Tyr Thr Ser Lys Leu Ala Pro 1 5 10 15 Asn Asp Pro Lys Arg Lys Met Leu Asn Asp Thr Phe Asn Trp Leu Asp 20 25 30 His Ala Tyr Lys Val Phe Phe Asp Val Ser Val Ala Leu Phe Gly Gly 35 40 45 Ile Asp Tyr Glu Ala Ala Glu Glu Leu Ile Asp Glu Lys Ser Thr Phe 50 55 60 Asp Ala Asp Leu Leu Cys Ala Ile Met Trp Phe Arg Leu Glu Glu Lys 65 70 75 80 Ser Asn Asn Pro Gly Pro Leu Gln Thr Thr Glu Gln Arg Thr Arg Leu 85 90 95 Phe Gln Lys Tyr Ser Gly His Glu Pro Ser Ser Phe Ala Gln Glu Tyr 100 105 110 Ile Lys Gly Asn Thr Asp Thr Glu Lys Tyr Glu Trp Val Asp Cys Arg 115 120 125 Leu Lys Phe Ala Asp Leu Ala Arg Asn Ile His Thr Thr Gln Glu Ser 130 135 140 Leu Lys Thr Asp Ala Tyr Thr Leu Phe Met Asn Lys Leu Ile Pro Val 145 150 155 160 Ser Lys Asp Asp Glu Phe Asn Ala Tyr Gly Phe Ile Ser Gln Leu Phe 165 170 175 Gly Thr Gly Lys Lys Glu Asp Arg Ser Val Lys Ala Ser Met Leu Glu 180 185 190 Glu Ile Ser Asn Ile Ile Glu Asp Lys Lys Pro Asn Thr Trp Glu Glu 195 200 205 Tyr Gln Asp Leu Ile Lys Lys Thr Phe Asn Val Ser Asn Tyr Lys Glu 210 215 220 Leu Lys Glu Lys Leu Ser Ala Gly Ser Ser Gly Arg Asp Gly Ser Leu 225 230 235 240 Val Ile Asp Leu Lys Glu Glu Lys Thr Gly Leu Leu Gln Pro Asn Phe 245 250 255 Ile Lys Asn Arg Ile Val Lys Phe Arg Glu Asp Ala Asp Lys Lys Arg 260 265 270 Thr Val Phe Ser Leu Pro Asn Arg Met Lys Leu Arg Glu Phe Ile Ser 275 280 285 Ser Gln Ile Gly Pro Phe Glu Gln Asn Ser Trp Ser Ala Val Leu Asn 290 295 300 Arg Ser Met Ala Ala Ile Gln Ser Lys Asn Ser Ser Asn Ile Leu Tyr 305 310 315 320 Thr Asn Gln Lys Gln Glu Arg Asn Asn Glu Ile Gln Glu Leu Leu Lys 325 330 335 Glu Asp Ile Leu Ser Ala Ala Ser Ile Leu Asn Asp Phe Arg Arg Gly 340 345 350

Glu Phe Asn Ser Ser Val Val Ser Lys Asn His Leu Gly Ser Arg Leu 355 360 365 Asn Glu Leu Phe Glu Met Trp Gln Ala Leu Lys Met Asn Asp Gly Ile 370 375 380 Glu Lys Tyr Thr Asp Leu Cys Lys Asp Asn Phe Ser Arg Arg Pro Val 385 390 395 400 Ser Ala Leu Leu Gln Tyr Ile Tyr Pro Tyr Phe Asp Lys Ile Thr Ala 405 410 415 Lys Gln Phe Leu Asp Ala Ala Ser Tyr Asn Thr Leu Val Glu Thr Asn 420 425 430 Asn Arg Lys Lys Ile His Pro Thr Val Thr Gly Pro Thr Val Cys Asn 435 440 445 Trp Gly Pro Lys Ser Thr Ile Asn Gly Ser Ile Thr Pro Pro Asn Gln 450 455 460 Met Val Lys Asp Arg Pro Ala Gly Ser His Gly Met Ile Trp Val Thr 465 470 475 480 Met Thr Val Arg Asp Asn Gly Arg Trp Val Lys His His Leu Pro Phe 485 490 495 His Asn Ser Arg Tyr Tyr Glu Glu His Tyr Cys Tyr Arg Glu Gly Leu 500 505 510 Pro Thr Lys Asn Gln Pro Arg Thr Lys Gln Leu Gly Thr Gln Val Gly 515 520 525 Ser Ile Ile Ser Ala Pro Ser Leu Ala Ile Leu Lys Ser Gln Glu Glu 530 535 540 Gln Asp Arg Arg Asn Asp Arg Lys Ser Arg Phe Lys Ala His Lys Ser 545 550 555 560 Ile Ile Arg Ser Gln Glu Asn Ile Lys Tyr Asn Val Ala Phe Asp Lys 565 570 575 Ser Thr Asn Phe Asp Val Thr Arg Lys Asn Gly Glu Phe Phe Ile Thr 580 585 590 Ile Ser Ser Arg Val Thr Thr Pro Lys Tyr Ser His Lys Leu Asn Val 595 600 605 Gly Asp Ile Ile Met Gly Leu Asp Asn Asn Gln Thr Ala Pro Cys Thr 610 615 620 Tyr Ser Ile Trp Arg Ile Val Glu Lys Asp Thr Glu Gly Ser Phe Phe 625 630 635 640 His Asn Lys Ile Trp Leu Gln Leu Val Thr Asp Gly Lys Ile Thr Ser 645 650 655 Ile Val Asp Asn Asn Arg Gln Val Asp Gln Leu Ser Tyr Ala Gly Val 660 665 670 Glu Tyr Ser Asn Phe Ala Glu Trp Arg Lys Asp Arg Arg Gln Phe Leu 675 680 685 Arg Ser Ile Asn Glu Asp Tyr Val Lys Lys Ser Asp Asn Trp Leu Asn 690 695 700 Met Asn Leu Tyr Gln Trp Asn Ala Glu Tyr Ser Arg Leu Leu Leu Gly 705 710 715 720 Val Met Lys Asp Asn Lys Asp Lys Asn Ile Gln Asn Thr Phe Arg Ala 725 730 735 Glu Ile Glu Glu Leu Ile Cys Gly Lys Phe Gly Ile Arg Leu Gly Ser 740 745 750 Leu Ser His His Ser Leu Gln Phe Leu Thr Asn Cys Lys Ser Leu Ile 755 760 765 Ser Ser Tyr Phe Met Leu Asn Asn Lys Lys Glu Glu His Asp Gln Glu 770 775 780 Ser Phe Asp Ser Asp Phe Phe Arg Leu Met Arg Ser Ile Asp Asp Lys 785 790 795 800 Arg Ile Arg Lys Arg Lys Glu Lys Ser Ser Arg Ile Ser Ser Ser Val 805 810 815 Leu Gln Ile Ala Arg Glu Asn Asn Val Lys Ser Leu Cys Val Glu Gly 820 825 830 Asp Leu Pro Thr Ala Thr Lys Lys Thr Lys Pro Lys Gln Asn Gln Lys 835 840 845 Ser Ile Asp Trp Cys Ala Arg Ala Val Val Lys Lys Leu Asn Asp Gly 850 855 860 Cys Lys Val Leu Gly Ile Asn Leu Gln Ala Ile Asp Pro Arg Asp Thr 865 870 875 880 Ser His Leu Asp Pro Phe Val Tyr Tyr Gly Lys Lys Ser Thr Lys Val 885 890 895 Gly Lys Glu Ala Arg Tyr Val Ile Val Glu Pro Ser Asn Ile Lys Glu 900 905 910 Tyr Met Thr Lys Lys Phe Thr Asp Trp His Arg Gly Val Ser Lys Lys 915 920 925 Ser Lys Lys Gly Asp Val Gln Thr Ser Thr Thr Ala Pro Leu Tyr Gln 930 935 940 Glu Ala Leu Lys Gln Phe Ala Asp His Tyr Lys Leu Asp Phe Asp Ser 945 950 955 960 Leu Pro Lys Met Lys Phe Tyr Glu Leu Ala Lys Ile Leu Glu Asp His 965 970 975 Lys Gln Val Ile Ile Pro Cys Arg Gly Gly Arg Ala Tyr Leu Ser Thr 980 985 990 Tyr Pro Ile Thr Lys Asp Ser Ser Lys Ile Asn Phe Asn Gly Arg Glu 995 1000 1005 Arg Trp Tyr Asn Gln Ser Asp Val Val Ala Ala Val Asn Ile Val 1010 1015 1020 Leu Arg Gly Ile Arg Asp Glu Asn 1025 1030 <210> SEQ ID NO 18 <211> LENGTH: 1066 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 18 Met Pro Asp Pro Ile Lys Ser Tyr Lys Ser Pro Ile Ile Ile Asp Pro 1 5 10 15 Asn Asn Ala His Asp Val Glu Lys Leu Asp Phe Leu Arg Glu Thr Phe 20 25 30 Val Tyr Leu Ser Asn Gly Thr Lys Cys Phe Met His Val Phe Leu Ser 35 40 45 Leu Leu Gly Gly Met Asn Glu Thr Leu Ala Lys Lys Ile Val Ser Leu 50 55 60 Glu Thr Pro Lys Lys Glu Lys Lys Lys Lys Ser Asn Lys Pro Ser His 65 70 75 80 Lys Ile Glu Leu Phe Leu Ala Ile Cys Trp Phe Arg Leu Val Lys Ile 85 90 95 Ser Lys Asn Glu Ser Ser Val Leu Pro Ala Leu Leu Gly Asn Arg Phe 100 105 110 Glu Lys Tyr Phe Gly Ala Lys Ala Thr Pro Glu Val Met Glu Tyr Phe 115 120 125 Ser Ala Asn Tyr Asp Glu Ala Thr Tyr Ala Trp Lys Asp Met Arg Glu 130 135 140 Glu Phe Val Ser Leu Lys Ser Lys Leu Lys Val Ser Glu Lys Asp Leu 145 150 155 160 Ile Ser Asp Ile Gly Ser Met Ile Asn Glu Arg Tyr Ile Gly Leu Lys 165 170 175 Phe Gly Lys Pro Trp Gly Ile Ile Ser Gly Leu Phe Gly Glu Gly Lys 180 185 190 Lys Val Asp Arg Ser Leu Lys Val Glu Leu Leu Lys Asn Val Leu Glu 195 200 205 Glu Ile Glu Lys Asn Pro Pro Lys Thr Lys Asp Gln Leu Ala Lys Met 210 215 220 Ile Leu Lys Cys Ala Asp Cys Lys Asn Gly Gln Glu Ile His Ala Lys 225 230 235 240 Cys Gly Lys Ile Gly Arg Met Ser Ser Val Ser Asn Trp Ala Asp Glu 245 250 255 Val Gly Ser Glu Lys Glu Ile Val Leu Ser Phe Val Lys Ser Lys Ile 260 265 270 Ser Gln Asp Leu Ala Lys Gln Ser Asn Glu Arg Asn Trp Lys Cys Val 275 280 285 Asn Ala Leu Lys Ser Tyr Ile Leu Ser Glu Ile Gly Asn Cys Phe Asp 290 295 300 Gln Ser Ser Trp Ser Glu Met Leu Asn Asn Ser Leu Ser Val Ile Gln 305 310 315 320 Ser Lys Thr Thr Arg Asn Tyr Asn Phe Cys Ile Glu Gln Leu Glu Glu 325 330 335 Lys Lys Asn Leu Asn Gln Asn His Arg Lys Phe Gly Thr Met Ile Glu 340 345 350 Asp Tyr Phe Ser Ser Arg Phe Phe Thr Gly Glu Asn Lys Phe Ile Ile 355 360 365 Cys Asn Phe His Val Gly Asp Lys Asp Lys Val Ser Ala Leu Leu Ala 370 375 380 Ser Cys Glu Gly Leu Ser Glu Glu Glu Leu Glu Glu Lys Ile Gln Asn 385 390 395 400 Phe Cys Glu Ser Gln Lys Gln Glu Ser Lys Met Pro Ile Pro Ala Leu 405 410 415 Leu Met Tyr Leu Asn Ser Leu Lys Asp Ser Ile Thr Val Asp Gln Met 420 425 430 Phe Gln Gly Ile Leu Tyr Asn Lys Ile Arg Asp Lys Ile Glu Arg Gln 435 440 445 Lys Leu His Pro Ile Val Pro Asn Asn Asp Ser Phe Asp Trp Gly Met 450 455 460 Ser Ser Lys Ile Asn Gly Arg Ile Ile Ser Pro Lys Glu Lys Ala Lys 465 470 475 480 His Asn Ala Gln Asn Asn Arg Ser Leu Tyr Asp Ser Gly Ile Trp Ile 485 490 495 Glu Ile Ser Val Leu Lys Asn Lys Glu Trp Ala Lys His His Tyr Lys 500 505 510 Ile Ser Asn Thr Arg Phe Val Glu Glu Phe Tyr Tyr Pro Ser Ser Asn 515 520 525 Asp Glu Asn Ser Leu Asp Gln Val Phe Arg Thr Gly Arg Asn Gly Phe 530 535 540 Asn Asn Pro Ala Lys Asn Asn Leu Ser Leu Glu Gln Val Ser Asn Ile 545 550 555 560 Lys Asn Ala Pro Lys Asn Arg Arg Arg Ala Ile Lys Arg Gln Met Arg 565 570 575 Val Glu Ala Ala His Gln Gln Asn Val Leu Pro His Val Lys Trp Asp

580 585 590 Asp Asn Tyr Cys Ile Thr Ile Ser Lys Tyr Gly Asp Lys Phe Val Thr 595 600 605 Phe Ile Ser Lys Lys Phe Lys Ser Lys Lys Ser Lys Glu Tyr Val Val 610 615 620 Phe Leu Gly Phe Asp Gln Asn Gln Thr Ala Ser His Thr Phe Ala Ala 625 630 635 640 Val Gln Ile Cys Asp Ser Lys Asp Glu Asn Val Ile Pro Tyr Cys Gly 645 650 655 Leu Phe Val Lys Pro Leu Glu Cys Gly His Ile Thr Ser Val Gln Lys 660 665 670 Val Lys Asp Arg Ser Ile Asp Gln Leu Ser Tyr Ser Gly Leu Pro Trp 675 680 685 Lys Asp Phe Ile Ser Trp Ser Gln Glu Arg Lys Glu Phe Val Ser Lys 690 695 700 Trp Arg Met Val Glu Val Lys Thr Arg Asn Gly Glu Lys Leu Asp Asp 705 710 715 720 Leu Thr Val Lys Ile Asn Lys Leu Asp Glu Asn Lys His Gly Leu Tyr 725 730 735 Ala Tyr Asn Ser Lys Tyr Phe Trp Tyr Leu Lys Ser Ile Met Arg Lys 740 745 750 Lys Thr Lys Asp Glu Leu Phe Glu Ile Arg Lys Glu Leu Leu Thr Val 755 760 765 Ile Lys Thr Gly Arg Leu Cys Val Leu Arg Leu Ser Ser Leu Asn His 770 775 780 Ser Ser Phe Leu Met Leu Lys Asn Ala Lys Ser Ala Ile Ser Cys Tyr 785 790 795 800 Phe Asn Asn Leu Leu Lys Gly Val Ser Asn Asp Gln Glu Lys Tyr Glu 805 810 815 Ala Asp Pro Glu Met Phe Glu Leu Arg Arg Glu Val Glu Ala Lys Arg 820 825 830 Gln Asn Lys Cys Met Ser Lys Lys Asn Leu Ile Ser Ser Gln Ile Val 835 840 845 Ser Lys Ala Ile Glu Leu Arg Gly Asn Tyr Gly Ser Val Ala Ile Ile 850 855 860 Gly Glu Asp Leu Ser Asp Tyr Val Pro Asp Lys Gly Lys Lys Ser Thr 865 870 875 880 Gln Asn Ala Asn Leu Leu Asp Trp Leu Ser Arg Gly Val Ala Asn Lys 885 890 895 Val Lys Gln Ile Ala Asn Met His Asp Asn Ile Ser Phe Lys Asp Val 900 905 910 Ser Pro Gln Trp Thr Ser His Gln Asp Ser Phe Val Asp Arg Asn Pro 915 920 925 Asn Ser Ala Leu Arg Val Arg Phe Gly Ser Cys Asp Pro Glu Glu Met 930 935 940 Tyr Glu Lys Asp Phe Glu Ser Leu Ile Lys Phe Leu Lys Glu Asp Cys 945 950 955 960 Gly His Tyr Thr Asn Ser Met Asn Asp Phe Leu Ser His Tyr Gly Val 965 970 975 Ser Arg Lys Asp Met Leu Glu Ile Lys Phe Ser Ala Phe Lys Ile Leu 980 985 990 Met Lys Asn Ile Leu Asn Lys Thr Gly Glu Lys Ser Leu Leu Tyr Pro 995 1000 1005 Lys Arg Gly Gly Arg Leu Tyr Leu Ala Thr His Lys Leu Gly Gln 1010 1015 1020 Cys Thr Arg Arg Thr Tyr Asn Gly Val Asp Phe Trp Glu Cys Asp 1025 1030 1035 Ala Asp Cys Val Ala Ala Phe Asn Ile Ala Leu Ser Gly Ile Arg 1040 1045 1050 Lys Tyr Tyr Gly Ile Lys Ser Glu Ala Val Ser Pro Val 1055 1060 1065 <210> SEQ ID NO 19 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 19 ctagcaatga cctaatagtg tgtccttagt tgacat 36 <210> SEQ ID NO 20 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 20 tctcaacgat agtcagacat gtgtcctcag tgacac 36 <210> SEQ ID NO 21 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 21 cctacaatac ctaagaaatc cgtcctaagt tgacgg 36 <210> SEQ ID NO 22 <211> LENGTH: 37 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 22 gtagcaatca gtacatattg tgcctttcat tggcaca 37 <210> SEQ ID NO 23 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 23 gtagcaatca gtacatattg tgcctttcat tggcac 36 <210> SEQ ID NO 24 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 24 gttggaatga ctaatttttg tgcccaccgt tggcac 36 <210> SEQ ID NO 25 <400> SEQUENCE: 25 000 <210> SEQ ID NO 26 <400> SEQUENCE: 26 000 <210> SEQ ID NO 27 <400> SEQUENCE: 27 000 <210> SEQ ID NO 28 <400> SEQUENCE: 28 000 <210> SEQ ID NO 29 <400> SEQUENCE: 29 000 <210> SEQ ID NO 30 <400> SEQUENCE: 30 000 <210> SEQ ID NO 31 <400> SEQUENCE: 31 000 <210> SEQ ID NO 32 <400> SEQUENCE: 32 000 <210> SEQ ID NO 33 <400> SEQUENCE: 33 000 <210> SEQ ID NO 34 <400> SEQUENCE: 34 000 <210> SEQ ID NO 35 <400> SEQUENCE: 35

000 <210> SEQ ID NO 36 <400> SEQUENCE: 36 000 <210> SEQ ID NO 37 <400> SEQUENCE: 37 000 <210> SEQ ID NO 38 <400> SEQUENCE: 38 000 <210> SEQ ID NO 39 <400> SEQUENCE: 39 000 <210> SEQ ID NO 40 <400> SEQUENCE: 40 000 <210> SEQ ID NO 41 <400> SEQUENCE: 41 000 <210> SEQ ID NO 42 <400> SEQUENCE: 42 000 <210> SEQ ID NO 43 <400> SEQUENCE: 43 000 <210> SEQ ID NO 44 <400> SEQUENCE: 44 000 <210> SEQ ID NO 45 <400> SEQUENCE: 45 000 <210> SEQ ID NO 46 <400> SEQUENCE: 46 000 <210> SEQ ID NO 47 <400> SEQUENCE: 47 000 <210> SEQ ID NO 48 <400> SEQUENCE: 48 000 <210> SEQ ID NO 49 <400> SEQUENCE: 49 000 <210> SEQ ID NO 50 <400> SEQUENCE: 50 000 <210> SEQ ID NO 51 <400> SEQUENCE: 51 000 <210> SEQ ID NO 52 <400> SEQUENCE: 52 000 <210> SEQ ID NO 53 <400> SEQUENCE: 53 000 <210> SEQ ID NO 54 <400> SEQUENCE: 54 000 <210> SEQ ID NO 55 <400> SEQUENCE: 55 000 <210> SEQ ID NO 56 <400> SEQUENCE: 56 000 <210> SEQ ID NO 57 <400> SEQUENCE: 57 000 <210> SEQ ID NO 58 <400> SEQUENCE: 58 000 <210> SEQ ID NO 59 <400> SEQUENCE: 59 000 <210> SEQ ID NO 60 <400> SEQUENCE: 60 000 <210> SEQ ID NO 61 <400> SEQUENCE: 61 000 <210> SEQ ID NO 62 <400> SEQUENCE: 62 000 <210> SEQ ID NO 63 <400> SEQUENCE: 63 000 <210> SEQ ID NO 64 <400> SEQUENCE: 64 000 <210> SEQ ID NO 65 <400> SEQUENCE: 65 000 <210> SEQ ID NO 66 <400> SEQUENCE: 66 000 <210> SEQ ID NO 67 <400> SEQUENCE: 67 000 <210> SEQ ID NO 68 <400> SEQUENCE: 68 000 <210> SEQ ID NO 69 <400> SEQUENCE: 69 000 <210> SEQ ID NO 70 <400> SEQUENCE: 70 000 <210> SEQ ID NO 71 <400> SEQUENCE: 71

000 <210> SEQ ID NO 72 <400> SEQUENCE: 72 000 <210> SEQ ID NO 73 <400> SEQUENCE: 73 000 <210> SEQ ID NO 74 <400> SEQUENCE: 74 000 <210> SEQ ID NO 75 <400> SEQUENCE: 75 000 <210> SEQ ID NO 76 <400> SEQUENCE: 76 000 <210> SEQ ID NO 77 <400> SEQUENCE: 77 000 <210> SEQ ID NO 78 <400> SEQUENCE: 78 000 <210> SEQ ID NO 79 <400> SEQUENCE: 79 000 <210> SEQ ID NO 80 <400> SEQUENCE: 80 000 <210> SEQ ID NO 81 <400> SEQUENCE: 81 000 <210> SEQ ID NO 82 <400> SEQUENCE: 82 000 <210> SEQ ID NO 83 <400> SEQUENCE: 83 000 <210> SEQ ID NO 84 <400> SEQUENCE: 84 000 <210> SEQ ID NO 85 <400> SEQUENCE: 85 000 <210> SEQ ID NO 86 <400> SEQUENCE: 86 000 <210> SEQ ID NO 87 <400> SEQUENCE: 87 000 <210> SEQ ID NO 88 <400> SEQUENCE: 88 000 <210> SEQ ID NO 89 <400> SEQUENCE: 89 000 <210> SEQ ID NO 90 <400> SEQUENCE: 90 000 <210> SEQ ID NO 91 <400> SEQUENCE: 91 000 <210> SEQ ID NO 92 <400> SEQUENCE: 92 000 <210> SEQ ID NO 93 <400> SEQUENCE: 93 000 <210> SEQ ID NO 94 <400> SEQUENCE: 94 000 <210> SEQ ID NO 95 <400> SEQUENCE: 95 000 <210> SEQ ID NO 96 <400> SEQUENCE: 96 000 <210> SEQ ID NO 97 <400> SEQUENCE: 97 000 <210> SEQ ID NO 98 <400> SEQUENCE: 98 000 <210> SEQ ID NO 99 <400> SEQUENCE: 99 000 <210> SEQ ID NO 100 <211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 100 auuuuugugc ccaucguugg cac 23 <210> SEQ ID NO 101 <211> LENGTH: 23 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 101 agaaauccgu cuuucauuga cgg 23 <210> SEQ ID NO 102 <400> SEQUENCE: 102 000 <210> SEQ ID NO 103 <400> SEQUENCE: 103 000 <210> SEQ ID NO 104 <400> SEQUENCE: 104 000 <210> SEQ ID NO 105

<400> SEQUENCE: 105 000 <210> SEQ ID NO 106 <400> SEQUENCE: 106 000 <210> SEQ ID NO 107 <400> SEQUENCE: 107 000 <210> SEQ ID NO 108 <400> SEQUENCE: 108 000 <210> SEQ ID NO 109 <400> SEQUENCE: 109 000 <210> SEQ ID NO 110 <400> SEQUENCE: 110 000 <210> SEQ ID NO 111 <400> SEQUENCE: 111 000 <210> SEQ ID NO 112 <400> SEQUENCE: 112 000 <210> SEQ ID NO 113 <400> SEQUENCE: 113 000 <210> SEQ ID NO 114 <400> SEQUENCE: 114 000 <210> SEQ ID NO 115 <400> SEQUENCE: 115 000 <210> SEQ ID NO 116 <400> SEQUENCE: 116 000 <210> SEQ ID NO 117 <400> SEQUENCE: 117 000 <210> SEQ ID NO 118 <400> SEQUENCE: 118 000 <210> SEQ ID NO 119 <400> SEQUENCE: 119 000 <210> SEQ ID NO 120 <400> SEQUENCE: 120 000 <210> SEQ ID NO 121 <400> SEQUENCE: 121 000 <210> SEQ ID NO 122 <400> SEQUENCE: 122 000 <210> SEQ ID NO 123 <400> SEQUENCE: 123 000 <210> SEQ ID NO 124 <400> SEQUENCE: 124 000 <210> SEQ ID NO 125 <400> SEQUENCE: 125 000 <210> SEQ ID NO 126 <400> SEQUENCE: 126 000 <210> SEQ ID NO 127 <400> SEQUENCE: 127 000 <210> SEQ ID NO 128 <400> SEQUENCE: 128 000 <210> SEQ ID NO 129 <400> SEQUENCE: 129 000 <210> SEQ ID NO 130 <400> SEQUENCE: 130 000 <210> SEQ ID NO 131 <400> SEQUENCE: 131 000 <210> SEQ ID NO 132 <400> SEQUENCE: 132 000 <210> SEQ ID NO 133 <400> SEQUENCE: 133 000 <210> SEQ ID NO 134 <400> SEQUENCE: 134 000 <210> SEQ ID NO 135 <400> SEQUENCE: 135 000 <210> SEQ ID NO 136 <400> SEQUENCE: 136 000 <210> SEQ ID NO 137 <400> SEQUENCE: 137 000 <210> SEQ ID NO 138 <400> SEQUENCE: 138 000 <210> SEQ ID NO 139 <400> SEQUENCE: 139 000 <210> SEQ ID NO 140 <400> SEQUENCE: 140 000 <210> SEQ ID NO 141

<400> SEQUENCE: 141 000 <210> SEQ ID NO 142 <400> SEQUENCE: 142 000 <210> SEQ ID NO 143 <400> SEQUENCE: 143 000 <210> SEQ ID NO 144 <400> SEQUENCE: 144 000 <210> SEQ ID NO 145 <400> SEQUENCE: 145 000 <210> SEQ ID NO 146 <400> SEQUENCE: 146 000 <210> SEQ ID NO 147 <400> SEQUENCE: 147 000 <210> SEQ ID NO 148 <400> SEQUENCE: 148 000 <210> SEQ ID NO 149 <400> SEQUENCE: 149 000 <210> SEQ ID NO 150 <211> LENGTH: 106 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 150 cuagcaauga ccuaauagug uguccuuagu ugacaunnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn cuagcaauga ccuaauagug uguccuuagu ugacau 106 <210> SEQ ID NO 151 <211> LENGTH: 107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(71) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 151 cuagcaauga ccuaauagug uguccuuagu ugacaunnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn ncuagcaaug accuaauagu guguccuuag uugacau 107 <210> SEQ ID NO 152 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 152 ucucaacgau agucagacau guguccucag ugacacnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnucucaacg auagucagac auguguccuc agugacac 108 <210> SEQ ID NO 153 <211> LENGTH: 107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(71) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 153 ccuacaauac cuaagaaauc cguccuaagu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nccuacaaua ccuaagaaau ccguccuaag uugacgg 107 <210> SEQ ID NO 154 <211> LENGTH: 107 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (38)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 154 guagcaauca guacauauug ugccuuucau uggcacannn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn guagcaauca guacauauug ugccuuucau uggcaca 107 <210> SEQ ID NO 155 <211> LENGTH: 106 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 155 guagcaauca guacauauug ugccuuucau uggcacnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn guagcaauca guacauauug ugccuuucau uggcac 106 <210> SEQ ID NO 156 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 156 guuggaauga cuaauuuuug ugcccaccgu uggcacnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nnguuggaau gacuaauuuu ugugcccacc guuggcac 108 <210> SEQ ID NO 157 <211> LENGTH: 84 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (25)..(60) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 157 aauuuuugug cccaucguug gcacnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60 aauuuuugug cccaucguug gcac 84 <210> SEQ ID NO 158 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 158 cccacaauac cugagaaauc cguccuacgu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nncccacaau accugagaaa uccguccuac guugacgg 108 <210> SEQ ID NO 159 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE:

<221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 159 cccacaauac cugagaaauc cguccuacgu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nncccacaau accugagaaa uccguccuac guugacgg 108 <210> SEQ ID NO 160 <211> LENGTH: 108 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(72) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 160 cucucaaugc cuuagaaauc cguccuuggu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn nncucucaau gccuuagaaa uccguccuug guugacgg 108 <210> SEQ ID NO 161 <211> LENGTH: 106 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(70) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 161 cccacaauac cugagaaauc cguccuacgu ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnnnn cccacaauac cugagaaauc cguccuacgu ugacgg 106 <210> SEQ ID NO 162 <211> LENGTH: 92 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (35)..(58) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 162 gcaacaccua agaaauccgu cuuucauuga cgggnnnnnn nnnnnnnnnn nnnnnnnngc 60 aacaccuaag aaauccgucu uucauugacg gg 92 <210> SEQ ID NO 163 <211> LENGTH: 103 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (37)..(67) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 163 guugcaaaac ccaagaaauc cgucuuucau ugacggnnnn nnnnnnnnnn nnnnnnnnnn 60 nnnnnnnguu gcaaaaccca agaaauccgu cuuucauuga cgg 103 <210> SEQ ID NO 164 <400> SEQUENCE: 164 000 <210> SEQ ID NO 165 <400> SEQUENCE: 165 000 <210> SEQ ID NO 166 <400> SEQUENCE: 166 000 <210> SEQ ID NO 167 <400> SEQUENCE: 167 000 <210> SEQ ID NO 168 <400> SEQUENCE: 168 000 <210> SEQ ID NO 169 <400> SEQUENCE: 169 000 <210> SEQ ID NO 170 <400> SEQUENCE: 170 000 <210> SEQ ID NO 171 <400> SEQUENCE: 171 000 <210> SEQ ID NO 172 <400> SEQUENCE: 172 000 <210> SEQ ID NO 173 <400> SEQUENCE: 173 000 <210> SEQ ID NO 174 <400> SEQUENCE: 174 000 <210> SEQ ID NO 175 <400> SEQUENCE: 175 000 <210> SEQ ID NO 176 <400> SEQUENCE: 176 000 <210> SEQ ID NO 177 <400> SEQUENCE: 177 000 <210> SEQ ID NO 178 <400> SEQUENCE: 178 000 <210> SEQ ID NO 179 <400> SEQUENCE: 179 000 <210> SEQ ID NO 180 <400> SEQUENCE: 180 000 <210> SEQ ID NO 181 <400> SEQUENCE: 181 000 <210> SEQ ID NO 182 <400> SEQUENCE: 182 000 <210> SEQ ID NO 183 <400> SEQUENCE: 183 000 <210> SEQ ID NO 184 <400> SEQUENCE: 184 000 <210> SEQ ID NO 185 <400> SEQUENCE: 185 000 <210> SEQ ID NO 186 <400> SEQUENCE: 186 000

<210> SEQ ID NO 187 <400> SEQUENCE: 187 000 <210> SEQ ID NO 188 <400> SEQUENCE: 188 000 <210> SEQ ID NO 189 <400> SEQUENCE: 189 000 <210> SEQ ID NO 190 <400> SEQUENCE: 190 000 <210> SEQ ID NO 191 <400> SEQUENCE: 191 000 <210> SEQ ID NO 192 <400> SEQUENCE: 192 000 <210> SEQ ID NO 193 <400> SEQUENCE: 193 000 <210> SEQ ID NO 194 <400> SEQUENCE: 194 000 <210> SEQ ID NO 195 <400> SEQUENCE: 195 000 <210> SEQ ID NO 196 <400> SEQUENCE: 196 000 <210> SEQ ID NO 197 <400> SEQUENCE: 197 000 <210> SEQ ID NO 198 <400> SEQUENCE: 198 000 <210> SEQ ID NO 199 <400> SEQUENCE: 199 000 <210> SEQ ID NO 200 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: /replace="Thr" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: /replace="Leu" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: /replace="Ser" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (7)..(7) <223> OTHER INFORMATION: /replace="Leu" <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)..(7) <223> OTHER INFORMATION: /note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> SEQUENCE: 200 Ser Ser His Gln Asp Pro Phe 1 5 <210> SEQ ID NO 201 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: /replace="Gly" or "Ser" <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (2)..(2) <223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (4)..(4) <223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (6)..(6) <223> OTHER INFORMATION: /replace="Ile" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (7)..(7) <223> OTHER INFORMATION: /replace="Ser" or "Val" <220> FEATURE: <221> NAME/KEY: MOD_RES <222> LOCATION: (8)..(10) <223> OTHER INFORMATION: Any amino acid <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: /replace="Ala" <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)..(11) <223> OTHER INFORMATION: /note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> SEQUENCE: 201 Ala Xaa Asp Xaa Asn Gln Thr Xaa Xaa Xaa Thr 1 5 10 <210> SEQ ID NO 202 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(11) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 202 ccgucnnnnn nugacgg 17 <210> SEQ ID NO 203 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(11) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 203 gugccnnnnn nuggcac 17 <210> SEQ ID NO 204 <211> LENGTH: 17 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (6)..(11) <223> OTHER INFORMATION: a, c, u, g, unknown or other <220> FEATURE: <221> NAME/KEY: misc_feature <222> LOCATION: (11)..(11) <223> OTHER INFORMATION: May or may not be present <400> SEQUENCE: 204 gugucnnnnn nugacay 17 <210> SEQ ID NO 205 <211> LENGTH: 14 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 205 ucyuwvruug acgg 14

<210> SEQ ID NO 206 <211> LENGTH: 14 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 206 ccywycruug gcac 14 <210> SEQ ID NO 207 <400> SEQUENCE: 207 000 <210> SEQ ID NO 208 <400> SEQUENCE: 208 000 <210> SEQ ID NO 209 <400> SEQUENCE: 209 000 <210> SEQ ID NO 210 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (1)..(1) <223> OTHER INFORMATION: /replace="Phe" or "Ile" or "Leu" or "Met" or "Pro" or "Val" or "Trp" or "Tyr" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (2)..(2) <223> OTHER INFORMATION: /replace="Phe" or "Ile" or "Leu" or "Met" or "Pro" or "Arg" or "Val" or "Trp" or "Tyr" <220> FEATURE: <221> NAME/KEY: VARIANT <222> LOCATION: (3)..(3) <223> OTHER INFORMATION: /replace="Phe" or "Gly" or "Ile" or "Leu" or "Met" or "Pro" or "Val" or "Trp" or "Tyr" <220> FEATURE: <221> NAME/KEY: SITE <222> LOCATION: (1)..(4) <223> OTHER INFORMATION: /note="Variant residues given in the sequence have no preference with respect to those in the annotations for variant positions" <400> SEQUENCE: 210 Cys Cys Cys Glu 1 <210> SEQ ID NO 211 <400> SEQUENCE: 211 000 <210> SEQ ID NO 212 <400> SEQUENCE: 212 000 <210> SEQ ID NO 213 <400> SEQUENCE: 213 000 <210> SEQ ID NO 214 <400> SEQUENCE: 214 000 <210> SEQ ID NO 215 <400> SEQUENCE: 215 000 <210> SEQ ID NO 216 <400> SEQUENCE: 216 000 <210> SEQ ID NO 217 <400> SEQUENCE: 217 000 <210> SEQ ID NO 218 <400> SEQUENCE: 218 000 <210> SEQ ID NO 219 <400> SEQUENCE: 219 000 <210> SEQ ID NO 220 <400> SEQUENCE: 220 000 <210> SEQ ID NO 221 <400> SEQUENCE: 221 000 <210> SEQ ID NO 222 <400> SEQUENCE: 222 000 <210> SEQ ID NO 223 <400> SEQUENCE: 223 000 <210> SEQ ID NO 224 <400> SEQUENCE: 224 000 <210> SEQ ID NO 225 <400> SEQUENCE: 225 000 <210> SEQ ID NO 226 <400> SEQUENCE: 226 000 <210> SEQ ID NO 227 <400> SEQUENCE: 227 000 <210> SEQ ID NO 228 <400> SEQUENCE: 228 000 <210> SEQ ID NO 229 <400> SEQUENCE: 229 000 <210> SEQ ID NO 230 <400> SEQUENCE: 230 000 <210> SEQ ID NO 231 <400> SEQUENCE: 231 000 <210> SEQ ID NO 232 <400> SEQUENCE: 232 000 <210> SEQ ID NO 233 <400> SEQUENCE: 233 000 <210> SEQ ID NO 234 <400> SEQUENCE: 234 000 <210> SEQ ID NO 235 <400> SEQUENCE: 235 000

<210> SEQ ID NO 236 <400> SEQUENCE: 236 000 <210> SEQ ID NO 237 <400> SEQUENCE: 237 000 <210> SEQ ID NO 238 <400> SEQUENCE: 238 000 <210> SEQ ID NO 239 <400> SEQUENCE: 239 000 <210> SEQ ID NO 240 <400> SEQUENCE: 240 000 <210> SEQ ID NO 241 <400> SEQUENCE: 241 000 <210> SEQ ID NO 242 <400> SEQUENCE: 242 000 <210> SEQ ID NO 243 <400> SEQUENCE: 243 000 <210> SEQ ID NO 244 <400> SEQUENCE: 244 000 <210> SEQ ID NO 245 <400> SEQUENCE: 245 000 <210> SEQ ID NO 246 <400> SEQUENCE: 246 000 <210> SEQ ID NO 247 <400> SEQUENCE: 247 000 <210> SEQ ID NO 248 <400> SEQUENCE: 248 000 <210> SEQ ID NO 249 <400> SEQUENCE: 249 000 <210> SEQ ID NO 250 <400> SEQUENCE: 250 000 <210> SEQ ID NO 251 <400> SEQUENCE: 251 000 <210> SEQ ID NO 252 <400> SEQUENCE: 252 000 <210> SEQ ID NO 253 <400> SEQUENCE: 253 000 <210> SEQ ID NO 254 <400> SEQUENCE: 254 000 <210> SEQ ID NO 255 <400> SEQUENCE: 255 000 <210> SEQ ID NO 256 <400> SEQUENCE: 256 000 <210> SEQ ID NO 257 <400> SEQUENCE: 257 000 <210> SEQ ID NO 258 <400> SEQUENCE: 258 000 <210> SEQ ID NO 259 <400> SEQUENCE: 259 000 <210> SEQ ID NO 260 <400> SEQUENCE: 260 000 <210> SEQ ID NO 261 <400> SEQUENCE: 261 000 <210> SEQ ID NO 262 <400> SEQUENCE: 262 000 <210> SEQ ID NO 263 <400> SEQUENCE: 263 000 <210> SEQ ID NO 264 <400> SEQUENCE: 264 000 <210> SEQ ID NO 265 <400> SEQUENCE: 265 000 <210> SEQ ID NO 266 <400> SEQUENCE: 266 000 <210> SEQ ID NO 267 <400> SEQUENCE: 267 000 <210> SEQ ID NO 268 <400> SEQUENCE: 268 000 <210> SEQ ID NO 269 <400> SEQUENCE: 269 000 <210> SEQ ID NO 270 <400> SEQUENCE: 270 000 <210> SEQ ID NO 271 <400> SEQUENCE: 271 000

<210> SEQ ID NO 272 <400> SEQUENCE: 272 000 <210> SEQ ID NO 273 <400> SEQUENCE: 273 000 <210> SEQ ID NO 274 <400> SEQUENCE: 274 000 <210> SEQ ID NO 275 <400> SEQUENCE: 275 000 <210> SEQ ID NO 276 <400> SEQUENCE: 276 000 <210> SEQ ID NO 277 <400> SEQUENCE: 277 000 <210> SEQ ID NO 278 <400> SEQUENCE: 278 000 <210> SEQ ID NO 279 <400> SEQUENCE: 279 000 <210> SEQ ID NO 280 <400> SEQUENCE: 280 000 <210> SEQ ID NO 281 <400> SEQUENCE: 281 000 <210> SEQ ID NO 282 <400> SEQUENCE: 282 000 <210> SEQ ID NO 283 <400> SEQUENCE: 283 000 <210> SEQ ID NO 284 <400> SEQUENCE: 284 000 <210> SEQ ID NO 285 <400> SEQUENCE: 285 000 <210> SEQ ID NO 286 <400> SEQUENCE: 286 000 <210> SEQ ID NO 287 <400> SEQUENCE: 287 000 <210> SEQ ID NO 288 <400> SEQUENCE: 288 000 <210> SEQ ID NO 289 <400> SEQUENCE: 289 000 <210> SEQ ID NO 290 <400> SEQUENCE: 290 000 <210> SEQ ID NO 291 <400> SEQUENCE: 291 000 <210> SEQ ID NO 292 <400> SEQUENCE: 292 000 <210> SEQ ID NO 293 <400> SEQUENCE: 293 000 <210> SEQ ID NO 294 <400> SEQUENCE: 294 000 <210> SEQ ID NO 295 <400> SEQUENCE: 295 000 <210> SEQ ID NO 296 <400> SEQUENCE: 296 000 <210> SEQ ID NO 297 <400> SEQUENCE: 297 000 <210> SEQ ID NO 298 <400> SEQUENCE: 298 000 <210> SEQ ID NO 299 <400> SEQUENCE: 299 000 <210> SEQ ID NO 300 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Simian virus 40 <400> SEQUENCE: 300 Pro Lys Lys Lys Arg Lys Val 1 5 <210> SEQ ID NO 301 <211> LENGTH: 16 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: Nucleoplasmin bipartite NLS sequence" <400> SEQUENCE: 301 Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> SEQ ID NO 302 <211> LENGTH: 9 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: c-Myc NLS sequence" <400> SEQUENCE: 302 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> SEQ ID NO 303 <211> LENGTH: 11 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: c-Myc NLS sequence" <400> SEQUENCE: 303 Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10

<210> SEQ ID NO 304 <211> LENGTH: 38 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 304 Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15 Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro 20 25 30 Arg Asn Gln Gly Gly Tyr 35 <210> SEQ ID NO 305 <211> LENGTH: 42 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: IBB domain from importin-alpha sequence" <400> SEQUENCE: 305 Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu 1 5 10 15 Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys 20 25 30 Asp Glu Gln Ile Leu Lys Arg Arg Asn Val 35 40 <210> SEQ ID NO 306 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: Myoma T protein sequence" <400> SEQUENCE: 306 Val Ser Arg Lys Arg Pro Arg Pro 1 5 <210> SEQ ID NO 307 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: Myoma T protein sequence" <400> SEQUENCE: 307 Pro Pro Lys Lys Ala Arg Glu Asp 1 5 <210> SEQ ID NO 308 <211> LENGTH: 8 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 308 Pro Gln Pro Lys Lys Lys Pro Leu 1 5 <210> SEQ ID NO 309 <211> LENGTH: 12 <212> TYPE: PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 309 Ser Ala Leu Ile Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 <210> SEQ ID NO 310 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Influenza virus <400> SEQUENCE: 310 Asp Arg Leu Arg Arg 1 5 <210> SEQ ID NO 311 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Influenza virus <400> SEQUENCE: 311 Pro Lys Gln Lys Lys Arg Lys 1 5 <210> SEQ ID NO 312 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Hepatitis virus <400> SEQUENCE: 312 Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1 5 10 <210> SEQ ID NO 313 <211> LENGTH: 10 <212> TYPE: PRT <213> ORGANISM: Mus sp. <400> SEQUENCE: 313 Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 <210> SEQ ID NO 314 <211> LENGTH: 20 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 314 Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15 Lys Ser Lys Lys 20 <210> SEQ ID NO 315 <211> LENGTH: 17 <212> TYPE: PRT <213> ORGANISM: Homo sapiens <400> SEQUENCE: 315 Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys <210> SEQ ID NO 316 <400> SEQUENCE: 316 000 <210> SEQ ID NO 317 <400> SEQUENCE: 317 000 <210> SEQ ID NO 318 <400> SEQUENCE: 318 000 <210> SEQ ID NO 319 <400> SEQUENCE: 319 000 <210> SEQ ID NO 320 <400> SEQUENCE: 320 000 <210> SEQ ID NO 321 <400> SEQUENCE: 321 000 <210> SEQ ID NO 322 <400> SEQUENCE: 322 000 <210> SEQ ID NO 323 <400> SEQUENCE: 323 000 <210> SEQ ID NO 324 <400> SEQUENCE: 324 000 <210> SEQ ID NO 325 <400> SEQUENCE: 325 000 <210> SEQ ID NO 326 <400> SEQUENCE: 326 000 <210> SEQ ID NO 327 <400> SEQUENCE: 327 000 <210> SEQ ID NO 328 <400> SEQUENCE: 328

000 <210> SEQ ID NO 329 <400> SEQUENCE: 329 000 <210> SEQ ID NO 330 <400> SEQUENCE: 330 000 <210> SEQ ID NO 331 <400> SEQUENCE: 331 000 <210> SEQ ID NO 332 <400> SEQUENCE: 332 000 <210> SEQ ID NO 333 <400> SEQUENCE: 333 000 <210> SEQ ID NO 334 <400> SEQUENCE: 334 000 <210> SEQ ID NO 335 <400> SEQUENCE: 335 000 <210> SEQ ID NO 336 <400> SEQUENCE: 336 000 <210> SEQ ID NO 337 <400> SEQUENCE: 337 000 <210> SEQ ID NO 338 <400> SEQUENCE: 338 000 <210> SEQ ID NO 339 <400> SEQUENCE: 339 000 <210> SEQ ID NO 340 <400> SEQUENCE: 340 000 <210> SEQ ID NO 341 <400> SEQUENCE: 341 000 <210> SEQ ID NO 342 <400> SEQUENCE: 342 000 <210> SEQ ID NO 343 <400> SEQUENCE: 343 000 <210> SEQ ID NO 344 <400> SEQUENCE: 344 000 <210> SEQ ID NO 345 <400> SEQUENCE: 345 000 <210> SEQ ID NO 346 <400> SEQUENCE: 346 000 <210> SEQ ID NO 347 <400> SEQUENCE: 347 000 <210> SEQ ID NO 348 <400> SEQUENCE: 348 000 <210> SEQ ID NO 349 <400> SEQUENCE: 349 000 <210> SEQ ID NO 350 <400> SEQUENCE: 350 000 <210> SEQ ID NO 351 <400> SEQUENCE: 351 000 <210> SEQ ID NO 352 <400> SEQUENCE: 352 000 <210> SEQ ID NO 353 <400> SEQUENCE: 353 000 <210> SEQ ID NO 354 <400> SEQUENCE: 354 000 <210> SEQ ID NO 355 <400> SEQUENCE: 355 000 <210> SEQ ID NO 356 <400> SEQUENCE: 356 000 <210> SEQ ID NO 357 <400> SEQUENCE: 357 000 <210> SEQ ID NO 358 <400> SEQUENCE: 358 000 <210> SEQ ID NO 359 <400> SEQUENCE: 359 000 <210> SEQ ID NO 360 <400> SEQUENCE: 360 000 <210> SEQ ID NO 361 <400> SEQUENCE: 361 000 <210> SEQ ID NO 362 <400> SEQUENCE: 362 000 <210> SEQ ID NO 363 <400> SEQUENCE: 363 000 <210> SEQ ID NO 364 <400> SEQUENCE: 364

000 <210> SEQ ID NO 365 <400> SEQUENCE: 365 000 <210> SEQ ID NO 366 <400> SEQUENCE: 366 000 <210> SEQ ID NO 367 <400> SEQUENCE: 367 000 <210> SEQ ID NO 368 <400> SEQUENCE: 368 000 <210> SEQ ID NO 369 <400> SEQUENCE: 369 000 <210> SEQ ID NO 370 <400> SEQUENCE: 370 000 <210> SEQ ID NO 371 <400> SEQUENCE: 371 000 <210> SEQ ID NO 372 <400> SEQUENCE: 372 000 <210> SEQ ID NO 373 <400> SEQUENCE: 373 000 <210> SEQ ID NO 374 <400> SEQUENCE: 374 000 <210> SEQ ID NO 375 <400> SEQUENCE: 375 000 <210> SEQ ID NO 376 <400> SEQUENCE: 376 000 <210> SEQ ID NO 377 <400> SEQUENCE: 377 000 <210> SEQ ID NO 378 <400> SEQUENCE: 378 000 <210> SEQ ID NO 379 <400> SEQUENCE: 379 000 <210> SEQ ID NO 380 <400> SEQUENCE: 380 000 <210> SEQ ID NO 381 <400> SEQUENCE: 381 000 <210> SEQ ID NO 382 <400> SEQUENCE: 382 000 <210> SEQ ID NO 383 <400> SEQUENCE: 383 000 <210> SEQ ID NO 384 <400> SEQUENCE: 384 000 <210> SEQ ID NO 385 <400> SEQUENCE: 385 000 <210> SEQ ID NO 386 <400> SEQUENCE: 386 000 <210> SEQ ID NO 387 <400> SEQUENCE: 387 000 <210> SEQ ID NO 388 <400> SEQUENCE: 388 000 <210> SEQ ID NO 389 <400> SEQUENCE: 389 000 <210> SEQ ID NO 390 <400> SEQUENCE: 390 000 <210> SEQ ID NO 391 <400> SEQUENCE: 391 000 <210> SEQ ID NO 392 <400> SEQUENCE: 392 000 <210> SEQ ID NO 393 <400> SEQUENCE: 393 000 <210> SEQ ID NO 394 <400> SEQUENCE: 394 000 <210> SEQ ID NO 395 <400> SEQUENCE: 395 000 <210> SEQ ID NO 396 <400> SEQUENCE: 396 000 <210> SEQ ID NO 397 <400> SEQUENCE: 397 000 <210> SEQ ID NO 398 <400> SEQUENCE: 398 000 <210> SEQ ID NO 399 <400> SEQUENCE: 399 000 <210> SEQ ID NO 400 <211> LENGTH: 131

<212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 400 gggaauuuuu gugcccaucg uuggcacccu aaugcggaag uaguggguaa cccggaauuu 60 uugugcccau cguuggcacu ccgcaagaau ugauuggcuc caauucuaau uuuugugccc 120 aucguuggca c 131 <210> SEQ ID NO 401 <211> LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 401 aauuuuugug cccaucguug gcac 24 <210> SEQ ID NO 402 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 402 ccuaaugcgg aaguaguggg uaacccgg 28 <210> SEQ ID NO 403 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 403 uccgcaagaa uugauuggcu ccaauucu 28 <210> SEQ ID NO 404 <211> LENGTH: 131 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 404 gggaauuuuu gugcccaucg uuggcacagg caucaucagc auuaaccacg caaacaauuu 60 uugugcccau cguuggcacg cgugcuggau ugcuucgaug gucugcgaau uuuugugccc 120 aucguuggca c 131 <210> SEQ ID NO 405 <211> LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 405 aauuuuugug cccaucguug gcac 24 <210> SEQ ID NO 406 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 406 aggcaucauc agcauuaacc acgcaaac 28 <210> SEQ ID NO 407 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 407 gcgugcugga uugcuucgau ggucugcg 28 <210> SEQ ID NO 408 <211> LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 408 catgtggacc acattaggct gcaaaactgc gcatttacga aaacgcgaaa gtttgcgtgg 60 ttaatgctga tgatgcctta acaatgccga ttcgcggtgc ggatgaacgt aatttctcga 120 ggcgtatt 128 <210> SEQ ID NO 409 <211> LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 409 catgtggacc acattaggct tggttgttgc tgccgacgac ggtgtgatgc cgcagaccat 60 cgaagcaatc cagcacgcga aagcggcgca ggtaccggtg gtggttgcgt aatttctcga 120 ggcgtatt 128 <210> SEQ ID NO 410 <211> LENGTH: 128 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 410 aatacgcctc gagaaattac aaagtgatgc aggcgtttcc aggtgctttc cctaatgcgg 60 aagtagtggg taacccggtg cgtaccgatg tgttggcgct gccgttgcag cctaatgtgg 120 tccacatg 128 <210> SEQ ID NO 411 <211> LENGTH: 171 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 411 gtgccaacga tgggcacaaa aattagaatt ggagccaatc aattcttgcg gagtgccaac 60 gatgggcaca aaaattagaa ttggagccaa tcaattcttg cggagtgcca acgatgggca 120 caaaaattcc ctatagtgag tcgtattact cgagggatcc ttattacatt t 171 <210> SEQ ID NO 412 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 412 taatacgact cactatag 18 <210> SEQ ID NO 413 <211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 413 gtgccaacga tgggcacaaa aattagaatt ggagccaatc aattcttgcg ga 52 <210> SEQ ID NO 414 <211> LENGTH: 171 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 414 gtgccaacga tgggcacaaa aattcgcaga ccatcgaagc aatccagcac gcgtgccaac 60 gatgggcaca aaaattgttt gcgtggttaa tgctgatgat gcctgtgcca acgatgggca 120 caaaaattcc ctatagtgag tcgtattact cgagggatcc ttattacatt t 171 <210> SEQ ID NO 415 <211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 415 taatacgact cactatag 18 <210> SEQ ID NO 416

<211> LENGTH: 52 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic primer" <400> SEQUENCE: 416 gtgccaacga tgggcacaaa aattcgcaga ccatcgaagc aatccagcac gc 52 <210> SEQ ID NO 417 <400> SEQUENCE: 417 000 <210> SEQ ID NO 418 <400> SEQUENCE: 418 000 <210> SEQ ID NO 419 <400> SEQUENCE: 419 000 <210> SEQ ID NO 420 <400> SEQUENCE: 420 000 <210> SEQ ID NO 421 <400> SEQUENCE: 421 000 <210> SEQ ID NO 422 <400> SEQUENCE: 422 000 <210> SEQ ID NO 423 <400> SEQUENCE: 423 000 <210> SEQ ID NO 424 <400> SEQUENCE: 424 000 <210> SEQ ID NO 425 <400> SEQUENCE: 425 000 <210> SEQ ID NO 426 <400> SEQUENCE: 426 000 <210> SEQ ID NO 427 <400> SEQUENCE: 427 000 <210> SEQ ID NO 428 <400> SEQUENCE: 428 000 <210> SEQ ID NO 429 <400> SEQUENCE: 429 000 <210> SEQ ID NO 430 <400> SEQUENCE: 430 000 <210> SEQ ID NO 431 <400> SEQUENCE: 431 000 <210> SEQ ID NO 432 <400> SEQUENCE: 432 000 <210> SEQ ID NO 433 <400> SEQUENCE: 433 000 <210> SEQ ID NO 434 <400> SEQUENCE: 434 000 <210> SEQ ID NO 435 <400> SEQUENCE: 435 000 <210> SEQ ID NO 436 <400> SEQUENCE: 436 000 <210> SEQ ID NO 437 <400> SEQUENCE: 437 000 <210> SEQ ID NO 438 <400> SEQUENCE: 438 000 <210> SEQ ID NO 439 <400> SEQUENCE: 439 000 <210> SEQ ID NO 440 <400> SEQUENCE: 440 000 <210> SEQ ID NO 441 <400> SEQUENCE: 441 000 <210> SEQ ID NO 442 <400> SEQUENCE: 442 000 <210> SEQ ID NO 443 <400> SEQUENCE: 443 000 <210> SEQ ID NO 444 <400> SEQUENCE: 444 000 <210> SEQ ID NO 445 <400> SEQUENCE: 445 000 <210> SEQ ID NO 446 <400> SEQUENCE: 446 000 <210> SEQ ID NO 447 <400> SEQUENCE: 447 000 <210> SEQ ID NO 448 <400> SEQUENCE: 448 000 <210> SEQ ID NO 449 <400> SEQUENCE: 449 000 <210> SEQ ID NO 450 <400> SEQUENCE: 450 000

<210> SEQ ID NO 451 <400> SEQUENCE: 451 000 <210> SEQ ID NO 452 <400> SEQUENCE: 452 000 <210> SEQ ID NO 453 <400> SEQUENCE: 453 000 <210> SEQ ID NO 454 <400> SEQUENCE: 454 000 <210> SEQ ID NO 455 <400> SEQUENCE: 455 000 <210> SEQ ID NO 456 <400> SEQUENCE: 456 000 <210> SEQ ID NO 457 <400> SEQUENCE: 457 000 <210> SEQ ID NO 458 <400> SEQUENCE: 458 000 <210> SEQ ID NO 459 <400> SEQUENCE: 459 000 <210> SEQ ID NO 460 <400> SEQUENCE: 460 000 <210> SEQ ID NO 461 <400> SEQUENCE: 461 000 <210> SEQ ID NO 462 <400> SEQUENCE: 462 000 <210> SEQ ID NO 463 <400> SEQUENCE: 463 000 <210> SEQ ID NO 464 <400> SEQUENCE: 464 000 <210> SEQ ID NO 465 <400> SEQUENCE: 465 000 <210> SEQ ID NO 466 <400> SEQUENCE: 466 000 <210> SEQ ID NO 467 <400> SEQUENCE: 467 000 <210> SEQ ID NO 468 <400> SEQUENCE: 468 000 <210> SEQ ID NO 469 <400> SEQUENCE: 469 000 <210> SEQ ID NO 470 <400> SEQUENCE: 470 000 <210> SEQ ID NO 471 <400> SEQUENCE: 471 000 <210> SEQ ID NO 472 <400> SEQUENCE: 472 000 <210> SEQ ID NO 473 <400> SEQUENCE: 473 000 <210> SEQ ID NO 474 <400> SEQUENCE: 474 000 <210> SEQ ID NO 475 <400> SEQUENCE: 475 000 <210> SEQ ID NO 476 <400> SEQUENCE: 476 000 <210> SEQ ID NO 477 <400> SEQUENCE: 477 000 <210> SEQ ID NO 478 <400> SEQUENCE: 478 000 <210> SEQ ID NO 479 <400> SEQUENCE: 479 000 <210> SEQ ID NO 480 <400> SEQUENCE: 480 000 <210> SEQ ID NO 481 <400> SEQUENCE: 481 000 <210> SEQ ID NO 482 <400> SEQUENCE: 482 000 <210> SEQ ID NO 483 <400> SEQUENCE: 483 000 <210> SEQ ID NO 484 <400> SEQUENCE: 484 000 <210> SEQ ID NO 485 <400> SEQUENCE: 485 000 <210> SEQ ID NO 486 <400> SEQUENCE: 486 000

<210> SEQ ID NO 487 <400> SEQUENCE: 487 000 <210> SEQ ID NO 488 <400> SEQUENCE: 488 000 <210> SEQ ID NO 489 <400> SEQUENCE: 489 000 <210> SEQ ID NO 490 <400> SEQUENCE: 490 000 <210> SEQ ID NO 491 <400> SEQUENCE: 491 000 <210> SEQ ID NO 492 <400> SEQUENCE: 492 000 <210> SEQ ID NO 493 <400> SEQUENCE: 493 000 <210> SEQ ID NO 494 <400> SEQUENCE: 494 000 <210> SEQ ID NO 495 <400> SEQUENCE: 495 000 <210> SEQ ID NO 496 <400> SEQUENCE: 496 000 <210> SEQ ID NO 497 <400> SEQUENCE: 497 000 <210> SEQ ID NO 498 <400> SEQUENCE: 498 000 <210> SEQ ID NO 499 <400> SEQUENCE: 499 000 <210> SEQ ID NO 500 <211> LENGTH: 212 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 500 gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgat 60 ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120 gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180 tttttcgcaa cgggtttgcc gccagaacac ag 212 <210> SEQ ID NO 501 <211> LENGTH: 3375 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 501 atgaaaatcg aagaaggtaa aggtcaccat caccatcacc acatgtctaa caaggagaag 60 aatgccagcg agacccggaa ggcctacacc acaaagatga tccccaggag ccacgaccgc 120 atgaagctgc tgggcaactt tatggactat ctgatggatg gcacccctat cttctttgag 180 ctgtggaatc agttcggcgg cggcatcgac agagatatca tcagcggcac agccaacaag 240 gataagatct ccgacgatct gctgctggcc gtgaactggt ttaaagtgat gccaatcaat 300 tctaagcccc agggcgtgtc cccttctaac ctggccaatc tgttccagca gtacagcgga 360 tccgagcctg acatccaggc acaggagtat ttcgcctcca actttgacac cgagaagcac 420 cagtggaagg atatgcgggt ggagtacgag agactgctgg ccgagctgca gctgtctagg 480 agcgacatgc atcacgatct gaagctgatg tacaaggaga agtgcatcgg cctgtccctg 540 tctaccgccc actatatcac aagcgtgatg tttggcaccg gcgccaagaa caatcgccag 600 acaaagcacc agttctattc caaagtgatc cagctgctgg aggagagcac ccagatcaat 660 tccgtggagc agctggcctc catcatcctg aaggccggcg actgcgattc ttacaggaag 720 ctgaggatca ggtgttcccg caagggagca accccatcta tcctgaagat cgtgcaggac 780 tatgagctgg gcacaaacca cgacgatgaa gtgaatgtgc cctccctgat cgccaacctg 840 aaggagaagc tgggcaggtt tgagtacgag tgcgagtgga agtgtatgga gaagatcaag 900 gccttcctgg cctctaaagt gggcccttac tatctgggca gctattccgc catgctggag 960 aatgccctga gcccaatcaa gggcatgacc acaaagaact gtaagttcgt gctgaagcag 1020 atcgacgcca agaacgatat caagtacgag aatgagccct ttggcaagat cgtggagggc 1080 ttctttgact ctccttattt cgagagcgat accaatgtga agtgggtgct gcaccctcac 1140 cacatcggcg agtctaacat caagacactg tgggaggacc tgaatgccat ccacagcaag 1200 tacgaggagg acatcgcctc tctgagcgag gataagaagg agaagcggat caaggtgtac 1260 cagggcgatg tgtgccagac catcaacaca tattgtgagg aagtgggcaa ggaggccaag 1320 accccactgg tgcagctgct gaggtacctg tattcccgca aggacgatat cgccgtggac 1380 aagatcatcg atggcatcac attcctgtct aagaagcaca aggtggagaa gcagaagatc 1440 aacccagtga tccagaagta ccccagcttc aattttggca acaattccaa gctgctgggc 1500 aagatcatca gcccaaagga caagctgaag cacaacctga agtgcaacag aaatcaggtg 1560 gataattaca tctggatcga gatcaaggtg ctgaacacca agacaatgcg gtgggagaag 1620 caccactatg ccctgagctc caccagattt ctggaggagg tgtactatcc cgccacatcc 1680 gagaatccac ctgacgcact ggcagcacgg ttcagaacca agacaaacgg ctacgagggc 1740 aagccagccc tgtctgccga gcagatcgag cagatcagga gcgcaccagt gggactgaga 1800 aaggtgaaga agcggcagat gagactggag gcagcaaggc agcagaatct gctgccacgc 1860 tatacctggg gcaaggattt taacatcaat atctgtaaga ggggcaacaa tttcgaggtg 1920 accctggcca caaaggtgaa gaagaagaag gagaagaact acaaggtggt gctgggctat 1980 gacgccaaca tcgtgcgcaa gaatacctac gcagcaatcg aggcacacgc aaacggcgat 2040 ggcgtgatcg actataatga tctgcctgtg aagccaatcg agtctggctt tgtgacagtg 2100 gagagccagg tgagggacaa gtcctacgat cagctgtctt ataacggcgt gaagctgctg 2160 tactgcaagc ctcacgtgga gagccggaga tccttcctgg agaagtatcg gaacggcacc 2220 atgaaggaca atagaggcaa caatatccag atcgacttca tgaaggattt tgaggccatc 2280 gccgacgatg agacaagcct gtactacttc aacatgaagt actgtaagct gctgcagtct 2340 agcatccgca accactcctc tcaggccaag gagtataggg aggagatctt cgagctgctg 2400 cgcgatggca agctgtccgt gctgaagctg agctccctgt ctaatctgag cttcgtgatg 2460 tttaaggtgg ccaagtctct gatcggcacc tactttggcc acctgctgaa gaagcctaag 2520 aactccaagt ctgacgtgaa ggccccaccc atcacagacg aggataagca gaaggccgat 2580 ccagagatgt tcgcactgcg gctggcactg gaggagaaga gactgaataa ggtgaagagc 2640 aagaaggaag tgatcgccaa caagatcgtg gccaaggcac tggagctgag ggacaagtac 2700 ggaccagtgc tgatcaaggg cgagaatatc agcgatacca caaagaaggg caagaagtct 2760 agcaccaatt ccttcctgat ggactggctg gccagaggcg tggccaacaa ggtgaaggag 2820 atggtcatga tgcaccaggg cctggagttc gtggaggtga accccaattt tacctcccac 2880 caggatcctt tcgtgcacaa gaacccagag aataccttcc gggcaaggta cagcaggtgc 2940 accccttccg agctgacaga gaagaaccgc aaggagatcc tgtccttcct gtctgacaag 3000 cccagcaagc ggcctactaa cgcctactat aatgagggcg ccatggcctt tctggccaca 3060 tatggcctga agaagaatga cgtgctgggc gtgtccctgg agaagttcaa gcagatcatg 3120 gccaacatcc tgcaccagcg gtccgaggat cagctgctgt ttccctctag aggcggcatg 3180 ttctacctgg ccacctataa gctggacgcc gatgccacaa gcgtgaactg gaatggcaag 3240 cagttttggg tgtgtaacgc cgacctggtg gccgcctaca atgtgggcct ggtggacatc 3300 cagaaggatt tcaagaagaa gaaaaggccg gcggccacga aaaaggccgg ccaggcaaaa 3360 aagaaaaagt aataa 3375 <210> SEQ ID NO 502 <211> LENGTH: 3258 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 502 atgaaaatcg aagaaggtaa aggtcaccat caccatcacc acatgagctc cgccatcaag 60

tcctacaagt ctgtgctgcg gccaaacgag agaaagaatc agctgctgaa gagcaccatc 120 cagtgcctgg aggacggctc cgccttcttt ttcaagatgc tgcagggcct gtttggcggc 180 atcacccccg agatcgtgag attcagcaca gagcaggaga agcagcagca ggatatcgcc 240 ctgtggtgtg ccgtgaattg gttcaggcct gtgagccagg actccctgac ccacacaatc 300 gcctccgata acctggtgga gaagtttgag gagtactatg gcggcacagc cagcgacgcc 360 atcaagcagt acttcagcgc ctccatcggc gagtcctact attggaatga ctgccgccag 420 cagtactatg atctgtgtcg ggagctgggc gtggaggtgt ctgacctgac ccacgatctg 480 gagatcctgt gccgggagaa gtgtctggcc gtggccacag agagcaacca gaacaattct 540 atcatcagcg tgctgtttgg caccggcgag aaggaggata ggtctgtgaa gctgcgcatc 600 acaaagaaga tcctggaggc catcagcaac ctgaaggaga tcccaaagaa tgtggccccc 660 atccaggaga tcatcctgaa tgtggccaag gccaccaagg agacattcag acaggtgtac 720 gcaggaaacc tgggagcacc atccaccctg gagaagttta tcgccaagga cggccagaag 780 gagttcgatc tgaagaagct gcagacagac ctgaagaaag tgatccgggg caagtctaag 840 gagagagatt ggtgctgtca ggaggagctg aggagctacg tggagcagaa taccatccag 900 tatgacctgt gggcctgggg cgagatgttc aacaaggccc acaccgccct gaagatcaag 960 tccacaagaa actacaattt tgccaagcag aggctggagc agttcaagga gatccagtct 1020 ctgaacaatc tgctggtggt gaagaagctg aacgactttt tcgatagcga gtttttctcc 1080 ggcgaggaga cctacacaat ctgcgtgcac cacctgggcg gcaaggacct gtccaagctg 1140 tataaggcct gggaggacga tcccgccgat cctgagaatg ccatcgtggt gctgtgcgac 1200 gatctgaaga acaattttaa gaaggagcct atcaggaaca tcctgcgcta catcttcacc 1260 atccgccagg agtgtagcgc acaggacatc ctggcagcag caaagtacaa tcagcagctg 1320 gatcggtata agagccagaa ggccaaccca tccgtgctgg gcaatcaggg ctttacctgg 1380 acaaacgccg tgatcctgcc agagaaggcc cagcggaacg acagacccaa ttctctggat 1440 ctgcgcatct ggctgtacct gaagctgcgg caccctgacg gcagatggaa gaagcaccac 1500 atcccattct acgatacccg gtttttccag gagatctatg ccgccggcaa tagccctgtg 1560 gacacctgtc agtttaggac accccgcttc ggctatcacc tgcctaagct gaccgatcag 1620 acagccatcc gcgtgaacaa gaagcacgtg aaggcagcaa agaccgaggc acggatcaga 1680 ctggccatcc agcagggcac actgccagtg tccaatctga agatcaccga gatctccgcc 1740 acaatcaact ctaagggcca ggtgcgcatc cccgtgaagt ttgacgtggg aaggcagaag 1800 ggaaccctgc agatcggcga ccggttctgc ggctacgatc agaaccagac agcctctcac 1860 gcctatagcc tgtgggaggt ggtgaaggag ggccagtacc acaaggagct gggctgtttt 1920 gtgcgcttca tctctagcgg cgacatcgtg tccatcaccg agaaccgggg caatcagttt 1980 gatcagctgt cttatgaggg cctggcctac ccccagtatg ccgactggag aaagaaggcc 2040 tccaagttcg tgtctctgtg gcagatcacc aagaagaaca agaagaagga gatcgtgaca 2100 gtggaggcca aggagaagtt tgacgccatc tgcaagtacc agcctaggct gtataagttc 2160 aacaaggagt acgcctatct gctgcgggat atcgtgagag gcaagagcct ggtggagctg 2220 cagcagatca ggcaggagat ctttcgcttc atcgagcagg actgtggagt gacccgcctg 2280 ggatctctga gcctgtccac cctggagaca gtgaaggccg tgaagggcat catctactcc 2340 tatttttcta cagccctgaa tgcctctaag aacaatccca tcagcgacga gcagcggaag 2400 gagtttgatc ctgagctgtt cgccctgctg gagaagctgg agctgatcag gactcggaag 2460 aagaagcaga aggtggagag aatcgccaat agcctgatcc agacatgcct ggagaacaat 2520 atcaagttca tcaggggcga gggcgacctg tccaccacaa acaatgccac caagaagaag 2580 gccaactcta ggagcatgga ttggctggcc agaggcgtgt ttaataagat ccggcagctg 2640 gccccaatgc acaacatcac cctgttcggc tgcggcagcc tgtacacatc ccaccaggac 2700 cctctggtgc acagaaaccc agataaggcc atgaagtgta gatgggcagc aatcccagtg 2760 aaggacatcg gcgattgggt gctgagaaag ctgtcccaga acctgagggc caagaatatc 2820 ggcaccggcg agtactatca ccagggcgtg aaggagttcc tgtctcacta tgagctgcag 2880 gacctggagg aggagctgct gaagtggcgg tctgatagaa agagcaacat cccttgctgg 2940 gtgctgcaga atagactggc cgagaagctg ggcaacaagg aggccgtggt gtacatccca 3000 gtgaggggcg gccgcatcta ttttgcaacc cacaaggtgg caacaggagc cgtgagcatc 3060 gtgttcgacc agaagcaagt gtgggtgtgt aatgcagatc acgtggcagc agcaaacatc 3120 gcactgaccg tgaagggcat cggcgagcag tcctctgacg aggagaaccc cgatggctcc 3180 aggatcaagc tgcagctgac atctaaaagg ccggcggcca cgaaaaaggc cggccaggca 3240 aaaaagaaaa agtaataa 3258 <210> SEQ ID NO 503 <211> LENGTH: 228 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polynucleotide" <400> SEQUENCE: 503 cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga 60 ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt 120 gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg 180 attgggaaga caatagcagg catgctgggg atgcggtggg ctctatgg 228 <210> SEQ ID NO 504 <400> SEQUENCE: 504 000 <210> SEQ ID NO 505 <400> SEQUENCE: 505 000 <210> SEQ ID NO 506 <400> SEQUENCE: 506 000 <210> SEQ ID NO 507 <400> SEQUENCE: 507 000 <210> SEQ ID NO 508 <400> SEQUENCE: 508 000 <210> SEQ ID NO 509 <400> SEQUENCE: 509 000 <210> SEQ ID NO 510 <400> SEQUENCE: 510 000 <210> SEQ ID NO 511 <400> SEQUENCE: 511 000 <210> SEQ ID NO 512 <400> SEQUENCE: 512 000 <210> SEQ ID NO 513 <400> SEQUENCE: 513 000 <210> SEQ ID NO 514 <400> SEQUENCE: 514 000 <210> SEQ ID NO 515 <400> SEQUENCE: 515 000 <210> SEQ ID NO 516 <400> SEQUENCE: 516 000 <210> SEQ ID NO 517 <400> SEQUENCE: 517 000 <210> SEQ ID NO 518 <400> SEQUENCE: 518 000 <210> SEQ ID NO 519 <400> SEQUENCE: 519 000 <210> SEQ ID NO 520 <400> SEQUENCE: 520 000 <210> SEQ ID NO 521 <400> SEQUENCE: 521

000 <210> SEQ ID NO 522 <400> SEQUENCE: 522 000 <210> SEQ ID NO 523 <400> SEQUENCE: 523 000 <210> SEQ ID NO 524 <400> SEQUENCE: 524 000 <210> SEQ ID NO 525 <400> SEQUENCE: 525 000 <210> SEQ ID NO 526 <400> SEQUENCE: 526 000 <210> SEQ ID NO 527 <400> SEQUENCE: 527 000 <210> SEQ ID NO 528 <400> SEQUENCE: 528 000 <210> SEQ ID NO 529 <400> SEQUENCE: 529 000 <210> SEQ ID NO 530 <400> SEQUENCE: 530 000 <210> SEQ ID NO 531 <400> SEQUENCE: 531 000 <210> SEQ ID NO 532 <400> SEQUENCE: 532 000 <210> SEQ ID NO 533 <400> SEQUENCE: 533 000 <210> SEQ ID NO 534 <400> SEQUENCE: 534 000 <210> SEQ ID NO 535 <400> SEQUENCE: 535 000 <210> SEQ ID NO 536 <400> SEQUENCE: 536 000 <210> SEQ ID NO 537 <400> SEQUENCE: 537 000 <210> SEQ ID NO 538 <400> SEQUENCE: 538 000 <210> SEQ ID NO 539 <400> SEQUENCE: 539 000 <210> SEQ ID NO 540 <400> SEQUENCE: 540 000 <210> SEQ ID NO 541 <400> SEQUENCE: 541 000 <210> SEQ ID NO 542 <400> SEQUENCE: 542 000 <210> SEQ ID NO 543 <400> SEQUENCE: 543 000 <210> SEQ ID NO 544 <400> SEQUENCE: 544 000 <210> SEQ ID NO 545 <400> SEQUENCE: 545 000 <210> SEQ ID NO 546 <400> SEQUENCE: 546 000 <210> SEQ ID NO 547 <400> SEQUENCE: 547 000 <210> SEQ ID NO 548 <400> SEQUENCE: 548 000 <210> SEQ ID NO 549 <400> SEQUENCE: 549 000 <210> SEQ ID NO 550 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 550 accccctttc caaagcccat tccctctttt cgagccgggg tgtgc 45 <210> SEQ ID NO 551 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 551 accccctttc caaagcccat tccctctttt tgagccgggg tgtgc 45 <210> SEQ ID NO 552 <211> LENGTH: 45 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 552 accccctttc caaagcccat tccctgttta tgagccgggg tgtgc 45 <210> SEQ ID NO 553 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide"

<400> SEQUENCE: 553 accccctttc caaagcccat tccctcttta agagccgggg tgtg 44 <210> SEQ ID NO 554 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 554 accccctttc caaagcccat tacctcttta agagccgggg tgtg 44 <210> SEQ ID NO 555 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 555 accccctttc caaagcccat tccctctgta agagccgggg tgtg 44 <210> SEQ ID NO 556 <400> SEQUENCE: 556 000 <210> SEQ ID NO 557 <400> SEQUENCE: 557 000 <210> SEQ ID NO 558 <400> SEQUENCE: 558 000 <210> SEQ ID NO 559 <400> SEQUENCE: 559 000 <210> SEQ ID NO 560 <400> SEQUENCE: 560 000 <210> SEQ ID NO 561 <400> SEQUENCE: 561 000 <210> SEQ ID NO 562 <400> SEQUENCE: 562 000 <210> SEQ ID NO 563 <400> SEQUENCE: 563 000 <210> SEQ ID NO 564 <400> SEQUENCE: 564 000 <210> SEQ ID NO 565 <400> SEQUENCE: 565 000 <210> SEQ ID NO 566 <400> SEQUENCE: 566 000 <210> SEQ ID NO 567 <400> SEQUENCE: 567 000 <210> SEQ ID NO 568 <400> SEQUENCE: 568 000 <210> SEQ ID NO 569 <400> SEQUENCE: 569 000 <210> SEQ ID NO 570 <400> SEQUENCE: 570 000 <210> SEQ ID NO 571 <400> SEQUENCE: 571 000 <210> SEQ ID NO 572 <400> SEQUENCE: 572 000 <210> SEQ ID NO 573 <400> SEQUENCE: 573 000 <210> SEQ ID NO 574 <400> SEQUENCE: 574 000 <210> SEQ ID NO 575 <400> SEQUENCE: 575 000 <210> SEQ ID NO 576 <400> SEQUENCE: 576 000 <210> SEQ ID NO 577 <400> SEQUENCE: 577 000 <210> SEQ ID NO 578 <400> SEQUENCE: 578 000 <210> SEQ ID NO 579 <400> SEQUENCE: 579 000 <210> SEQ ID NO 580 <400> SEQUENCE: 580 000 <210> SEQ ID NO 581 <400> SEQUENCE: 581 000 <210> SEQ ID NO 582 <400> SEQUENCE: 582 000 <210> SEQ ID NO 583 <400> SEQUENCE: 583 000 <210> SEQ ID NO 584 <400> SEQUENCE: 584 000 <210> SEQ ID NO 585 <400> SEQUENCE: 585 000 <210> SEQ ID NO 586 <400> SEQUENCE: 586 000 <210> SEQ ID NO 587

<400> SEQUENCE: 587 000 <210> SEQ ID NO 588 <400> SEQUENCE: 588 000 <210> SEQ ID NO 589 <400> SEQUENCE: 589 000 <210> SEQ ID NO 590 <400> SEQUENCE: 590 000 <210> SEQ ID NO 591 <400> SEQUENCE: 591 000 <210> SEQ ID NO 592 <400> SEQUENCE: 592 000 <210> SEQ ID NO 593 <400> SEQUENCE: 593 000 <210> SEQ ID NO 594 <400> SEQUENCE: 594 000 <210> SEQ ID NO 595 <400> SEQUENCE: 595 000 <210> SEQ ID NO 596 <400> SEQUENCE: 596 000 <210> SEQ ID NO 597 <400> SEQUENCE: 597 000 <210> SEQ ID NO 598 <400> SEQUENCE: 598 000 <210> SEQ ID NO 599 <400> SEQUENCE: 599 000 <210> SEQ ID NO 600 <211> LENGTH: 2 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 600 Gly Ser 1 <210> SEQ ID NO 601 <211> LENGTH: 7 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 601 Gly Ser Gly Gly Gly Gly Ser 1 5 <210> SEQ ID NO 602 <211> LENGTH: 15 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 602 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> SEQ ID NO 603 <211> LENGTH: 18 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 603 Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly Gly Ser Gly 1 5 10 15 Gly Ser <210> SEQ ID NO 604 <211> LENGTH: 32 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 604 Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr 1 5 10 15 Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser 20 25 30 <210> SEQ ID NO 605 <211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 605 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> SEQ ID NO 606 <211> LENGTH: 167 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 606 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu

130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> SEQ ID NO 607 <211> LENGTH: 198 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 607 Met Asp Ser Leu Leu Met Asn Arg Arg Lys Phe Leu Tyr Gln Phe Lys 1 5 10 15 Asn Val Arg Trp Ala Lys Gly Arg Arg Glu Thr Tyr Leu Cys Tyr Val 20 25 30 Val Lys Arg Arg Asp Ser Ala Thr Ser Phe Ser Leu Asp Phe Gly Tyr 35 40 45 Leu Arg Asn Lys Asn Gly Cys His Val Glu Leu Leu Phe Leu Arg Tyr 50 55 60 Ile Ser Asp Trp Asp Leu Asp Pro Gly Arg Cys Tyr Arg Val Thr Trp 65 70 75 80 Phe Thr Ser Trp Ser Pro Cys Tyr Asp Cys Ala Arg His Val Ala Asp 85 90 95 Phe Leu Arg Gly Asn Pro Asn Leu Ser Leu Arg Ile Phe Thr Ala Arg 100 105 110 Leu Tyr Phe Cys Glu Asp Arg Lys Ala Glu Pro Glu Gly Leu Arg Arg 115 120 125 Leu His Arg Ala Gly Val Gln Ile Ala Ile Met Thr Phe Lys Asp Tyr 130 135 140 Phe Tyr Cys Trp Asn Thr Phe Val Glu Asn His Glu Arg Thr Phe Lys 145 150 155 160 Ala Trp Glu Gly Leu His Glu Asn Ser Val Arg Leu Ser Arg Gln Leu 165 170 175 Arg Arg Ile Leu Leu Pro Leu Tyr Glu Val Asp Asp Leu Arg Asp Ala 180 185 190 Phe Arg Thr Leu Gly Leu 195 <210> SEQ ID NO 608 <211> LENGTH: 208 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 608 Met Thr Asp Ala Glu Tyr Val Arg Ile His Glu Lys Leu Asp Ile Tyr 1 5 10 15 Thr Phe Lys Lys Gln Phe Phe Asn Asn Lys Lys Ser Val Ser His Arg 20 25 30 Cys Tyr Val Leu Phe Glu Leu Lys Arg Arg Gly Glu Arg Arg Ala Cys 35 40 45 Phe Trp Gly Tyr Ala Val Asn Lys Pro Gln Ser Gly Thr Glu Arg Gly 50 55 60 Ile His Ala Glu Ile Phe Ser Ile Arg Lys Val Glu Glu Tyr Leu Arg 65 70 75 80 Asp Asn Pro Gly Gln Phe Thr Ile Asn Trp Tyr Ser Ser Trp Ser Pro 85 90 95 Cys Ala Asp Cys Ala Glu Lys Ile Leu Glu Trp Tyr Asn Gln Glu Leu 100 105 110 Arg Gly Asn Gly His Thr Leu Lys Ile Trp Ala Cys Lys Leu Tyr Tyr 115 120 125 Glu Lys Asn Ala Arg Asn Gln Ile Gly Leu Trp Asn Leu Arg Asp Asn 130 135 140 Gly Val Gly Leu Asn Val Met Val Ser Glu His Tyr Gln Cys Cys Arg 145 150 155 160 Lys Ile Phe Ile Gln Ser Ser His Asn Gln Leu Asn Glu Asn Arg Trp 165 170 175 Leu Glu Lys Thr Leu Lys Arg Ala Glu Lys Arg Arg Ser Glu Leu Ser 180 185 190 Ile Met Ile Gln Val Lys Ile Leu His Thr Thr Lys Ser Pro Ala Val 195 200 205 <210> SEQ ID NO 609 <211> LENGTH: 229 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic polypeptide" <400> SEQUENCE: 609 Met Ser Ser Glu Thr Gly Pro Val Ala Val Asp Pro Thr Leu Arg Arg 1 5 10 15 Arg Ile Glu Pro His Glu Phe Glu Val Phe Phe Asp Pro Arg Glu Leu 20 25 30 Arg Lys Glu Thr Cys Leu Leu Tyr Glu Ile Asn Trp Gly Gly Arg His 35 40 45 Ser Ile Trp Arg His Thr Ser Gln Asn Thr Asn Lys His Val Glu Val 50 55 60 Asn Phe Ile Glu Lys Phe Thr Thr Glu Arg Tyr Phe Cys Pro Asn Thr 65 70 75 80 Arg Cys Ser Ile Thr Trp Phe Leu Ser Trp Ser Pro Cys Gly Glu Cys 85 90 95 Ser Arg Ala Ile Thr Glu Phe Leu Ser Arg Tyr Pro His Val Thr Leu 100 105 110 Phe Ile Tyr Ile Ala Arg Leu Tyr His His Ala Asp Pro Arg Asn Arg 115 120 125 Gln Gly Leu Arg Asp Leu Ile Ser Ser Gly Val Thr Ile Gln Ile Met 130 135 140 Thr Glu Gln Glu Ser Gly Tyr Cys Trp Arg Asn Phe Val Asn Tyr Ser 145 150 155 160 Pro Ser Asn Glu Ala His Trp Pro Arg Tyr Pro His Leu Trp Val Arg 165 170 175 Leu Tyr Val Leu Glu Leu Tyr Cys Ile Ile Leu Gly Leu Pro Pro Cys 180 185 190 Leu Asn Ile Leu Arg Arg Lys Gln Pro Gln Leu Thr Phe Phe Thr Ile 195 200 205 Ala Leu Gln Ser Cys His Tyr Gln Arg Leu Pro Pro His Ile Leu Trp 210 215 220 Ala Thr Gly Leu Lys 225 <210> SEQ ID NO 610 <211> LENGTH: 4 <212> TYPE: PRT <213> ORGANISM: Unknown <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Unknown: KDEL motif sequence" <400> SEQUENCE: 610 Lys Asp Glu Leu 1 <210> SEQ ID NO 611 <211> LENGTH: 5 <212> TYPE: PRT <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic peptide" <400> SEQUENCE: 611 Glu Ala Ala Ala Lys 1 5 <210> SEQ ID NO 612 <211> LENGTH: 44 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(44) <223> OTHER INFORMATION: a, c, t, g, unknown or other <400> SEQUENCE: 612 atttttgtgc ccatcgttgg cacnnnnnnn nnnnnnnnnn nnnn 44 <210> SEQ ID NO 613 <211> LENGTH: 44 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <220> FEATURE: <221> NAME/KEY: modified_base <222> LOCATION: (24)..(44) <223> OTHER INFORMATION: a, c, u, g, unknown or other <400> SEQUENCE: 613 agaaauccgu cuuucauuga cggnnnnnnn nnnnnnnnnn nnnn 44 <210> SEQ ID NO 614 <211> LENGTH: 36 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 614 ttcgcgtgct ggattgcttc gatggtctgc ggcatc 36 <210> SEQ ID NO 615 <211> LENGTH: 36 <212> TYPE: DNA

<213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 615 gatgccgcag accatcgaag caatccagca cgcgaa 36 <210> SEQ ID NO 616 <211> LENGTH: 28 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 616 gcgugcugga uugcuucgau ggucugcg 28 <210> SEQ ID NO 617 <211> LENGTH: 34 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 617 gcaacaccua agaaauccgu cuuucauuga cggg 34 <210> SEQ ID NO 618 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 618 guugcaaaac ccaagaaauc cgucuuucau ugacgg 36 <210> SEQ ID NO 619 <211> LENGTH: 24 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 619 aauuuuugug cccaucguug gcac 24 <210> SEQ ID NO 620 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 620 cucucaaugc cuuagaaauc cguccuuggu ugacgg 36 <210> SEQ ID NO 621 <211> LENGTH: 36 <212> TYPE: RNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <221> NAME/KEY: source <223> OTHER INFORMATION: /note="Description of Artificial Sequence: Synthetic oligonucleotide" <400> SEQUENCE: 621 cccacaauac cugagaaauc cguccuacgu ugacgg 36

Claims

1. An engineered, non-naturally occurring Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated (Cas) system comprising: (a) an RNA guide or a nucleic acid encoding the RNA guide, wherein the RNA guide comprises a direct repeat sequence and a spacer sequence, wherein the direct repeat sequence comprises 5'-CCGUCNNNNNNUGACGG-3' (SEQ ID NO: 202), wherein N is any nucleobase; and (b) a CRISPR-Cas effector protein or a nucleic acid encoding the CRISPR-Cas effector protein, wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to a target nucleic acid.

2. The system of claim 1, wherein SEQ ID NO: 202 is proximal to the 3' end of the direct repeat sequence.

3. The system of claim 1, wherein the direct repeat sequence comprises a stem-loop structure proximal to a 3' end of the direct repeat sequence, wherein the stem-loop structure comprises: (a) a first stem nucleotide strand 5 nucleotides in length; (b) a second stem nucleotide strand 5 nucleotides in length, wherein the first and second stem nucleotide strands bind with each other; and (c) a loop nucleotide strand arranged between the first and second stem nucleotide strands, wherein the loop nucleotide strand comprises 6, 7, or 8 nucleotides.

4. The system of claim 1, wherein the direct repeat sequence comprises an RNA transcript of a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 9 or SEQ ID NO: 10.

5. The system of claim 1, wherein the direct repeat sequence comprises an RNA transcript of a nucleotide sequence set forth in SEQ ID NO: 9 or SEQ ID NO: 10.

6. The system of claim 1, wherein the RNA guide comprises a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 162 or SEQ ID NO: 163 or a fragment thereof.

7. The system of claim 1, wherein the RNA guide comprises a nucleotide sequence set forth in to SEQ ID NO: 162 or SEQ ID NO: 163 or a fragment thereof.

8. The system of claim 1, wherein the RNA guide comprises a nucleotide sequence with at least 95% sequence identity to SEQ ID NO: 101.

9. The system of claim 1, wherein the RNA guide comprises a nucleotide sequence set forth in SEQ ID NO: 101.

10. The system of claim 1, wherein the spacer sequence comprises between 15 and 47 nucleotides in length.

11. The system of claim 10, wherein the spacer sequence comprises between 24 and 38 nucleotides in length or between 20 and 33 nucleotides in length.

12. The system of claim 1, wherein the spacer sequence has at least 90%, 95%, or 100% complementarity to the target nucleic acid.

13. The system of claim 1, wherein the system does not include a tracrRNA.

14. The system of claim 1, wherein the CRISPR-Cas effector protein comprises one or more of: (a) a RuvC domain comprising the amino acid sequence X.sub.1SHX.sub.4DX.sub.6X.sub.7(SEQ ID NO: 200), wherein X.sub.1 is S or T, X.sub.4 is Q or L, X.sub.6 is P or S, and X, is F or L, (b) a RuvC domain comprising the amino acid sequence X.sub.1XDXNX.sub.6X.sub.7XXXX.sub.11 (SEQ ID NO: 201), wherein X, is A, G, or S, X is any amino acid, X.sub.6 is Q or I, X, is T, S, or V, and X.sub.10 is T or A; and (c) a RuvC domain comprising the amino acid sequence X.sub.1X.sub.2X.sub.3E (SEQ ID NO: 210), wherein X.sub.1 is C, F, I, L, M, P, V, W, or Y, X.sub.2 is C, F, I, L, M, P, R, V, W, or Y, and X.sub.3 is C, F, G, I, L, M, P, V, W, or Y.

15. The system of claim 1, wherein the CRISPR-Cas effector protein comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 5.

16. The system of claim 1, wherein the CRISPR-Cas effector protein comprises the amino acid sequence set forth in SEQ ID NO: 5.

17. The system of claim 1, wherein the CRISPR-Cas effector protein recognizes a protospacer adjacent motif (PAM) sequence, wherein the PAM sequence comprises a nucleotide sequence set forth as 5'-TTN-3', wherein N is any nucleotide.

18. The system of claim 17, wherein the PAM sequence comprises a nucleotide sequence set forth as 5'-TTY-3', wherein Y is C or T, or 5'-TTH-3', wherein H is A or C or T.

19. The system of claim 1, wherein the CRISPR-Cas effector protein further comprises at least one nuclear localization signal (NLS), at least one nuclear export signal (NES), or at least one NLS and at least one NES.

20. The system of claim 1, wherein the CRISPR-Cas effector protein further comprises a peptide tag, a fluorescent protein, a base-editing domain, a DNA methylation domain, a histone residue modification domain, a localization factor, a transcription modification factor, a light-gated control factor, a chemically inducible factor, or a chromatin visualization factor.

21. The system of claim 1, wherein the nucleic acid encoding the CRISPR-Cas effector protein is codon-optimized for expression in a cell.

22. The system of claim 1, wherein the nucleic acid encoding the CRISPR-Cas effector protein is operably linked to a promoter.

23. The system of claim 1, wherein the nucleic acid encoding the CRISPR-Cas effector protein is in a vector.

24. The system of claim 23, wherein the vector comprises a retroviral vector, a lentiviral vector, a phage vector, an adenoviral vector, an adeno-associated vector, or a herpes simplex vector.

25. The system of claim 1, wherein the system is present in a delivery system comprising a nanoparticle, a liposome, an exosome, a microvesicle, or a gene-gun.

26. A cell comprising the system of claim 1.

27. The cell of claim 26, wherein the cell is a prokaryotic cell or a eukaryotic cell.

28. The cell of claim 26, wherein the cell is a mammalian cell or a plant cell.

29. The cell of claim 28, wherein the cell is a human cell.

30. A method of binding the system of claim 1 to the target nucleic acid in a cell comprising: (a) providing the system; and (b) delivering the system to the cell, wherein the cell comprises the target nucleic acid, wherein the CRISPR-Cas effector protein binds to the RNA guide, and wherein the spacer sequence binds to the target nucleic acid.

31. The method of claim 30, wherein the target nucleic acid is a single-stranded DNA or a double-stranded DNA.

32. The method of claim 30, wherein binding the system to the target nucleic acid results in cleavage of the target nucleic acid.

33. The method of claim 32, wherein cleavage of the target nucleic acid results in formation of an insertion or a deletion in the target nucleic acid.