Targeted enrichment via endonuclease protection

Abstract

The present invention relates to a method for enriching target nucleic acid fragments from a nucleic acid sample, the method comprising the following steps: cutting the nucleic acid sample with a first and a second RNA or DNA-guided nuclease complex, preferably a first and a second gRNA-CAS complex, thereby producing a target nucleic acid fragment and at least one non-target nucleic acid fragment. The generated fragments are then contacted with an exonuclease, wherein the exonuclease only digests the non-target nucleic acid fragments. The present invention also relates to the use of the enriched target nucleic acid fragments for preparing adapter-connected target nucleic acid fragments and sequencing target nucleic acid fragments.

Description

Technical Field

[0001] The present invention belongs to the field of genetic research, more specifically, to the field of targeted nucleic acid isolation, such as library preparation for further analysis or processing in genetic research. New methods and compositions for reducing the complexity of nucleic acid samples or enriching target nucleic acids in nucleic acid samples are disclosed. Background of the Invention

[0003] An important component of genetic research is the sequence analysis of defined DNA loci. This can be genotyping of known variants, or identification of sequence changes or variants. Such analysis usually needs to be completed in a multiplex manner, such as the need to analyze a specific locus group in a large number of samples. The ideal test for this is flexible in terms of the number of samples and loci to be screened, high precision and suitable for different sequencing platforms. An attempt is made to provide a test including an enrichment step but ideally without amplification. For example, US2014 / 0134610 describes a method for reducing complexity, using a type II restriction enzyme to fragment nucleic acids in a sample, then connecting a protective linker and subsequently degrading all non-capture nucleic acids with an exonuclease. In WO2016 / 028887, this method is improved as follows: using a programmable endonuclease, i.e., CRISPR-endonuclease, to fragment nucleic acids in a sample.

[0004] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a locus containing multiple shorter direct repeats and is found in 40% of sequenced bacteria and 90% of sequenced archaea. CRISPR repeats form an acquired bacterial immune system to defend against genetic pathogens such as phages and plasmids. When bacteria are attacked by pathogens, a small segment of the pathogen genome is processed by CRISPR-associated proteins (CAS) and incorporated into the bacterial genome between CRISPR repeats. The CRISPR locus is then transcribed and processed to form the so-called crRNA, which contains about 30bp of the same sequence as the pathogen genome. These RNA molecules form the basis for identifying pathogens after subsequent infection and cause pathogen genetic factors to be silenced by directly digesting the pathogen genome. CAS protein Cas9 is the main component of the type II CRISPR-CAS system from Streptococcus pyogenes (S.pyogenes), and forms an endonuclease when combined with crRNA and a second RNA called trans-activating crRNA (tracrRNA), which targets the invading pathogenic DNA to be degraded by introducing a DNA double-strand break (DSB) at the genomic position defined by crRNA. This type II CRISPR-Cas9 system proves to be a convenient and effective tool in biochemistry, and can introduce modifications at sites of interest in eukaryotic genomes by targeting the introduction of double-stranded gaps and subsequent activation of endogenous repair mechanisms. Jinek et al. (2012, Science 337: 816-820) demonstrated that single-stranded chimeric RNA (single guide RNA, sRNA, sgRNA) can form functional endonucleases in conjunction with Cas9, and the single-stranded chimeric RNA is generated by combining crRNA and tracrRNA basic sequences into a single RNA molecule. Many different CRISPR-CAS systems have been identified from different bacterial populations (Zetsche et al. 2015 Cell 163, 759-771; Kim et al. 2017, Nat. Commun. 8, 1-7; Ran et al. 2015. Nature 520, 186-191).

[0005] In addition to the CRISPR-CAS system in which RNA guidance is used to guide the endonuclease to a specific location on a nucleic acid molecule, other endonucleases that use DNA or RNA guidance are known in the art (Doxzen et al. 2017, PLOS ONE 12(5):e0177097; Kaya et al. 2016, PNAS Vol. 113 No. 15, 4057-4062).

[0006] There remains a great need in the art for flexible and accurate methods for reducing nucleic acid complexity. In particular, there is a need in the art for general methods for enriching samples for one or more target nucleic acid fragments, for example for subsequent analysis or processing for genetic studies.

[0007] The present invention, as described in detail below, allows for highly simplified library preparation methods for downstream processing and / or analysis. SUMMARY OF THE INVENTION

[0009] In a first aspect, the present invention relates to a method for enriching target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps:

[0010] a) providing a sample comprising nucleic acid molecules, wherein the nucleic acid molecules comprise a sequence of interest;

[0011] b) cleaving the nucleic acid molecule with at least a first and a second RNA or DNA-guided endonuclease complex, thereby generating a target nucleic acid fragment comprising a sequence of interest and at least one non-target nucleic acid fragment;

[0012] c) contacting the cleaved nucleic acid molecule obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment; and

[0013] d) Optionally, purifying the target nucleic acid fragments comprising the sequence of interest from the digest obtained in step c).

[0014] Preferably, the RNA or DNA guided endonuclease complex is a gRNA-CAS complex. Therefore, the present invention preferably relates to a method for enriching target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps:

[0015] a) providing a sample comprising nucleic acid molecules, wherein the nucleic acid molecules comprise a sequence of interest;

[0016] b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, thereby generating a target nucleic acid fragment comprising a sequence of interest and at least one non-target nucleic acid fragment;

[0017] c) contacting the cleaved nucleic acid molecule obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment; and

[0018] d) Optionally, purifying the target nucleic acid fragments comprising the sequence of interest from the digest obtained in step c).

[0019] Preferably, step b) is performed as follows: the first and second gRNA-CAS complexes are incubated with the nucleic acid molecule at about 10-90° C., preferably about 37° C., for about 1 minute to about 18 hours, preferably about 60 minutes.

[0020] Preferably, step c) is performed by incubating the cleaved nucleic acid molecule with the exonuclease at about 10-90°C, preferably about 37°C, for about 1 minute to about 12 hours, preferably 30 minutes.

[0021] Preferably, at least one of the first and second gRNA-CAS complexes comprises a Cas9 protein.

[0022] Preferably, at least one of the first and second gRNA-CAS complexes comprises an sgRNA.

[0023] Preferably, at least one of the first and second gRNA-CAS complexes comprises crRNA and tracrRNA as different molecules.

[0024] Preferably, at least one of the first and second gRNA-CAS complexes is capable of inducing DSB.

[0025] Preferably, both the first and second gRNA-CAS complexes are capable of inducing DSB.

[0026] Preferably, in step b), at least one of the first and second gRNA-CAS complexes nicks one strand of the nucleic acid molecule, and the nucleic acid molecule is contacted with at least a third gRNA-CAS complex, which nicks the complementary strand substantially at a complementary position to the position of the nick formed by the first or second gRNA-CAS complex.

[0027] In a second aspect, the present invention relates to a method for preparing adaptor-ligated target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps:

[0028] a) providing a sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest;

[0029] b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, thereby generating a target nucleic acid fragment comprising a sequence of interest and at least one non-target nucleic acid fragment;

[0030] c) contacting the cleaved nucleic acid molecule obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment;

[0031] d) optionally, purifying the target nucleic acid fragments comprising the sequence of interest from the digest obtained in step c; and

[0032] e) ligating the adapter to the target nucleic acid fragment.

[0033] Preferably, the linker is a sequence linker.

[0034] In a third aspect, the present invention relates to a method for sequencing target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps:

[0035] a) providing a sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest;

[0036] b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, thereby generating a target nucleic acid fragment comprising the sequence of interest and at least one non-target nucleic acid fragment;

[0037] c) contacting the cleaved nucleic acid molecule obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment;

[0038] d) optionally, purifying the target nucleic acid fragment containing the sequence of interest from the digest obtained in step c;

[0039] e) optionally, ligating an adapter to the target nucleic acid fragment; and

[0040] f) sequencing the at least one target nucleic acid fragment.

[0041] Preferably, the methods defined herein are performed on a plurality of nucleic acid samples in parallel.

[0042] Preferably, the nucleic acid molecule is genomic DNA.

[0043] Preferably, the nucleic acid molecule is a nucleic acid molecule obtainable from plants, animals, humans or microorganisms.

[0044] In a fourth aspect, the present invention relates to a kit of parts for enriching target nucleic acid fragments from nucleic acid molecules, the kit comprising:

[0045] - at least a first and a second gRNA-CAS complex as defined herein and

[0046] -Exonucleases.

[0047] In a fifth aspect, the present invention relates to the use of the first and second gRNA-CAS complexes defined herein or the kit defined herein for enriching at least one target nucleic acid fragment from a nucleic acid molecule.

[0048] definition

[0049] Various terms related to methods, compositions, applications and other aspects of the present invention are used throughout this specification and claims. Unless otherwise indicated, such terms are given with the ordinary meaning of the field to which the present invention belongs. Other specifically defined terms are interpreted in a manner consistent with the definitions provided herein. Although any methods and materials similar or equivalent to those described herein can be used to implement the present invention's testing, preferred methods and materials are as described herein.

[0050] Methods for implementing the routine techniques used in the present invention are obvious to the skilled person. Routine techniques in molecular biology, biochemistry, computational chemistry, cell culture, recombinant DNA, bioinformatics, genomics, sequencing and related fields are well known to those skilled in the art and are discussed in, for example, the following references: Sambrook et al. Molecular Cloning. A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989; Ausubel et al. Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and regularly updated; and the series Methods in Enzymology, Academic Press, San Diego.

[0051] Unless the context clearly dictates otherwise, "a," "an," and "the" include plural referents. Thus, for example, reference to "a cell" includes a combination of 2 or more cells, and the like.

[0052] The term "about" as used herein is used to describe and explain small changes. For example, the term can refer to less than or equal to ±10%, such as less than or equal to ±5%, less than or equal to ±4%, less than or equal to ±3%, less than or equal to ±2%, less than or equal to ±1%, less than or equal to ±0.5%, less than or equal to ±0.1% or less than or equal to ±0.05%. In addition, amounts, ratios and other numerical values ​​are sometimes expressed in the form of ranges herein. It should be understood that this range form is used for convenience and simplicity, and it should be flexibly understood to include numerical values ​​clearly specified as limit ranges, and also include all individual numerical values ​​or sub-intervals covered by the range, just as each numerical value and sub-interval are clearly specified. For example, the ratio in the range of about 1-about 200 should be understood to include clearly listed limit values ​​of about 1 and about 200, and also include single ratios such as about 2, about 3 and about 4, and sub-intervals such as about 10-about 50, about 20-about 100, and the like.

[0053] As used herein, the term "joint" is a single-stranded, double-stranded, partially double-stranded, Y-shaped or hairpin nucleic acid molecule that can attach and preferably connect other nucleic acid ends, such as one or two chains of a double-stranded DNA molecule, and preferably has a limited length, such as about 10-about 200, or about 10-about 100 bases, or about 10-about 80, or about 10-about 50, or about 10-about 30 base pairs in length, and is preferably chemically synthesized. The double-stranded structure of the joint can be formed by two different oligonucleotide molecules that are base-paired with each other, or by a hairpin structure of a single oligonucleotide chain. Obviously, the adhesive end of the joint can be designed to be compatible with or optionally connected to an overhanging portion, the overhanging portion is prepared by cutting with a restriction enzyme and / or a programmable nuclease, can be designed to be compatible with the overhanging portion generated after adding a non-templated extension reaction (such as 3'-A addition), or can have a blunt end.

[0054] “And / or”: The term “and / or” refers to a situation where one or more of the circumstances described therein may occur alone, or in combination with at least one of the circumstances described, up to all of the circumstances described.

[0055] For use with nucleic acids or nucleic acid reactions, "amplification" refers to an in vitro method for preparing copies of a specific nucleic acid, such as a target nucleic acid or a tagged nucleic acid. Various methods for amplifying nucleic acids are known in the art, and nucleic acid reactions include polymerase chain reaction, ligase chain reaction, strand displacement amplification reaction, rolling circle amplification reaction, transcription-mediated amplification such as NASBA (e.g., U.S. Pat. No. 5,409,818), loop-mediated amplification (e.g., "LAMP" amplification using looped sequences, such as described in U.S. Pat. No. 6,410,278), and isothermal amplification reactions. The amplified nucleic acid can be DNA, including, consisting of, or derived from: DNA or RNA or a mixture of DNA and RNA, including modified DNA and / or RNA. Regardless of whether the starting nucleic acid is DNA, RNA, or both, the product obtained from the amplification of one or more nucleic acid molecules (i.e., "amplification product") can be DNA or RNA, or a mixture of DNA and RNA nucleosides or nucleotides, or it can include modified DNA or RNA nucleosides or nucleotides.

[0056] A "copy" may be, but is not limited to, a sequence that has full sequence complementarity or full sequence identity with a specific sequence. Alternatively, a copy need not have perfect sequence complementarity or identity with the specific sequence, for example allowing for a certain degree of sequence variation. For example, a copy can include nucleotide analogs such as deoxyinosine or deoxyuridine, internal sequence variations (such as sequence variations introduced by primers that contain sequences that are hybridizable but not complementary to the specific sequence), and / or sequence errors that occur during amplification.

[0057] The term "complementarity" is defined herein as the sequence identity of a sequence to a fully complementary strand (e.g., a second or reverse strand). For example, a 100% complementary (or fully complementary) sequence is understood herein to have 100% sequence identity to the complementary strand, and, for example, an 80% complementary sequence is understood herein to have 80% sequence identity to the (full) complementary strand.

[0058] "Comprising": This term is interpreted as inclusive and open, and not exclusive. In particular, this term and its variations refer to the inclusion of specific features, steps or components. These terms should not be interpreted as excluding the presence of other features, steps or components.

[0059] "Construct" or "nucleic acid construct" or "vector": This refers to an artificial nucleic acid molecule, resulting from the use of recombinant DNA technology and can be used to deliver foreign DNA to a host cell, usually with the purpose of expressing the DNA region contained on the construct in the host cell. The vector backbone of the construct can be, for example, a plasmid, into which the (chimeric) gene is integrated, or if appropriate transcription regulatory sequences (e.g. (inducible) promoters) are already present, only the desired nucleotide sequence (e.g. coding sequence) is integrated downstream of said transcription regulatory sequences. The vector may contain further genetic factors to facilitate its use in molecular cloning, such as selectable markers, multiple cloning sites, etc.

[0060] The terms "double-stranded" and "duplex" as used herein describe two complementary polynucleotides that are base paired, ie, hybridized together. Complementary nucleotide strands are also known in the art as reverse complements.

[0061] As used herein, the term "effective amount" refers to an amount of a biologically active agent sufficient to cause a desired biological effect. For example, in some embodiments, an effective amount of an exonuclease may refer to an amount of an exonuclease sufficient to induce cleavage of unprotected nucleic acids. It will be appreciated by those skilled in the art that the effective amount of an agent may vary according to a variety of factors, such as the substance used, the conditions under which the substance is used, and the desired biological effect, such as the degree of nuclease cleavage to be detected.

[0062] “Exemplary”: This term means “serving as an example, instance, or illustration” and should not be interpreted to exclude other configurations disclosed herein.

[0063] "Expression": This refers to the process in which a DNA region operably linked to appropriate regulatory regions, especially a promoter, is transcribed into RNA, which in turn can be translated into a protein or peptide.

[0064] "Guide sequence" is understood herein as a sequence that guides an RNA or DNA-guided endonuclease to a specific site on an RNA or DNA molecule. In the context of a gRNA-CAS complex, "guide sequence" is further understood herein as a portion of an sgRNA or crRNA that is required to target a gRNA-CAS complex to a specific site on double-stranded DNA.

[0065] A gRNA-CAS complex is understood herein as a CAS protein complexed or hybridized with a guide RNA, also referred to as a CRISPR-endonuclease or CRISPR-nuclease, wherein the guide RNA may be a crRNA and / or a tracrRNA or a sgRNA.

[0066] "Identity" and "similarity" can be easily calculated by known methods. "Sequence identity" and "sequence similarity" can be determined by comparing two peptides or two nucleotide sequences with an overall or local alignment algorithm, depending on the length of the two sequences. Sequences of similar length are preferably compared with an overall alignment algorithm (such as Needleman Wunsch), which preferably compares sequences over the complete length, while sequences of significantly different lengths are preferably compared with a local alignment algorithm (such as Smith Waterman). When sequences (preferably compared with default parameters by, for example, programs GAP or BESTFIT) have at least a certain minimum percentage of sequence identity (as defined below), the sequences can be subsequently referred to as "substantially identical" or "substantially similar". GAP uses the Needleman and Wunsch overall alignment algorithm to compare two sequences over their complete length (full length), maximizing the number of matches and minimizing the number of gaps. When two sequences have similar lengths, an overall alignment is applicable to determining sequence identity. Typically, the default parameters of GAP are used, with a gap creation penalty of 50 (nucleotides) / 8 (proteins) and a gap extension penalty of 3 (nucleotides) / 2 (proteins). For nucleotides, the default scoring matrix used is nwsgapdna, and for proteins, the default scoring matrix is ​​Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and percentages of sequence identity can be determined using computer programs such as the GCG Wisconsin package, version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software such as the program "needle" (using the overall NeedlemanWunsch algorithm) or "water" (using the local Smith Waterman algorithm), EmbossWIN version 2.10.0, using the same parameters as GAP above, or using default settings (both for "needle" and "water" and for protein and DNA alignments, the default gap penalty is 10.0 and the default gap extension penalty is 0.5; the default scoring matrix is ​​Blosum62 for proteins and DNAFull for DNA). When the sequences differ significantly in length, local algorithms such as those using the Smith Waterman algorithm are preferred.

[0067] Alternatively, the percentage of similarity or identity can be determined by searching against public databases using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the invention can be further used as a "query sequence" to perform searches against public databases, e.g., to identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can be performed using the NBLAST program, score = 100, word length = 12, to obtain nucleotide sequence homologs of the nucleic acid molecules of the invention. BLAST protein searches can be performed using the BLASTx program, score = 50, word length = 3, to obtain amino acid sequence homologs of the protein molecules of the invention. To obtain gapped alignments for comparison purposes, gapped BLAST can be used as described in Altschul et al. (1997) Nucleic Acids Res. 25 (17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of each program (eg, BLASTx and BLASTn) can be used. See the National Center for Biotechnology Information webpage at http: / / www.ncbi.nlm.nih.gov / .

[0068] The term "nucleotide" includes, but is not limited to, naturally occurring nucleotides, including guanine, cytosine, adenine, and thymine (G, C, A, and T, respectively). The term "nucleotide" is also intended to include those parts that contain not only the known purine and pyrimidine bases, but also other modified heterocyclic bases. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated ribose or other heterocycles. In addition, the term "nucleotide" includes those parts that contain haptens or fluorescent labels and may contain not only conventional ribose and deoxyribose, but also other sugars. Modified nucleosides or nucleotides also include modifications of the sugar moiety, for example, where one or more hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, and the like.

[0069] The terms "nucleic acid", "polynucleotide" and "nucleic acid molecule" are used interchangeably herein to describe polymers of any length, such as greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, up to about 10,000 or more bases composed of nucleotides such as deoxynucleotides or ribonucleotides, and can be produced enzymatically or synthetically (e.g., PNA, as described in U.S. Pat. No. 5,948,902 and references cited therein). The nucleic acid can hybridize with naturally occurring nucleic acids in a sequence-specific manner similar to two naturally occurring nucleic acids, such as being able to participate in Watson-Crick base pairing interactions. In addition, nucleic acids and polynucleotides can be isolated (and optionally subsequently fragmented) from cells, tissues and / or body fluids. The nucleic acid can be, for example, genomic DNA (gDNA), mitochondrial, cell-free DNA (cfDNA), DNA from a library, and / or RNA from a library.

[0070] As used herein, the term "nucleic acid sample" or "sample comprising nucleic acid" refers to any sample containing nucleic acid, wherein the sample refers to a material or a mixture of materials, usually (although not necessarily) in liquid form, comprising one or more target nucleotide sequences of interest. The nucleic acid sample used as the starting material in the method of the present invention can be from any source, such as a whole genome, a set of chromosomes, a single chromosome, one or more regions from one or more chromosomes or transcribed genes, and can be directly purified from a biological source or a laboratory source such as a nucleic acid library. The nucleic acid sample can be obtained from the same individual, which can be a human or other species (such as plants, bacteria, fungi, algae, archaea, etc.), or from different individuals of the same species, or different individuals of different species. For example, the nucleic acid sample can be from a cell, a tissue, a biopsy, a body fluid, a genomic DNA library, a cDNA library and / or an RNA library.

[0071] The terms "sequence of interest", "target nucleotide sequence of interest" and "target sequence" are used interchangeably herein and include, but are not limited to, any gene sequence preferably present in a cell, such as a gene, a partial gene, or a non-coding sequence within or adjacent to a gene. The target sequence of interest may be present in a chromosome, an episome, an organelle genome such as a mitochondrial or chloroplast genome, or genetic material that can exist independently of the main body of genetic material, such as an infectious viral genome, a plasmid, an episome, such as a transposon. The sequence of interest may be within the coding sequence of a gene, within a transcribed non-coding sequence, such as a leader sequence, a trailer sequence, or an intron. The nucleic acid sequence of interest may be present in a double-stranded nucleic acid or a single-stranded nucleic acid.

[0072] The sequence of interest may be, but is not limited to, a sequence having or suspected of having a polymorphism such as a SNP.

[0073] As used herein, the term "oligonucleotide" refers to a single-stranded polymer of nucleotides, preferably about 2-200 nucleotides in length, or up to 500 nucleotides. Oligonucleotides can be synthesized or enzymatically prepared, and in some embodiments, have a length of about 10-50 nucleotides. Oligonucleotides can contain ribonucleotide monomers (i.e., oligoribonucleotides) or deoxyribonucleotide monomers. For example, an oligonucleotide can be about 10-20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-100, 100-150, 150-200, or about 200-250 nucleotides in length.

[0074] "Plant": This includes plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, plant clumps, and plant cells intact in plants or plant parts such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, husks, stalks, roots, root tips, anthers, grains, etc. Non-limiting examples of plants include crops and cultivated plants such as barley, cabbage, canola, cassava, cauliflower, chicory, cotton, cucumber, eggplant, grapes, peppers, lettuce, corn, melon, oilseed rape, potatoes, pumpkin, rice, rye, sorghum, squash, sugarcane, sugar beets, sunflowers, sweet peppers, tomatoes, watermelons, wheat, and zucchini.

[0075] A "protospacer sequence" is a sequence that recognizes or can hybridize to a guide sequence within a guide RNA, more specifically the crRNA or, in the case of an sgRNA, the crRNA portion of a guide RNA, and is located in, at, or near a target sequence.

[0076] "Endonucleases" are enzymes that hydrolyze at least one strand of a double-stranded DNA or one strand of an RNA molecule after binding to its target or recognition site, and endonucleases are understood herein as site-specific endonucleases and the terms "endonuclease" and "nuclease" are used interchangeably herein. Restriction endonucleases are understood herein as endonucleases that hydrolyze both strands of a duplex simultaneously to introduce a double-stranded gap in the DNA. "Nick" endonucleases are endonucleases that hydrolyze only one strand of a duplex to generate a "nicked" rather than a cut DNA molecule.

[0077] An "exonuclease" is defined herein as any enzyme that cleaves one or more nucleotides from a polynucleotide end (exo).

[0078] "Reducing complexity" or "complexity reduction" is understood herein as the reduction of complex nucleic acid samples, such as samples derived from genomic DNA, cfDNA derived from liquid biopsies, isolated RNA samples, etc. Complexity reduction can result in enrichment of one or more specific target sequences or target nucleic acid fragments (also named target fragments herein) contained in the complex starting material and / or the generation of a sample subset, wherein the subset comprises or consists of one or more specific target sequences or fragments contained in the complex starting material, and the amount of non-target sequences or fragments is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% compared to the amount of non-target sequences or fragments in the starting material, i.e., before the complexity reduction. Reducing complexity is generally performed before further analysis or method steps, such as amplification, barcoding, sequencing, determining epigenetic variations, etc. The complexity reduction is preferably a reproducible complexity reduction, meaning that when the same sample is reduced in complexity by the same method, the same or at least comparable subsets are obtained, as opposed to random complexity reduction. Examples of complexity reduction methods include, for example (Keygene NV, the Netherlands; see, e.g., EP 0 534858), arbitrarily primed PCR amplification, capture probe hybridization, the method described by Dong (see, e.g., WO 03 / 012118, WO 00 / 24939) and index linking (Unrau P. and Deugau KV (1994) Gene 145:163-169), WO2006 / 137733; WO2007 / 037678; WO2007 / 073165; WO2007 / 073171, US 2005 / 260628, WO 03 / 010328, methods described in US2004 / 10153, genome partitioning (see, e.g., WO 2004 / 022758), serial analysis of gene expression (SAGE; see, e.g., Velculescu et al., 1995, supra and Matsumura et al., 1999, The Plant Genet 145:163-169). Journal, Vol. 20(6):719-726) and SAGE improvements (see, e.g., Powell, 1998, Nucleic Acids Research, Vol. 26(14):3445-3446; and Kenzelmann and 1999, Nucleic Acids Research, Volume 27(3):917-918), MicroSAGE (see, e.g., Datson et al., 1999, Nucleic Acids Research, Volume 27(5):1300-1307), massively parallel signature sequencing (MPSS; see, e.g., Brenner et al., 2000, Nature Biotechnology, Volume 18:630-634 and Brenner et al., 2000, PNAS, Volume 97(4):1665-1670), self-subtracted cDNA libraries (Laveder et al., 2002, Nucleic Acids Research, Volume 30(9):e38), real-time multiplex ligation-dependent probe amplification (RT-MLPA; see, e.g., Eldering et al., 2003, Volume 31(23):e153), high coverage expression profiling (HiCEP; see, e.g., Fukumura et al., 2003, Nucleic Acids Research, vol. 31(16):e94), the general microarray system disclosed by Roth et al. (Roth et al., 2004, Nature Biotechnology, vol. 22(4):418-426), transcriptome subtraction (see, e.g., Li et al., Nucleic Acids Research, vol. 33(16):e136), and fragment display (see, e.g., Metsis et al., 2004, Nucleic Acids Research, vol. 32(16):e127).

[0079] "Sequence" or "nucleotide sequence": This refers to the order of nucleotides of or within a nucleic acid. In other words, any sequence of nucleotides in a nucleic acid can be referred to as a sequence or nucleic acid sequence. For example, a target sequence is the order of nucleotides contained in a single strand of a DNA duplex.

[0080] As used herein, the term "sequencing" refers to a method for obtaining at least 10 consecutive nucleotide features (e.g., at least 20, at least 50, at least 100, or at least 200 or more consecutive nucleotide features) in a polynucleotide. The term "second-generation sequencing" refers to so-called parallel synthesis sequencing or ligation platform sequencing, such as those currently used by Illumina, Life Technologies, PacBio, and Roche. Second-generation sequencing methods may also include nanopore sequencing methods, such as those commercialized by Oxford Nanopore Technologies, or electronic detection-based methods such as ion torrent technology commercialized by Life Technologies.

[0081] A "target nucleic acid fragment" or "target fragment" can be a small or longer stretch or selected portion of a nucleic acid, single or double stranded, comprising or consisting of a sequence of interest, which is preferably the object of further analysis or action, such as but not limited to replication, amplification, sequencing and / or other nucleic acid detection processes. Prior to complexity reduction, the target nucleic acid fragment is preferably contained within a larger nucleic acid molecule, such as that present in the sample to be analyzed.

[0082] The sequence of interest can be any sequence in the sample nucleic acid, such as a gene, a gene complex, a locus, a pseudogene, a regulatory region, a high repeat region, a polymorphic region or a part thereof. The sequence of interest can also be a region containing genetic or epigenetic variation, and the variation indicates a phenotype or disease. In some aspects, a group of target nucleic acid fragments are selected for enrichment, and the fragments include one or more sequences of interest, or are composed of them. Optionally, the group is composed of structural or functional related target nucleic acid fragments. One or more target fragments can include natural or non-natural, artificial or non-classical nucleotides, including but not limited to DNA, RNA, BNA (bridging nucleic acid), LNA (locked nucleic acid), PNA (peptide nucleic acid), morpholino nucleic acid, ethylene glycol nucleic acid, threose nucleic acid, epigenetically modified nucleotides such as methylated DNA and analogs and combinations thereof. Preferably, these sequences of interest are small or longer continuous nucleotide extension fragments (i.e., polynucleotides) of single-stranded DNA chains in double-stranded DNA, wherein the double-stranded DNA also includes a sequence complementary to the target sequence in the double-stranded DNA complementary chain. The double-stranded DNA composed of the sequence of interest and its complementary chain is also named as target nucleic acid fragment double-stranded DNA in this article. Preferably, the double-stranded DNA is genomic DNA (gDNA) and / or cell-free DNA (cfDNA). DETAILED DESCRIPTION OF THE INVENTION

[0084] The inventors discovered that the functional gRNA-CAS complex has an unexpected protective effect on the cleaved fragments. In fact, it appears that after cleavage, the cleaved fragments are protected against exonuclease cleavage. Without wishing to be bound by theory, this protection is attributed to the complex that remains bound to the ends of the cleaved fragments during exonuclease treatment. Thus, the methods of the present invention unexpectedly show that amplification-free methods for target enrichment, such as those disclosed herein, do not require the attachment of protective adapters.

[0085] In a first aspect, a method for enriching at least one target nucleic acid fragment from a sample comprising nucleic acid molecules is provided. Preferably, the target nucleic acid fragment comprises a sequence of interest. Preferably, the nucleic acid fragment is contained in a nucleic acid molecule present in the sample before the enrichment step as described in detail below. Therefore, preferably, the target nucleic acid fragment is a fragment of a nucleic acid molecule in the sample.

[0086] Preferably, the present invention relates to a method for enriching target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps:

[0087] a) providing a sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises a sequence of interest;

[0088] b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, thereby generating a target nucleic acid fragment comprising a sequence of interest and at least one non-target nucleic acid fragment;

[0089] c) contacting the cleaved nucleic acid molecule obtained in step b) with an exonuclease and allowing the exonuclease to digest the at least one non-target nucleic acid fragment; and

[0090] d) Optionally, purifying the target nucleic acid fragments comprising the sequence of interest from the digest obtained in step c).

[0091] Preferably, the RNA or DNA guided endonuclease complex in step b) is at least one of a gRNA-CAS complex, a gRNA-argonaute complex and a gDNA-argonaute complex. Preferably, the RNA or DNA guided endonuclease complex in step b) is a gRNA-CAS complex.

[0092] Preferably, in step c), the at least first and second gRNA-CAS complexes bind to the target nucleic acid fragment.

[0093] Preferably, in step c), the at least first and second gRNA-CAS complexes remain bound to the target nucleic acid fragment during step c) or at least part of step c).

[0094] Preferably, in step c), the target nucleic acid fragment is not digested by exonuclease, ie, in step c), the target nucleic acid fragment is protected from exonuclease digestion.

[0095] Preferably, in step c), only one or more non-target nucleic acid fragments are digested by the exonuclease.

[0096] In step b), the nucleic acid molecule is cleaved with at least the first and second gRNA-CAS complexes. Optionally, step b) can be further described in the steps of contacting the nucleic acid molecule with the first and second gRNA-CAS complexes and allowing the complexes to cleave the nucleic acid molecule. Therefore, in one embodiment, step b) can be further described as follows:

[0097] b1) contacting the nucleic acid molecule with a first and a second gRNA-CAS complex, wherein the gRNA of the first complex guides the first complex to a sequence upstream of the sequence of interest, and wherein the gRNA of the second complex guides the second complex to a sequence downstream of the sequence of interest; and

[0098] b2) allowing the first and second gRNA-CAS complexes to cleave nucleic acid molecules, wherein at least one cleaved nucleic acid molecule is a target nucleic acid fragment, and at least 1, preferably 2, cleaved nucleic acid molecules are non-target nucleic acid fragments.

[0099] The inventor unexpectedly found that exonuclease was added to the digest of step b, and no further measures were taken to protect the target nucleic acid fragment, causing the enrichment of the fragment of interest. In other words, unexpectedly, it is not necessary to protect the target nucleic acid fragment from exonuclease degradation by, for example, further protection of connecting an inert joint. Therefore, the inventive method preferably does not include the following further steps: before the exonuclease treatment step, the target nucleic acid fragment, or the end of the target nucleic acid fragment. In a preferred embodiment, the method defined herein does not add a protective joint before the exonuclease treatment. Under this background, a protective joint should be understood herein as a joint specially designed to protect the target nucleic acid fragment captured by a joint for exonuclease digestion. This joint preferably provides protection for exonuclease degradation by incorporating a chemical moiety or a blocking group (such as thiophosphate) or lacking a terminal nucleotide (a hairpin or a stem-loop joint or a cyclizable joint).

[0100] The method of the present invention is for example used to enrich a nucleic acid sample, preferably to assist in downstream processing or analysis of one or more target nucleic acid fragments in the sample. Enrichment causes a reduction in the complexity of the nucleic acid sample used as starting material in step a) of the method of the present invention and / or the generation of one or more target nucleic acid fragment subsets of the nucleic acid sample used as starting material in step a) of the method of the present invention.

[0101] Therefore, the first aspect of the present invention also provides at least:

[0102] i) A method for reducing the complexity of a nucleic acid sample comprising a sequence of interest, comprising steps a) to c) as defined above and optionally step d);

[0103] ii) a method for providing a subset of a nucleic acid sample, comprising steps a) to c) and optionally step d) as defined above, wherein the subset comprises one or more target nucleic acid fragments; and

[0104] iii) A method for isolating or obtaining a fragment comprising a sequence of interest (from a nucleic acid molecule comprising said sequence of interest), ie a target nucleic acid fragment, comprising steps a) to c) as defined above and optionally step d).

[0105] Reducing nucleic acid sample complexity has particular utility in nucleic acid sequencing applications, especially in samples where the target nucleic acid fragment is a minor species within a complex sample (e.g., but not limited to a genome). Enrichment or complexity reduction can significantly reduce the cost of the sequencing data generated because a large portion of the complex sample is removed prior to sequencing, while the target nucleic acid fragment is selectively retained, thereby generating a higher percentage of sequence reads from the sequence of interest.

[0106] In a preferred embodiment, the enriched target nucleic acid fragments generated by the methods herein are used in a single molecule, real-time sequencing reaction, such as that available from Pacific Biosciences, Menlo Park, CA. Sequencing. Other sequencing technologies are also contemplated, such as nanopore sequencing (e.g., from Oxford Nanopore), Sequencing (Enomina), tSMS TM Sequencing (Helicos), Ion Sequencing (Life Technologies Corporation), pyrosequencing (such as from Roche / 454), Sequencing (Life Technologies, Inc.), microarray sequencing (such as from Affymetrix), Sanger sequencing, etc. Preferably, the sequencing method is capable of sequencing long template molecules, such as> 1000-10,000 bases or more. Preferably, the sequencing method is capable of detecting sequencing base modifications during the reaction, such as by monitoring the kinetic detection of the sequencing reaction. Preferably, the sequencing method can analyze the sequence of a single template molecule, such as real-time analysis. More applications that benefit from the reduced complexity of the method of the present invention include, but are not limited to, cloning, amplification, diagnosis, prediction, therapeutic diagnosis, genetic screening, etc., and optionally, for polymorphism detection, such as but not limited to cancer diagnostic tests. Optionally, the enriched nucleic acids generated by the methods herein are used to evaluate epigenetic variations such as DNA methylation tests. DNA methylation can be evaluated by any appropriate test known in the art, such as a combination of bisulfite conversion assay and sequencing. Bisulfite conversion, also known as bisulfite treatment, is used to deaminize unmethylated cytosine to generate uracil in DNA, which is used in downstream applications to evaluate DNA methylation status. Methylated cytosine is protected from conversion to uracil, allowing the use of direct sequencing to determine the position of unmethylated cytosine and 5-methylcytosine at single nucleotide resolution. Alternatively or additionally, when analyzing unamplified and optionally unmodified DNA, DNA modifications can be directly detected from sequencing data without the need for additional specific tests. An example of detecting DNA modifications in unamplified and unmodified DNA is the use of SMRT sequencing technology from Pacific Biosciences. The method may thus further include the step of reporting detected mutations or diagnoses to human subjects. The method may thus further include the step of generating a report, the report comprising the findings obtained using the method of the present invention.

[0107] The at least first and second gRNA-CAS complexes are understood herein as CRISPR-associated (CAS) proteins or CRISPR nucleases, each of which is compounded with a guide RNA. The CRISPR nuclease comprises a nuclease domain and at least one domain that interacts with a guide RNA. When compounded with a guide RNA, the guide RNA directs the CRISPR nuclease to a specific nucleic acid sequence. The guide RNA interacts with the CRISPR nuclease and a specific target nucleic acid sequence, so that once the guide sequence is directed to a site containing a specific nucleic acid sequence, the CRISPR nuclease can introduce a break at the target site. Preferably, in the case where one or two domains of the nuclease are catalytically active, the CRISPR nuclease can introduce a single or double-strand break at the target site, respectively. The technician clearly understands how to design a guide RNA in a manner that, when combined with a CRISPR nuclease, a single or double-strand break is introduced at a predetermined site of the nucleic acid molecule.

[0108] Based on the core element content and sequence, CRISPR nucleases can generally be divided into 6 main types (types I-VI), which are further subdivided into subtypes (Makarova et al., 2011, Nat Rev Microbiol 9: 467-77 and Wright et al., 2016, Cell 164 (1-2): 29-44). In general, the two key elements of the CRISPR-CAS system complex are CRISPR nuclease and crRNA. CrRNA consists of short repeat sequences interspersed with spacer sequences derived from invading DNA. CAS protein has a variety of activities such as nuclease activity. Therefore, the gRNA-CAS complex provides a mechanism for targeting specific sequences and certain enzymatic activities according to the sequence.

[0109] Type I CRISPR-CAS systems typically include Cas 3 proteins with separate helicase and DNase activities. For example, in type I-E systems, crRNA is incorporated into a multi-subunit effector complex called Cascade (CRISPR-associated complex for antiviral defense) (Brouns et al., 2008, Science 321: 960-4), which specifically binds to double-helical DNA and triggers degradation by Cas3 protein (Sinkunas et al., 2011, EMSO J 30: 1335-1342; Beloglazova et al., 2011, EMBO J 30: 616-627).

[0110] Type II CRISPR-CAS system includes characteristic Cas9 protein, which is a single protein (about 160KDa) that can produce crRNA and specifically cut double helix DNA.Cas9 protein generally includes 2 nuclease domains, i.e., RuvC-like nuclease domain near the amino terminus and HNH (or McrA-like) nuclease domain near the middle of the protein.Each nuclease domain of Cas9 protein is specifically used to cut a chain of double helix (Jinek et al., 2012, Science 337 (6096): 816-821).Cas9 protein is an example of CAS protein of type II CRISPR / -CAS system and forms endonuclease, when combined with crRNA and a second RNA called trans-activating crRNA (tracrRNA), it targets invading pathogen DNA, and degrades by introducing DNA double-strand breaks (DSBs) at positions in the pathogen genome defined by crRNA. Jinek et al. (2012, Science 337:816-820) demonstrated that a single-stranded chimeric guide RNA (hereinafter "sgRNA") generated by fusing the essential parts of crRNA and tracrRNA can combine with Cas9 protein to form a functional nuclease.

[0111] Type III CRISPR-CAS systems include polymerase and RAMP components. Type III systems can be further divided into subtypes III-A and III-B. Type III-A CRISPR-CAS systems show targeting plasmids, and polymerase-like proteins of type III-A systems are involved in specific DNA cleavage (Marraffini and Sontheimer, 2008, Science 322: 1843-1845). Type III-B CRISPR-CAS systems also show targeting RNA (Hale et al., 2009, Cell 139: 945-956).

[0112] Type IV CRISPR-Cas systems contain Csf1, an uncharacterized protein proposed to form part of a Cascade-like complex, although these systems are typically found as isolated cas genes without an associated CRISPR array.

[0113] A type V CRISPR-CAS system, clustered regularly interspaced short palindromic repeats 1, or CRISPR / Cpf1, from Prevotella and Francisella was recently described. The Cpf1 gene is associated with the CRISPR locus and encodes an endonuclease that uses crRNA to target DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, which may overcome some of the limitations of the CRISPR-Cas9 system. Cpf1 is a single RNA-guided endonuclease, has no tracrRNA, and uses a T-rich protospacer adjacent motif. Cpf1 cleaves DNA via staggered DNA double-strand breaks (Zetsche et al. (2015) Cell 163(3):759–771). The type V CRISPR-CAS system preferably comprises at least one of Cpf1, C2c1, and C2c3.

[0114] Type VI CRISPR-CAS system may include Cas13a protein, which includes RNase A activity. In the case where the target nucleic acid fragment is RNA, at least the first and second gRNA-CAS complexes of the inventive method may include Cas13a, such as but not limited to Cas13a from Leptotreichia wadee (LwCas13a) or Leptotrichia shahii (LshCas13a), as described in Gootenberg et al., Science. April 28, 2017; 356(6336): 438-442.

[0115] The first and second gRNA-CAS complexes of the method of the present invention may comprise any CRISPR nuclease defined above. Preferably, at least one of the first and second gRNA-CAS complexes of the method of the present invention comprises a type II CRISPR nuclease such as Cas9 (e.g., a protein of SEQ ID NO: 1, which is encoded by SEQ ID NO: 2, or a protein of SEQ ID NO: 19) or a type V CRISPR nuclease such as Cpf1 (e.g., a protein of SEQ ID NO: 3, which is encoded by SEQ ID NO: 4) or Mad7 (e.g., a protein of SEQ ID NO: 20 or 21), or a derivative protein thereof, preferably at least about 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the protein over its entire length.

[0116] Preferably, at least one of the first and second gRNA-CAS complexes of the method of the present invention comprises a type II CRISPR nuclease, preferably a Cas9 nuclease.

[0117] The skilled person knows how to prepare the different components of the CRISPR-CAS system, including the CRISPR nuclease. In the prior art, there are many reports on its design and application. See, for example, Haeussler et al. (J Genet Genomics. (2016) 43 (5): 239-50. doi: 10.1016 / j.jgg.2016.04.008.) for a recent review on the design of guide RNAs and their combined use with CAS proteins (originally obtained from Streptococcus pyogenes (S. pyogenes)), or Lee et al. (Plant Biotechnology Journal (2016) 14 (2) 448–462).

[0118] Generally, CRISPR nucleases such as Cas9 contain two catalytically active nuclease domains. For example, the Cas9 protein can contain a RuvC-like nuclease domain and a HNH-like nuclease domain. RuvC and HNH domains work together to cut single strands to produce double-strand breaks in DNA (Jinek et al., Science, 337: 816-821). The inactivated CRISPR nuclease contains modifications so that no nuclease domain shows cutting activity. The CRISPR nuclease of at least one of the first and second gRNA-CAS complexes used in the method of the present invention can be a CRISPR nuclease variant, in which one of the nuclease domains is mutated so that it no longer has function (i.e., lacks nuclease activity), thereby producing a nickase. An example is a SpCas9 variant with a D10A or H840A mutation. Preferably, at least one of the nucleases of the first and second gRNA-CAS complexes is not an inactivated nuclease. Preferably, the CRISPR nuclease of the first gRNA-CAS complex is a nickase or (endo) nuclease. Preferably, the CRISPR nuclease of the second gRNA-CAS complex is a nickase or an (endo)nuclease.

[0119] At least the first and second gRNA-CAS complexes of the methods of the present invention may comprise or consist of a complete Cas9 protein or variant, or may comprise fragments thereof. Preferably, such fragments do bind crRNA and tracrRNA or sgRNA, but may lack one or more residues required for nuclease activity.

[0120] Preferably, at least one of the first and second gRNA-CAS complexes comprises a Cas9 protein. Optionally, both the first and second gRNA-CAS complexes of the method of the present invention comprise a Cas9 protein. Cas9 protein can be derived from Streptococcus pyogenes (SpCas9; NCBI Reference Sequence NC_017053.1; UniProtKB-Q99ZW2), Geobacillus thermodenitrificans (UniProtKB-A0A178TEJ9), Corynebacterium ulcerous (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Ref: NC_021284.1); Prevotella intermedia (NCBI Ref: NC_017861.1;Spiroplasma taiwanense (NCBIRef: NC_021846.1); Streptococcus iniae (NCBI Ref: NC_021314.1); Belliella baltica (NCBI Ref: NC_018010.1); Psychroflexustorquisl (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBIRef: YP_820832.1); Listeria innocua (NCBI Ref: NP_472073.1); Campylobacter jejuni (NCBI Ref: YP_002344900.1); or Neisseria meningitidis (NCBI Ref: YP_002342100.1). Cas9 variants from these are contemplated, with inactivated HNH or RuvC domains homologous to SpCas9, such as SpCas9_D10A or SpCas9_H840A, or Cas9 with equivalent substitutions at positions corresponding to D10 or H840 in the SpCas9 protein, producing nickases.

[0121] According to a preferred embodiment, the programmable nuclease can be derived from Cpf1, such as Cpf1 from Acidaminococcus sp; UniProtKB-U2UMQ6. The variant can be a Cpf1-nickase with an inactivated RuvC or NUC domain, wherein the RuvC or NUC domain no longer has nuclease activity. The skilled person is well aware of the techniques available in the art, such as site-directed mutagenesis, PCR-mediated mutagenesis and total gene synthesis, which allow inactivated nucleases such as inactivated RuvC or NUC domains. An example of a Cpf1 nickase with an inactivated NUC domain is Cpf1 R1226A (see Gao et al. Cell Research (2016) 26: 901–913, Yamano et al. Cell (2016) 165 (4): 949–962). In this variant, there is an arginine to alanine (R1226A) transition within the NUC domain, which renders the NUC domain inactive.

[0122] The at least first and second gRNA-CAS complexes also include CRISPR nuclease-related guide RNAs that direct the complex to a defined site in a nucleic acid sample, also referred to as pre-spacer sequences. The guide RNA includes a guide sequence that targets the gRNA-CAS complex to a pre-spacer sequence, which is preferably near a sequence of interest in a nucleic acid molecule, at a sequence of interest in a nucleic acid molecule, or inside a sequence of interest in a nucleic acid molecule, and can be a combination of sgRNA or crRNA and tracrRNA (such as for Cas9) or only crRNA (such as in the case of Cpf1). Optionally, more than one type of guide RNA can be used in the same experiment, for example, for 2 or more different sequences of interest, or even for the same sequence of interest.

[0123] It is understood herein that the sequence of interest is present in the nucleic acid sample before cutting with at least the first and second gRNA-CAS complexes. Cutting the nucleic acid sample can produce at least 2 or more nucleic acid fragments, wherein at least one nucleic acid fragment is a target nucleic acid fragment and at least one nucleic acid fragment is a non-target nucleic acid fragment. The target nucleic acid fragment comprises or consists of a sequence of interest. Therefore, before cutting the nucleic acid sample, it is clear to the technician that the nucleic acid sample covers the target nucleic acid fragment and that the target nucleic acid fragment is released from the nucleic acid sample after cutting. The inventors have found that the nucleic acid fragments cut by the gRNA-CAS complex are protected from digestion, preferably exonuclease digestion.

[0124] The method of the present invention requires that the gRNA of the first gRNA-CAS complex guides the first complex to a sequence in a nucleic acid sample, so that the first gRNA-CAS complex cuts the nucleic acid sample upstream of the sequence of interest, and the gRNA of the second complex guides the second gRNA-CAS complex to a sequence in the nucleic acid sample, so that the second gRNA-CAS complex cuts the nucleic acid sample downstream of the sequence of interest.

[0125] Preferably, the gRNA-CAS complex comprises a CRISPR nuclease that cleaves the nucleic acid within the protoplasmic spacer sequence. The preferred CRISPR nuclease is Cas9.

[0126] The pre-spacer sequence bound by the first gRNA-CAS complex can be a sequence in the target nucleic acid fragment and / or the non-target nucleic acid fragment. Similarly, the pre-spacer sequence bound by the second gRNA-CAS complex can be a sequence in the target nucleic acid fragment and / or the non-target nucleic acid fragment. Preferably, the pre-spacer sequence is a sequence overlapping with the target nucleic acid fragment and the non-target nucleic acid fragment, that is, the cleavage site of the gRNA-CAS complex is within the pre-spacer sequence.

[0127] Preferably, the position of the pre-spacer sequence depends on the CRISPR nuclease used in the method of the present invention. As a non-limiting example, the CRISPR nuclease SpCAS9 cuts the nucleic acid within the pre-spacer sequence. Therefore, when CAS9 is used in the method of the present invention, it is preferred that the pre-spacer sequence is partially located in the target nucleic acid fragment and partially located in the non-target fragment, that is, the pre-spacer sequence overlaps between the target nucleic acid fragment and the non-target nucleic acid fragment. Thus, preferably, the gRNA guide sequence of at least one of the first and second gRNA-CAS complexes is capable of hybridizing with a pre-spacer sequence selected from the group consisting of:

[0128] A) hybridization with a pre-spacer sequence contained in the target nucleic acid fragment;

[0129] B) hybridizing with a protospacer sequence contained in a non-target nucleic acid fragment; and

[0130] C) A protospacer sequence overlapping between the target nucleic acid fragment and the non-target nucleic acid fragment.

[0131] A) In one embodiment, the gRNA guide sequence of at least one of the first gRNA-CAS complex and the second gRNA-CAS complex is capable of hybridizing with a sequence that is a sequence of a target nucleic acid fragment or a portion thereof, or hybridizing with its complementary sequence in the opposite strand, for example, in the case where the nucleic acid fragment is double-stranded. In other words, in this embodiment, the pre-spacer sequence targeted by at least one of the first and second gRNA-CAS complexes is a sequence of the target nucleic acid fragment or is located therein. Preferably, the pre-spacer sequence targeted by at least the first gRNA-CAS complex is the 5'-end of the target nucleic acid fragment sequence or is adjacent to it, or its complementary sequence, and preferably the pre-spacer sequence targeted by at least the second gRNA-CAS complex is the 3'-end of the target nucleic acid fragment sequence or is adjacent to it, or its complementary sequence. Adjacent can be directly adjacent, or preferably the distance is no more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500 or 1000 consecutive nucleotides. The number of nucleotides may depend on the CRISPR nuclease used in the methods of the invention.

[0132] B) In one embodiment, the gRNA guide sequence of at least one of the first gRNA-CAS complex and the second gRNA-CAS complex is capable of hybridizing with a sequence that will form or form part of a non-target nucleic acid fragment, or hybridize with its complementary sequence in the opposite strand, in the case where the nucleic acid sample is a double-stranded nucleic acid. In other words, in this embodiment, the position of the pre-spacer sequence targeted by at least one of the first and second gRNA-CAS complexes is almost adjacent to or directly adjacent to the sequence that will form the target nucleic acid fragment after cleavage. Preferably, when the fragment is present in the nucleic acid sample or its complementary sequence, the pre-spacer sequence targeted by the first gRNA-CAS complex is almost flanked by the 5'-end of the target nucleic acid fragment, preferably directly flanked by the 5'-end. Preferably, when the fragment is present in the nucleic acid sample or its complementary sequence, the pre-spacer sequence targeted by the second gRNA-CAS complex is flanked by the 3'-end of the target nucleic acid fragment, or directly flanked by the 3'-end. Preferably, the distance between the pre-spacer sequence and each 5' end or 3' end of the target nucleic acid fragment sequence in the nucleic acid sample does not exceed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides. The number of nucleotides may depend on the CRISPR nuclease used in the method of the present invention.

[0133] C) In a preferred embodiment, the guide sequence of at least one of the first gRNA-CAS complex and the second gRNA-CAS complex is capable of hybridizing with a sequence that overlaps between the non-target nucleic acid fragment and the target nucleic acid fragment. Preferably, the guide sequence of at least the first or second gRNA-CAS complex is capable of hybridizing with a sequence that overlaps between the 3' end of the non-target nucleic acid fragment and the 5' end of the target nucleic acid fragment. Preferably, the guide sequence of at least the first or second gRNA-CAS complex is capable of hybridizing with a sequence that overlaps between the 5' end of the non-target nucleic acid fragment and the 3' end of the target nucleic acid fragment. In other words, in this embodiment, it is preferred that the pre-spacer sequence targeted by at least the first or second gRNA-CAS complex overlaps between the 3' end of the non-target nucleic acid fragment and the 5' end of the target nucleic acid fragment (when the fragment is present in the nucleic acid sample, i.e., before the nucleic acid sample is cut).

[0134] As a non-limiting example, SpCas9 can cut between positions 3 and 4 within a 20 nt pre-spacer sequence. Thus, the target nucleic acid fragment at its 3'-end can include 3 nt of the pre-spacer sequence and the non-target nucleic acid fragment at its 5'-end can include 17 nt of the pre-spacer sequence. Likewise, if the pre-spacer sequence is on the complementary strand, the target nucleic acid fragment at its 3'-end can include 17 nt of the pre-spacer sequence and the non-target nucleic acid fragment at its 5'-end can include 3 nt of the pre-spacer sequence. Thus, in the example where the pre-spacer sequence is 20 consecutive nucleotides, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides of the pre-spacer sequence may be present at the 3'-end of the non-target nucleic acid fragment, and respectively 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of the pre-spacer sequence may be present at the 5'-end of the target sequence, depending on the type of CRISPR nuclease used in the method of the present invention.

[0135] Preferably, the pre-spacer sequence targeted by at least the first or second gRNA-CAS complex overlaps between the 5'-end of the non-target nucleic acid fragment and the 3'-end of the target nucleic acid fragment (when the fragment is present in the nucleic acid sample, i.e., before the nucleic acid sample is cut). As a non-limiting example of a pre-spacer sequence of 20 nucleotides, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 nucleotides of the pre-spacer sequence may be present at the 5'-end of the non-target nucleic acid fragment, and each of 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 nucleotides of the pre-spacer sequence may be present at the 3-end of the target sequence, depending on the type of CRISPR nuclease used in the method of the present invention.

[0136] In a preferred embodiment, at least one of the first and second gRNA-CAS complexes binds to a sequence within the target nucleic acid fragment. Preferably, both the first and second gRNA-CAS complexes bind to a sequence within the target nucleic acid fragment.

[0137] Alternatively or additionally, at least one of the first and second gRNA-CAS complexes binds to a sequence within a non-target nucleic acid fragment. Preferably, both the first and second gRNA-CAS complexes bind to a sequence within a non-target nucleic acid fragment.

[0138] Alternatively or additionally, at least one of the first and second gRNA-CAS complexes binds to a sequence overlapping between the target nucleic acid fragment and the non-target nucleic acid fragment. Preferably, both the first and second gRNA-CAS complexes bind to a sequence overlapping between the target nucleic acid fragment and the non-target nucleic acid fragment.

[0139] In a preferred embodiment, at least one of the first and second gRNA-CAS complexes still binds to the 5'-end or 3'-end of the target nucleic acid fragment, respectively, after cleavage. Preferably, after cleavage, at least one gRNA-CAS complex remains bound to the 5'-end of the target nucleic acid fragment and one gRNA-CAS complex remains bound to the 3'-end of the target nucleic acid fragment. In contrast, the gRNA-CAS complex is preferably flanked on both sides of the target nucleic acid fragment.

[0140] Because in addition to the pre-spacer sequence, the gRNA-CAS complex also requires a pre-spacer adjacent motif (PAM) sequence for recognition, the gRNA should be designed so that the targeted pre-spacer sequence is adjacent to such a PAM sequence, depending on the gRNA-CAS complex used. The PAM sequence is essential for CRISPR / Cas nuclease activity and is relatively short, so it usually exists multiple times in any given sequence of a certain length. For example, the PAM motif of the Streptococcus pyogenes Cas9 protein is NGG, which ensures that for any given genomic sequence, there are multiple PAM motifs and many different guide RNAs can be designed. In addition, guide RNAs can also be designed to target the opposite strand of the same double-stranded sequence. The sequence directly adjacent to the PAM is incorporated into the guide RNA. Depending on the CRISPR-CAS complex used, it may be of different lengths. For example, the optimal length for targeting sequences in Cas9 sgRNA is 20nt. Depending on the CRISPR / Cas nuclease used, the complex then induces nicks in the two DNA chains at different distances from the PAM. For example, the Streptococcus pyogenes Cas9 protein induces a nick in both DNA strands 3 bp upstream of the PAM sequence to generate a blunt DNA DSB. Depending on, for example, the CRISPR-CAS complex used, the PAM site used to cleave the nucleic acid sample may be present in the generated nucleic acid fragments or the generated non-target nucleic acid fragments.

[0141] Preferably, the sequence of interest in the nucleic acid sample is flanked by a PAM sequence or comprises a PAM sequence preferably near the end of the sequence of interest, which is known to interact with the CRISPR-system nuclease of the complex defined herein (see, for example, Ran et al. 2015, Nature 520: 186-191). Additionally or alternatively, the PAM sequence is preferably flanked by a pre-spacer sequence targeted by at least one of the first and second gRNA-CAS complexes.

[0142] For example, if the CRISPR nuclease is Streptococcus pyogenes Cas9, the PAM sequence may have the sequence 5'-NGG-3'. For example, for thermodenitrifying Bacillus T12 Cas9 (see, for example, WO2016 / 198361), the PAM sequence may have the sequence 5'-NNNNCNNA-3'. More known PAM sequences for Cas9 endonucleases are: type IIA 5'-NGGNNNN-3' (Streptococcus pyogenes), 5'-NNGTNNN-3' (Streptococcus pasteurianus), 5'-NNGGAAN-3' (Streptococcus thermophilus), 5'-NNGGGNN-3' (Staphylococcus aureus) and type IIC 5'-NGGNNNN-3' (Corynebacterium difteriae), 5'-NNGGGTN-3' (Campylobacter lari), 5'-NNNCATN-3' (Parvobaculum lavamentivorans) and 5'-NNNNGTA-3' (Neiseria cinerea). One skilled in the art is thus able to design gRNAs to fragment target sequences from sample nucleic acids.

[0143] Molecules suitable for use as crRNA and tracrRNA as gRNA in gRNA-CAS complexes are well known in the art (see, for example, WO2013142578 and Jinek et al., Science (2012) 337, 816-821).

[0144] In one embodiment, at least one of the crRNAs comprises a sequence that can hybridize with or near a sequence of interest, preferably a sequence of interest as defined herein. Therefore, preferably, at least one of the crRNAs comprises a sequence that is completely complementary to a sequence in a sequence of interest, i.e., the sequence of interest comprises a pre-spacer sequence.

[0145] In one embodiment, the at least one crRNA comprises a sequence that can hybridize to or near the complementary sequence of a sequence of interest, preferably a sequence of interest as defined herein. Thus preferably, at least one of the crRNAs comprises a nucleotide sequence that has complete sequence identity to the sequence of interest or a portion of the sequence of interest.

[0146] Preferably, one or more crRNAs can also be complexed with tracrRNA. At least one of the crRNAs used in the method of the present invention can contain or consist of unmodified or naturally occurring nucleotides. Alternatively or additionally, at least one crRNA can contain or consist of modified or non-naturally occurring nucleotides, preferably such chemically modified nucleotides are used to protect crRNA from degradation. In one embodiment, at least 2 or all crRNAs used in the method of the present invention can contain or consist of modified or non-naturally occurring nucleotides.

[0147] In one embodiment of the invention, the at least one crRNA may comprise ribonucleotides and non-ribonucleotides. The at least one crRNA can comprise one or more ribonucleotides and one or more deoxyribonucleotides.

[0148] The at least one crRNA may include one or more non-naturally occurring nucleotides or nucleotide analogs, such as nucleotides with thiophosphate linkages, locked nucleic acids (LNA) nucleotides containing a methylene bridge between the 2' and 4' carbons of the ribose ring, bridged nucleic acids (BNA), 2'-O-methyl analogs, 2'-deoxy analogs, 2'-fluoro analogs, or combinations thereof. The modified nucleotides may include a modified base selected from, but not limited to, the group consisting of 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, and 7-methylguanosine.

[0149] The at least one crRNA may be chemically modified as follows: 2'-O-methyl (M), 2'-O-methyl 3' thiophosphate (MS), 2'-O-methyl 3' thioPACE (phosphonoacetate) (MSP) or a combination thereof is incorporated at one or more terminal nucleotides. This chemically modified crRNA can include stability and / or activity increased compared to unmodified crRNA (Hendel et al., 2015, Nat Biotechnol. 33 (9); 985-989). In certain embodiments, the at least one crRNA comprises ribonucleotides in the region hybridized with the pre-spacer sequence. In one embodiment of the present invention, the deoxyribonucleotides and / or nucleotide analogs can be incorporated into an engineered crRNA structure, for example, but not limited to, in a sequence hybridized with the pre-spacer sequence, in a sequence interacting with the tracrRNA, or between these sequences.

[0150] Alternatively or additionally, the chemically modified nucleotide can be located 5' and / or 3' to the sequence that hybridizes to the pre-spacer sequence. The chemically modified sequence can further be located 5' and / or 3' to the sequence that interacts with the tracrRNA.

[0151] In a preferred embodiment, the length of the at least one crRNA can be at least about 15, 20, 25, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleotides in length. In some preferred embodiments, the length of the at least one crRNA is less than about 75, 50, 45, 40, 35, 30, 25 or about 20 nucleotides. Preferably, the crRNA length used for the method of the present invention is about 20-100, 25-80, 30-60 or about 35-50 nucleotides in length.

[0152] The portion of the crRNA sequence hybridized with the pre-spacer sequence is designed to have sufficient complementarity with the pre-spacer sequence to hybridize with the pre-spacer sequence and guide the sequence-specific binding of the compounded nuclease. The pre-spacer sequence is preferably adjacent to the pre-spacer sequence adjacent motif (PAM) sequence, which can interact with the CRISPR nuclease in the RNA-guided CRISPR system nuclease endonuclease complex defined herein. For example, in the case where the CRISPR nuclease is Streptococcus pyogenes Cas9, the PAM sequence is preferably 5'-NGG-3', wherein N can be any of T, G, A or C. The technician can transform the crRNA to target any desired sequence, preferably by transforming the sequence to be at least partially complementary to any desired pre-spacer sequence, thereby hybridizing with it. Preferably, the complementarity between the partial crRNA sequence and its corresponding pre-spacer sequence is at least about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% when optimally aligned with an appropriate alignment algorithm. The partial crRNA sequence complementary to the pre-spacer sequence can be at least 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. In some preferred embodiments, the sequence complementary to the DNA target sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably, the sequence complementary to the DNA sequence is at least 17 nucleotides in length. Preferably, the complementary crRNA sequence is about 10-30 nucleotides in length, about 17-25 nucleotides in length, or about 15-21 nucleotides in length. The portion of the crRNA complementary to the pre-spacer sequence is preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length, preferably 20 or 21 nucleotides, preferably 20 nucleotides.

[0153] The portion of the crRNA that interacts with tracrRNA is designed to have sufficient complementarity with tracrRNA to hybridize with tracrRNA and guide the complexed nuclease to the pre-spacer sequence. Preferably, the complementarity between this partial crRNA sequence and its corresponding portion of tracrRNA is at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% when optimally aligned with an appropriate alignment algorithm. The partial crRNA that interacts with tracrRNA is preferably at least about 5, 10, 15, 20, 22, 25, 30, 35, 40, 45 or more nucleotides in length. In some preferred embodiments, the partial crRNA that interacts with tracrRNA is less than about 60, 55, 50, 45, 40, 35, 30 or 35 nucleotides in length. Preferably, the portion of crRNA that interacts with tracrRNA is about 5-40, 10-35, 15-30, 20-28 nucleotides in length. The portion of crRNA that interacts with tracrRNA is preferably 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.

[0154] In one embodiment, at least the first and second gRNA-Cas complexes used in the methods of the present invention comprise the first and second crRNAs, respectively. However, the first and second gRNA-Cas complexes may comprise the same tracrRNA.

[0155] The tracrRNA preferably comprises one or more structural motifs that can interact with the CRISPR system nuclease of the complex defined herein. Preferably, the tracrRNA can also interact with the crRNA defined herein. The tracrRNA and crRNA can hybridize by base pairing between crRNA and tracrRNA. The tracrRNA is preferably capable of forming a complex with the CRISPR system nuclease and crRNA. The crRNA can compound the tracrRNA and can hybridize with the target sequence, thereby guiding the nuclease to the target sequence.

[0156] The tracrRNA may comprise one or more stem-loop structures, such as 1, 2, 3 or more stem-loop structures.

[0157] The tracrRNA can comprise or consist of unmodified or naturally occurring nucleotides. Alternatively or additionally, the tracrRNA can comprise or consist of modified or non-naturally occurring nucleotides, preferably such chemically modified nucleotides are used to protect the tracrRNA from degradation.

[0158] In one embodiment of the present invention, the tracrRNA comprises ribonucleotides and non-ribonucleotides. The tracrRNA can comprise one or more ribonucleotides and one or more deoxyribonucleotides.

[0159] The tracrRNA may comprise one or more non-naturally occurring nucleotides or nucleotide analogs, such as nucleotides with thiophosphate linkages, locked nucleic acids (LNA) nucleotides comprising a methylene bridge between the 2' and 4' carbons of the ribose ring, bridged nucleic acids (BNA), 2'-O-methyl analogs, 2'-deoxy analogs, 2'-fluoro analogs, or combinations thereof. The modified nucleotides may comprise a modified base selected from, but not limited to, the group consisting of 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, and 7-methylguanosine.

[0160] The tracrRNA may be chemically modified by incorporating 2'-O-methyl (M), 2'-O-methyl 3' phosphorothioate (MS), 2'-O-methyl 3' thioPACE (phosphonoacetate) (MSP) or a combination thereof at one or more terminal nucleotides. Such chemically modified tracrRNAs can comprise increased stability and / or activity compared to unmodified tracrRNAs (Hendel et al., 2015, Nat Biotechnol. 33(9); 985-989). In certain embodiments, the tracrRNA comprises ribonucleotides in the region that interacts with the crRNA.

[0161] In one embodiment of the present invention, the deoxyribonucleotides and / or nucleotide analogs can be incorporated into the modified tracrRNA structure, for example but not limited to, in the sequence interacting with crRNA, in the sequence interacting with the CRISPR system nuclease, or between these sequences.

[0162] Alternatively or additionally, the chemically modified nucleotides can be located 5' and / or 3' to the sequence that interacts with the crRNA. The chemically modified nucleotides can further be located 5' and / or 3' to the sequence that interacts with the CRISPR system nuclease.

[0163] In a preferred embodiment, the tracrRNA length can be about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150 or more nucleotides in length. In some preferred embodiments, the tracrRNA is less than about 200, 180, 160, 140, 120, 100, 95, 90, 85, 80 or 75 nucleotides in length. The tracrRNA length is preferably about 30-120, 40-100, 50-90 or about 60-80 nucleotides in length.

[0164] The portion of the tracrRNA sequence that interacts with the CRISPR system nuclease is designed to be sufficient to guide the composite nuclease to the target sequence. The portion of the tracrRNA sequence that interacts with the CRISPR system nuclease can be about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 72, 75, 80, 85, 90, 95, 100 or more nucleotides in length. In some preferred embodiments, the sequence that interacts with the CRISPR system nuclease is less than about 120, 100, 80, 72, 70, 60, 55, 50, 45, 40, 30 or 20 nucleotides in length. Preferably, the portion of the tracrRNA sequence that interacts with the CRISPR system nuclease is about 20-90, 30-85, 35-80, 40–75 or 50-72 nucleotides in length. Preferably, the portion of the tracrRNA that interacts with the CRISPR system nuclease is about 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or 76 nucleotides in length.

[0165] The portion of the tracrRNA sequence that interacts with crRNA is designed to have sufficient complementarity with crRNA to hybridize with crRNA and guide the complexed nuclease to the target sequence. Preferably, the complementarity between this portion of the tracrRNA sequence and its corresponding portion of the crRNA is at least about 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 100% when optimally aligned with an appropriate alignment algorithm. The portion of the tracrRNA that interacts with crRNA is preferably at least about 5, 10, 15, 20, 22, 25, 30, 35, 40, 45 or more nucleotides in length. In some preferred embodiments, the portion of the tracrRNA that interacts with crRNA is less than about 60, 55, 50, 45, 40, 35, 30 or 35 nucleotides in length. In a preferred embodiment, the portion of tracrRNA that interacts with crRNA is about 5-40, 10-35, 15-30, 20-28 nucleotides in length. Preferably, the portion that interacts with crRNA is about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides in length.

[0166] Preferably, crRNA and tracrRNA are linked together to form sgRNA. CrRNA and tracrRNA can be linked, preferably covalently linked, using any conventional method known in the art. For example, the conventional connection of crRNA and tracrRNA is described in Jinek et al. (supra) and WO13 / 176772, which are incorporated herein by reference. CrRNA and tracrRNA can be covalently linked, using, for example, linker nucleotides or directly covalently linked to the 5' end of tracrRNA via the 3' end of crRNA. Preferably, the gRNA of the at least first and second gRNA-CAS complex is designed to cut the target nucleic acid fragment contained in the nucleic acid from the nucleic acid sample after the nucleic acid sample is incubated with at least the first and second gRNA-CAS complexes. In addition, it is preferred that the first gRNA is designed to bind the target nucleic acid fragment after the nucleic acid sample is cut. In addition, it is preferred that the second gRNA is designed to bind the target nucleic acid fragment after the nucleic acid sample is cut. Preferably, when the target nucleic acid fragment is present in the nucleic acid sample, the flank is at least one non-target nucleic acid fragment. Preferably, when the target nucleic acid fragment is present in the nucleic acid sample, both sides are flanked by non-target nucleic acid fragments, that is, one non-target nucleic acid fragment is directly present at 5' of the target nucleic acid fragment and one non-target nucleic acid fragment is directly present at 3' of the target nucleic acid fragment.

[0167] Preferably, at least one of the first and second gRNA-CAS complexes of the method of the present invention comprises an sgRNA for targeting a CRISPR nuclease, preferably Cas9, to a sequence in a target nucleic acid fragment. Optionally, both the first and second gRNA-CAS complexes of the method of the present invention comprise an sgRNA for targeting each of the first and second gRNA-CAS complexes to a sequence in a target nucleic acid fragment. At least one of the first and second gRNA-CAS complexes of the method of the present invention preferably comprises an sgRNA for targeting a CRISPR nuclease, preferably Cas9, to a sequence adjacent to, preferably directly adjacent to, a target nucleic acid fragment, when the fragment is contained in a nucleic acid sample. Optionally, both the first and second gRNA-CAS complexes of the method of the present invention comprise an sgRNA for targeting each of the first or second gRNA-CAS complexes to a sequence adjacent to, preferably directly adjacent to, a target nucleic acid fragment, wherein the target nucleic acid is contained in a nucleic acid sample.

[0168] Preferably, at least one of the first and second gRNA-CAS complexes of the method of the present invention comprises an sgRNA for targeting a sequence overlapping between a target nucleic acid fragment and a non-target nucleic acid fragment, preferably Cas9, by a CRISPR nuclease, when the fragment is contained in a nucleic acid sample. Optionally, both the first and second gRNA-CAS complexes of the method of the present invention comprise an sgRNA for targeting a sequence overlapping between each first or second gRNA-CAS complex and a target nucleic acid fragment, wherein the target nucleic acid is contained in a nucleic acid sample. Optionally, both the first and second gRNA-CAS complexes of the method of the present invention comprise an sgRNA for targeting a sequence overlapping between each first or second gRNA-CAS complex, respectively, the 5'-end of the target nucleic acid fragment and the 3'-end of the non-target nucleic acid fragment and the 5'-end of the target nucleic acid fragment, when the target nucleic acid is contained in a nucleic acid sample.

[0169] Alternatively, at least one of the first and second gRNA-CAS complexes of the present invention comprises a dual guide RNA to target a sequence in a nucleic acid sample, i.e., a pre-spacer sequence present in a target nucleic acid fragment or a non-target nucleic acid fragment. Dual guide RNA (dgRNA) is understood herein to include crRNA and tracrRNA as separate but preferably hybridized molecules, or to be composed thereof. Optionally, the first and second gRNA-CAS complexes of the present invention both comprise dgRNA for targeting the pre-spacer sequence to each first or second gRNA-CAS complex.

[0170] Preferably, at least one of the first and second gRNA-CAS complexes is capable of inducing a double strand break (DSB). Preferably, both the first and second gRNA-CAS complexes are capable of inducing a double strand break (DSB) in a nucleic acid sample.

[0171] Alternatively, at least one of the first and second gRNA-CAS complexes is a nickase, represented herein as the first or second gRNA-CAS-nickase complex, which is capable of producing a gap on only one strand of the double-stranded DNA. In this embodiment of the invention, in step b), an additional, i.e., third gRNA-CAS complex is added, which is capable of producing a gap on the complementary strand of the double-stranded DNA, roughly at the complementary position of the first or second gRNA-CAS-nickase complex producing the gap. Producing a gap roughly at the complementary position preferably causes a double-stranded (i.e., blunt or staggered) break in the nucleic acid sample.

[0172] As a non-limiting example, for example, the pre-spacer sequence of the third gRNA-CAS-nickase is preferably a sequence in the complementary chain that is complementary to the pre-spacer sequence targeted by the first gRNA-CAS-nickase complex, or a sequence within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 nucleotides moved in the upstream or downstream direction of the complementary chain. For example, in the case where the first gRNA-CAS complex is a gRNA-CAS-nickase complex, the third gRNA-CAS-nickase complex can be added in step b, resulting in a double-strand break induced by the first and third gRNA-CAS-nickase complexes on one side of the sequence of interest, which can be a blunt end, in which case the actual opposite position is nicked by the first and third complexes, or can be staggered, in which case the position of the nick formed by the first and third complexes is not completely opposite. Similarly, in addition to the first and third gRNA-CAS-nickase complexes, the use of a second and more such as a fourth gRNA-CAS-nickase complex can produce two blunt or staggered ends of the target nucleic acid fragment obtained in step b) of the method of the present invention. In some cases, for example in the case of subsequent directional adaptor ligation, it may be desirable to generate staggered ends at one or both ends of the target nucleic acid fragments generated in step b of the method of the invention.

[0173] Step b) of the method of the present invention can be performed as follows: incubating the at least first and second gRNA-CAS complexes with the nucleic acid sample under conditions and time suitable for the gRNA-CAS complex to induce at least one single-strand break, optionally a double-strand break, such as but not limited to the conditions detailed in the Examples provided herein. Optionally, the incubation is performed at about 10-90° C., preferably about 37° C. for about 1 minute to about 18 hours, preferably about 60 minutes.

[0174] The inventors found that the target nucleic acid fragments cut by gRNA-CAS are protected from exonuclease treatment. Therefore, after the target nucleic acid fragments are cut from the nucleic acid, an exonuclease is immediately added to digest one or more non-target nucleic acids. The target nucleic acid fragments are protected from degradation, while the unprotected fragments are degraded, causing enrichment of the target fragments or reduction in complexity. Thus, the method of the present invention adopts a method of removing unwanted (non-target) nucleic acid sample portions, rather than removing the portion of interest, thereby avoiding complex affinity selection schemes.

[0175] The exonuclease can be exonuclease I, III, V, VII, VIII or related enzymes, or any combination thereof. Exonuclease III recognizes the nick and extends the nick to the vacancy until a section of ssDNA is formed. Exonuclease VII can degrade this ssDNA. Exonuclease I also degrades ssDNA. ExoIII and ExoVII are a preferred combination of exonucleases for use in step c) of the method of the present invention.

[0176] Exonuclease V is capable of degrading ssDNA and dsDNA in the 3' to 5' and 5' to 3' directions. Therefore, in a preferred embodiment, the exonuclease in step c) of the method of the present invention is an exonuclease capable of degrading ssDNA and dsDNA in the 3' to 5' and 5' to 3' directions, preferably exonuclease V.

[0177] More information on degrading non-target sequences is provided in U.S. Patent Publication No. 2014 / 0134610, which is incorporated herein by reference in its entirety for all purposes.

[0178] In addition, endonucleases, i.e., restriction enzymes, can be used to degrade unprotected fragments, together with, before, after, or in any combination thereof, the exonuclease digestion of step c) of the method of the present invention. It should be understood herein that the restriction enzymes used in the method of the present invention are preferably selected based on one or more target sequences of interest, and the sequences are enriched by the method of the present invention, because one or more restriction enzymes preferably should not have a recognition site present in one or more target sequences of interest, but preferably should have a recognition site present at one or more positions in the remaining nucleic acid sample, i.e., one or more non-target nucleic acid fragments. Prior to the exonuclease treatment of step c) of the method of the present invention or even prior to the cleavage reaction of step b), the benefit of restriction enzyme digestion is that such digestion produces fragments that are more easily digested by the exonuclease of step c) if the fragments are not protected by the gRNA-CAS complex.

[0179] Step c) and the optional endonuclease step are performed under conditions and time sufficient to allow the exonuclease (and the optional endonuclease) to degrade substantially all of the unprotected fragments, such as, but not limited to, the conditions detailed in the Examples provided herein. Preferably, step c is performed under conditions and time sufficient to allow the exonuclease (and the optional endonuclease) to degrade all of the unprotected fragments. Step c) is performed at about 10-90° C., preferably about 37° C., preferably for about 1 minute to about 12 hours, preferably 30 minutes.

[0180] After step c), the exonuclease and optionally the endonuclease may be inactivated, for example, but not limited to, by treatment with at least one protease such as proteinase K or heat inactivation. Such techniques are standard in the art and the skilled person directly understands how to inactivate the exonuclease and optionally the endonuclease. A preferred inactivation step is heating the sample at a temperature of about 50-90° C., preferably about 75° C., for about 1–120 minutes, preferably about 10 minutes. The inactivation step is preferably between steps c) and d) of the method of the invention.

[0181] After step c) of the present invention, the sample enriched with one or more target nucleic acid fragments can be subjected to a purification step such as an AMPure bead-based purification process to remove complexes, enzymes, free nucleotides, possible free adapters, and possible small, non-target nucleic acid fragments. The target nucleic acid fragments can be recovered after purification and subjected to further processing and / or analysis such as single molecule sequencing.

[0182] The method of the present invention may further comprise a size selection step. Optionally, the size selection step is performed before step b), between steps b) and c), or after step c) of the method of the present invention.

[0183] The target nucleic acid fragments can vary in length, but are preferably at least 200, 500, 1000, 3000, 5000, 7000, 10,000, 15,000 or 20,000 (up to at least 100,000) bases in length. The length depends primarily on the intended use and, in some optimal embodiments, is based on the average read length of the particular sequencing technology to be used.

[0184] It should be understood herein that an effective amount of components is used in the method of the present invention. For example, at least the first and second gRNA-CAS complexes added in step b) are provided in an amount sufficient to induce cleavage of one or more nucleic acid molecules in the sample. In addition, the amount of exonuclease added in step c) is sufficient to degrade at least about 75%, 80%, 85%, 90%, 95% or 100% of the non-target nucleic acid fragments in the sample or starting material.

[0185] The method of the invention may comprise one or more purification steps, preferably after step c) as defined herein. An optional purification step is a proteinase K treatment. Alternatively or additionally, the purification may comprise the following steps:

[0186] I. exposing the digested nucleic acid sample obtained after step c) to one or more solid supports that specifically and effectively bind one or more target nucleic acid fragments; and optionally,

[0187] II. Washing the one or more solid supports and eluting the target nucleic acid fragments from the one or more solid supports.

[0188] The one or more solid supports may be, but are not limited to, Ampure beads.Since at least one isolated target nucleic acid fragment is obtained after purification, the method defined herein may also be viewed as a method for isolating one or more target nucleic acid fragments from a nucleic acid sample.

[0189] The method of the invention may be followed by a step of sequencing one or more target nucleic acid fragments.Thus, the method defined herein may also be viewed as a method of sequencing one or more target nucleic acid fragments from a nucleic acid sample.

[0190] Optionally, the method of the present invention further comprises an amplification step. Preferably, this amplification is performed after the exonuclease treatment, i.e. step c) as defined herein. Amplification can be performed by PCR or any amplification method known in the art.

[0191] The method of the present invention may also include the step of connecting one or more adapters to the target nucleic acid fragment. Preferably, such adapter connection is performed after step c) as defined herein. These one or more adapters may comprise a functional domain, preferably selected from a restriction site domain, a capture domain, a sequencing primer binding site, an amplification primer binding site, a detection domain, a barcode sequence, a transcription promoter domain and a PAM sequence or any combination thereof. The barcode may be, but is not limited to, a sample barcode or a unique molecular identifier (UMI).

[0192] In particularly preferred embodiments, the one or more adapters are sequencing adapters, for example comprising functional domains allowing Roche 454A and 454B sequencing, ILLUMINA TM SOLEXA TM Sequencing, Applied Biosystems' SOLID TM Sequencing, SMRT at Pacific Biosciences TM sequencing, Pollonator Polony sequencing, Oxford Nanopore technology, or whole genome sequencing.

[0193] According to the design of the joint, the joint can be a single strand, a double strand, a partially double strand, a Y-type, a hairpin or a circularizable joint. Optionally, one or more joints can be used. Optionally, one or more groups of 2 joints can be used, wherein the first joint of one group is intended to connect to the 5' terminal side of the target nucleic acid fragment and the second joint of the group is intended to connect to the 3' terminal side of the target nucleic acid fragment. The first and second joints in the group preferably each comprise a compatible primer binding sequence, so that the fragment connected by the joint is easy to amplify or sequence-check with a compatible primer.

[0194] In a preferred embodiment, the method of the present invention does not have an amplification and / or cloning step. Reducing the amplification step is beneficial because epigenetic information (e.g., 5-mC, 6-mA, etc.) can be lost in the amplicon. Further amplification can introduce changes in the amplicon (such as errors during amplification) so that its nucleotide sequence does not reflect the initial sample. Similarly, the target region is cloned into another organism and does not usually maintain the modifications present in the initial sample nucleic acid. Therefore, in a preferred embodiment, the target sequence to be enriched for further analysis is usually not amplified and / or cloned in the method herein.

[0195] A stem-loop or hairpin adapter is single-stranded, but its ends are complementary, so that the adapter folds back on itself to produce a double-stranded portion and a single-stranded loop. Stem-loop adapters can connect linear, double-stranded nucleic acid ends. For example, a stem-loop adapter connects the ends of a double-stranded target nucleic acid fragment so that there are no terminal nucleotides (e.g., any gaps are filled and ligated, using a polymerase and a ligase, respectively), and the resulting molecule lacks a terminal nucleotide, rather than carrying a single-stranded loop at each end.

[0196] The target nucleic acid fragments can be ligated to circularizable adapters. In this aspect, the target sequence-containing fragments can be circularized by self-circularization on either side of the fragments through compatible structures (which can be caused by adapter ligation or restriction enzyme digestion of the ligated adapters), or by hybridization to selection probes complementary to the desired fragment ends. The extension and final ligation steps form a covalently closed circular, optionally double-stranded polynucleotide.

[0197] It should be understood herein that the nucleic acid sample comprises at least one target nucleic acid fragment. Differently, the nucleic acid sample may thus comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more target nucleic acid fragments, for example at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000 or more target nucleic acid fragments, wherein each target nucleic acid fragment in the sample preferably has a different sequence. The method of the present invention can provide simultaneous enrichment of these target nucleic acid fragments from a nucleic acid sample. Therefore, optionally, in step b) of the method of the present invention, multiple groups of at least the first and second gRNA-CAS complexes are added to enrich, separate or sequence multiple target nucleic acid fragments from a nucleic acid sample. Preferably, these multiple groups of first and second gRNA-CAS complexes may comprise the same CRISPR nuclease, but their gRNAs are different. For example, for each target nucleic acid fragment, two different gRNA molecules can be used, such as one gRNA incorporated into a first gRNA-CAS complex and another gRNA incorporated into a second gRNA-CAS complex. For example, at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000 or more target nucleic acid fragments, preferably at least about 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 750, 1000 or more groups of gRNA molecules, preferably at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000 or more different gRNA molecules can be used in the methods of the invention.

[0198] Optionally, the method of the present invention is multiplexed, i.e., applied to multiple nucleic acid samples simultaneously, for example, for at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 500, 1000 or more nucleic acid samples. The method can be implemented in parallel with respect to multiple samples, wherein "parallel" is understood herein to mean almost simultaneously, but each sample is processed in a separate reaction tube or container. Additionally or alternatively, one or more steps of the method of the present invention can be performed on a merged sample. To trace back the enrichment, separation and / or sequencing fragments to the initial sample, the fragments can be labeled with identifiers and then the samples are merged. Such identifiers can be any detectable entity, such as, but not limited to, radioactive or fluorescent labels, but preferably specific nucleotide sequences or combinations of nucleotide sequences, preferably with defined lengths. Additionally or alternatively, samples can be merged using smart confounding strategies, such as, but not limited to, 2D and 3D merging strategies, so that after merging, each sample is contained in at least 2 or 3 pools, respectively. Specific target fragments can be traced back to the initial sample, using the coordinates of each pool containing specific enrichment, separation and / or sequencing target fragments.

[0199] The nucleic acid sample of the method of the present invention can be from any source, such as human, animal, plant, microorganism, and can be of any kind, such as endogenous or exogenous, for example, genomic DNA, chromosomal DNA, artificial chromosome, plasmid DNA or free DNA, cDNA, RNA, mitochondria, or artificial libraries such as BAC or YAC, etc. The DNA can be nuclear or organelle DNA. The DNA is preferably chromosomal DNA, preferably endogenous.

[0200] In another aspect, the present invention provides a kit for use in the method as defined above. Preferably, the kit comprises at least one of:

[0201] - one or more vials comprising at least a first and a second gRNA-CAS complex as defined herein;

[0202] - one or more vials comprising at least a first and a second gRNA for complexing with a CRISPR-CAS protein to form a gRNA-CAS complex, and another vial comprising the CRISPR-CAS protein;

[0203] - another vial containing one or more exonucleases to degrade non-target nucleic acids; and

[0204] -Optionally, a vial containing one or more restriction enzymes to degrade non-target nucleic acids.

[0205] Optionally, the kit further comprises one or more linkers as defined herein, with one or more vials as shown above or in separate vials. The kit preferably comprises at least 2, 4, 10, 20, 30 or 50 vials, containing one or more gRNAs as defined herein. The volume of any vial in the kit preferably does not exceed 100 mL, 50 mL, 20 mL, 10 mL, 5 mL, 4 mL, 3 mL, 2 mL or 1 mL.

[0206] The reagents may be present in lyophilized form or dissolved in an appropriate buffer. The kit may also contain any other components required to complete the invention, such as buffers, pipettes, microtiter plates, and written instructions. Such other components for use in the kit of the invention are known to the skilled person.

[0207] Finally, the use of at least the first and second gRNA-CAS complexes or kits as defined herein for enriching at least one target nucleic acid fragment from a nucleic acid sample is provided. More specifically, the use of the at least first and second gRNA-CAS complexes for protecting the target nucleic acid fragment from exonuclease degradation is provided. Sequence Listing <110> Master Gene Co., Ltd. <120> Targeted enrichment via endonuclease protection <130> p6080445pct <150> 18208936.7 <151> 2018-11-28 <160> twenty one <170> PatentIn version 3.5 <210> 1 <211> 1368 <212> PRT <213> artificial sequence <220> <223> Cas9 <400> 1 Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val 1 5 10 15 Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30 Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Asn Leu Ile 35 40 45 Gly Ala Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60 Lys Arg Thr Ala Arg Arg Tyr Thr Arg Arg Lys Asn Arg With Cys 65 70 75 80 Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Asp Ser 85 90 95 Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110 His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125 His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Leu Val Asp 130 135 140 Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His 145 150 155 160 Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175 Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190 Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205 Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220 Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn 225 230 235 240 Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255 Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270 Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285 Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300 Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser 305 310 315 320 Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335 Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350 Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365 Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380 Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg 385 390 395 400 Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415 Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430 Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445 Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460 Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu 465 470 475 480 Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495 Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510 Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525 Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540 Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr 545 550 555 560 Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575 Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590 Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605 Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620 Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala 625 630 635 640 His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655 Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670 Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685 Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700 Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu 705 710 715 720 His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735 Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750 Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765 Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780 Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro 785 790 795 800 Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815 Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830 Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845 Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860 Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys 865 870 875 880 Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895 Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910 Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925 Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940 Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser 945 950 955 960 Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975 Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990 Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005 Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020 Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035 Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050 Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065 Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080 Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095 Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110 Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125 Light Light Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140 Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155 Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170 Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200 Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215 Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245 Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260 His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275 Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290 Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305 Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320 Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335 Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350 Gly Tyr Glu Thr Arg to Asp to Gln Ser to Gly Gly Asp 1355 1360 1365 <210> 2 <211> 4104 <212> DNA <213> artificial sequence <220> <223> sequence encoding Cas9 <400> 2 atggataaaa atatagcat tggtctggat attggtacca atagcgttgg ttgggcagtt 60 attaccgatg atataagt tccgagcaa aaatttaaag ttctggtaa taccgatcgt 120 catagcatta aaaaaaatct gattggtgca ctgctgtttg atagcggtga aaccgcagaa 180 gcaacccgtc tgaaacgtac cgcacgtcgt cgttataccc gtcgtaaaaa tcgtatttgt 240 tatctgcagg aaatttttag caatgaaatg gcaaaagttg atgatagctt ttttcatcgt 300 ctggaagaaa gctttctggt tgaagaagat aaaaaacatg aacgtcatcc gatttttggt 360 aatattgttg atgaagttgc atatcatgaa aaatatccga ccatttatca tctgcgtaaa 420 aaactggttg atagcaccga taaagcagat ctgcgtctga tttatctggc actggcacat 480 atgattaaat ttcgtggtca ttttctgatt gaaggtgatc tgaatccgga taatagcgat 540 gttgataaac tgtttattca gctggttcag acctataatc agctgtttga agaaaatccg 600 attaatgcaa gcggtgttga tgcaaaagca attctgagcg cacgtctgag caaaagccgt 660 cgtctggaaa atctgattgc acagctgccg ggtgaaaaaa aaaatggtct gtttggtaat 720 ctgattgcac tgagcctggg tctgaccccg aattttaaaa gcaattttga tctggcagaa 780 gatgcaaaac tgcagctgag caaagatacc tatgatgatg atctggataa tctgctggca 840 cagattggtg atcagtatgc agatctgttt ctggcagcaa aaaatctgag cgatgcaatt 900 ctgctgagcg atattctgcg tgttaatacc gaaattacca aagcaccgct gagcgcaagc 960 atgattaaac gttatgatga acatcatcag gatctgaccc tgctgaaagc actggttcgt 1020 cagcagctgc cggaaaaata taaagaaatt ttttttgatc agagcaaaaa tggttatgca 1080 ggttatattg atggtggtgc aagccaggaa gaattttata aatttattaa accgattctg 1140 gaaaaaatgg atggtaccga agaactgctg gttaaactga atcgtgaaga tctgctgcgt 1200 aaacagcgta cctttgataa tggtagcatt ccgcatcaga ttcatctggg tgaactgcat 1260 gcaattctgc gtcgtcagga agatttttat ccgtttctga aagataatcg tgaaaaaatt 1320 gaaaaaattc tgacctttcg tattccgtat tatgttggtc cgctggcacg tggtaatagc 1380 cgttttgcat ggatgacccg taaaagcgaa gaaaccatta ccccgtggaa ttttgaagaa 1440 gttgttgata aaggtgcaag cgcacagagc tttattgaac gtatgaccaa ttttgataaa 1500 aatctgccga atgaaaaagt tctgccgaaa catagcctgc tgtatgaata tttaccgtt 1560 tataatgaac tgaccaaagt taaatatgtt accgaaggta tgcgtaaacc ggcatttctg 1620 agcggtgaac agaaaaaagc aattgttgat ctgctgttta aaaccaatcg taaagttacc 1680 gttaaacagc tgaaagaaga ttattttaaa aaaattgaat gttttgatag cgttgaaatt 1740 agcggtgttg aagatcgttt taatgcaagc ctgggtacct atcatgatct gctgaaaatt 1800 attaaagata aagattttct ggataatgaa gaaaatgaag atattctgga agatattgtt 1860 ctgaccctga ccctgtttga agatcgtgaa atgattgaag aacgtctgaa aacctatgca 1920 catctgtttg atgataaagt tatgaaacag ctgaaacgtc gtcgttatac cggttggggt 1980 cgtctgagcc gtaaactgat taatggtatt cgtgataaac agagcggtaa aaccattctg 2040 gattttctga aaagcgatgg ttttgcaaat cgtaatttta tgcagctgat tcatgatgat 2100 agcctgacct ttaaagaaga tattcagaaa gcacaggtta gcggtcaggg tgatagcctg 2160 catgaacata ttgcaaatct ggcaggtagc ccggcaatta aaaaaggtat tctgcagacc 2220 gttaaagttg ttgatgaact ggttaaagtt atgggtcgtc ataaaccgga aaatattgtt 2280 attgaaatgg cacgtgaaaa tcagaccacc cagaaaggtc agaaaatag ccgtgaacgt 2340 atgaaacgta ttgaagaagg tattaaagaa ctgggtagcc agattctgaa agaacatccg 2400 gttgaaaata cccagctgca gaatgaaaaa ctgtatctgt attatctgca gaatggtcgt 2460 gatatgtatg ttgatcagga actggatatt aatcgtctga gcgattatga tgttgatcat 2520 attgttccgc agagctttct gaaagatgat agcattgata ataaagttct gacccgtagc 2580 gataaaaatc gtggtaaaag cgataatgtt ccgagcgaag aagttgttaa aaaaatgaaa 2640 aattattggc gtcagctgct gaatgcaaaa ctgattaccc agcgtaaatt tgataatctg 2700 accaaagcag aacgtggtgg tctgagcgaa ctggataaag caggttttat taaacgtcag 2760 ctggttgaaa cccgtcagat taccaaacat gttgcacaga ttctggatag ccgtatgaat 2820 accaaatatg atgaaaatga taaactgatt cgtgaagtta aagttattac cctgaaaagc 2880 aaactggtta gcgatttcg taaagatttt cagttttata aagttcgtga attaataat 2940 tatcatcatg cacatgatgc atatctgaat gcagttgttg gtaccgcact gattaaaaaa 3000 <h2 style=";text-align:left;direction:ltr">tatccgaaac tggaaagcga atttgtttat ggtgattata aagtttatga tgttcgtaaa 3060<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atgattgcaa aaagcgaaca ggaaattggt aaagcaaccg caaaatattt tttttatagc 3120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aatattatga atttttttaa aaccgaaatt accctggcaa atggtgaaat tcgtaaacgt 3180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccgctgattg aaaccaatgg tgaaaccggt gaaattgttt gggataaagg tcgtgatttt 3240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcaaccgttc gtaaagttct gagcatgccg caggttaata ttgttaaaaa aaccgaagtt 3300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagaccggtg gttttagcaa agaaagcatt ctgccgaaac gtaatagcga taaactgatt 3360<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcacgtaaaa aagattggga tccgaaaaaa tatggtggtt ttgatagccc gaccgttgca 3420<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tatagcgttc tggttgttgc aaaagttgaa aaaggtaaaa gcaaaaaact gaaaagcgtt 3480<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaagaactgc tgggtattac cattatggaa cgtagcagct ttgaaaaaaa tccgattgat 3540<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttctggaag caaaaggtta taaagaagtt aaaaaagatc tgattattaa actgccgaaa 3600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tatagcctgt ttgaactgga aaatggtcgt aaacgtatgc tggcaagcgc aggtgaactg 3660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagaaaggta atgaactggc actgccgagc aaatatgtta attttctgta tctggcaagc 3720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">cattatgaaa aactgaaagg tagcccggaa gataatgaac agaaacagct gtttgttgaa 3780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cagcataaac attatctgga tgaaattatt gaacagatta gcgaatttag caaacgtgtt 3840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> attctggcag atgcaaatct ggataaagtt ctgagcgcat ataataaaca tcgtgataaa 3900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccgattcgtg aacaggcaga aaatattatt catctgttta ccctgaccaa tctgggtgca 3960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccggcagcat ttaaatattt tgataccacc attgatcgta aacgttatac cagcaccaaa 4020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaagttctgg atgcaaccct gattcatcag agcattaccg gtctgtatga aacccgtatt 4080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatctgagcc agctgggtgg tgat 4104<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <210> 3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <211> 1300<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <212> PRT<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <213> artificial sequence<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <220><h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <223> FnCpfI<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> <400> 3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Met Ser Ile Tyr Gln Glu Phe Val Asn Lys Tyr Ser Leu Ser Lys Thr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 1 5 10 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Leu Arg Phe Glu Leu Ile Pro Gln Gly Lys Thr Leu Glu Asn Ile Lys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Arg Gly Leu Ile Leu Asp Asp Glu Lys Arg Ala Lys Asp Tyr Lys<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> Lys Ala Lys Gln Ile Ile Asp Lys Tyr His Gln Phe Phe Ile Glu Glu 50 55 60 Ile Leu Ser Ser Val Cys Ile Ser Glu Asp Leu Leu Gln Asn Tyr Ser 65 70 75 80 Asp Val Tyr Phe Lys Leu Lys Lys Ser Asp Asp Asp Asn Leu Gln Lys 85 90 95 Asp Phe Lys Ser Ala Lys Asp Thr Ile Lys Lys Gln Ile Ser Glu Tyr 100 105 110 Ile Lys Asp Ser Glu Lys Phe Lys Asn Leu Phe Asn Gln Asn Leu Ile 115 120 125 Asp Ala Lys Lys Gly Gln Glu Ser Asp Leu Ile Leu Trp Leu Lys Gln 130 135 140 Ser Lys Asp Asn Gly Ile Glu Leu Phe Lys Ala Asn Ser Asp Ile Thr 145 150 155 160 Asp Ile Asp Glu Ala Leu Glu Ile Ile Lys Ser Phe Lys Gly Trp Thr 165 170 175 Thr Tyr Phe Lys Gly Phe His Glu Asn Arg Lys Asn Val Tyr Ser Ser 180 185 190 Asn Asp Ile Pro Thr Ser Ile Ile Tyr Arg Ile Val Asp Asp Asn Leu 195 200 205 Pro Lys Phe Leu Glu Asn Lys Ala Lys Tyr Glu Ser Leu Lys Asp Lys 210 215 220 Ala Pro Glu Ala Ile Asn Tyr Glu Gln Ile Lys Lys Asp Leu Ala Glu 225 230 235 240 Glu Leu Thr Phe Asp Ile Asp Tyr Lys Thr Ser Glu Val Asn Gln Arg 245 250 255 Val Phe Ser Leu Asp Glu Val Phe Glu Ile Ala Asn Phe Asn Asn Tyr 260 265 270 Leu Asn Gln Ser Gly Ile Thr Lys Phe Asn Thr Ile Ile Gly Gly Lys 275 280 285 Phe Val Asn Gly Glu Asn Thr Lys Arg Lys Gly Ile Asn Glu Tyr Ile 290 295 300 Asn Leu Tyr Ser Gln Gln Ile Asn Asp Lys Thr Leu Lys Lys Tyr Lys 305 310 315 320 Met Ser Val Leu Phe Lys Gln Ile Leu Ser Asp Thr Glu Ser Lys Ser 325 330 335 Phe Val Ile Asp Lys Leu Glu Asp Asp Ser Asp Val Val Thr Thr Met 340 345 350 Gln Ser Phe Tyr Glu Gln Ile Ala Ala Phe Lys Thr Val Glu Glu Lys 355 360 365 Ser Ile Lys Glu Thr Leu Ser Leu Leu Phe Asp Asp Leu Lys Ala Gln 370 375 380 Lys Leu Asp Leu Ser Lys Ile Tyr Phe Lys Asn Asp Lys Ser Leu Thr 385 390 395 400 Asp Leu Ser Gln Gln Val Phe Asp Asp Tyr Ser Val Ile Gly Thr Ala 405 410 415 Val Leu Glu Tyr Ile Thr Gln Gln Ile Ala Pro Lys Asn Leu Asp Asn 420 425 430 Pro Ser Lys Lys Glu Gln Glu Leu Ile Ala Lys Lys Thr Glu Lys Ala 435 440 445 Lys Tyr Leu Ser Leu Glu Thr Ile Lys Leu Ala Leu Glu Glu Phe Asn 450 455 460 Lys His Arg Asp Ile Asp Lys Gln Cys Arg Phe Glu Glu Ile Leu Ala 465 470 475 480 Asn Phe Ala Ala Ile Pro Met Ile Phe Asp Glu Ile Ala Gln Asn Lys 485 490 495 Asp Asn Leu Ala Gln Ile Ser Ile Lys Tyr Gln Asn Gln Gly Lys Lys 500 505 510 Asp Leu Leu Gln Ala Ser Ala Glu Asp Asp Val Lys Ala Ile Lys Asp 515,520,525 Leu Leu Asp Gln Thr Asn Asn Leu Leu His Lys Leu Lys Ile Phe His 530 535 540 Ser Gln Ser Glu Asp Lys Ala Asn Ileu Asp Lys Asp Glu His 545 550 555 560 Phe Tyr Leu Val Phe Glu Glu Cys Tyr Phe Glu Leu Ala Asn Ile Val 565,570,575 Pro Leu Tyr Asn With Arg Asn Tyr With Thr Gln Lys Pro Tyr Ser 580,585,590 Asp Glu Lys Phe Lys Leu Asn Phe Glu Asn Ser Thr Leu Ala Asn Gly 595,600,605 Trp Asp Lys Asn Lys Glu Pro Asp Asn Thr Ala Ile Leu Phe Ile Lys 610 615 620 Asp Asp Lys Tyr Leu Gly Val Met Lys Lys Asn Asn Lys Ile 625 630 635 640 Phe Asp Asp Lys Ala Ile Lys Glu Asn Gly Glu Gly Tyr Lys Lys 645,650,655 Ile Val Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met Leu Pro Lys Val 660,665,670 Phe Phe Ser Ala Lys Ser Ile Lys Phe Tyr Asn Pro Ser Glu Asp Ile 675 680 685 Leu Arg Ile Arg Asn His Ser Thr His Thr Lys Asn Gly Ser Pro Gln 690 695 700 Lys Gly Tyr Glu Lys Phe Glu Phe Asn Ile Glu Asp Cys Arg Lys Phe 705 710 715 720 Ile Asp Phe Tyr Lys Gln Ser Ile Ser Lys His Pro Glu Trp Lys Asp 725 730 735 Phe Gly Phe Arg Phe Ser Asp Thr Gln Arg Tyr Asn Ser Ile Asp Glu 740 745 750 Phe Tyr Arg Glu Val Glu Asn Gln Gly Tyr Lys Leu Thr Phe Glu Asn 755 760 765 Ile Ser Glu Ser Tyr Ile Asp Ser Val Val Asn Gln Gly Lys Leu Tyr 770 775 780 Leu Phe Gln Ile Tyr Asn Lys Asp Phe Ser Ala Tyr Ser Lys Gly Arg 785 790 795 800 Pro Asn Leu His Thr Leu Tyr Trp Lys Ala Leu Phe Asp Glu Arg Asn 805 810 815 Leu Gln Asp Val Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr 820 825 830 Arg Lys Gln Ser Ile Pro Lys Lys Ile Thr His Pro Ala Lys Glu Ala 835 840 845 Ile Ala Asn Lys Asn Lys Asp Asn Pro Lys Lys Glu Ser Val Phe Glu 850 855 860 Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Glu Asp Lys Phe Phe Phe 865 870 875 880 His Cys Pro Ile Thr Ile Asn Phe Lys Ser Ser Gly Ala Asn Lys Phe 885 890 895 Asn Asp Glu Ile Asn Leu Leu Leu Lys Glu Lys Ala Asn Asp Val His 900 905 910 Ile Leu Ser Ile Asp Arg Gly Glu Arg His Leu Ala Tyr Tyr Thr Leu 915 920 925 Val Asp Gly Lys Gly Asn Ile Ile Lys Gln Asp Thr Phe Asn Ile Ile 930 935 940 Gly Asn Asp Arg Met Lys Thr Asn Tyr His Asp Lys Leu Ala Ala Ile 945 950 955 960 Glu Lys Asp Arg Asp Ser Ala Arg Lys Asp Trp Lys Lys Ile Asn Asn 965 970 975 Ile Lys Glu Met Lys Glu Gly Tyr Leu Ser Gln Val Val His Glu Ile 980 985 990 Only Lys Leu Will Have Glu Tyr Asn Only Will Have Phe Glu Asp Leu 995 1000 1005 Asn Phe Gly Phe Lys Arg Gly Arg Phe Lys Val Glu Lys Gln Val 1010 1015 1020 Tyr Gln Lys Leu Glue Met Leu And Leu Glue Asn Tyr Leu 1025 1030 1035 Val Phe Lys Asp Asn Glu Phe Asp Lys Thr Gly Gly Val Leu Arg 1040 1045 1050 Ala Tyr Gln Leu Thr Ala Pro Phe Glu Thr Phe Lys Lys Met Gly 1055 1060 1065 Lys Gln Thr Gly Ile Tyr Tyr Val Pro Ala Gly Phe Thr Ser 1070 1075 1080 Lys Ile Cys Pro Val Thr Gly Phe Val Asn Gln Leu Tyr Pro Lys 1085 1090 1095 Tyr Glu Ser Val Ser Lys Ser Gln Glu Phe Phe Ser Lys Phe Asp 1100 1105 1110 Lys Ile Cys Tyr Asn Leu Asp Lys Gly Tyr Phe Glu Phe Ser Phe 1115 1120 1125 Asp Tyr Lys Asn Phe Gly Asp Lys Ala Ala Lys Gly Lys Trp Thr 1130 1135 1140 Ile Ala Ser Phe Gly Ser Arg Leu Ile Asn Phe Arg Asn Ser Asp 1145 1150 1155 Lys Asn His Asn Trp Asp Thr Arg Glu Val Tyr Pro Thr Lys Glu 1160 1165 1170 Leu Glu Lys Leu Leu Lys Asp Tyr Ser Ile Glu Tyr Gly His Gly 1175 1180 1185 Glu Cys Ile Lys Ala Ala Ile Cys Gly Glu Ser Asp Lys Lys Phe 1190 1195 1200 Phe Ala Lys Leu Thr Ser Val Leu Asn Thr Ile Leu Gln Met Arg 1205 1210 1215 Asn Ser Lys Thr Gly Thr Glu Leu Asp Tyr Leu Ile Ser Pro Val 1220 1225 1230 Ala Asp Val Asn Gly Asn Phe Phe Asp Ser Arg Gln Ala Pro Lys 1235 1240 1245 Asn Met Pro Gln Asp Ala Asp Ala Asn Gly Ala Tyr His Ile Gly 1250 1255 1260 Leu Lys Gly Leu Met Leu Leu Gly Arg Ile Lys Asn Asn Gln Glu 1265 1270 1275 Gly Lys Lys Leu Asn Leu Val Ile Lys Asn Glu Glu Tyr Phe Glu 1280 1285 1290 Phe Val Gln Asn Arg Asn Asn 1295 1300 <210> 4 <211> 3900 <212> DNA <213> artificial sequence <220> <223> sequence encoding FnCpfI <400> 4 atgagcattt atcaggaatt tgttaataaa tatagcctga gcaaaaccct gcgttttgaa 60 ctgattccgc agggtaaaac cctggaaaat attaaagcac gtggtctgat tctggatgat 120 gaaaaacgtg caaaagatta taaaaaagca aaacagatta ttgataaata tcatcagttt 180 tttattgaag aaattctgag cagcgtttgt attagcgaag atctgctgca gaattatagc 240 gatgtttat ttaaactgaa aaaaagcgat gatgataatc tgcagaaaga tttaaaagc 300 gcaaaagata ccattaaaaa acagattagc gaatatatta aagatagcga aaaatttaaa 360 aatctgttta atcagaatct gattgatgca aaaaaaggtc aggaaagcga tctgattctg 420 tggctgaaac agagcaaaga taatggtatt gaactgttta aagcaaatag cgatattacc 480 gatattgatg aagcactgga aattattaaa agctttaaag gttggaccac ctattttaaa 540 ggttttcatg aaaatcgtaa aaatgttat agcagcaatg atattccgac cagcattatt 600 tatcgtattg ttgatgataa tctgccgaaa tttctggaaa ataaagcaaa atatgaaagc 660 ctgaaagata aagcaccgga agcaattaat tatgaacaga ttaaaaaaga tctggcagaa 720 gaactgacct ttgatattga ttataaaacc agcgaagtta atcagcgtgt ttttagcctg 780 gatgaagttt ttgaaattgc aaattttaat aattatctga atcagagcgg tattaccaaa 840 ttaatacca ttattggtgg taaatttgtt aatggtgaaa ataccaaacg taaaggtatt 900 aatgaatata ttaatctgta tagccagcag attaatgata aaaccctgaa aaatataaa 960 atgagcgttc tgtttaaaca gattctgagc gataccgaaa gcaaaagctt tgttattgat 1020 aaactggaag atgatagcga tgttgttacc accatgcaga gcttttatga acagattgca 1080 gcatttaaaa ccgttgaaga aaaaagcatt aaagaaaccc tgagcctgct gtttgatgat 1140 ctgaaagcac agaaactgga tctgagcaaa atttatttta aaaatgataa aagcctgacc 1200 gatctgagcc agcaggtttt tgatgatt agcgttattg gtaccgcagt tctggaatat 1260 attacccagc agattgcacc gaaaaatctg gataatccg gcaaaaaaga acaggaactg attgcaaaaa aaaccgaaaa agcaaaatat ctgagcctgg aaaccattaa actggcactg gagaattta ataaacatcg tgatattgat aaacagtgtc gttttgaaga aattctggca aattttgcag caattccgat gattttgat gaattgcac agaataaaga taatctggca cagattagca ttaaatatca gaatcagggt aaaaaagatc tgctgcaggc aagcgcagaa gatgatgtta aagcaattaa agatctgctg gatcagacca father gcataaactg aaaatttttc atattagcca gagcgaagat aaagcaaata ttctggataa agatgaacat ttttatctgg tttttgaaga atgttatttt gaactggcaa atattgttcc gctgtataat aaaattcgta attatattac ccagaaccg tatagcgatg aaaaatttaa actgaatttt gaaaatagca ccctggcaaa tggttgggat aaaaataag aaccggataa taccgcaatt ctgtttatta aagatgataa atattatctg ggtgttatga ataaaaaaaa taataaaatt tttgatgata aagcaattaa agaaaataaa ggtgaaggtt ataaaaaat tgtttataaa ctgctgccgg gtgcaaataa aatgctgccg aaagtttttt ttagcgcaaa aagcattaaa 2040 ttttataatc cgagcgaaga tattctgcgt attcgtaatc atagcaccca taccaaaaat 2100 ggtagcccgc agaaaggtta tgaaaaattt gaatttaata ttgaagattg tcgtaaattt 2160 attgattttt ataaacagag cattagcaaa catccggaat ggaaagatttt tggttttcgt 2220 tttagcgata cccagcgtta taatagcatt gatgaatttt atcgtgaagt tgaaaatcag 2280 ggttataaac tgacctttga aaatattagc gaaagctata ttgatagcgt tgttaatcag 2340 ggtaaactgt atctgtttca gatttataat aaagatttta gcgcatatag caaaggtcgt 2400 ccgaatctgc ataccctgta ttggaaagca ctgtttgatg aacgtaatct gcaggatgtt 2460 gtttataaac tgaatggtga agcagaactg tttatcgta aacagagcat tccgaaaaaa 2520 attacccatc cggcaaaaga agcaattgca aataaaaaata aagataatcc gaaaaaagaa 2580 agcgtttttg aatatgatct gattaaagat aaacgtttta ccgaagaataa atttttttt 2640 cattgtccga ttaccattaa tttaaaagc agcggtgcaa ataaatttaa tgatgaaatt 2700 aatctgctgc tgaaagaaaa agcaatgat gttcatattc tgagcattga tcgtggtgaa 2760 cgtcatctgg catattatac cctggttgat ggtaaaggta atattattaa acggatacc 2820 tttaatatta ttggtaatga tcgtatgaa accaattatc atgataact ggcagcaatt 2880 gaaaaagatc gtgatagcgc acgtaagat tggaaaaaa ttaataat taaagaatg 2940 aaagaaggtt atctgagcca ggttgttcat gaaattgca aactggttat tgaatataat 3000 gcaattgttg ttttgaga tctgaatttt ggttttaaac gtggtcgttt taaagttgaa 3060 aaacaggttt atcagaact ggaaaaaatg ctgattgaa aactgaatta tctggtttt 3120 aaagataatg aatttgataa aaccggtggt gttctgcgtg catatcagct gaccgcaccg 3180 tttgaaacct ttaaaaaaat gggtaacag accggtatta tttatgt tccggcaggt 3240 tttaccagca aaatttgtcc ggttaccggt ttgttaatc agctgtatcc gaaatatgaa 3300 agcgttagca aaagccagga atttttgc aaatttgata aaatttgtta taatctggat 3360 aaaggttatt ttgaatttag ctttgattat aaaaatttg gtgataaagc agcaaaggt 3420 aaatggacca ttgcaagctt tggtagccgt ctgattaatt ttcgtaatag cgataaaaat 3480 cataattggg atacccgtga agtttatccg accaaagaac tggaaaaact gctgaaagat 3540 tatagcattg aatatggtca tggtgaatgt attaaagcag caatttgtgg tgaaagcgat 3600 aaaaaattt ttgcaaaact gaccagcgtt ctgaatacca ttctgcagat gcgtaatagc 3660 aaaaccggta ccgaactgga ttatctgatt agcccggttg cagatgttaa tggtaattttt 3720 tttgatagcc gtcaggcacc gaaaaatatg ccgcaggatg cagatgcaaa tggtgcatat 3780 catattggtc tgaaaggtct gatgctgctg ggtcgtatta aaaataatca ggaaggtaaa 3840 aaactgaatc tggttattaa aaatgaagaa tattttgaat ttgttcagaa tcgtaataat 3900 <210> 5 <211> 48502 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 5 gggcggcgac ctcgcgggtt ttcgctattt atgaaaattt tccggtttaa ggcgtttccg 60 ttcttcttcg tcataactta atgtttttat ttaaaatacc ctctgaaaag aaaggaaacg 120 acaggtgctg aaagcgaggc tttttggcct ctgtcgtttc ctttctctgt ttttgtccgt 180 ggaatgaaca atggaagtca acaaaaagca gctggctgac attttcggtg cgagtatccg 240 taccattcag aactggcagg aacagggaat gcccgttctg cgaggcggtg gcaagggtaa 300 tgaggtgctt tatgactctg ccgccgtcat aaaatggtat gccgaaaggg atgctgaaat 360 tgagaacgaa aagctgcgcc gggaggttga agaactgcgg caggccagcg aggcagatct 420 ccagccagga actattgagt acgaacgcca tcgacttacg cgtgcgcagg ccgacgcaca 480 ggaactgaag aatgccagag actccgctga agtggtggaa accgcattct gtactttcgt 540 gctgtcgcgg atcgcaggtg aaattgccag tattctcgac gggctccccc tgtcggtgca 600 gcggcgtttt ccggaactgg aaaaccgaca tgttgatttc ctgaaacggg atatcatcaa 660 agccatgaac aaagcagccg cgctggatga actgataccg gggttgctga gtgaatatat 720 cgaacagtca ggttaacagg ctgcggcatt ttgtccgcgc cgggcttcgc tcactgttca 780 ggccggagcc acagaccgcc gttgaatggg cggatgctaa ttactatctc ccgaaagaat 840 ccgcatacca ggaagggcgc tgggaaacac tgccctttca gcgggccatc atgaatgcga 900 tgggcagcga ctacatccgt gaggtgaatg tggtgaagtc tgcccgtgtc ggttattcca 960 aaatgctgct gggtgtttat gcctacttta tagagcataa gcagcgcaac acccttatct 1020 ggttgccgac ggatggtgat gccgagaact ttatgaaaac ccacgttgag ccgactattc 1080 gtgatattcc gtcgctgctg gcgctggccc cgtggtatgg caaaaagcac cgggataaca 1140 cgctcaccat gaagcgtttc actaatgggc gtggcttctg gtgcctgggc ggtaaagcgg 1200 caaaaaacta ccgtgaaaag tcggtggatg tggcgggtta tgatgaactt gctgcttttg 1260 atgatgatat tgaacaggaa ggctctccga cgttcctggg tgacaagcgt attgaaggct 1320 cggtctggcc aaagtccatc cgtggctcca cgccaaaagt gagaggcacc tgtcagattg 1380 agcgtgcagc cagtgaatcc ccgcatttta tgcgttttca tgttgcctgc ccgcattgcg 1440 gggaggagca gtatcttaaa tttggcgaca aagacacgcc gtttggcctc aaatggacgc 1500 cggatgaccc ctccagcgtg tttatctct gcgagcataa tgcctgcgtc atccgccagc 1560 aggagctgga ctttactgat gcccgttata tctgcgaaaa gaccgggatc tggacccgtg 1620 atggcattct ctggttttcg tcatccggtg aagagattga gccacctgac agtgtgacct 1680 ttcacatctg gacagcgtac agcccgttca ccacctgggt gcagattgtc aaagactgga 1740 tgaaaacgaa aggggatacg ggaaaacgta aaaccttcgt aaacaccacg ctcggtgaga 1800 cgtgggaggc gaaaattggc gaacgtccgg atgctgaagt gatggcagag cggaaagagc 1860 attattcagc gcccgttcct gaccgtgtgg cttacctgac cgccggtatc gactcccagc 1920 tggaccgcta cgaaatgcgc gtatggggat gggggccggg tgaggaaagc tggctgattg 1980 accggcagat tattatgggc cgccacgacg atgaacagac gctgctgcgt gtggatgagg 2040 ccatcaataa aacctatacc cgccggaatg gtgcagaaat gtcgatatcc cgtatctgct 2100 gggatactgg cgggattgac ccgaccattg tgtatgaacg ctcgaaaaaa catgggctgt 2160 tccgggtgat ccccattaaa ggggcatccg tctacggaaa gccggtggcc agcatgccac 2220 gtaagcgaaa caaaaacggg gtttacctta ccgaaatcgg tacggatacc gcgaaagagc 2280 agatttataa ccgcttcaca ctgacgccgg aagggatga accgctccc ggtgccgttc 2340 acttcccgaa taacccggat atttttgatc tgaccgaagc gcagcagctg actgctgaag 2400 agcaggtcga aaatgggtg gatggcagga aaaaatact gtgggacagc aaaagcgac 2460 gcaatgaggc actcgactgc ttcgtttg cgctggcggc gctgcgcatc agtatttccc 2520 gctggcagct ggatctcagt gcgctgctgg cgagcctgca ggaagaggat ggtgcagcaa 2580 ccaacaagaa aacactggca gattacgccc gtgccttac cggaggat gatgacgcg 2640 acaggaagaa cttgccgctg cccgtgcggc actgcatgac ctgatgacag gtaaacgggt 2700 ggcacagta cagaaagacg gacgaagggt ggagtttacg gccactccg tgtctgacct 2760 gaaaaaatat attgcagagc tggagtgca gaccggcatg accagcgac gcaggggacc 2820 tgcaggattt tatgtatgaa aacgcccacc attcccacc ttctggggcc ggacggcatg 2880 acatcgctgc gcgaatatgc cggttatcac ggcggtggca gcggattgg agggcagttg 2940 cggtcgtgga acccaccgag tgaagtgtg gatgcagccc tgttgcccaa ctttacccgt 3000 ggcaatgccc gcgcagacga tctggtacgc aataacggct atgccgccaa cgccatccag 3060 ctgcatcagg atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc 3120 tatctgggca tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg 3180 aaagagtttg ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc 3240 atgatgattc gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc 3300 acctgggata ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag 3360 cgcatcagca acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt 3420 aatgacagcg gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg 3480 ccgcagaaat ggacatggat accccgtgag ttacccggcg ggcgcgcctc gttcattcac 3540 gtttttgaac ccgtggagga cgggcagact cgcggtgcaa atgtgtttta cagcgtgatg 3600 gagcagatga agatgctcga cacgctgcag aacacgcagc tgcagagcgc cattgtgaag 3660 gcgatgtatg ccgccaccat tgagagtgag ctggatacgc agtcagcgat ggattttatt 3720 ctgggcgcga acagtcagga gcagcgggaa aggctgaccg gctggattgg tgaaattgcc 3780 gcgtattacg ccgcagcgcc ggtccggctg ggaggcgcaa aagtaccgca cctgatgccg 3840 ggtgactcac tgaacctgca gacggctcag gatacggata acggctactc cgtgtttgag 3900 cagtcactgc tgcggtatat cgctgccggg ctgggtgtct cgtatgagca gctttcccgg 3960 aattacgccc agatgagcta ctccacggca cgggccagtg cgaacgagtc gtgggcgtac 4020 tttatggggc ggcgaaaatt cgtcgcatcc cgtcaggcga gccagatgtt tctgtgctgg 4080 ctggaagagg ccatcgttcg ccgcgtggtg acgttacctt caaaagcgcg cttcagtttt 4140 caggaagccc gcagtgcctg ggggaactgc gactggatag gctccggtcg tatggccatc 4200 gatggtctga aagaagttca ggaagcggtg atgctgatag aagccggact gagtacctac 4260 gagaaagagt gcgcaaaacg cggtgacgac tatcaggaaa tttttgccca gcaggtccgt 4320 gaaacgatgg agcgccgtgc agccggtctt aaaccgcccg cctgggcggc tgcagcattt 4380 gaatccgggc tgcgacaatc aacagaggag gagaagagtg acagcagagc tgcgtaatct 4440 cccgcatatt gccagcatgg cctttaatga gccgctgatg cttgaacccg cctatgcgcg 4500 ggttttcttt tgtgcgcttg caggccagct tgggatcagc agcctgacgg atgcggtgtc 4560 cggcgacagc ctgactgccc aggaggcact cgcgacgctg gcattatccg gtgatgatga 4620 cggaccacga caggcccgca gttatcaggt catgaacggc atcgccgtgc tgccggtgtc 4680 cggcacgctg gtcagccgga cgcgggcgct gcagccgtac tcggggatga ccggttacaa 4740 cggcattatc gcccgtctgc aacaggctgc cagcgatccg atggtggacg gcattctgct 4800 cgatatggac acgcccggcg ggatggtggc gggggcattt gactgcgctg acatcatcgc 4860 ccgtgtgcgt gacataaaac cggtatgggc gcttgccaac gacatgaact gcagtgcagg 4920 tcagttgctt gccagtgccg cctcccggcg tctggtcacg cagaccgccc ggacaggctc 4980 catcggcgtc atgatggctc acagtaatta cggtgctgcg ctggagaaac agggtgtgga 5040 aatcacgctg atttacagcg gcagccataa ggtggatggc aacccctaca gccatcttcc 5100 ggatgacgtc cgggagacac tgcagtcccg gatggacgca acccgccaga tgtttgcgca 5160 gaaggtgtcg gcatataccg gcctgtccgt gcaggttgtg ctggataccg aggctgcagt 5220 gtacagcggt caggaggcca ttgatgccgg actggctgat gaacttgtta acagcaccga 5280 tgcgatcacc gtcatgcgtg atgcactgga tgcacgtaaa tcccgtctct caggagggcg 5340 aatgaccaaa gagactcaat caacaactgt ttcagccact gcttcgcagg ctgacgttac 5400 tgacgtggtg ccagcgacgg agggcgagaa cgccagcgcg gcgcagccgg acgtgaacgc 5460 gcagatcacc gcagcggttg cggcagaaaa cagccgcatt atggggatcc tcaactgtga 5520 ggaggctcac ggacgcgaag aacaggcacg cgtgctggca gaaacccccg gtatgaccgt 5580 gaaaacggcc cgccgcattc tggccgcagc accacagagt gcacaggcgc gcagtgacac 5640 tgcgctggat cgtctgatgc agggggcacc ggcaccgctg gctgcaggta acccggcatc 5700 tgatgccgtt aacgatttgc tgaacacacc agtgtaaggg atgtttatga cgagcaaaga 5760 aacctttacc cattaccagc cgcagggcaa cagtgacccg gctcataccg caaccgcgcc 5820 cggcggattg agtgcgaaag cgcctgcaat gaccccgctg atgctggaca cctccagccg 5880 taagctggtt gcgtgggatg gcaccaccga cggtgctgcc gttggcattc ttgcggttgc 5940 tgctgaccag accagcacca cgctgacgtt ctacaagtcc ggcacgttcc gttatgagga 6000 tgtgctctgg ccggaggctg ccagcgacga gacgaaaaaa cggaccgcgt ttgccggaac 6060 ggcaatcagc atcgtttaac tttacccttc atcactaaag gccgcctgtg cggctttttt 6120 tacgggattt ttttatgtcg atgtacacaa ccgcccaact gctggcggca aatgagcaga 6180 aatttaagtt tgatccgctg tttctgcgtc tctttttccg tgagagctat cccttcacca 6240 cggagaaagt ctatctctca caaattccgg gactggtaaa catggcgctg tacgtttcgc 6300 cgattgtttc cggtgaggtt atccgttccc gtggcggctc cacctctgaa tttacgccgg 6360 gatatgtcaa gccgaagcat gaagtgaatc cgcagatgac cctgcgtcgc ctgccggatg 6420 aagatccgca gaatctggcg gacccggctt accgccgccg tcgcatcatc atgcagaaca 6480 tgcgtgacga agagctggcc attgctcagg tcgaagagat gcaggcagtt tctgccgtgc 6540 ttaagggcaa atacaccatg accggtgaag ccttcgatcc ggttgaggtg gatatgggcc 6600 gcagtgagga gaataacatc acgcagtccg gcggcacgga gtggagcaag cgtgacaagt 6660 ccacgtatga cccgaccgac gatatcgaag cctacgcgct gaacgccagc ggtgtggtga 6720 atatcatcgt gttcgatccg aaaggctggg cgctgttccg ttccttcaaa gccgtcaagg 6780 agaagctgga tacccgtcgt ggctctaatt ccgagctgga gacagcggtg aaagacctgg 6840 gcaaagcggt gtcctataag gggatgtatg gcgatgtggc catcgtcgtg tattccggac 6900 agtacgtgga aaacggcgtc aaaaagaact tcctgccgga caacacgatg gtgctgggga 6960 acactcaggc acgcggtctg cgcacctatg gctgcattca ggatgcggac gcacagcgcg 7020 aaggcattaa cgcctctgcc cgttacccga aaaactgggt gaccaccggc gatccggcgc 7080 gtgagttcac catgattcag tcagcaccgc tgatgctgct ggctgaccct gatgagttcg 7140 tgtccgtaca actggcgtaa tcatggccct tcggggccat tgtttctctg tggaggagtc 7200 catgacgaaa gatgaactga ttgcccgtct ccgctcgctg ggtgaacaac tgaaccgtga 7260 tgtcagcctg acggggacga aagaagaact ggcgctccgt gtggcagagc tgaaagagga 7320 gcttgatgac acggatgaaa ctgccggtca ggacacccct ctcagccggg aaaatgtgct 7380 gaccggacat gaaaatgagg tgggatcagc gcagccggat accgtgattc tggatacgtc 7440 tgaactggtc acggtcgtgg cactggtgaa gctgcatact gatgcacttc acgccacgcg 7500 ggatgaacct gtggcatttg tgctgccggg aacggcgttt cgtgtctctg ccggtgtggc 7560 agccgaaatg acagagcgcg gcctggccag aatgcaataa cgggaggcgc tgtggctgat 7620 ttcgataacc tgttcgatgc tgccattgcc cgcgccgatg aaacgatacg cgggtacatg 7680 ggaacgtcag ccaccattac atccggtgag cagtcaggtg cggtgatacg tggtgttttt 7740 gatgaccctg aaaatatcag ctatgccgga cagggcgtgc gcgttgaagg ctccagcccg 7800 tccctgtttg tccggactga tgaggtgcgg cagctgcggc gtggagacac gctgaccatc 7860 ggtgaggaaa atttctgggt agatcgggtt tcgccggatg atggcggaag ttgtcatctc 7920 tggcttggac ggggcgtacc gcctgccgtt aaccgtcgcc gctgaaaggg ggatgtatgg 7980 ccataaaagg tcttgagcag gccgttgaaa acctcagccg tatcagcaaa acggcggtgc 8040 ctggtgccgc cgcaatggcc attaaccgcg ttgcttcatc cgcgatatcg cagtcggcgt 8100 cacaggttgc ccgtgagaca aaggtacgcc ggaaactggt aaaggaaagg gccaggctga 8160 aaagggccac ggtcaaaaat ccgcaggcca gaatcaaagt taaccggggg gatttgcccg 8220 taatcaagct gggtaatgcg cgggttgtcc tttcgcgccg caggcgtcgt aaaaaggggc 8280 agcgttcatc cctgaaaggt ggcggcagcg tgcttgtggt gggtaaccgt cgtattcccg 8340 gcgcgtttat tcagcaactg aaaaatggcc ggtggcatgt catgcagcgt gtggctggga 8400 aaaaccgtta ccccattgat gtggtgaaaa tcccgatggc ggtgccgctg accacggcgt 8460 ttaaacaaaa tattgagcgg atacggcgtg aacgtcttcc gaaagagctg ggctatgcgc 8520 tgcagcatca actgaggatg gtaataaagc gatgaaacat actgaactcc gtgcagccgt 8580 actggatgca ctggagaagc atgacaccgg ggcgacgttt tttgatggtc gccccgctgt 8640 ttttgatgag gcggattttc cggcagttgc cgtttatctc accggcgctg aatacacggg 8700 cgaagagctg gacagcgata cctggcaggc ggagctgcat atcgaagttt tcctgcctgc 8760 tcaggtgccg gattcagagc tggatgcgtg gatggagtcc cggatttatc cggtgatgag 8820 cgatatcccg gcactgtcag atttgatcac cagtatggtg gccagcggct atgactaccg 8880 gcgcgacgat gatgcgggct tgtggagttc agccgatctg acttatgtca ttacctatga 8940 aatgtgagga cgctatgcct gtaccaaatc ctacaatgcc ggtgaaaggt gccgggacca 9000 ccctgtgggt ttataagggg agcggtgacc cttacgcgaa tccgctttca gacgttgact 9060 ggtcgcgtct ggcaaaagtt aaagacctga cgcccggcga actgaccgct gagtcctatg 9120 acgacagcta tctcgatgat gaagatgcag actggactgc gaccgggcag gggcagaaat 9180 ctgccggaga taccagcttc acgctggcgt ggatgcccgg agagcagggg cagcaggcgc 9240 tgctggcgtg gtttaatgaa ggcgataccc gtgcctataa aatccgcttc ccgaacggca 9300 cggtcgatgt gttccgtggc tgggtcagca gtatcggtaa ggcggtgacg gcgaaggaag 9360 tgatcacccg cacggtgaaa gtcaccaatg tgggacgtcc gtcgatggca gaagatcgca 9420 gcacggtaac agcggcaacc ggcatgaccg tgacgcctgc cagcacctcg gtggtgaaag 9480 ggcagagcac cacgctgacc gtggccttcc agccggaggg cgtaaccgac aagagctttc 9540 gtgcggtgtc tgcggataaa acaaaagcca ccgtgtcggt cagtggtatg accatcaccg 9600 tgaacggcgt tgctgcaggc aaggtcaaca ttccggttgt atccggtaat ggtgagtttg 9660 ctgcggttgc agaaattacc gtcaccgcca gttaatccgg agagtcagcg atgttcctga 9720 aaaccgaatc atttgaacat aacggtgtga ccgtcacgct ttctgaactg tcagccctgc 9780 agcgcattga gcatctcgcc ctgatgaaac ggcaggcaga acaggcggag tcagacagca 9840 accggaagtt tactgtggaa gacgccatca gaaccggcgc gtttctggtg gcgatgtccc 9900 tgtggcataa ccatccgcag aagacgcaga tgccgtccat gaatgaagcc gttaaacaga 9960 ttgagcagga agtgcttacc acctggccca cggaggcaat ttctcatgct gaaaacgtgg 10020 tgtaccggct gtctggtatg tatgagtttg tggtgaataa tgcccctgaa cagacagagg 10080 acgccgggcc cgcagagcct gtttctgcgg gaaagtgttc gacggtgagc tgagttttgc 10140 cctgaaactg gcgcgtgaga tggggcgacc cgactggcgt gccatgcttg ccgggatgtc 10200 atccacggag tatgccgact ggcaccgctt ttacagtacc cattattttc atgatgttct 10260 gctggatatg cacttttccg ggctgacgta caccgtgctc agcctgtttt tcagcgatcc 10320 ggatatgcat ccgctggatt tcagtctgct gaaccggcgc gaggctgacg aagagcctga 10380 agatgatgtg ctgatgcaga aagcggcagg gcttgccgga ggtgtccgct ttggcccgga 10440 cgggaatgaa gttatccccg cttccccgga tgtggcggac atgacggagg atgacgtaat 10500 gctgatgaca gtatcagaag ggatcgcagg aggagtccgg tatggctgaa ccggtaggcg 10560 atctggtcgt tgatttgagt ctggatgcgg ccagatttga cgagcagatg gccagagtca 10620 ggcgtcattt ttctggtacg gaaagtgatg cgaaaaaaac agcggcagtc gttgaacagt 10680 cgctgagccg acaggcgctg gctgcacaga aagcggggat ttccgtcggg cagtataaag 10740 ccgccatgcg tatgctgcct gcacagttca ccgacgtggc cacgcagctt gcaggcgggc 10800 aaagtccgtg gctgatcctg ctgcaacagg gggggcaggt gaaggactcc ttcggcggga 10860 tgatccccat gttcaggggg cttgccggtg cgatcaccct gccgatggtg ggggccacct 10920 cgctggcggt ggcgaccggt gcgctggcgt atgcctggta tcagggcaac tcaaccctgt 10980. ccgatttcaa caaaacgctg gtcctttccg gcaatcaggc gggactgacg gcagatcgta tgctggtcct gtccagagcc gggcaggcgg cagggctgac gtttaaccag accagcgagt cactcagcgc actggttaag gcgggggtaa gcggtgaggc tcagattgcg tccatcagcc aggtgtggc gcgtttctcc tctgcatccg gcgtggaggt ggacaaggtc gctgaagcct tcgggaagct gaccacagac ccgacgtcgg ggctgacggc gatggctcgc cagttccata acgtgtcggc ggagcagatt gcgtatgttg ctcagttgca gcgttccggc gatgaagccg gggcattgca ggcggcgac gaggccgca cgaaagggtt tgatgaccag acccgccgcc tgaagagaa catgggcacg ctggagacct gggcagacag gactgcgcgg gcattcaaat ccatgtggga tgcggtgctg gatattggtc gtcctgatac cgcgcaggag atgctgatta 11520 aggcagaggc tgcgtataag aaagcagacg acatctggaa tctgcgcaag gatgattatt ttgttaacga tgaagcgcgg gcgcgttact gggatgatcg tgaaaaggcc cgtcttgcgc 11640 ttgaagccgc ccgaaagaag gctgagcagc agactcaaca ggacaaaaat gcgcagcagc 11700 agagcgatac cgaagcgtca cggctgaaat ataccgaaga ggcgcagaag gcttacgaac 11760 ggctgcagac gccgctggag aaatataccg cccgtcagga agaactgaac aaggcactga 11820 aagacgggaa aatcctgcag gcggattaca acacgctgat ggcggcggcg aaaaaggatt 11880 atgaagcgac gctgaaaaag ccgaaacagt ccagcgtgaa ggtgtctgcg ggcgatcgtc 11940 aggaagacag tgctcatgct gccctgctga cgcttcaggc agaactccgg acgctggaga 12000 agcatgccgg agcaaatgag aaaatcagcc agcagcgccg ggatttgtgg aaggcggaga 12060 gtcagttcgc ggtactggag gaggcggcgc aacgtcgcca gctgtctgca caggagaaat 12120 ccctgctggc gcataaagat gagacgctgg agtacaaacg ccagctggct gcacttggcg 12180 acaaggttac gtatcaggag cgcctgaacg cgctggcgca gcaggcggat aaattcgcac 12240 agcagcaacg ggcaaaacgg gccgccattg atgcgaaaag ccgggggctg actgaccggc 12300 aggcagaacg ggaagccacg gaacagcgcc tgaaggaaca gtatggcgat aatccgctgg 12360 cgctgaataa cgtcatgtca gagcagaaaa agacctgggc ggctgaagac cagcttcgcg 12420 ggaactggat ggcaggcctg aagtccggct ggagtgagtg ggaagagagc gccacggaca 12480 gtatgtcgca ggtaaaaagt gcagccacgc agacctttga tggtattgca cagaatatgg 12540 cggcgatgct gaccggcagt gagcagaact ggcgcagctt cacccgttcc gtgctgtcca 12600 tgatgacaga aattctgctt aagcaggcaa tggtggggat tgtcgggagt atcggcagcg 12660 ccattggcgg ggctgttggt ggcggcgcat ccgcgtcagg cggtacagcc attcaggccg 12720 ctgcggcgaa attccatttt gcaaccggag gatttacggg aaccggcggc aaatatgagc 12780 cagcggggat tgttcaccgt ggtgagtttg tcttcacgaa ggaggcaacc agccggattg 12840 gcgtggggaa tctttaccgg ctgatgcgcg gctatgccac cggcggttat gtcggtacac 12900 cgggcagcat ggcagacagc cggtcgcagg cgtccgggac gtttgagcag aataaccatg 12960 tggtgattaa caacgacggc acgaacgggc agataggtcc ggctgctctg aaggcggtgt 13020 atgacatggc ccgcaagggt gcccgtgatg aaattcagac acagatgcgt gatggtggcc 13080 tgttctccgg aggtggacga tgaagacctt ccgctggaaa gtgaaacccg gtatggatgt 13140 ggcttcggtc ccttctgtaa gaaaggtgcg ctttggtgat ggctattctc agcgagcgcc 13200 tgccgggctg aatgccaacc tgaaaacgta cagcgtgacg ctttctgtcc cccgtgagga 13260 ggccacggta ctggagtcgt ttctggaaga gcacgggggc tggaaatcct ttctgtggac 13320 gccgccttat gagtggcggc agataaaggt gacctgcgca aaatggtcgt cgcgggtcag 13380 tatgctgcgt gttgagttca gcgcagagtt tgaacaggtg gtgaactgat gcaggatatc 13440 cggcaggaaa cactgaatga atgcacccgt gcggagcagt cggccagcgt ggtgctctgg 13500 gaaatcgacc tgacagaggt cggtggagaa cgttattttt tctgtaatga gcagaacgaa 13560 aaaggtgagc cggtcacctg gcaggggcga cagtatcagc cgtatcccat tcaggggagc 13620 ggttttgaac tgaatggcaa aggcaccagt acgcgcccca cgctgacggt ttctaacctg 13680 tacggtatgg tcaccgggat ggcggaagat atgcagagtc tggtcggcgg aacggtggtc 13740 cggcgtaagg tttacgcccg ttttctggat gcggtgaact tcgtcaacgg aaacagttac 13800 gccgatccgg agcaggaggt gatcagccgc tggcgcattg agcagtgcag cgaactgagc 13860 gcggtgagtg cctcctttgt actgtccacg ccgacggaaa cggatggcgc tgtttttccg 13920 ggacgtatca tgctggccaa cacctgcacc tggacctatc gcggtgacga gtgcggttat 13980 agcggtccgg ctgtcgcgga tgaatatgac cagccaacgt ccgatatcac gaaggataaa 14040 tgcagcaaat gcctgagcgg ttgtaagttc cgcaataacg tcggcaactt tggcggcttc 14100 ctttccatta acaaactttc gcagtaaatc ccatgacaca gacagaatca gcgattctgg 14160 cgcacgcccg gcgatgtgcg ccagcggagt cgtgcggctt cgtggtaagc acgccggagg 14220 gggaaagata tttcccctgc gtgaatatct ccggtgagcc ggaggctatt tccgtatgtc 14280 gccggaagac tggctgcagg cagaaatgca gggtgagatt gtggcgctgg tccacagcca 14340 ccccggtggt ctgccctggc tgagtgaggc cgaccggcgg ctgcaggtgc agagtgattt 14400 gccgtggtgg ctggtctgcc gggggacgat tcataagttc cgctgtgtgc cgcatctcac 14460 cgggcggcgc tttgagcacg gtgtgacgga ctgttacaca ctgttccggg atgcttatca 14520 tctggcgggg attgagatgc cggactttca tcgtgaggat gactggtggc gtaacggcca 14580 gaatctctat ctggataatc tggaggcgac ggggctgtat caggtgccgt tgtcagcggc 14640 acagccgggc gatgtgctgc tgtgctgttt tggttcatca gtgccgaatc acgccgcaat 14700 ttactgcggc gacggcgagc tgctgcacca tattcctgaa caactgagca aacgagagag 14760 gtacaccgac aaatggcagc gacgcacaca ctccctctgg cgtcaccggg catggcgcgc 14820 atctgccttt acggggattt acaacgattt ggtcgccgca tcgaccttcg tgtgaaaacg 14880 ggggctgaag ccatccgggc actggccaca cagctcccgg cgtttcgtca gaaactgagc 14940 gacggctggt atcaggtacg gattgccggg cgggacgtca gcacgtccgg gttaacggcg 15000 cagttacatg agactctgcc tgatggcgct gtaattcata ttgttcccag agtcgccggg 15060 gccaagtcag gtggcgtatt ccagattgtc ctgggggctg ccgccattgc cggatcattc 15120 tttaccgccg gagccaccct tgcagcatgg ggggcagcca ttggggccgg tggtatgacc 15180 ggcatcctgt tttctctcgg tgccagtatg gtgctcggtg gtgtggcgca gatgctggca 15240 ccgaaagcca gaactccccg tatacagaca acggataacg gtaagcagaa cacctatttc 15300 tcctcactgg ataacatggt tgcccagggc aatgttctgc ctgttctgta cggggaaatg 15360 cgcgtggggt cacgcgtggt ttctcaggag atcagcacgg cagacgaagg ggacggtggt 15420 caggttgtgg tgattggtcg ctgatgcaaa atgttttatg tgaaaccgcc tgcgggcggt 15480 tttgtcattt atggagcgtg aggaatgggt aaaggaagca gtaaggggca taccccgcgc 15540 gaagcgaagg acaacctgaa gtccacgcag ttgctgagtg tgatcgatgc catcagcgaa 15600 gggccgattg aaggtccggt ggatggctta aaaagcgtgc tgctgaacag tacgccggtg 15660 ctggacactg aggggaatac caadatacc ggtgtcacgg tggtgttccg ggctggtgag 15720 caggagcaga ctccgccgga gggatttgaa tcctccggct ccgagacggt gctgggtacg gagtgaat atgacacgcc gatcacccgc accattacgt ctgcaaacat cgaccgtctg cgctttacct tcggtgtaca ggcactggtg gaaaccacct caaagggtga caggaatccg tcggaagtcc gcctgctggt tcagatacaa cgtaacggtg gctgggtgac ggaaaaagac atcaccatta agggcaaaac cacctcgcag tatctggcct cggtggtgat gggtaacctg ccgccgcgcc cgtttaatat ccggatgcgc aggatgacgc cggacagcac cacagaccag ctgcagaaca aaacgctctg gtcgtcatac actgaatca tcgatgtgaa acagtgctac ccgaacacgg cactggtcgg cgtgcaggtg gactcggagc agttcggcag ccagcaggtg agccgtaatt atcatctgcg cggggcgtatt ctgcaggtgc cgtcgaacta taacccgcag acgcggcaat acagcggtat ctgggacgga acgtttaaac cggcatacag caacaacatg gcctggtgtc tgtgggatat gctgacccat ccgcgctacg gcatgggga acgtcttggt 16380 gcggcggatg tggataaatg ggcgctgtat gtcatcggcc agtactgcga ccagtcagtg ccggacggct ttggcggcac ggagccgcgc atcacctgta atgcgtacct gaccacacag 16500. cgtaaggcgt gggatgtgct cagcgatttc tgctcggcga tgcgctgtat gccggtatgg 16560 aacgggcaga cgctgacgtt cgtgcaggac cgaccgtcgg ataagacgtg gacctataac cgcagtaatg tggtgatgcc ggatgatggc gcgccgttcc gctacagctt cagcgccctg 16680 aaggaccgcc ataatgccgt tgaggtgac tggattgacc cgaacaacgg ctgggagacg gcgacagagc ttgttgaaga tacgcaggcc attgcccgtt acggtcgtaa tgttacgaag atggatgcct ttggctgtac cagccggggg caggcacacc gcgccgggct gtggctgatt 16860 aaaacagaac tgctggaac gcagaccgtg gatttcagcg tcggcgcaga agggcttcgc catgtaccgg gcgatgttat tgaaatctgc gatgatgact atgccggtat cagcaccggt 16980 ggtcgtgtgc tggcggtgaa cagccagacc cggacgctga cgctcgaccg tgaaatcacg ctgccatcct ccggtaccgc gctgataagc ctggttgacg gaagtggcaa tccggtcagc 17100. gtggaggttc agtccgtcac cgacggcgtg aaggtaaaag tgagccgtgt tcctgacggt 17160 gttgctgaat acagcgtatg ggagctgaag ctgccgacgc tgcgccagcg actgttccgc 17220 tgcgtgagta tccgtgagaa cgacgacggc acgtatgcca tcaccgccgt gcagcatgtg 17280 ccggaaaaag aggccatcgt ggataacggg gcgcactttg acggcgaaca gagtggcacg 17340 gtgaatggtg tcacgccgcc agcggtgcag cacctgaccg cagaagtcac tgcagacagc 17400 ggggaatatc aggtgctggc gcgatgggac acaccgaagg tggtgaaggg cgtgagtttc 17460 ctgctccgtc tgaccgtaac agcggacgac ggcagtgagc ggctggtcag cacggcccgg 17520 acgacggaaa ccacataccg cttcacgcaa ctggcgctgg ggaactacag gctgacagtc 17580 cgggcggtaa atgcgtgggg gcagcagggc gatccggcgt cggtatcgtt ccggattgcc 17640 gcaccggcag caccgtcgag gattgagctg acgccgggct attttcagat aaccgccacg 17700 ccgcatcttg ccgtttatga cccgacggta cagtttgagt tctggttctc ggaaaagcag 17760 attgcggata tcagacaggt tgaaaccagc acgcgttatc ttggtacggc gctgtactgg 17820 atagccgcca gtatcaatat caaaccgggc catgattatt acttttatat ccgcagtgtg 17880 aacaccgttg gcaaatcggc attcgtggag gccgtcggtc gggcgagcga tgatgcggaa 17940 ggttacctgg attttttcaa aggcaagata accgaatccc atctcggcaa ggagctgctg 18000 gaaaaagtcg agctgacgga ggataacgcc agcagactgg aggagttttc gaaagagtgg 18060 aggatgcca gtgataagtg gaatgccatg tgggctgtca aaattgagca gaccaaagac 18120 ggcaaacatt atgtcgcggg tattggcctc agcatggagg acacggagga aggcaaactg 18180 agccagtttc tggttgccgc caatcgtatc gcatttattg acccggcaaa cgggaatgaa 18240 acgccgatgt ttgtggcgca gggcaaccag atattcatga acgacgtgtt cctgaagcgc 18300 ctgacggccc ccaccattac cagcggcggc aatcctccgg ccttttccct gacaccggac 18360 ggaaagctga ccgctaaaaa tgcggatatc agtggcagtg tgaatgcgaa ctccgggacg 18420 ctcagtaatg tgacgatagc tgaaaactgt acgataaacg gtacgctgag ggcggaaaaa 18480 atcgtcgggg acattgtaaa ggcggcgagc gcggcttttc cgcgccagcg tgaaagcagt 18540 gtggactggc cgtcaggtac ccgtactgtc accgtgaccg atgaccatcc ttttgatcgc 18600 cagatagtgg tgcttccgct gacgtttcgc ggaagtaagc gtactgtcag cggcaggaca 18660 acgtattcga tgtgttatct gaaagtactg atgaacggtg cggtgattta tgatggcgcg 18720 gcgaacgagg cggtacaggt gttctcccgt attgttgaca tgccagcggg tcggggaaac 18780 gtgatcctga cgttcacgct tacgtccaca cggcattcgg cagatattcc gccgtatacg 18840 tttgccagcg atgtgcaggt tatggtgatt aagaaacagg cgctgggcat cagcgtggtc 18900 tgagtgtgtt acagaggttc gtccgggaac gggcgtttta ttataaaaca gtgagaggtg 18960 aacgatgcgt aatgtgtgta ttgccgttgc tgtctttgcc gcacttgcgg tgacagtcac 19020 tccggcccgt gcggaaggtg gacatggtac gtttacggtg ggctattttc aagtgaaacc 19080 gggtacattg ccgtcgttgt cgggcgggga taccggtgtg agtcatctga aagggattaa 19140 cgtgaagtac cgttatgagc tgacggacag tgtgggggtg atggcttccc tggggttcgc 19200 cgcgtcgaaa aagagcagca cagtgatgac cggggaggat acgtttcact atgagagcct 19260 gcgtggacgt tatgtgagcg tgatggccgg accggtttta caaatcagta agcaggtcag 19320 tgcgtacgcc atggccggag tggctcacag tcggtggtcc ggcagtacaa tggattaccg 19380 taagacggaa atcactcccg ggtatatgaa agagacgacc actgccaggg acgaaagtgc 19440 aatgcggcat acctcagtgg cgtggagtgc aggtatacag attaatccgg cagcgtccgt 19500 cgttgttgat attgcttatg aaggctccgg cagtggcgac tggcgtactg acggattcat 19560 cgttggggtc ggttataaat tctgattagc caggtaacac agtgttatga cagcccgccg 19620 gaaccggtgg gcttttttgt ggggtgaata tggcagtaaa gatttcagga gtcctgaaag 19680 acggcacagg aaaaccggta cagaactgca ccattcagct gaaagccaga cgtaacagca 19740 ccacggtggt ggtgaacacg gtgggctcag agaatccgga tgaagccggg cgttacagca 19800 tggatgtgga gtacggtcag tacagtgtca tcctgcaggt tgacggtttt ccaccatcgc 19860 acgccgggac catcaccgtg tatgaagatt caaaccggg gacgctgaat gattttctct 19920 gtgccatgac ggaggatgat gcccggccgg aggtgctgcg tcgtcttgaa ctgatggtgg 19980 aagaggtggc gcgtaacgcg tccgtggtgg cacagagtac ggcagacgcg aagaaatcag 20040 ccggcgatgc cagtgcatca gctgctcagg tcgcggccct tgtgactgat gcaactgact 20100 cagcacgcgc cgccagcacg tccgccggac aggctgcatc gtcagctcag gaagcgtcct 20160 ccggggcgaga agcggcatca gcaaaggcca ctgaagcgga aaaaagtgcc gcagccgcag 20220 agtcctcaaa aaacgcggcg gccaccagtg ccggtgcggc gaaacgtca gaaacgaatg 20280 ctgcagcgtc aaacaatca gccgccacgt ctgcctccac cgcggccacg aaagcgtcag 20340 aggccgccac ttcagcacga gatgcggtgg cctcaaaaga ggcagcaaaa tcatcagaaa 20400 cgaacgcatc atcaagtgcc ggtcgtgcag cttctcggc aacggcggca gaaaattctg 20460 ccagggcggc aaaaacgtcc gagacgaatg ccaggtcatc tgaaacagca gccgaacgga 20520 gcgcctctgc cgcggcagac gcaaaaacag cggcggcggg gagtgcgtca acggcatcca 20580 cgaaggcgac agaggctgcg ggaagtgcgg tatcagcatc gcagagcaaa agtgcggcag 20640 aagcggcggc aatacgtgca aaaaattcgg caaaacgtgc agaagatata gcttcagctg 20700 tcgcgcttga ggatgcggac acaacgagaa aggggatagt gcagctcagc agtgcaacca 20760 acagcacgtc tgaaacgctt gctgcaacgc caaaggcggt taaggtggta atggatgaaa 20820 cgaacagaaa agcccactgg acagtccggc actgaccgga acgccaacag caccaaccgc 20880 gctcagggga acaaacaata cccagattgc gaacaccgct tttgtactgg ccgcgattgc 20940 agatgttatc gacgcgtcac ctgacgcact gaatacgctg aatgaactgg ccgcagcgct 21000 cgggaatgat ccagattttg ctaccaccat gactaacgcg cttgcgggta aacaaccgaa 21060 gaatgcgaca ctgacggcgc tggcagggct ttccacggcg aaaaataaat taccgtattt 21120 tgcggaaaat gatgccgcca gcctgactga actgactcag gttggcaggg atattctggc 21180 aaaaaattcc gttgcagatg ttcttgaata ccttggggcc ggtgagaatt cggccttttcc 21240 ggcaggtgcg ccgatcccgt ggccatcaga tatcgttccg tctggctacg tcctgatgca 21300 ggggcaggcg tttgacaaat cagcctaccc aaaacttgct gtcgcgtatc catcgggtgt 21360 gcttcctgat atgcgaggct ggacaatcaa ggggaaaccc gccagcggtc gtgctgtatt 21420 gtctcaggaa caggatggaa ttaagtcgca cacccacagt gccagtgcat ccggtacgga 21480 tttggggacg aaaaccacat cgtcgtttga ttacgggacg aaaacaacag gcagtttcga 21540 ttacggcacc aaatcgacga ataacacggg ggctcatgct cacagtctga gcggttcaac 21600 aggggccgcg ggtgctcatg cccacacaag tggtttaagg atgaacagtt ctggctggag 21660 tcagtatgga acagcaacca ttacaggaag tttatccaca gttaaaggaa ccagcacaca 21720 gggtattgct tatttatcga aaacggacag tcagggcagc cacagtcact cattgtccgg 21780 tacagccgtg agtgccggtg cacatgcgca tacagttggt attggtgcgc accagcatcc 21840 ggttgttatc ggtgctcatg cccattcttt cagtattggt tcacacggac acaccatcac 21900 cgttaacgct gcgggtaacg cggaaaacac cgtcaaaaac attgcattta actatattgt 21960 gaggcttgca taatggcatt cagaatgagt gaacaaccac ggaccataaa aattataat 22020 ctgctggccg gaactaatga atttattggt gaaggtgacg catatattcc gcctcatacc 22080 ggtctgcctg caaacagtac cgatattgca ccgccagata ttccggctgg ctttgtggct 22140 gttttcaaca gtgatgaggc atcgtggcat ctcgttgaag accatcgggg taaaaccgtc 22200 tatgacgtgg cttccggcga cgcgttattt atttctgaac tcggtccgtt accggaaaat 22260 tttacctggt tatcgccggg aggggaatat cagaagtgga acggcacagc ctgggtgaag 22320 gatacggaag cagaaaaact gttccggatc cgggaggcgg aagaaacaaa aaaaagcctg 22380 atgcaggtag ccagtgagca tattgcgccg cttcaggatg ctgcagatct ggaaattgca 22440 acgaaggaag aaacctcgtt gctggaagcc tggaagaagt atcgggtgtt gctgaaccgt 22500 gttgatacat caactgcacc tgatattgag tggcctgctg tccctgttat ggagtaatcg 22560 ttttgtgata tgccgcagaa acgttgtatg aaataacgtt ctgcggttag ttagtatatt 22620 gtaaagctga gtattggttt atttggcgat tattatcttc aggagaataa tggaagttct 22680 atgactcaat tgttcatagt gttacatca ccgccaattg cttttaagac tgaacgcatg 22740 aaattggtt ttcgtcatg ttttgagtct gctgttgata tttctaaagt cggttttttt 22800 tcttcgtttt ctctaactat tttccatgaa atacattttt gattattatt tgaatcaatt 22860 ccaattacct gaagtctttc atctataatt ggcattgtat gtattggttt attggagtag 22920 atgcttgctt ttctgagcca tagctctgat atccaaatga agccataggc atttgttatt 22980 ttggctctgt cagctgcata acgccaaaaa atatatttat ctgcttgatc ttcaaatgtt 23040 gtattgatta aatcaattgg atggaattgt ttatcataaa aaattaatgt ttgaatgtga 23100 taaccgtcct ttaaaaaagt cgtttctgca agcttggctg tatagtcaac taactcttct 23160 gtcgaagtga tatttttagg cttatctacc agttttagac gctctttaat atcttcagga 23220 attattttat tgtcatattg tatcatgcta aatgacaatt tgcttatgga gtaatctttt 23280 aattttaaat aagttattct cctggcttca tcaaataaag agtcgaatga tgttggcgaa 23340 atcacatcgt cacccattgg attgtttatt tgtatgccaa gagagttaca gcagttatac 23400 attctgccat agattatagc taaggcatgt aataattcgt aatcttttag cgtattagcg 23460 acccatcgtc tttctgattt aataatagat gattcagtta aatatgaagg taatttcttt 23520 tgtgcaagtc tgactaactt ttttatacca atgtttaaca tactttcatt tgtaataaac 23580 tcaatgtcat tttcttcaat gtaagatgaa ataagagtag cctttgcctc gctatacatt 23640 tctaaatcgc cttgtttttc tatcgtattg cgagaatttt tagcccaagc cattaatgga 23700 tcatttttcc atttttcaat aacattattg ttataccaaa tgtcatatcc tataatctgg 23760 tttttgtttt tttgaataat aaatgttact gttcttgcgg tttggaggaa ttgattcaaa 23820 ttcaagcgaa ataattcagg gtcaaaatat gtatcaatgc agcatttgag caagtgcgat 23880 aaatctttaa gtcttctttc ccatggtttt ttagtcataa aactctccat tttgataggt 23940 tgcatgctag atgctgatat atttagagg tgataaaatt aactgcttaa ctgtcaatgt 24000 aatacaagtt gtttgatctt tgcaatgatt cttatcagaa accatatagt aaattagtta 24060 cacaggaaat ttttaatatt attattatca ttcattatgt attaaaatta gagttgtggc 24120 ttggctctgc taacacgttg ctcataggag atatggtaga gccgcagaca cgtcgtatgc 24180 aggaacgtgc tgcggctggc tggtgaactt ccgatagtgc gggtgttgaa tgatttccag 24240 ttgctaccga ttttacatat tttttgcatg agagaatttg taccacctcc caccgaccat 24300 ctatgactgt acgccactgt ccctaggact gctatgtgcc ggagcggaca ttacaaacgt 24360 ccttctcggt gcatgccact gttgccaatg acctgcctag gaattggtta gcaagttact 24420 accggatttt gtaaaaacag ccctcctcat ataaaaagta ttcgttcact tccgataagc 24480 gtcgtaattt tctatctttc atcatattct agatccctct gaaaaaatct tccgagtttg 24540 ctaggcactg atacataact cttttccaat aattggggaa gtcattcaaa tctataatag 24600 gtttcagatt tgcttcaata aattctgact gtagctgctg aaacgttgcg gttgaactat 24660 atttccttat aacttttacg aaagagtttc tttgagtaat cacttcactc aagtgcttc 24720 ctgcctccaa acgataccctg ttagcaat ttaatagctt gaatgatga agagctctgt 24780 gtttgtcttc ctgcctccag ttcgccgggc attcaacata aaactgata gcaccggag 24840 ttccggaac gaaatttgca tatacccatt gctcacgaaa aaaaatgtcc ttgtcgatat 24900 agggatgaat cgcttggtgt acccatcta ctgcgaaaac ttgacctttc tctcccatat 24960 tgcagtcgcg gcacgatgga actaaattaa taggcatcac cgaaaattca ggataatgtg 25020 caataggaag aaaatgatct atattttg tctgtcctat atcaccacaa atggacatt 25080 tttcacctga tgaaacaagc atgtcatcgt atatgttct agcgggttg tttttactc 25140 ggagattatt ttcataagc tttctatt taacctttgt caggttacca actactaagg 25200 ttgtaggctc aagagggtgt gtcctgtcgt agtaaataa ctgacctgtc gagcttaata 25260 ttctatattg ttgttctttc tgcaaaaag tgggaagtg agtaatgaaa ttatttctaa 25320 catttatctg catcatacct tccgagcatt tattaagcat ttcgctataa gttctcgctg 25380 gaagaggtag ttttcatt gtactttacc ttcattctg ttcattca tcgcttttta 25440 aacggttcga ccttctaatc ctatctgacc attataattt tttagaatgg ttcataaga 25500 aagctctgaa tcacggact gcgataataa gtggtggtat ccagaatttg tcactcag 25560 taaaaacacc tcacgagtta aaacaccta gttctcaccg atgtctca tatccggacg 25620 gataatattt attgctctc ttgaccgtag gactttccac atgcaggatt ttggaacctc 25680 ttgcagtact actggggaat gagttgcaat tattgctaca ccattgcgtg catcgagtaa 25740 gtcgcttaat gttcgtaaaa aagcagagag caaggtgga tgcagatgaa cctctggttc 25800 atcgaataaa actaatgact ttcgccaac gatactact aatcttgtga tgtaataa 25860 aacaattgca tgtccagagc tcattcgaag cagatatttc tggatattgt cataaaacaa 25920 tttagtgaat ttcatcgt ccacttgaat ctgtggttca ttacgtctta actctcata 25980 tttagaaatg aggctgatga gttccatatt tgaaaagtttt tcatcactac ttagtttttt 26040 gatagcttca agccagagtt gtctttttct atctactctc atacaaccaa taaatgctga 26100 aatgaattct aagcggagat cgcctagtga ttttaaacta ttgctggcag cattcttgag 26160 tccaataataa aagtattgtg taccttttgc tgggtcaggt tgttctttag gaggagtaaa 26220 26280 tgactcgata agtctattat tttcagagaa aaaatattca ttgttttctg ggttggtgat 26340 tgcaccaatc attccattca aaattgttgt tttaccacac ccattccgcc cgataaaagc 26400 atgaatgttc gtgctggggca tagaattaac cgtcacctca aaaggtatag ttaaatcact 26460 gaatccggga gcactttttc tattaaatga aaagtggaaa tctgcaatt ctggcaaacc 26520 atttaacaca cgtgcgaact gtccatgaat ttctgaaaga gttacccctc tagtaatga 26580 ggtgttaagg acgctttcat tttcaatgtc ggctaatcga tttggccata ctactaaatc 26640 ctgaatagct ttaagaaggt tatgtttaaa accatcgctt aatttgctga gattaacata 26700 gtagtcaatg ctttcaccta aggaaaaaaa catttcaggg agttgactga atttttatc 26760 tattaatgaa taagtgctta cttctcttt ttgacctaca aaaccaattt taacatttcc 26820 gatatcgcat tttcaccat gctcatcaa gatagtaga windowacattg window 26880 atagtcattc caccacattg ctcgtaggaa tgcctttt tttctactg caggaatata 26940 cccgccctt tcaatacac taaactccaa catatagtaa cccttaattt tattaaaata 27000 accgcaattt atttggcggc aacacaggat ctctcttta agttactc tattacatac 27060 gttttccatc taaaaattag tagtattga cttaacgggg catcgtattg tagttttcca 27120 tattagctt tctgcttcct ttggatac cactgttat tcatgttgca tggtgcactg 27180 tttataccaa cgatatagtc tattaatgca tatatagtat cgccgaacga ttagctctc 27240 aggctctga agaagcgttt caagtactaa taagccgata gatagccacg gactcgtag 27300 ccattttca taagtgttaa cttccgctcc tcgctcataa cagacattca ctacagttat 27360 ggcggaaagg tatgcatgct gggtgtgggg aagtcgtgaa agaaagaag tcagctgcgt 27420 cgtttgacat cactgctatc ttcttactgg ttgcaggt cgtagtggt ggcacacaaa 27480 gctttgcact ggattgcgag gctttgtgct tctctggagt gcgacaggtt tgatgacaaa 27540 aaatgcgc aagaagacaa aaatcacctt gcgctaatgc tctgttacag gtcactaata 27600 ccatctaagt agttgattca tagtgactgc atatgttgtg ttttacagta ttatgtagtc 27660 tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt 27720 tcagctttt tatactaagt tggcattata aaaaagcatt gcttatcaat ttgttgcaac 27780 gaacaggtca ctatcagtca aaataaaatc attatttgat ttcaattttg tcccactccc 27840 tgcctctgtc atcacgatac tgtgatgcca tggtgtccga cttatgccg agaagatgtt 27900 gagcaaactt atcgcttatc tgcttctcat agagtcttgc agacaaactg cgcaactcgt 27960 gaaaggtagg cggatcccct tcgaaggaaa gacctgatgc tttcgtgcg cgcataaaat 28020 accttgatac tgtgccggat gaaagcggtt cgcgacgagt agatgcaatt atggttctc 28080 cgccaagaat ctctttgcat tttcaagtg tttccttcat tgatattccg agagcatcaa 28140 tatgcaatgc tgttgggatg gcaattttta cgcctgtttt gctttgctcg acataaagat 28200 atccatctac gatatcagac cacttcattt cgcataaatc accaactcgt tgcccggtaa 28260 caacagccag ttccattgca agtctgagcc aacatggtga tgattctgct gcttgataaa 28320 ttttcaggta ttcgtcagcc gtaagtcttg atctccttac ctctgatttt gctgcgcgag 28380 tggcagcgac atggtttgtt gttatatggc cttcagctat tgcctctcgg aatgcatcgc 28440 tcagtgttga tctgattaac ttggctgacg ccgccttgcc ctcgtctatg tatccattga 28500 gcattgccgc aatttctttt gtggtgatgt cttcaagtgg agcatcaggc agacccctcc 28560 ttattgcttt aattttgctc atgtaattta tgagtgtctt ctgcttgatt cctctgctgg 28620 ccaggatttt ttcgtagcga tcaagccatg aatgtaacgt aacggaatta tcactgttga 28680 ttctcgctgt cagaggcttg tgtttgtgtc ctgaaaataa ctcaatgttg gcctgtatag 28740 cttcagtgat tgcgattcgc ctgtctctgc ctaatccaaa ctctttaccc gtccttgggt 28800 ccctgtagca gtaatatcca ttgtttctta tataaaggtt agggggtaaa tcccggcgct 28860 catgacttcg ccttcttccc atttctgatc ctcttcaaaa ggccacctgt tactggtcga 28920 tttaagtcaa cctttaccgc tgattcgtgg aacagatact ctcttccatc cttaaccgga 28980 ggtgggaata tcctgcattc ccgaacccat cgacgaactg tttcaaggct tcttggacgt 29040 cgctggcgtg cgttccactc ctgaagtgtc aagtacatcg caaagtctc gcaattacac 29100 gcaagaaaaa accgccatca ggcggcttgg tgttctttca gttcttcaat tcgaatattg 29160 gttacgtctg catgtgctat ctgcgcccat atcatccagt ggtcgtagca gtcgttgatg 29220 ttctccgctt cgataactct gttgaatggc tctccattcc attctcctgt gactcggaag 29280 tgcatttatc atctccataa aacaaaaccc gccgtagcga gttcagataa aataaatccc 29340 cgcgagtgcg aggattgtta tgtaatattg ggtttaatca tctatatgtt ttgtacagag 29400 agggcaagta tcgtttccac cgtactcgtg ataataattt tgcacggtat cagtcatttc 29460 tcgcacattg cagaatgggg atttgtcttc attagactta taaaccttca tggaatattt 29520 gtatgccgac tctatatcta taccttcatc tacataaaca ccttcgtgat gtctgcatgg 29580 agacaagaca ccggatctgc acaacattga taacgcccaa tctttttgct cagactctaa 29640 ctcattgata ctcatttata aactccttgc aatgtatgtc gtttcagcta aacggtatca 29700 gcaatgttta tgtaaagaaa cagtaagata atactcaacc cgatgtttga gtacggtcat 29760 catctgacac tacagactct ggcatcgctg tgaagacgac gcgaaattca gcattttcac 29820 aagcgttatc ttttacaaaa ccgatctcac tctcctttga tgcgaatgcc agcgtcagac 29880 atcatatgca gatactcacc tgcatcctga acccattgac ctccaacccc gtaatagcga 29940 tgcgtaatga tgtcgatagt tactaacggg tcttgttcga ttaactgccg cagaaactct 30000 tccaggtcac cagtgcagtg cttgataaca ggagtcttcc caggatggcg aacaacaaga 30060 aactggtttc cgtcttcacg gacttcgttg ctttccagtt tagcaatacg cttactccca 30120 tccgagataa caccttcgta atactcacgc tgctcgttga gttttgattt tgctgtttca 30180 agctcaacac gcagtttccc tactgttagc gcaatatcct cgttctcctg gtcgcggcgt 30240 ttgatgtatt gctggtttct ttcccgttca tccagcagtt ccagcacaat cgatggtgtt 30300 accaattcat ggaaaaggtc tgcgtcaaat ccccagtcgt catgcattgc ctgctctgcc 30360 gcttcacgca gtgcctgaga gttaatttcg ctcacttcga acctctctgt ttactgataa 30420 gttccagatc ctcctggcaa cttgcacaag tccgacaacc ctgaacgacc aggcgtcttc 30480 gttcatctat cggatcgcca cactcacaac aatgagtggc agatatagcc tggtggttca 30540 ggcggcgcat ttttattgct gtgttgcgct gtaattcttc tatttctgat gctgaatcaa 30600 tgatgtctgc catctttcat taatccctga actgttggtt aatacgcttg agggtgaatg 30660 cgaataataa aaaaggagcc tgtagctccc tgatgatttt gcttttcatg ttcatcgttc 30720 cttaaagacg ccgtttaaca tgccgattgc caggcttaaa tgagtcggtg tgaatcccat 30780 cagcgttacc gtttcgcggt gcttcttcag tacgctacgg caaatgtcat cgacgttttt 30840 atccggaaac tgctgtctgg ctttttttga tttcagaatt agcctgacgg gcaatgctgc 30900 gaagggcgtt ttcctgctga ggtgtcattg aacaagtccc atgtcggcaa gcataagcac 30960 acagaatatg aagcccgctg ccagaaaaat gcattccgtg gttgtcatac ctggtttctc 31020 tcatctgctt ctgctttcgc caccatcatt tccagctttt gtgaaaggga tgcggctaac 31080 gtatgaaatt cttcgtctgt ttctactggt attggcacaa acctgattcc aatttgagca 31140 aggctatgtg ccatctcgat actcgttctt aactcaacag aagatgcttt gtgcatacag 31200 cccctcgttt attatttatc tcctcagcca gccgctgtgc tttcagtgga tttcggataa 31260 cagaaaggcc gggaaatacc cagcctcgct ttgtaacgga gtagacgaaa gtgattgcgc 31320 ctacccggat attatcgtga ggatgcgtca tcgccattgc tccccaaata caaaaccaat 31380 ttcagccagt gcctcgtcca ttttttcgat gaactccggc acgatctcgt caaaactcgc 31440 catgtacttt tcatcccgct caatcacgac ataatgcagg ccttcacgct tcatacgcgg 31500 gtcatagttg gcaaagtacc aggcattttt tcgcgtcacc cacatgctgt actgcacctg 31560 ggccatgtaa gctgacttta tggcctcgaa accaccgagc cggaacttca tgaaatcccg 31620 ggaggtaaac gggcatttca gttcaaggcc gttgccgtca ctgcataaac catcgggaga 31680 gcaggcggta cgcatacttt cgtcgcgata gatgatcggg gattcagtaa cattcacgcc 31740 ggaagtgaat tcaaacaggg ttctggcgtc gttctcgtac tgttttcccc aggccagtgc 31800 tttagcgtta acttccggag ccacaccggt gcaaacctca gcaagcaggg tgtggaagta 31860 ggacattttc atgtcaggcc acttctttcc ggagcggggt tttgctatca cgttgtgaac 31920 ttctgaagcg gtgatgacgc cgagccgtaa tttgtgccac gcatcatccc cctgttcgac 31980 agctctcaca tcgatcccgg tacgctgcag gataatgtcc ggtgtcatgc tgccaccttc 32040 tgctctgcgg ctttctgttt caggaatcca agagctttta ctgcttcggc ctgtgtcagt 32100 tctgacgatg cacgaatgtc gcggcgaaat atctgggaac agagcggcaa taagtcgtca 32160 tcccatgttt tatccagggc gatcagcaga gtgttaatct cctgcatggt ttcatcgtta 32220 accggagtga tgtcgcgttc cggctgacgt tctgcagtgt atgcagtatt ttcgacaatg 32280 cgctcggctt catccttgtc atagatacca gcaaatccga aggccagacg ggcacactga 32340 atcatggctt tatgacgtaa catccgtttg ggatgcgact gccacggccc cgtgatttct 32400 ctgccttcgc gagttttgaa tggttcgcgg cggcattcat ccatccattc ggtaacgcag 32460 atcggatgat tacggtcctt gcggtaaatc cggcatgtac aggattcatt gtcctgctca 32520 aagtccatgc catcaaactg ctggttttca ttgatgatgc gggaccagcc atcaacgccc 32580 accaccggaa cgatgccatt ctgcttatca ggaaaggcgt aaatttcttt cgtccacgga 32640 ttaaggccgt actggttggc aacgatcagt aatgcgatga actgcgcatc gctggcatca 32700 cctttaaatg ccgtctggcg aagagtggtg atcagttcct gtgggtcgac agaatccatg 32760 ccgacacgtt cagccagctt cccagccagc gttgcgagtg cagtactcat tcgttttata 32820 cctctgaatc aatatcaacc tggtggtgag caatggtttc aaccatgtac cggatgtgtt 32880 ctgccatgcg ctcctgaaac tcaacatcgt catcaaacgc acgggtaatg gattttttgc 32940 tggccccgtg gcgttgcaaa tgatcgatgc atagcgattc aaacaggtgc tggggcaggc 33000 ctttttccat gtcgtctgcc agttctgcct ctttctcttc acggcgagc tgctggtagt 33060 gacgcgccca gctctgagcc tcaagacgat cctgaatgta ataagcgttc atggctgaac tcctgaata gctgtgaaaa tatcgcccgc gaaatgccgg gctgattagg aaaacagga agggggttag tgaatgcttt tgcttgatct cagtttcagt attaatcc attttttata agcgtcga gcttcacga acatcttttc atcgccaata aaagtggcga tagtgaattt agtctggata gccataagtg tttgatccat tctttggggac tcctggctga ttaagtatgt cgataaggcg tttccatccg tcacgtaatt tacgggtgat tcgttcaagt aaagattcgg aagggcagcc agcaacaggc caccctgcaa tggcatattg catggtgtgc tccttattta 33540. tacataacga aaaacgcctc gagtgaagcg ttattggtat gcggtaaaac cgcactcagg cggccttgat agtcatatca tctgaatcaa atattcctga tgtatcgata tcggtaattc ttattccttc gctaccatcc attggaggcc atccttcctg accatttcca tcattccagt 33660 cgaactcaca cacaacacca tatgcattta agtcgcttga aattgctata agcagagcat 33720 gttgcgccag catgattaat acagcattta atacagagcc gtgtttattg agtcggtatt 33780 cagagtctga ccagaaatta ttaatctggt gaagtttttc ctctgtcatt acgtcatggt 33840 cgatttcaat ttctattgat gctttccagt cgtaatcaat gatgtatttt ttgatgtttg 33900 acatctgttc atatcctcac agataaaaaa tcgccctcac actggagggc aaagaagatt 33960 tccaataatc agaacaagtc ggctcctgtt tagttacgag cgacattgct ccgtgtattc 34020 actcgttgga atgaatacac agtgcagtgt ttattctgtt atttatgcca aaaataaagg 34080 ccactatcag gcagctttgt tgttctgttt accaagttct ctggcaatca ttgccgtcgt 34140 tcgtattgcc catttatcga catatttccc atcttccatt acaggaaaca tttcttcagg 34200 cttaaccatg cattccgatt gcagcttgca tccattgcat cgcttgaatt gtccacacca 34260 ttgattttta tcaatagtcg tagtcatacg gatagtcctg gtattgttcc atcacatcct 34320 gaggatgctc ttcgaactct tcaaattctt cttccatata tcaccttaaa tagtggattg 34380 cggtagtaaa gattgtgcct gtcttttaac cacatcaggc tcggtggttc tcgtgtaccc 34440 ctacagcgag aaatcggata aactattaca acccctacag tttgatgagt atagaaatgg 34500 atccactcgt tattctcgga cgagtgttca gtaatgaacc tctggagaga accatgtata 34560 tgatcgttat ctgggttgga cttctgcttt taagcccaga taactggcct gaatatgtta 34620 atgagagaat cggtattcct catgtgtggc atgtttttcgt ctttgctctt gcattttcgc 34680 tagcaattaa tgtgcatcga ttatcagcta ttgccagcgc cagatataag cgatttaagc 34740 taagaaaacg cattaagatg caaaacgata aagtgcgatc agtaattcaa aaccttacag 34800 aagagcaatc tatggttttg tgcgcagccc ttaatgaagg caggaagtat gtggttacat 34860 caaaacaatt cccacatatt agtgagttga ttgagcttgg tgtgttgaac aaaacttttt 34920 cccgatggaa tggaaagcat atattattcc ctattgagga tatttactgg actgaattag 34980 ttgccagcta tgatccatat aatattgaga taaagccaag gccaatatct aagtaactag 35040 ataagaggaa tcgattttcc cttaattttc tggcgtccac tgcatgttat gccgcgttcg 35100 ccaggcttgc tgtaccatgt gcgctgattc ttgcgctcaa tacgttgcag gttgctttca 35160 atctgtttgt ggtattcagc cagcactgta aggtctatcg gatttagtgc gctttctact 35220 cgtgatttcg gtttgcgatt cagcgagaga atagggcggt taactggttt tgcgcttacc 35280 ccaaccaaca ggggatttgc tgctttccat tgagcctgtt tctctgcgcg acgttcgcgg 35340 cggcgtgttt gtgcatccat ctggattctc ctgtcagtta gctttggtgg tgtgtggcag 35400 ttgtagtcct gaacgaaaac cccccgcgat tggcacattg gcagctaatc cggaatcgca 35460 cttacggcca atgcttcgtt tcgtatcaca caccccaaag ccttctgctt tgaatgctgc 35520 ccttcttcag ggcttaattt ttaagagcgt caccttcatg gtggtcagtg cgtcctgctg 35580 atgtgctcag tatcaccgcc agtggtattt atgtcaacac cgccagagat aatttatcac 35640 cgcagatggt tatctgtatg ttttttatat gaatttattt tttgcagggg ggcattgttt 35700 ggtaggtgag agatctgaat tgctatgttt agtgagttgt atctatttat ttttcaataa 35760 atacaattgg ttatgtgttt tgggggcgat cgtgaggcaa agaaaacccg gcgctgaggc 35820 cgggttattc ttgttctctg gtcaaattat atagttggaa aacaaggatg catatatgaa 35880 tgaacgatgc agaggcaatg ccgatggcga tagtgggtat catgtagccg cttatgctgg 35940 aaagaagcaa taacccgcag aaaaacaaag ctccaagctc aacaaaacta agggcataga 36000 caataactac cgatgtcata tacccatact ctctaatctt ggccagtcgg cgcgttctgc 36060 ttccgattag aaacgtcaag gcagcaatca ggattgcaat catggttcct gcatatgatg 36120 acaatgtcgc cccaagacca tctctatgag ctgaaaaaga aacaccagga atgtagtggc 36180 ggaaaaggag atagcaaatg cttacgataa cgtaaggaat tattactatg taaacaccag 36240 gcatgattct gttccgcata attactcctg ataattaatc cttaactttg cccacctgcc 36300 ttttaaaaca ttccagtata tcacttttca ttcttgcgta gcaatatgcc atctcttcag 36360 ctatctcagc attggtgacc ttgttcagag gcgctgagag atggcctttt tctgatagat 36420 aatgttctgt taaaatatct ccggcctcat cttttgcccg caggctaatg tctgaaaatt 36480 gaggtgacgg gttaaaaata atatccttgg caaccttttt tatatccctt ttaaattttg 36540 gcttaatgac tatatccaat gagtcaaaaa gctccccttc aatatctgtt gcccctaaga 36600 cctttaatat atcgccaaat acaggtagct tggcttctac cttcaccgtt gttcggccga 36660 tgaaatgcat atgcataaca tcgtctttgg tggttcccct catcagtggc tctatctgaa 36720 cgcgctctcc actgcttaat gacattcctt tcccgattaa aaaatctgtc agatcggatg 36780 tggtcggccc gaaaacagtt ctggcaaaac caatggtgtc gccttcaaca aacaaaaaag 36840 atgggaatcc caatgattcg tcatctgcga ggctgttctt aatatcttca actgaagctt 36900 tagagcgatt tatcttctga accagactct tgtcatttgt tttggtaaag agaaaagttt 36960 ttccatcgat tttatgaata tacaaataat tggagccaac ctgcaggtga tgattatcag 37020 ccagcagaga attaaggaaa acagacaggt ttattgagcg cttatctttc cctttatttt 37080 tgctgcggta agtcgcataa aaaccattct tcataattca atccatttac tatgttatgt 37140 tctgagggga gtgaaaattc ccctaattcg atgaagattc ttgctcaatt gttatcagct 37200 atgcgccgac cagaacacct tgccgatcag ccaaacgtct cttcaggcca ctgactagcg 37260 ataactttcc ccacaacgga acaactctca ttgcatggga tcattgggta ctgtgggttt 37320 agtggttgta aaaacacctg accgctatcc ctgatcagtt tcttgaaggt aaactcatca 37380 cccccaagtc tggctatgca gaaatcacct ggctcaacag cctgctcagg gtcaacgaga 37440 attaacattc cgtcaggaaa gcttggcttg gagcctgttg gtgcggtcat ggaattacct 37500 tcaacctcaa gccagaatgc agaatcactg gcttttttgg ttgtgcttac ccatctctcc 37560 gcatcacctt tggtaaaggt tctaagctca ggtgagaaca tccctgcctg aacatgagaa 37620 aaaacagggt actcatactc acttctaagt gacggctgca tactaaccgc ttcatacatc 37680 tcgtagattt ctctggcgat tgaagggcta aattcttcaa cgctaacttt gagaattttt 37740 gcaagcaatg cggcgttata agcatttaat gcattgatgc cattaaataa agcaccaacg 37800 cctgactgcc ccatccccat cttgtctgcg acagattcct gggataagcc aagttcattt 37860 ttctttttt cataaattgc tttaggcga cgtgcgtcct caagctgctc ttgtgttaat 37920 ggttcttttt ttgtgctcat acgttaaatc tatcaccgca agggataaat atctaacacc 37980 gtgcgtgttg actatttac ctctggcggt gataatggtt gcatgtacta aggaggttgt 38040 atggacaac gcataccct gaagatt gcaatgcgct tggggcaac cacacagct 38100 aagatctcg gcgtatatca aagcgcgatc aacaaggcca ttcatgcagg ccgaagatt 38160 tttttaacta taaacgctga tggaagcgtt tatgcggaag aggtaaagcc cttcccgagt 38220 aaaaaaaaa cacacata ataaccccg ctcttacaca ttccagccct gaaaaagggc 38280 atcaattaa accacaccta tggtgtatgc atttattgc atacattca tcaattgtta 38340 tctaggaaa tacttacata tggttcgtgc aaaaaacgc aacgaggctc tacgaatcga 38400 gagtgcgttg cttaacaaaa tcgcaatgct tggactgag aagacagcgg aagctgtggg 38460 cgttgataag tcgcagatca gcaggtggaa gagggactgg attccaagt tctcaatgct 38520 gcttgctgtt cttgaatggg gggtcgttga cgacgacatg gctcgattgg cgcgacaagt 38580 tgctgcgatt ctcaccaata aaaaacgccc ggcggcaacc gagcgttctg aacaaatcca gatggagttc tgaggtcatt actggatcta tcaacaggag tcattatgac aaatacagca aaaatactca acttcggcag aggtaacttt gccggacagg agcgtaatgt ggcagatctc 38760. 38820. gatgatggtt acgccagact atcaaatatg ctgcttgagg cttattcggg cgcagatctg accaagcgac agtttaaagt gctgcttgcc attctgcgta aaacctatgg gtggaataaa ccaatggaca gaatcaccga ttctcaactt agcgagatta caaagttacc tgtcaaacgg tgcaatgaag ccaagttaga actcgtcaga atgaatta tcaagcagca aggcggcatg tttggaccaa ataaaaacat ctcagaatgg tgcatccctc aaaacgaggg aaaatcccct aaaacgaggg ataaaacatc cctcaaattg ggggattgct atccctcaaa acagggggac acaaaagaca ctattacaaa agaaaaaaga aaagattatt cgtcagaga ttctggcgaa tcctctgacc agccagaaaa cgacctttct gtggtgaaac cggatgctgc aattcagagc 39240 ggcagcaagt gggggacagc agaagacctg accgccgcag agtggatgtt tgacatggtg 39300 aagactatcg caccatcagc cagaaaaccg aattttgctg ggtgggctaa cgatatccgc 39360 ctgatgcgtg aacgtgacgg acgtaaccac cgcgacatgt gtgtgctgtt ccgctgggca 39420 tgccaggaca acttctggtc cggtaacgtg ctgagcccgg ccaaactccg cgataagtgg 39480 acccaactcg aaatcaaccg taacaagcaa caggcaggcg tgacagccag caaaccaaaa 39540 ctcgacctga caaacacaga ctggatttac ggggtggatc tatgaaaaac atcgccgcac 39600 agatggttaa ctttgaccgt gagcagatgc gtcggatcgc caacaacatg ccggaacagt 39660 acgacgaaaa gccgcaggta cagcaggtag cgcagatcat caacggtgtg ttcagccagt 39720 tactggcaac tttcccggcg agcctggcta accgtgacca gaacgaagtg aacgaaatcc 39780 gtcgccagtg ggttctggct tttcgggaaa acgggatcac cacgatggaa caggttaacg 39840 caggaatgcg cgtagcccgt cggcagaatc gaccatttct gccatcaccc gggcagtttg 39900 ttgcatggtg ccgggaagaa gcatccgtta ccgccggact gccaaacgtc agcgagctgg 39960 ttgatatggt ttacgagtat tgccggaagc gaggcctgta tccggatgcg gagtcttatc 40020 cgtggaaatc aaacgcgcac tactggctgg ttaccaacct gtatcagaac atgcgggcca 40080 atgcgcttac tgatgcggaa ttacgccgta aggccgcaga tgagcttgtc catatgactg 40140 cgagaattaa ccgtggtgag gcgatccctg aaccagtaaa acaacttcct gtcatgggcg 40200 gtagacctct aaatcgtgca caggctctgg cgaagatcgc agaaatcaaa gctaagttcg 40260 gactgaaagg agcaagtgta tgacgggcaa agaggcaatt attcattacc tggggacgca 40320 taatagcttc tgtgcgccgg acgttgccgc gctaacaggc gcaacagtaa ccagcataaa 40380 tcaggccgcg gctaaaatgg cacgggcagg tcttctggtt atcgaaggta aggtctggcg 40440 aacggtgtat taccggtttg ctaccaggga agaacgggaa ggaaagatga gcacgaacct 40500 ggtttttaag gagtgtcgcc agagtgccgc gatgaaacgg gtattggcgg tatatggagt 40560 taaagatga ccatctacat tactgagcta fathercaggcc tgctggtaat cgcaggcctt tttatttggg ggagaggga gtcatgaaaa aactacctt tgaaattcga tctccagcac atcagcaaaa cgctattcac gcagtacagc aaatccttcc agacccaacc aaaccaatcg tagtaaccat tcaggaacgc aaccgcagct tagaccaaaa caggaagcta tgggcctgct taggtgacgt ctctcgtcag gttgaatggc atggtcgctg gctggatgca gaaagctgga agtgtgtgtt taccgcagca ttaaagcagc agtgttgt tcctaacctt gccgggaatg gctttgtggt aataggccag tcaaccagca ggatgcgtgt aggcgaattt gcggagctat 40980. taggcttat acaggcattc ggtacagagc gtggcgttaa gtggtcagac gaagcgagac 41040 tggctctgga gtggaaagcg agatggggag acagggctgc atgataaatg tcgttagttt ctccggtggc aggacgtcag catatttgct ctggctaatg gagcaaaagc gacgggcagg taaagacgtg cattacgttt tcatggatac aggttgtga catccaatga catatcggtt tgtcaggga gttgtgaagt tctgggatat accgctcacc gtattgcagg ttgatatcaa cccggagctt ggacagccaa atggttatac ggtatgggaa ccaaaggata ttcagacgcg 41340 aatgcctgtt ctgaagccat ttatcgatat ggtaaagaaa tatggcactc catacgtcgg 41400 cggcgcgttc tgcactgaca gattaaaact cgttcccttc accaaatact gtgatgacca 41460 tttcgggcga gggaattaca ccacgtggat tggcatcaga gctgatgaac cgaagcggct 41520 aaagccaaag cctggaatca gatatcttgc tgaactgtca gactttgaga aggaagatat 41580 cctcgcatgg tggaagcaac aaccattcga tttgcaaata ccggaacatc tcggtaactg 41640 catattctgc attaaaaaat caacgcaaaa aatcggactt gcctgcaaag atgaggaggg 41700 attgcagcgt gtttttaatg aggtcatcac gggatcccat gtgcgtgacg gacatcggga 41760 aacgccaaag gagattatgt accgaggaag aatgtcgctg gacggtatcg cgaaaatgta 41820 ttcagaaaat gattatcaag ccctgtatca ggacatggta cgagctaaaa gattcgatac 41880 cggctcttgt tctgagtcat gcgaaatatt tggagggcag cttgatttcg acttcgggag 41940 ggaagctgca tgatgcgatg ttatcggtgc ggtgaatgca aagagataa ccgcttccga 42000 ccaaatcaac cttactggaa tcgatggtgt ctccggtgtg aagaacacc aacaggggtg 42060 ttaccactac cgcaggaaaa ggaggacgtg tggcgagaca gcgacgaagt atcaccgaca 42120 taatctgcga aaactgcaaa taccttccaa cgaaacgcac cagaataa cccaagccaa 42180 tcccaaaga atctgacgta aaaaccttca actacacggc tcacctgtgg gatatccggt 42240 ggctaagacg tcgtgcgagg aaaaaaaggt gattgaccaa atcgaagtt acgaacaaga 42300 aagcgtcgag cgagctttaa cgtgcgctaa ctgcggtcag aagctgcatg tgctggaagt 42360 tcacgtgtgt gagcactgct gcgcagaact gatgagcgat ccgaatagct cgatgcacga 42420 ggagagat gatggctaaa ccaggccgaa gacgatgtaa aaacgatgaa tgccgggaat 42480 gtttcaccc tgcattcgct aatcagtggt ggtgctctcc agtgtgga accagatag 42540 cactcgaacg agaagtaaaaagcgaaaaagcgaaaaagcgaaag aagaaacgac 42600 gacgagagga gcagaaacag aagaataac ttaagattcg aaaactcgcc ttaaagcccc 42660 gcagttactg gattaaacaa gcccaacaag ccgtaaacgc cttcatcaga gaaagagacc gcgacttacc atgtatctcg tgcggaacgc tcacgtctgc tcagtgggat gccggacatt 42780. accggacaac tgctgcggca cctcaactcc gatttaatga accgcaatatt cacaagcaat gcgtggtgtg caccagcac aaaagcgga atctcgttcc gtatcgcgtc gaactgatta gccgcatcgg gcaggaagca gtagacgaaa tcgaatcaaa ccataaccgc catcgctgga ctatcgaaga gtgcaaggcg atcaaggcag agtaccaaca gaactcaaa gacctgcga atagcagaag tgaggccgca tgacgttctc agtaaaacc attccagaca tgctcgttga agcatacgga aatcagacag aagtagcacg cagactgaaa tgtagtcgcg gtacggtcag aaaatacgtt gatgataaag acggaaat gcacgccatc gtcaacgacg ttctcatggt tcatcgcgga tggagtgaaa gagatgcgct attacgaaaa aattgatggc agcaaatacc gaaatatttg ggtagttggc gatctgcacg gatgctacac gaacctgatg aacaaactgg atacgattgg attcgacaac aaaaaagacc tgcttatctc ggtgggcgat ttggttgatc 43380 gtggtgcaga gaacgttgaa tgcctggaat taatcacatt cccctggttc agagctgtac 43440 gtggaaacca tgagcaaatg atgattgatg gcttatcaga gcgtggaaac gttaatcact 43500 ggctgcttaa tggcggtggc tggttcttta atctcgatta cgacaaagaa attctggcta 43560 aagctcttgc ccataaagca gatgaacttc cgttaatcat cgaactggtg agcaaagata 43620 aaaaatatgt tatctgccac gccgattatc cctttgacga atacgagttt ggaaagccag 43680 ttgatcatca gcaggtaatc tggaaccgcg aacgaatcag caactcacaa aacgggatcg 43740 tgaaagaaat caaaggcgcg gacacgttca tctttggtca tacgccagca gtgaaaccac 43800 tcaagtttgc caaccaaatg tatatcgata ccggcgcagt gttctgcgga aacctaacat 43860 tgattcaggt acagggagaa ggcgcatgag actcgaaagc gtagctaaat ttcattcgcc 43920 aaaaagcccg atgatgagcg actcaccacg ggccacggct tctgactctc tttccggtac 43980 tgatgtgatg gctgctatgg ggatggcgca atcacaagcc ggattcggta tggctgcatt 44040 ctgcggtaag cacgaactca gccagaacga caaacaaaag gctatcaact atctgatgca 44100 atttgcacac aaggtatcgg ggaaataccg tggtgtggca aagcttgaag gaaatactaa 44160 ggcaaaggta ctgcaagtgc tcgcaacatt cgcttatgcg gattattgcc gtagtgccgc 44220 gacgccgggg gcaagatgca gagattgcca tggtacaggc cgtgcggttg atattgccaa 44280 aacagagctg tgggggagag ttgtcgagaa agagtgcgga agatgcaaag gcgtcggcta 44340 ttcaaggatg ccagcaagcg cagcatatcg cgctgtgacg atgctaatcc caaaccttac 44400 ccaacccacc tggtcacgca ctgttaagcc gctgtatgac gctctggtgg tgcaatgcca 44460 caaagaagag tcaatcgcag acaacatttt gaatgcggtc acacgttagc agcatgattg 44520 ccacggatgg caacatatta acggcatgat attgacttat tgaataaaat tgggtaaatt 44580 tgactcaacg atgggttaat tcgctcgttg tggtagtgag atgaaaagag gcggcgctta 44640 ctaccgattc cgcctagttg gtcacttcga cgtatcgtct ggaactccaa ccatcgcagg 44700 cagagaggtc tgcaaaatgc aatcccgaaa cagttcgcag gtaatagtta gagcctgcat aacggtttcg ggatttttta tatctgcaca acaggtaaga gcattgagtc gataatcgtg aagagtcggc gagcctggtt agccagtgct ctttccgttg tgctgaatta agcgaatacc ggagcaga ccggatcacc aaatgcgtac aggcgtcatc gccgcccagc aacagcacaa cccaaactga gccgtagcca ctgtctgtcc tgaattcatt agtaatagtt acgctgcggc cttttacaca tgaccttcgt gaaagcgggt ggcaggaggt cgcgctaaca acctcctgcc gttttgcccg tgcatatcgg tcacgaacaa atctgattac taaacacagt agcctggatt tgttctatca gtaatcgacc ttattcctaa ttaatagag caaatcccct tattgggggt aagacatgaa gatgccagaa aaacatgacc tgttggccgc cattctcgcg gcaaaggaac aaggcatcgg ggcaatcctt gcgtttgcaa tggcgtacct tcgcggcaga fatherggcg gtgcgtttac aaaaacagta atcgacgcaa cgatgtgcgc cattatcgcc tggttcattc gtgaccttct cgacttcgcc ggactaagta gcaatctcgc ttatataacg agcgtgttta tcggctacat cggtactgac tcgattggtt cgcttatcaa acgcttcgct gctaaaaaag 45480 ccggagtaga agatggtaga aatcaataat caacgtaagg cgttcctcga tatgctggcg 45540 tggtcggagg gaactgataa cggacgtcag aaaaccagaa atcatggtta tgacgtcatt 45600 gtaggcggag agctatttac tgattactcc gatcaccctc gcaaacttgt cacgctaaac 45660 ccaaaactca aatcaacagg cgccggacgc taccagcttc tttcccgttg gtgggatgcc 45720 taccgcaagc agcttggcct gaaagacttc tctccgaaaa gtcaggacgc tgtggcattg 45780 cagcagatta aggagcgtgg cgctttacct atgattgatc gtggtgatat ccgtcaggca 45840 atcgaccgtt gcagcaatat ctgggcttca ctgccgggcg ctggttatgg tcagttcgag 45900 cataaggctg acagcctgat tgcaaaattc aaagaagcgg gcggaacggt cagagagatt 45960 gatgtatgag cagagtcacc gcgattatct ccgctctggt tatctgcatc atcgtctgcc 46020 tgtcatgggc tgttaatcat taccgtgata acgccattac ctacaaagcc cagcgcgaca 46080 aaaatgccag agaactgaag ctggcgaacg cggcaattac tgacatgcag atgcgtcagc 46140 gtgatgttgc tgcgctcgat gcaaaataca cgaaggagtt agctgatgct aaagctgaaa 46200 atgatgctct gcgtgatgat gttgccgctg gtcgtcgtcg gttgcacatc aaagcagtct 46260 gtcagtcagt gcgtgaagcc accaccgcct ccggcgtgga taatgcagcc tccccccgac 46320 tggcagacac cgctgaacgg gattatttca ccctcagaga gaggctgatc actatgcaaa 46380 aacaactgga aggaacccag aagtatatta atgagcagtg cagatagagt tgcccatatc 46440 gatgggcaac tcatgcaatt attgtgagca atacacacgc gcttccagcg gagtataaat 46500 gcctaaagta ataaaaccga gcaatccatt tacgaatgtt tgctgggttt ctgttttaac 46560 aacattttct gcgccgccac aaattttggc tgcatcgaca gttttcttct gcccaattcc 46620 agaaacgaag aaatgatggg tgatggtttc ctttggtgct actgctgccg gtttgttttg 46680 aacagtaaac gtctgttgag cacatcctgt aataagcagg gccagcgcag tagcgagtag 46740 catttttttc atggtgttat tcccgatgct ttttgaagtt cgcagaatcg tatgtgtaga 46800 aaattaaaca aaccctaaac aatgagttga aatttcatat tgttaatatt tattaatgta 46860 tgtcaggtgc gatgaatcgt cattgtattc ccggattaac tatgtccaca gccctgacgg 46920 ggaacttctc tgcgggagtg tccgggaata attaaaacga tgcacacagg gtttagcgcg 46980 tacacgtatt gcattatgcc aacgccccgg tgctgacacg gaagaaaccg gacgttatga 47040 tttagcgtgg aaagatttgt gtagtgttct gaatgctctc agtaaatagt aatgaattat 47100 caaaggtata gtaatatctt ttatgttcat ggatatttgt aacccatcgg aaaactcctg 47160 ctttagcaag attttccctg tattgctgaa atgtgatttc tcttgatttc aacctatcat 47220 aggacgtttc tataagatgc gtgtttcttg agaatttaac atttacaacc tttttaagtc 47280 cttttattaa cacggtgtta tcgttttcta acacgatgtg aatattatct gtggctagat 47340 agtaaatata atgtgagacg ttgtgacgtt ttagttcaga ataaacaat tcacagtcta 47400 aatcttttcg cacttgatcg aatatttctt taaaaatggc aacctgagcc attggtaaaa 47460 ccttccatgt gatacgaggg cgcgtagttt gcattatcgt tttatcgtt tcaatctggt 47520 ctgacctcct tgtgttttgt tgatgattta tgtcaaatat taggaatgtt ttcacttaat 47580 agtattggtt gcgtaacaaa gtgcggtcct gctggcattc tggagggaaa tacaaccgac 47640 agatgtatgt aaggccaacg tgctcaaatc ttcatacaga aagatttgaa gtaatatttt 47700 aaccgctaga tgaagagcaa gcgcatggag cgacaaaatg aataagaac aatctgctga 47760 tgatccctcc gtggatctga ttcgtgtaaa aaatatgctt aatagcacca tttctatgag 47820 ttaccctgat gttgtaattg catgtataga acataaggtg tctctggaag cattcagagc 47880 aattgaggca gcgttggtga agcacgataa taatatgaag gattattccc tggtggttga 47940 ctgatcacca taactgctaa tcattcaaac tatttagtct gtgacagagc caacacgcag 48000 tctgtcactg tcaggaaagt ggtaaaactg caactcaatt actgcaatgc cctcgtaatt 48060 aagtgaattt aaatatcgt cctgttcgga gggaagaacg cgggatgttc attcttcatc 48120 acttttaatt gatgtatatg ctctcttttc tgacgttagt ctccgacggc aggcttcaat 48180 gacccaggct gagaaattcc cggacccttt ttgctcaaga gcgatgttaa tttgttcaat 48240 catttggtta ggaaagcgga tgttgcgggt tgttgttctg cgggttctgt tcttcgttga 48300 catgaggttg ccccgtattc agtgtcgctg atttgtattg tctgaagttg tttttacgtt 48360 aagttgatgc agatcaatta atacgatacc tgcgtcataa ttgattattt gacgtggttt 48420 gatggcctcc acgcacgttg tgatatgtag atgataatca ttatcacttt acgggtcctt 48480 tccggtgatc cgacaggtta cg 48502 <210> 6 <211> 628 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 6 gggcggcgac ctcgcgggtt ttcgctattt atgaaaattt tccggtttaa ggcgtttccg 60 ttcttcttcg tcataactta atgtttttat ttaaaatacc ctctgaaaag aaaggaaacg 120 acaggtgctg aaagcgaggc tttttggcct ctgtcgtttc ctttctctgt ttttgtccgt 180 ggaatgaaca atggaagtca acaaaaagca gctggctgac attttcggtg cgagtatccg 240 taccattcag aactggcagg aacagggaat gcccgttctg cgaggcggtg gcaagggtaa 300 tgaggtgctt tatgactctg ccgccgtcat aaaatggtat gccgaaaggg atgctgaaat 360 tgagaacgaa aagctgcgcc gggaggttga agaactgcgg caggccagcg aggcagatct 420 ccagccagga actattgagt acgaacgcca tcgacttacg cgtgcgcagg ccgacgcaca 480 ggaactgaag aatgccagag actccgctga agtggtggaa accgcattct gtactttcgt 540 gctgtcgcgg atcgcaggtg aaattgccag tattctcgac gggctccccc tgtcggtgca 600 gcggcgtttt ccggaactgg aaaaccga 628 <210> 7 <211> 38767 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 7 catgttgatt tcctgaaacg ggatatcatc aaagccatga acaaagcagc cgcgctggat 60 gaactgatac cggggttgct gagtgaatat atcgaacagt caggttaaca ggctgcggca 120 ttttgtccgc gccgggcttc gctcactgtt caggccggag ccacagaccg ccgttgaatg 180 ggcggatgct aattactatc tcccgaaaga atccgcatac caggaagggc gctgggaaac 240 actgcccttt cagcgggcca tcatgaatgc gatgggcagc gactacatcc gtgaggtgaa 300 tgtggtgaag tctgcccgtg tcggttattc caaaatgctg ctgggtgttt atgcctactt 360 tatagagcat aagcagcgca acacccttat ctggttgccg acggatggtg atgccgagaa 420 ctttatgaaa acccacgttg agccgactat tcgtgatatt ccgtcgctgc tggcgctggc 480 cccgtggtat ggcaaaaagc accgggataa cacgctcacc atgaagcgtt tcactaatgg 540 gcgtggcttc tggtgcctgg gcggtaaagc ggcaaaaaac taccgtgaaa agtcggtgga 600 tgtggcgggt tatgatgaac ttgctgcttt tgatgatgat attgaacagg aaggctctcc 660 gacgttcctg ggtgacaagc gtattgaagg ctcggtctgg ccaaagtcca tccgtggctc 720 cacgccaaaa gtgagaggca cctgtcagat tgagcgtgca gccagtgaat ccccgcattt 780 tatgcgtttt catgttgcct gcccgcattg cggggaggag fộttctta aatttggcga 840 caaagagacg ccgtttggcc tcaaatggac gccggatgac ccctccagcg tgttttatct 900 ctgcgagcat aatgcctgcg tcatccgcca gcaggagctg gactttactg atgcccgtta 960 tatctgcgaa aagaccggga tctggacccg tgatggcatt ctctggtttt cgtcatccgg 1020 tgaagagatt gagccacctg acagtgtgac ctttcacatc tggacagcgt acagcccgtt 1080 caccacctgg gtgcagattg tcaaagactg gatgaaaacg aaaggggata cgggaaaacg 1140 taaaaccttc gtaaacacca cgctcggtga gacgtgggag gcgaaaattg gcgaacgtcc 1200 ggatgctgaa gtgatggcag agcggaaaga gcattattca gcgcccgttc ctgaccgtgt 1260 ggcttacctg accgccggta tcgactccca gctggaccgc tacgaaatgc gcgtatgggg 1320 atgggggccg ggtgaggaaa gctggctgat tgaccggcag attattatgg gccgccacga 1380 cgatgaacag acgctgctgc gtgtggatga ggccatcaat aaaacctata cccgccggaa 1440 tggtgcagaa atgtcgatat cccgtatctg ctgggatact ggcgggattg acccgaccat 1500 tgtgtatgaa cgctcgaaaa aacatgggct gttccgggtg atccccatta aaggggcatc 1560 cgtctacgga aagccggtgg ccagcatgcc acgtaagcga aacaaaaacg gggtttacct 1620 taccgaaatc ggtacggata ccgcgaaaga gcagatttat aaccgcttca cactgacgcc 1680 ggaaggggat gaaccgcttc ccggtgccgt tcacttcccg aataacccgg atatttttga 1740 tctgaccgaa gcgcagcagc tgactgctga agagcaggtc gaaaaatggg tggatggcag 1800 gaaaaaaata ctgtgggaca gcaaaaagcg acgcaatgag gcactcgact gcttcgttta 1860 tgcgctggcg gcgctgcgca tcagtatttc ccgctggcag ctggatctca gtgcgctgct 1920 ggcgagcctg caggaagagg atggtgcagc aaccaacaag aaaacactgg cagattacgc 1980 ccgtgcctta tccggagagg atgaatgacg cgacaggaag aacttgccgc tgcccgtgcg 2040 gcactgcatg acctgatgac aggtaaacgg gtggcaacag tacagaaaga cggacgaagg 2100 gtggagttta cggccacttc cgtgtctgac ctgaaaaaat atattgcaga gctggaagtg 2160 cagaccggca tgacacagcg acgcagggga cctgcaggat tttatgtatg aaaacgccca 2220 ccattcccac ccttctgggg ccggacggca tgacatcgct gcgcgaatat gccggttatc 2280 acggcggtgg cagcggattt ggagggcagt tgcggtcgtg gaacccaccg agtgaaagtg 2340 tggatgcagc cctgttgccc aactttaccc gtggcaatgc ccgcgcagac gatctggtac 2400 gcaataacgg ctatgccgcc aacgccatcc agctgcatca ggatcatatc gtcgggtctt 2460 ttttccggct cagtcatcgc ccaagctggc gctatctggg catcggggag gaaagccc 2520 gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct 2580 gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc 2640 acgcctttaa cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt 2700 tccggacaca gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg 2760 acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt 2820 acgtcagcga ggacgggtat cctggctgga tgccgcagaa atggacatgg atacccccgtg 2880 agttacccgg cgggcgcgcc tcgttcattc acgtttttga acccgtggag gacgggcaga 2940 ctcgcggtgc aaatgtgttt tacagcgtga tggagcagat gaagatgctc gacacgctgc 3000 agaacacgca gctccagagc gccattgtga aggcgatgta tgccgccacc attgagagtg 3060 agctggatac gcagtcagcg atggatttta ttctgggcgc gaacagtcag gagcagcggg 3180. aaaggctgac cggctggatt ggtgaaattg ccgcgtatta cgccgcagcg ccggtccggc tgggaggcgc aaaagtaccg cacctgatc cgggtgactc actgaacctg cagacggctc 3240 aggatacgga taacggctac tccgtgtttg agcagtcact gctgcggtat atcgctgccg ggctgggtgt ctcgtatgag cagctttccc ggaattacgc ccagatgagc tactccacgg 3360 cacgggccag tgcgaacgag tcgtgggcgt actttatggg gcggcgaaaa ttcgtcgcat 3420. cccgtcaggc gagccagatg tttctgtgct ggctggaga ggccatcgtt cgccgcgtgg 3480 3540. tgacgttacc ttcaaaagcg cgcttcagtt ttcaggaagc ccgcagtgcc tgggggaact gcgactggat aggctccggt cgtatggcca tcgatggtct gaaagaagtt caggaagcgg tgatgctgat agaagccgga ctgagtacct acgagaaaga gtgcgcaaaa cgcggtgacg 3660 actatcagga aatttttgcc cagcaggtcc gtgaaacgat ggagcgccgt gcagccggtc ttaaccgcc cgcctgggcg gctgcagcat ttgaatccgg gctgcgacaa tcaacagagg aggagagag tgacagcaga gctgcgtaat ctcccgcata ttgccagcat ggcctttaat gagccgctga tgcttgaacc cgcctatgcg cggggttttct tttgtgcgct tgcaggccag 3900 cttgggatca gcagcctgac ggatgcggtg tccggcgaca gcctgactgc ccaggaggca 3960. ctcgcgacgc tggcattatc cggtgatgat gacggaccac gacaggcccg cagttatcag gtcatgaacg gcatcgccgt gctgccggtg tccggcacgc tggtcagccg gacgcggggcg 4080. ctgcagccgt actcggggat gaccggttac aacggcatta tcgcccgtct gcaacaggct gccagcgatc cgatggtgga cggcattctg ctcgatatgg acacgcccgg cgggatggtg 4200 gcggggggcat ttgactgcgc tgacatcatc gcccgtgtgc gtgacataaa accggtatgg 4260 gcgcttgcca acgacatgaa ctgcagtgca ggtcagttgc ttgccagtgc cgcctcccgg cgtctggtca cgcagaccgc ccggacaggc tccatcggcg tcatgatggc tcacagtaat 4380 4440. tacggtgctg cgctggagaa acagggtgtg gaaatcacgc tgatttacag cggcagccat aaggtggatg gcaaccccta cagccatctt ccggatgacg tccgggagac actgcagtcc 4500. cggatggacg caacccgcca gatgtttgcg cagaaggtgt cggcatatac cggcctgtcc 4560 gtgcaggttg tgctggatac cgaggctgca gtgtacagcg gtcaggaggc cattgatgcc 4620 ggactggctg atgaacttgt taacagcacc gatgcgatca ccgtcatgcg tgatgcactg 4680 gatgcacgta aatcccgtct ctcaggaggg cgaatgacca aagagactca atcaacaact 4740 gtttcagcca ctgcttcgca ggctgacgtt actgacgtgg tgccagcgac ggagggcgag 4800 aacgccagcg cggcgcagcc ggacgtgaac gcgcagatca ccgcagcggt tgcggcagaa 4860 aacagccgca ttatggggat cctcaactgt gaggaggctc acggacgga agaacaggca 4920 cgcgtgctgg cagaaacccc cggtatgacc gtgaaaacgg cccgccgcat tctggccgca 4980 gcaccacaga gtgcacaggc gcgcagtgac actgcgctgg atcgtctgat gcaggggca 5040 ccggcaccgc tggctgcagg taacccggca tctgatgccg ttaacgattt gctgaacaca 5100 ccagtgtaag ggatgtttat gacgagcaaa gaaaccttta cccattacca gccgcagggc 5160 aacagtgacc cggctcatac cgcaaccgcg cccggcggat tgagtgcgaa agcgcctgca 5220 atgaccccgc tgatgctgga cacctccagc cgtaagctgg ttgcgtggga tggcaccacc 5280 gacggtgctg ccgttggcat tcttgcggtt gctgctgacc agaccagcac cacgctgacg 5340 ttctacaagt ccggcacgtt ccgttatgag gatgtgctct ggccggaggc tgccagcgac 5400 gagacgaaaa aacggaccgc gtttgccgga acggcaatca gcatcgttta actttaccct 5460 tcatcactaa aggccgcctg tgcggctttt tttacgggat ttttttatgt cgatgtacac 5520 aaccgcccaa ctgctggcgg caaatgagca gaaatttaag tttgatccgc tgtttctgcg 5580 tctctttttc cgtgagagct atcccttcac cacggagaaa gtctatctct cacaaattcc 5640 gggactggta aacatggcgc tgtacgtttc gccgattgtt tccggtgagg ttatccgttc 5700 ccgtggcggc tccacctctg aatttacgcc gggatatgtc aagccgaagc atgaagtgaa 5760 tccgcagatg accctgcgtc gcctgccgga tgaagatccg cagaatctgg cggacccggc 5820 ttaccgccgc cgtcgcatca tcatgcagaa catgcgtgac gaagagctgg ccattgctca 5880 ggtcgaagag atgcaggcag tttctgccgt gcttaagggc aaatacacca tgaccggtga 5940 agccttcgat ccggttgagg tggatatggg ccgcagtgag gagaataaca tcacgcagtc 6000 cggcggcacg gagtggagca agcgtgacaa gtccacgtat gacccgaccg acgatatcga 6060 agcctacgcg ctgaacgcca gcggtgtggt gaatatcatc gtgttcgatc cgaaaggctg 6120 ggcgctgttc cgttccttca aagccgtcaa ggagaagctg gatacccgtc gtggctctaa 6180 ttccgagctg gagacagcgg tgaaagacct gggcaaagcg gtgtcctata aggggatgta 6240 tggcgatgtg gccatcgtcg tgtattccgg acagtacgtg gaaaacggcg tcaaaaagaa 6300 cttcctgccg gacaacacga tggtgctggg gaacactcag gcacgcggtc tgcgcaccta 6360 tggctgcatt caggatgcgg acgcacagcg cgaaggcatt aacgcctctg cccgttaccc 6420 gaaaaactgg gtgaccaccg gcgatccggc gcgtgagttc accatgattc agtcagcacc 6480 gctgatgctg ctggctgacc ctgatgagtt cgtgtccgta caactggcgt aatcatggcc 6540 cttcggggcc attgtttctc tgtggaggag tccatgacga aagatgaact gattgcccgt 6600 ctccgctcgc tgggtgaaca actgaaccgt gatgtcagcc tgacggggac gaaagaagaa 6660 ctggcgctcc gtgtggcaga gctgaaagag gagcttgatg acacggatga aactgccggt 6720 caggacaccc ctctcagccg ggaaaatgtg ctgaccggac atgaaaatga ggtgggatca 6780 gcgcagccgg ataccgtgat tctggatacg tctgaactgg tcacggtcgt ggcactggtg 6840 aagctgcata ctgatgcact tcacgccacg cgggatgaac ctgtggcatt tgtgctgccg 6900 ggaacggcgt ttcgtgtctc tgccggtgtg gcagccgaaa tgacagagcg cggcctggcc 6960 agaatgcaat aacgggaggc gctgtggctg atttcgataa cctgttcgat gctgccattg 7020 cccgcgccga tgaaacgata cgcgggtaca tgggaacgtc agccaccatt acatccggtg 7080 agcagtcagg tgcggtgata cgtggtgttt ttgatgaccc tgaaaatatc agctatgccg 7140 gacagggcgt gcgcgttgaa ggctccagcc cgtccctgtt tgtccggact gatgaggtgc 7200 ggcagctgcg gcgtggagac acgctgacca tcggtgagga aaatttctgg gtagatcggg 7260 tttcgccgga tgatggcgga agttgtcatc tctggcttgg acggggcgta ccgcctgccg 7320 ttaaccgtcg ccgctgaaag ggggatgtat ggccataaaa ggtcttgagc aggccgttga 7380 aaacctcagc cgtatcagca aaacggcggt gcctggtgcc gccgcaatgg ccattaaccg 7440 cgttgcttca tccgcgatat cgcagtcggc gtcacaggtt gcccgtgaga caaaggtacg 7500 ccggaaactg gtaaaggaaa gggccaggct gaaaagggcc acggtcaaaa atccgcaggc 7560 cagaatcaaa gttaaccggg gggatttgcc cgtaatcaag ctgggtaatg cgcgggttgt 7620 cctttcgcgc cgcaggcgtc gtaaaaaggg gcagcgttca tccctgaaag gtggcggcag 7680 cgtgcttgtg gtgggtaacc gtcgtattcc cggcgcgttt attcagcaac tgaaaaatgg 7740 ccggtggcat gtcatgcagc gtgtggctgg gaaaaaccgt taccccattg atgtggtgaa 7800 aatcccgatg gcggtgccgc tgaccacggc gtttaaacaa aatattgagc ggatacggcg 7860 tgaacgtctt ccgaaagagc tgggctatgc gctgcagcat caactgagga tggtaataaa 7920 gcgatgaaac atactgaact ccgtgcagcc gtactggatg cactggagaa gcatgacacc 7980 ggggcgacgt tttttgatgg tcgccccgct gtttttgatg aggcggattt tccggcagtt 8040 gccgtttatc tcaccggcgc tgaatacacg ggcgaagagc tggacagcga tacctggcag 8100 gcggagctgc atatcgaagt tttcctgcct gctcaggtgc cggattcaga gctggatgcg 8160 tggatggagt cccggattta tccggtgatg agcgatatcc cggcactgtc agatttgatc 8220 accagtatgg tggccagcgg ctatgactac cggcgcgacg atgatgcggg cttgtggagt 8280 tcagccgatc tgacttatgt cattacctat gaaatgtgag gacgctatgc ctgtaccaaa 8340 tcctacaatg ccggtgaaag gtgccgggac caccctgtgg gtttataagg ggagcggtga 8400 cccttacgcg aatccgcttt cagacgttga ctggtcgcgt ctggcaaaag ttaaagacct 8460 gacgcccggc gaactgaccg ctgagtccta tgacgacagc tatctcgatg atgaagatgc 8520 agactggact gcgaccgggc aggggcagaa atctgccgga gataccagct tcacgctggc 8580 gtggatgccc ggagagcagg ggcagcaggc gctgctggcg tggtttaatg aaggcgatac 8640 ccgtgcctat aaaatccgct tcccgaacgg cacggtcgat gtgttccgtg gctgggtcag 8700 cagtatcggt aaggcggtga cggcgaagga agtgatcacc cgcacggtga aagtcaccaa 8760 tgtgggacgt ccgtcgatgg cagaagatcg cagcacggta acagcggcaa ccggcatgac 8820 cgtgacgcct gccagcacct cggtggtgaa agggcagagc accacgctga ccgtggcctt 8880 ccagccggag ggcgtaaccg acaagagctt tcgtgcggtg tctgcggata aaacaaaagc 8940 caccgtgtcg gtcagtggta tgaccatcac cgtgaacggc gttgctgcag gcaaggtcaa 9000 cattccggtt gtatccggta atggtgagtt tgctgcggtt gcagaaatta ccgtcaccgc 9060 cagttaatcc ggagagtcag cgatgttcct gaaaaccgaa tcatttgaac ataacggtgt 9120 gaccgtcacg ctttctgaac tgtcagccct gcagcgcatt gagcatctcg ccctgatgaa 9180 acggcaggca gaacaggcgg agtcagacag caaccggaag tttactgtgg aagacgccat 9240 cagaaccggc gcgtttctgg tggcgatgtc cctgtggcat aaccatccgc agaagacgca 9300 gatgccgtcc atgaatgaag ccgttaaaca gattgagcag gaagtgctta ccacctggcc 9360 cacggaggca atttctcatg ctgaaaacgt ggtgtaccgg ctgtctggta tgtatgagtt 9420 tgtggtgaat aatgcccctg aacagacaga ggacgccggg cccgcagagc ctgtttctgc 9480 gggaaagtgt tcgacggtga gctgagtttt gccctgaaac tggcgcgtga gatggggcga 9540 <h2 style=";text-align:left;direction:ltr">cccgactggc gtgccatgct tgccgggatg tcatccacgg agtatgccga ctggcaccgc 9600<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ttttacagta cccattattt tcatgatgtt ctgctggata tgcacttttc cgggctgacg 9660<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tacaccgtgc tcagcctgtt tttcagcgat ccggatatgc atccgctgga tttcagtctg 9720<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgaaccggc gcgaggctga cgaagagcct gaagatgatg tgctgatgca gaaagcggca 9780<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gggcttgccg gaggtgtccg ctttggcccg gacgggaatg aagttatccc cgcttccccg 9840<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatgtggcgg acatgacgga ggatgacgta atgctgatga cagtatcaga agggatcgca 9900<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggaggagtcc ggtatggctg aaccggtagg cgatctggtc gttgatttga gtctggatgc 9960<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ggccagattt gacgagcaga tggccagagt caggcgtcat ttttctggta cggaaagtga 10020<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tgcgaaaaaa acagcggcag tcgttgaaca gtcgctgagc cgacaggcgc tggctgcaca 10080<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaaagcgggg atttccgtcg ggcagtataa agccgccatg cgtatgctgc ctgcacagtt 10140<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> caccgacgtg gccacgcagc ttgcaggcgg gcaaagtccg tggctgatcc tgctgcaaca 10200<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gggggggcag gtgaaggact ccttcggcgg gatgatcccc atgttcaggg ggcttgccgg 10260<h2 style=";text-align:left;direction:ltr"> tgcgatcacc ctgccgatgg tgggggccac ctcgctggcg gtggcgaccg gtgcgctggc 10320 gtatgcctgg tatcagggca actcaaccct gtccgatttc aacaaaacgc tggtcctttc 10380. cggcaatcag gcgggactga cggcagatcg tatgctggtc ctgtccagag ccgggcaggc ggcagggctg acgtttaacc agaccagcga gtcactcagc gcactggtta aggcgggggt 10500. aagcggtgag gctcagattg cgtccatcag ccagagtgtg gcgcgtttct cctctgcatc 10560 cggcgtggag gtggacaagg tcgctgaagc cttcgggaag ctgaccacag acccgacgtc ggggctgacg gcgatggctc gccagttcca taacgtgtcg gcggagcaga ttgcgtatgt 10680 tgctcagttg cagcgttccg gcgatgaagc cggggcattg caggcggcga acgaggccgc 10740 aacgaaaggg tttgatgacc agacccgccg cctgaaagag aacatgggca cgctggagac ctgggcagac aggactgcgc gggcattcaa atccatgtgg gatgcggtgc tggatattgg 10860 tcgtcctgat accgcgcagg agatgctgat taaggcagag gctgcgtata agaaagcaga 10920 cgacatctgg aatctgcgca aggatgatta ttttgttaac gatgaagcgc gggcgcgtta 10980. ctgggatgat cgtgaaaagg cccgtcttgc gcttgaagcc gcccgaaaga aggctgagca gcagactcaa caggacaaaa atgcgcagca gcagagcgat accgaagcgt cacggctgaa atataccga gaggcgcaga aggcttacga acggctgcag acgccgctgg agaaatatac cgcccgtcag gaagaactga acaaggcact gaaagacggg aaaatcctgc aggcggatta caacacgctg atggcggcgg cgaaaagga ttatgaagcg acgctgaaaa agccgaaaca gtccagcgtg aaggtgtctg cgggcgatcg tcaggaagac agtgctcatg ctgccctgct gacgcttcag gcagaactcc ggacgctgga gagcatgcc ggagcaaatg agaaaatcag ccagcagcgc cgggatttgt ggaggcgga gagtcagttc gcggtactgg aggaggcggc 11460 gcaacgtcgc cagctgtctg cacaggaga atccctgctg gcgcataaag atgagacgct ggagtacaaa cgccagctgg ctgcacttgg cgacaaggtt acgtatcagg agcgcctgaa cgcgctggcg cagcaggcgg ataattcgc acagcagcaa cgggcaaaac gggccgccat tgatgcgaaa agccggggc tgactgaccg gcaggcagaa cgggaagcca cggaacagcg 11700 11760 aaagacctgg gcggctgaag accagcttcg cgggaactgg atggcaggcc tgaagtccgg 11820 ctggagtgag tgggaaga gcgccacgga foottgtcg caggtaaaaa gtgcagccac 11880 gcagaccttt gatggtattg cagaatat ggcggcgatg ctgaccggca gtgagcagaa 11940 ctggcgcagc ttcaccccgtt ccgtgctgtc catgatgaca gaaattctgc ttaagcaggc 12000 aatggtgggg attgtcggga gtatcggcag cgccattggc ggggctggtg gtggcggcgc 12060 atccgcgtca ggcggtacag ccattcaggc cgctgcggcg aaattccatt ttgcaaccgg 12120 aggatttacg ggaaccggcg gcaatatga gccagcgggg attgttcacc gtggtgagtt 12180 tgtcttcacg aaagggcaa ccagccggat tggcgtgggg aatctttacc ggctgatgcg 12240 cggctatgcc accggcggtt atgtcggtac accgggcagc atggcagaca gccggtcgca 12300 ggcgtccggg acgtttgagc agaataacca tgtggtgatt aacaacgacg gcacgaacgg 12360 gcagataggt ccggctgctc tgaaggcggt gtatgacatg gcccgcaagg gtgcccgtga 12420 tgaaattcag acacagatgc gtgatggtgg cctgttctcc ggaggtggac gatgaagacc 12480 ttccgctgga aagtgaaacc cggtatggat gtggcttcgg tcccttctgt aagaaaggtg 12540 cgctttggtg atggctattc tcagcgagcg cctgccgggc tgaatgccaa cctgaaaacg 12600 tacagcgtga cgctttctgt cccccgtgag gaggccacgg tactggagtc gtttctggaa 12660 gagcacgggg gctggaaatc ctttctgtgg acgccgcctt atgagtggcg gcagataaag 12720 gtgacctgcg caaaatggtc gtcgcgggtc agtatgctgc gtgttgagtt cagcgcagag 12780 tttgaacagg tggtgaactg atgcaggata tccggcagga aacactgaat gaatgcaccc 12840 gtgcggagca gtcggccagc gtggtgctct gggaaatcga cctgacagag gtcggtggag 12900 aacgttattt tttctgtaat gagcagaacg aaaaaggtga gccggtcacc tggcaggggc 12960 gacagtatca gccgtatccc attcagggga gcggttttga actgaatggc aaaggcacca 13020 gtacgcgccc cacgctgacg gtttctaacc tgtacggtat ggtcaccggg atggcggaag 13080 atatgcagag tctggtcggc ggaacggtgg tccggcgtaa ggtttacgcc cgttttctgg 13140 atgcggtgaa cttcgtcaac ggaaacagtt acgccgatcc ggagcaggag gtgatcagcc 13200 gctggcgcat tgagcagtgc agcgaactga gcgcggtgag tgcctccttt gtactgtcca 13260 cgccgacgga aacggatggc gctgtttttc cgggacgtat catgctggcc aacacctgca 13320 cctggaccta tcgcggtgac gagtgcggtt atagcggtcc ggctgtcgcg gatgaatatg 13380 accagccaac gtccgatatc acgaaggata aatgcagcaa atgcctgagc ggttgttaagt 13440 tccgcataa cgtcggcaac tttggcggct tcctttccat taacaaactt tcgcagtaaa 13500 tcccatgaca cagacagaat cagcgattct ggcgcacgcc cggcgatgtg cgccagcgga 13560 gtcgtgcggc ttcgtggtaa gcacgccgga gggggaaaga tatttcccct gcgtgaatat 13620 ctccggtgag ccggaggcta tttccgtatg tcgccggaag actggctgca ggcagaaatg 13680 cagggtgaga ttgtggcgct ggtccacagc caccccggtg gtctgccctg gctgagtgag 13740 gccgaccggc ggctgcaggt gcagagtgat ttgccgtggt ggctggtctg ccgggggacg 13800 attcataagt tccgctgtgt gccgcatctc accgggcggc gctttgagca cggtgtgacg 13860 gactgttaca cactgttccg ggatgcttat catctggcgg ggattgagat gccggacttt 13920 catcgtgagg atgactggtg gcgtaacggc cagaatctct atctggataa tctggaggcg 13980 acggggctgt atcaggtgcc gttgtcagcg gcacagccgg gcgatgtgct gctgtgctgt 14040 tttggttcat cagtgccgaa tcacgccgca atttactgcg gcgacggcga gctgctgcac 14100 catattcctg aacaactgag caaacgagag aggtacaccg acaaatggca gcgacgcaca 14160 cactccctct ggcgtcaccg ggcatggcgc gcatctgcct ttacggggat ttacaacgat 14220 ttggtcgccg catcgacctt cgtgtgaaaa cgggggctga agccatccgg gcactggcca 14280 cacagctccc ggcgtttcgt cagaaactga gcgacggctg gtatcaggta cggattgccg 14340 ggcgggacgt cagcacgtcc gggttaacgg cgcagttaca tgagactctg cctgatggcg 14400 ctgtaattca tattgttccc agagtcgccg gggccaagtc aggtggcgta ttccagattg 14460 tcctggggc tgccgccatt gccggatcat tctttaccgc cggagccacc cttgcagcat 14520 ggggggcagc cattggggcc ggtggtatga ccggcatcct gttttctctc ggtgccagta 14580 tggtgctcgg tggtgtggcg cagatgctgg caccgaaagc cagaactccc cgtatacaga 14640 caacggata cggtaagcag aacacctatt tctcctcact ggataacatg gttgcccagg 14700 gcaatgttct gcctgttctg tacgggggaaa tgcgcgtggg gtcacgcgtg gtttctcagg 14760 agatcagcac ggcaggaa ggggacggtg gtcaggttgt ggtgattggt cgctgatgca 14820 aaatgtttta tgtgaaaccg cctgcgggcg gttttgtcat ttatggagcg tgaggaatgg 14880 gtaaaggaag cattaagggg cataccccgc gcgaagcgaa ggacaacctg aagtccacgc 14940 agttgctgag tgtgatcgat gccatcagcg aagggccgat tgaaggtccg gtggatggct 15000 taaaaagcgt gctgctgaac agtacgccgg tgctggacac tgagggaat accaacatat 15060 ccggtgtcac ggtggtgttc cgggctggtg agcaggagca gactccgccg gagggatttg 15120 aatcctccgg ctccgagacg gtgctgggta cggaagtgaa atatgacacg ccgatcaccc 15180 gcaccattac gtctgcaaac atcgaccgtc tgcgctttac cttcggtgta caggcactgg 15240 tggaaaccac ctcaaagggt gacaggaatc cgtcggaagt ccgcctgctg gttcagatac 15300 aacgtaacgg tggctgggtg acggaaaaag acatcaccat taagggcaaa accacctcgc 15360 agtatctggc ctcggtggtg atgggtaacc tgccgccgcg cccgtttaat atccggatgc 15420 gcaggatgac gccggacagc accacagacc agctgcagaa caaaacgctc tggtcgtcat 15480 acactgaaat catcgatgtg aaacagtgct acccgaacac ggcactggtc ggcgtgcagg 15540 tggactcgga gcagttcggc agccagcagg tgagccgtaa ttatcatctg cgcgggcgta 15600 ttctgcaggt gccgtcgaac tataacccgc agacgcggca atacagcggt atctgggacg 15660 gaacgtttaa accggcatac agcaacaaca tggcctggtg tctgtgggat atgctgaccc 15720 atccgcgcta cggcatgggg aaacgtcttg gtgcggcgga tgtggataaa tgggcgctgt 15780 atgtcatcgg ccagtactgc gaccagtcag tgccggacgg ctttggcggc acggagccgc 15840 gcatcacctg taatgcgtac ctgaccacac agcgtaaggc gtgggatgtg ctcagcgatt 15900 tctgctcggc gatgcgctgt atgccggtat ggaacgggca gacgctgacg ttcgtgcagg 15960 accgaccgtc ggataagacg tggacctata accgcagtaa tgtggtgatg ccggatgatg 16020 gcgcgccgtt ccgctacagc ttcagcgccc tgaaggaccg ccataatgcc gttgaggtga 16080 actggattga cccgaacaac ggctgggaga cggcgacaga gcttgttgaa gatacgcagg 16140 ccattgcccg ttacggtcgt aatgttacga agatggatgc ctttggctgt accagccggg 16200 ggcaggcaca ccgcgccggg ctgtggctga ttaaaacaga actgctggaa acgcagaccg 16260 tggatttcag cgtcggcgca gaagggcttc gccatgtacc gggcgatgtt attgaaatct 16320 gcgatgatga ctatgccggt atcagcaccg gtggtcgtgt gctggcggtg aacagccaga 16380 cccggacgct gacgctcgac cgtgaaatca cgctgccatc ctccggtacc gcgctgataa 16440 gcctggttga cggaagtggc aatccggtca gcgtggaggt tcagtccgtc accgacggcg 16500 tgaaggtaaa agtgagccgt gttcctgacg gtgttgctga atacagcgta tgggagctga 16560 agctgccgac gctgcgccag cgactgttcc gctgcgtgag tatccgtgag aacgacgacg 16620 gcacgtatgc catcaccgcc gtgcagcatg tgccggaaaa agaggccatc gtggataacg 16680 gggcgcactt tgacggcgaa cagagtggca cggtgaatgg tgtcacgccg ccagcggtgc 16740 agcacctgac cgcagaagtc actgcagaca gcggggaata tcaggtgctg gcgcgatggg 16800 acacaccgaa ggtggtgaag ggcgtgagtt tcctgctccg tctgaccgta acagcggacg 16860 acggcagtga gcggctggtc agcacggccc ggacgacgga aaccacatac cgcttcacgc 16920 aactggcgct ggggaactac aggctgacag tccgggcggt aaatgcgtgg gggcagcagg 16980 gcgatccggc gtcggtatcg ttccggattg ccgcaccggc agcaccgtcg aggattgagc 17040 tgacgccggg ctattttcag ataaccgcca cgccgcatct tgccgtttat gacccgacgg 17100 tacagtttga gttctggttc tcggaaaagc agattgcgga tatcagacag gttgaaacca 17160 gcacgcgtta tcttggtacg gcgctgtact ggatagccgc cagtatcaat atcaaccgg 17220 gccatgatta ttacttat atccgcagtg tgaacaccgt tggcaatcg gcattcgtgg 17280 aggccgtcgg tcggcgagc gatgatgcgg aaggttacct ggatttttc aaggcaaga 17340 taaccgaatc ccatctcggc aaggagctgc tggaaaagt cgagctgacg gaggataacg 17400 cagcagact ggaggatttt tcgaagagt ggaaggatgc cagtgataag tggaatgcca 17460 tgtgggctgt caaattgag cagaccaaag acggcaaaca ttatgtcgcg ggtattggcc 17520 tcagcatgga ggacacggag gaagcaac tgagcagtt tctggttgcc gccaatcgta 17580 tcgcatttat tgacccggca aacgggaatg aaacgccgat gttgtggcg cagggcaacc 17640 agatattcat gaacgacgtg ttcctgaagc gcctgacggc ccccatt accagcggcg 17700 gcaatcctcc ggccttttcc ctgacaccgg acggaagct gaccctaa atgcggata 17760 tcagtggcag tgtgaatgcg aactccggga cgctcagtaa tgtgacgata gctgaaaact 17820 gtacgataaa cggtacgctg agggcggaa aaatcgtcgg ggacattgta aaggcggcga 17880 gcgcggcttt tccgcgccag cgtgaaagca gtgtggactg gccgtcaggt acccgtactg 17940 tcaccgtgac cgatgaccat ccttttgatc gccagatagt ggtgcttccg ctgacgtttc 18000 gcggaagtaa gcgtactgtc agcggcagga caacgtattc gatgtgttat ctgaaagtac 18060 tgatgaacgg tgcggtgatt tatgatggcg cggcgaacga ggcggtacag gtgttctccc 18120 gtattgttga catgccagcg ggtcggggaa acgtgatcct gacgttcacg cttacgtcca 18180 cacggcattc ggcagatatt ccgccgtata cgtttgccag cgatgtgcag gttatggtga 18240 ttaagaaaca ggcgctgggc atcagcgtgg tctgagtgtg ttacagaggt tcgtccggga 18300 acgggcgttt tattataaaa cagtgagagg tgaacgatgc gtaatgtgtg tattgccgtt 18360 gctgtctttg ccgcacttgc ggtgacagtc actccggccc gtgcggaagg tggacatggt 18420 acgtttacgg tgggctattt tcaagtgaaa ccgggtacat tgccgtcgtt gtcgggcggg 18480 gataccggtg tgagtcatct gaaagggatt aacgtgaagt accgttatga gctgacggac 18540 agtgtgggg tgatggcttc cctggggttc gccgcgtcga aaaagagcag cacagtgatg 18600 accgggggagg atacgtttca ctatgagagc ctgcgtggac gttatgtgag cgtgatggcc 18660 ggaccggttt tacaaatcag taagcaggtc agtgcgtacg ccatggccgg agtggctcac 18720 agtcggtggt ccggcagtac aatggattac cgtaagacgg aaatcactcc cgggtatatg 18780 aaaagagga ccactgccag ggacgaaagt gcaatgcggc atacctcagt ggcgtggagt 18840 gcaggtatac agattaatcc ggcagcgtcc gtcgttgttg atattgctta tgaaggctcc 18900 ggcagtggcg actggcgtac tgacggattc atcgttgggg tcggttataa attctgatta 18960 gccaggtaac acagtgttat gacagcccgc cggaaccggt gggcttttt gtggggtgaa 19020 tatggcagta aagatttcag gagtcctgaa agacggcaca ggaaaaccgg tacagaactg 19080 caccattcag ctgaaagcca gacgtaacag caccacggtg gtggtgaaca cggtgggctc 19140 agagaatccg gatgaagccg ggcgttacag catggatgtg gagtacggtc agtacagtgt 19200 catcctgcag gttgacggtt ttccaccatc gcacgccggg accatcaccg tgtatgaaga 19260 ttcacaaccg gggacgctga atgattttct ctgtgccatg acggaggatg atgcccggcc 19320 ggaggtgctg cgtcgtcttg aactgatggt ggaagaggtg gcgcgtaacg cgtccgtggt 19380 ggcacagagt acggcagacg cgaagaaatc agccggcgat gccagtgcat cagctgctca 19440 ggtcgcggcc cttgtgactg atgcaactga ctcagcacgc gccgccagca cgtccgccgg 19500 acaggctgca tcgtcagctc aggaagcgtc ctccggcgca gaagcggcat cagcaaaggc 19560 cactgaagcg gaaaaaagtg ccgcagccgc agagtcctca aaaaacgcgg cggccaccag 19620 tgccggtgcg gcgaaaacgt cagaaacgaa tgctgcagcg tcacaacaat cagccgccac 19680 gtctgcctcc accgcggcca cgaaagcgtc agaggccgcc acttcagcac gagatgcggt 19740 ggcctcaaaa gaggcagcaa aatcatcaga aacgaacgca tcatcaagtg ccggtcgtgc 19800 agcttcctcg gcaacggcgg cagaaaattc tgccagggcg gcaaaaacgt ccgagacgaa 19860 tgccaggtca tctgaaacag cagcggaacg gagcgcctct gccgcggcag acgcaaaaac 19920 agcggcggcg gggagtgcgt caacggcatc cacgaaggcg acagaggctg cgggaagtgc 19980 ggtatcagca tcgcagagca aaagtgcggc agaagcggcg gcaatacgtg caaaaaattc 20040 ggcaaaacgt gcaagagata tagcttcagc tgtcgcgctt gaggatgcgg acacaacgag 20100 aaaggggata gtgcagctca gcagtgcaac caacagcacg tctgaaacgc ttgctgcaac 20160 gccaaaggcg gttaaggtgg taatggatga aacgaacaga aaagcccact ggacagtccg 20220 gcactgaccg gaacgccaac agcaccaacc gcgctcaggg gaacaaacaa tacccagatt 20280 gcgaacaccg cttttgtact ggccgcgatt gcagatgtta tcgacgcgtc acctgacgca 20340 ctgaatacgc tgaatgaact ggccgcagcg ctcgggaatg atccagattt tgctaccacc 20400 atgactaacg cgcttgcggg taaacaaccg aagaatgcga cactgacgg gctggcaggg 20460 ctttccacgg cgaaaaataa attaccgtat tttgcggaaa atgatgccgc cagcctgact 20520 gaactgactc aggttggcag ggaattctg gcaaaaaatt ccgttgcaga tgttcttgaa 20580 taccttgggg ccggtgagaa ttcggccttt ccggcaggtg cgccgatccc gtggccatca 20640 gatatcgttc cgtctggcta cgtcctgatg caggggcagg cgtttgacaa atcagcctac 20700 ccaaaacttg ctgtcgcgta tccatcgggt gtgcttcctg atatgcgagg ctggacaatc 20760 aaggggaaac ccgccagcgg tcgtgctgta ttgtctcagg aacaggatgg aattaagtcg 20820 cacacccaca gtgccagtgc atccggtacg gatttgggga cgaaaaccac atcgtcgttt 20880 gattacggga cgaaaacaac aggcagtttc gattacggca ccaaatcgac gaataacacg 20940 ggggctcatg ctcacagtct gagcggttca acaggggccg cgggtgctca tgcccacaca 21000 agtggtttaa ggatgaacag ttctggctgg agtcagtatg gaacagcaac cattacagga 21060 agtttatcca cagttaaagg aaccagcaca cagggtattg cttatttatc gaaaacggac 21120 agtcagggca gccacagtca ctcattgtcc ggtacagccg tgagtgccgg tgcacatgcg 21180 catacagttg gtattggtgc gcaccagcat ccggttgtta tcggtgctca tgcccattct 21240 ttcagtattg gttcacacgg acacaccatc accgttaacg ctgcgggtaa cgcggaaaac 21300 accgtcaaaa acattgcatt taactatatt gtgaggcttg catatggca ttcagaatga gtgaacaacc aacggaccata aaaattata atctgctggc cggaactaat gaatttattg gtgaaggtga cgcatatatt ccgcctcata ccggtctgcc tgcaaacagt accgatattg 21540. caccgccaga tattccggct ggctttgtgg ctgttttcaa cagtgatgag gcatcgtggc atctcgttga agaccatcgg ggtaaaaccg tctatgacgt ggcttccggc gacgcgttat ttatttctga actcggtccg ttaccggaa attttacctg gttatcgccg ggaggggat atcagaagtg gaacggcaca gcctgggtga aggatacgga agcagaaaaa ctgttccgga tccgggaggc ggaagaaca aaaaaaagcc tgatgcaggt agccagtgag catattgcgc cgcttcagga tgctgcagat ctggaaattg caacgaagga agaaacctcg ttgctggaag cctggaagaa gtatcgggtg ttgctgaacc gtgttgatac atcaactgca cctgatattg agtggcctgc tgtccctgtt atggagtaat cgttttgtga tatgccgcag aaacgttgta tgaaataacg ttctgcggtt agttagtata ttgtaaagct gagtattggt tttttggcg attattatct tcaggagaat aatggaagtt ctatgactca attgttcata gtgtttacat 22080 caccgccaat tgcttttaag actgaacgca tgaaatatgg tttttcgtca tgttttgagt 22140 ctgctgttga tatttctaaa gtcggttttt tttcttcgtt ttctctaact attttccatg 22200 aaatacattt ttgattatta tttgaatcaa ttccaattac ctgaagtctt tcatctataa 22260 ttggcattgt atgtattggt ttattggagt agatgcttgc ttttctgagc catagctctg 22320 atatccaaat gaagccatag gcatttgtta ttttggctct gtcagctgca taacgccaaa 22380 aaatatattt atctgcttga tcttcaaatg ttgtattgat taaatcaatt ggatggaatt 22440 gtttatcata aaaaattaat gtttgaatgt gataaccgtc ctttaaaaaa gtcgtttctg 22500 caagcttggc tgtatagtca actaactctt ctgtcgaagt gatattttta ggcttatcta 22560 ccagttttag acgctcttta atatcttcag gaattatttt attgtcatat tgtatcatgc 22620 taaatgacaa tttgcttatg gagtaatctt ttaattttaa ataagttatt ctcctggctt 22680 catcaaataa agagtcgaat gatgttggcg aaatcacatc gtcacccatt ggattgttta 22740 tttgtatgcc aagagagtta cagcagttat acattctgcc atagattata gctaaggcat 22800 gtaataattc gtaatctttt agcgtattag cgacccatcg tctttctgat ttaataatag 22860 atgattcagt taaatatgaa ggtaatttct tttgtgcaag tctgactaac ttttttatac 22920 caatgtttaa catactttca tttgtaataa actcaatgtc attttcttca atgtaagatg 22980 aaataagagt agcctttgcc tcgctataca tttctaaatc gccttgtttt tctatcgtat 23040 tgcgagaatt tttagcccaa gccattaatg gatcattttt ccatttttca ataacattat 23100 tgtatacca aatgtcatat cctataatct ggtttttgtt ttttgaata ataaatgtta 23160 ctgttcttgc ggtttggagg aattgattca aattcaagcg aaatattca gggtcaaaat 23220 atgtatcaat gcagcatttg agcaagtgcg ataaatcttt aagtcttctt tcccatggtt 23280 ttttagtcat aaaactctcc atttgatag gttgcatgct agatgctgat atattttaga 23340 ggtgataaaa ttaactgctt aactgtcaat gtaatacaag ttgtttgatc tttgcaatga 23400 ttcttatcag aaaccatata gtaaattagt tacacaggaa atttttaata ttattattat 23460 cattcattat gtattaaaat tagagttgtg gcttggctct gctaacacgt tgctcatagg 23520 agatatggta gagccgcaga cacgtcgtat gcaggaacgt gctgcggctg gctggtgaac 23580 ttccgatagt gcgggtgttg aatgatttcc agttgctacc gattttacat atttttgca 23640 tgagagaatt tgtaccacct cccaccgacc atctatgact gtacgccact gtccctagga 23700 ctgctatgtg ccggagcgga cattacaaac gtccttctcg gtgcatgcca ctgttgccaa 23760 tgacctgcct aggaattggt tagcaagtta ctaccggatt ttgtaaaaac agccctcctc 23820 atataaaaag tattcgttca cttccgataa gcgtcgtaat tttctatctt tcatcatatt 23880 ctagatccct ctgaaaaaat cttccgagtt tgctaggcac tgatacataa ctcttttcca 23940 ataattgggg aagtcattca aatctataat aggtttcaga tttgcttcaa taaattctga 24000 ctgtagctgc tgaaacgttg cggttgaact atattcctt ataactttta cgaaagagtt 24060 tctttgagta atcacttcac tcaagtgctt ccctgcctcc aaacgatacc tgttagcaat 24120 atttaatagc ttgaaatgat gaagagctct gtgtttgtct tcctgcctcc agttcgccgg 24180 gcattcaaca taaaaactga tagcacccgg agttccggaa acgaaatttg catataccca 24240 ttgctcacga aaaaaaaatgt ccttgtcgat atagggatga atcgcttggt gtacctcatc 24300 tactgcgaaa acttgacctt tctctcccat attgcagtcg cggcacgatg gaactaaatt 24360 aataggcatc accgaaaatt caggataatg tgcaatagga agaaaatgat ctatattttt 24420 tgtctgtcct atatcaccac aaaatggaca tttttcacct gatgaaacaa gcatgtcatc 24480 gtaatatgtt ctagcgggtt tgtttttatc tcggagatta tttcataaa gctttcttaa 24540 tttaaccttt gtcaggttac caactactaa ggttgtaggc tcaagagggt gtgtcctgtc 24600 gtaggtaaat aactgacctg tcgagcttaa tattctatat tgttgttctt tctgcaaaaa 24660 agtgggggaag tgagtaatga aattattct aacatttatc tgcatcatac cttccgagca 24720 tttattaagc atttcgctat aagttctcgc tggaagaggt agttttttca ttgtacttta 24780 ccttcatctc tgttcattat catcgctttc aaaacggttc gaccttctaa tcctatctga 24840 ccattataat ttttagaat ggtttcataa gaaagctctg aatcaacgga ctgcgataat 24900 aagtggtggt atccagaatt tgtcacttca agtaaaaaca cctcacgagt taaaacacct 24960 aagttctcac cgaatgtctc aatatccgga cggataaatat ttattgcttc tcttgaccgt 25020 aggactttcc acatgcagga ttttggaacc tcttgcagta ctactgggga atgagttgca 25080 attattgcta caccattgcg tgcatcgagt aagtcgctta atgttcgtaa aaaagcagag 25140 agcaaaggtg gatgcagatg aacctctggt tcatcgaata aaactaatga cttttcgcca 25200 acgacatcta ctaatcttgt gatagtaaat aaaacaattg catgtccaga gctcattcga 25260 agcagatatt tctggatatt gtcataaaac aatttagtga attatcatc gtccacttga 25320 atctgtggtt cattacgtct taactcttca tatttagaaa tgaggctgat gagttccata 25380 tttgaaaagt ttcatcact acttagtttt ttgatagctt caagccagag ttgtcttttt 25440 ctatctactc tcatacaacc aataatgct gaaatgaatt ctaagcggag atcgcctagt gattttaaac tattgctggc agcattcttg agtccaatat aaaagtattg tgtacctttt gctgggtcag gttgttcttt aggaggagta aaaggatcaa atgcactaaa cgaaactgaa acaagcgatc gaaaatatcc ctttgggatt cttgactcga tagtctatt attttcagag aaaaaatatt cattgttttc tgggttggtg attgcaccaa tcattccatt caaattgtt gttttaccac acccattccg cccgataaaa gcatgaatgt tcgtgctggg catagaatta accgtcacct caaaaggtat agttaatca ctgaatccgg gagcactttt tctattaat gaaaagtgga aatctgacaa ttctggcaaa ccatttaaca cacgtgcgaa ctgtccatga atttctgaaa gagttacccc tctaagtaat gaggtgttaa ggacgctttc attttcaatg tcggctaatc gatttggcca tactactaa tcctgaatag ctttaagaag gttatgttta aaaccatcgc ttaatttgct tagtagc tagtagtca tgctttcacc tagaaaa aacatttcag ggagttgact gaattttta tctattaatg aatagtgct tacttcttct ttttgaccta caaaaccaat tttaacattt ccgatatcgc atttttcacc atgctcatca 26220 aagacagtaa gataaaacat tgtaacaaag gaatagtcat tccaaccatc tgctcgtagg 26280 aatgccttat ttttttctac tgcaggaata tacccgcctc tttcaataac actaaactcc 26340 aacatatagt aacccttaat tttattaaaa taaccgcaat ttatttggcg gcaacacagg 26400 atctctcttt taagttactc tctattacat acgttttcca tctaaaaatt agtagtattg 26460 aacttaacgg ggcatcgtat tgtagttttc catatttagc tttctgcttc cttttggata 26520 acccactgtt attcatgttg catggtgcac tgtttatacc aacgatatag tctattaatg 26580 catatagt atcgccgaac gattagctct tcaggcttct gaagaagcgt ttcaagtact 26640 aataagccga tagatagcca cggacttcgt agccattttt cataagtgtt aacttccgct 26700 cctcgctcat aacagacatt cactacagtt atggcggaaa ggtatgcatg ctgggtgtgg 26760 ggaagtcgtg aaagaaaaga agtcagctgc gtcgtttgac atcactgcta tcttcttact 26820 ggttatgcag gtcgtagtgg gtggcacaca aagctttgca ctggattgcg aggctttgtg 26880 cttctctgga gtgcgacagg tttgatgaca aaaaattagc gcaagaagac aaaaatcacc 26940 ttgcgctaat gctctgttac aggtcactaa taccatctaa gtagttgatt catagtgact 27000 gcatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata 27060 tattgatatt tatatcattt tacgtttctc gttcagcttt tttatactaa gttggcatta 27120 taaaaaagca ttgcttatca atttgttgca acgaacaggt cactatcagt caaaataaaa 27180 tcattattg atttcaattt tgtcccactc cctgcctctg tcatcacgat actgtgatgc 27240 catggtgtcc gacttatgcc cgagaagatg ttgagcaaac ttatcgctta tctgcttctc 27300 atagagtctt gcagacaaac tgcgcaactc gtgaaaggta ggcggatccc cttcgaagga 27360 aagacctgat gctttcgtg cgcgcataaa ataccttgat actgtgccgg atgaaagcgg 27420 ttcgcgacga gtagatgcaa ttatggttc tccgccaaga atctctttgc attatcaag 27480 tgtttccttc attgatattc cgagagcatc aatatgcaat gctgttggga tggcaatttt 27540 tacgcctgtt ttgctttgct cgacataaag atatccatct acgatatcag accacttcat 27600 ttcgcataaa tcaccaactc gttgcccggt aacaacagcc agttccattg caagtctgag 27660 ccaacatggt gatgattctg ctgcttgata aattttcagg tattcgtcag ccgtaagtct 27720 tgatctcctt acctctgatt ttgctgcgcg agtggcagcg acatggtttg ttgttatatg 27780 gccttcagct attgcctctc ggaatgcatc gctcagtgtt gatctgatta acttggctga 27840 cgccgccttg ccctcgtcta tgtatccatt gagcattgcc gcaatttctt ttgtggtgat 27900 gtcttcaagt ggagcatcag gcagacccct ccttattgct ttaattttgc tcatgtaatt 27960 tatgagtgtc ttctgcttga ttcctctgct ggccaggatt ttttcgtagc gatcaagcca 28020 tgaatgtaac gtaacggaat tatcactgtt gattctcgct gtcagaggct tgtgtttgtg 28080 tcctgaaaat aactcaatgt tggcctgtat agcttcagtg attgcgattc gcctgtctct 28140 gcctaatcca aactctttac ccgtccttgg gtccctgtag cagtaatatc cattgtttct 28200 tatataaagg ttagggggta aatcccggcg ctcatgactt cgccttcttc ccatttctga 28260 tcctcttcaa aaggccacct gttactggtc gatttaagtc aacctttacc gctgattcgt 28320 ggaacagata ctctcttcca tccttaaccg gaggtgggaa tatcctgcat tcccgaaccc 28380 atcgacgaac tgtttcaagg cttcttggac gtcgctggcg tgcgttccac tcctgaagtg 28440 tcaagtacat cgcaaagtct ccgcaattac acgcaagaaa aaaccgccat caggcggctt 28500 ggtgttcttt cagttcttca attcgaatat tggttacgtc tgcatgtgct atctgcgccc 28560 atatcatcca gtggtcgtag cagtcgttga tgttctccgc ttcgataact ctgttgaatg 28620 gctctccatt ccattctcct gtgactcgga agtgcattta tcatctccat aaaacaaaac 28680 ccgccgtagc gagttcagat aaaataaatc cccgcgagtg cgaggattgt tatgtaatat 28740 tgggtttaat catctatatg ttttgtacag agagggcaag tatcgtttcc accgtactcg 28800 tgataataat tttgcacggt atcagtcatt tctcgcacat tgcagaatgg ggatttgtct 28860 tcattagact tataaacctt catggaatat ttgtatgccg actctatatc tataccttca 28920 tctacataaa caccttcgtg atgtctgcat ggagacaaga caccggatct gcacaacatt 28980 gataacgccc aatctttttg ctcagactct aactcattga tactcattta taaactcctt 29040 gcaatgtatg tcgtttcagc taaacggtat cagcaatgtt tatgtaaaga aacagtaaga 29100 taatactcaa cccgatgttt gagtacggtc atcatctgac actacagact ctggcatcgc 29160 tgtgaagacg acgcgaaatt cagcattttc acaagcgtta tcttttacaa aaccgatctc 29220 actctccttt gatgcgaatg ccagcgtcag acatcatatg cagatactca cctgcatcct 29280 gaacccattg acctccaacc ccgtaatagc gatgcgtaat gatgtcgata gttactaacg 29340 ggtcttgttc gattaactgc cgcagaaact cttccaggtc accagtgcag tgcttgataa 29400 caggagtctt cccaggatgg cgaacaacaa gaaactggtt tccgtcttca cggacttcgt 29460 tgctttccag tttagcaata cgcttactcc catccgagat aacaccttcg taatactcac 29520 gctgctcgtt gagttttgat tttgctgttt caagctcaac acgcagtttc cctactgtta 29580 <h2 style=";text-align:left;direction:ltr">gcgcaatatc ctcgttctcc tggtcgcggc gtttgatgta ttgctggttt ctttcccgtt 29640<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> catccagcag ttccagcaca atcgatggtg ttaccaattc atggaaaagg tctgcgtcaa 29700<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atccccagtc gtcatgcatt gcctgctctg ccgcttcacg cagtgcctga gagttaattt 29760<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgctcacttc gaacctctct gtttactgat aagttccaga tcctcctggc aacttgcaca 29820<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agtccgacaa ccctgaacga ccaggcgtct tcgttcatct atcggatcgc cacactcaca 29880<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> acaatgagtg gcagatatag cctggtggtt caggcggcgc atttttattg ctgtgttgcg 29940<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctgtaattct tctatttctg atgctgaatc aatgatgtct gccatctttc attaatccct 30000<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gaactgttgg ttaatacgct tgagggtgaa tgcgaataat aaaaaaggag cctgtagctc 30060<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cctgatgatt ttgcttttca tgttcatcgt tccttaaaga cgccgtttaa catgccgatt 30120<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gccaggctta aatgagtcgg tgtgaatccc atcagcgtta ccgtttcgcg gtgcttcttc 30180<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> agtacgctac ggcaaatgtc atcgacgttt ttatccggaa actgctgtct ggcttttttt 30240<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gatttcagaa ttagcctgac gggcaatgct gcgaagggcg ttttcctgct gaggtgtcat 30300 tgaacaagtc ccatgtcggc aagcataagc acacagaata tgaagcccgc tgccagaaaa 30360 atgcattccg tggttgtcat acctggtttc tctcatctgc ttctgctttc gccaccatca 30420 tttccagctt ttgtgaaagg gatgcggcta acgtatgaaa ttcttcgtct gtttctactg 30480 gtattggcac aaacctgatt caatttgag caaggctatg tgccatctcg atactcgttc 30540 ttaactcaac agaagatgct ttgtgcatac agcccctcgt ttattattta tctcctcagc 30600 cagccgctgt gctttcagtg gatttcggat aacagaaagg ccgggaaata cccagcctcg 30660 ctttgtaacg gagtagacga aagtgattgc gcctacccgg atattatcgt gaggatgcgt 30720 catcgccatt gctccccaaa tacaaaacca atttcagcca gtgcctcgtc cattttttcg 30780 atgaactccg gcacgatctc gtcaaaactc gccatgtact tttcatcccg ctcaatcacg 30840 acataatgca ggccttcacg cttcatacgc gggtcatagt tggcaaagta ccaggcattt 30900 ttcgcgtca cccacatgct gtactgcacc tgggccatgt aagctgactt tatggcctcg 30960 aaaccaccga gccggaactt catgaaatcc cgggaggtaa acgggcattt cagttcaagg 31020 ccgttgccgt cactgcataa accatcggga gagcaggcgg tacgcatact ttcgtcgcga 31080 tagatgatcg gggattcagt aacattcacg ccggaagtga attcaaacag ggttctggcg 31140 tcgttctcgt actgttttcc ccaggccagt gctttagcgt taacttccgg agccacaccg 31200 gtgcaaacct cagcaagcag ggtgtggaag taggacattt tcatgtcagg ccacttcttt 31260 ccggagcggg gttttgctat cacgttgtga acttctgaag cggtgatgac gccgagccgt 31320 aatttgtgcc acgcatcatc cccctgttcg acagctctca catcgatccc ggtacgctgc 31380 aggataatgt ccggtgtcat gctgccacct tctgctctgc ggctttctgt ttcaggaatc 31440 caagagcttt tactgcttcg gcctgtgtca gttctgacga tgcacgaatg tcgcggcgaa 31500 atatctggga acagagcggc aataagtcgt catcccatgt tttatccagg gcgatcagca 31560 gagtgttaat ctcctgcatg gtttcatcgt taaccggagt gatgtcgcgt tccggctgac 31620 gttctgcagt gtatgcagta ttttcgacaa tgcgctcggc ttcatccttg tcatagatac 31680 cagcaaatcc gaaggccaga cgggcacact gaatcatggc tttatgacgt aacatccgtt 31740 tgggatgcga ctgccacggc cccgtgattt ctctgccttc gcgagttttg aatggttcgc 31800 ggcggcattc atccatccat tcggtaacgc agatcggatg attacggtcc ttgcggtaaa 31860 tccggcatgt acaggattca ttgtcctgct caaagtccat gccatcaaac tgctggtttt 31920 cattgatgat gcgggaccag ccatcaacgc ccaccaccgg aacgatgcca ttctgcttat 31980 caggaaaggc gtaaatttct ttcgtccacg gattaaggcc gtactggttg gcaacgatca 32040 gtaatgcgat gaactgcgca tcgctggcat cacctttaaa tgccgtctgg cgaagagtgg 32100 tgatcagttc ctgtgggtcg acagaatcca tgccgacacg ttcagccagc ttcccagcca 32160 gcgttgcgag tgcagtactc attcgtttta tacctctgaa tcaatatcaa cctggtggtg 32220 agcaatggtt tcaaccatgt accggatgtg ttctgccatg cgctcctgaa actcaacatc 32280 gtcatcaaac gcacgggtaa tggatttttt gctggccccg tggcgttgca aatgatcgat 32340 <h2 style=";text-align:left;direction:ltr">gcatagcgat tcaaacaggt gctggggcag gcctttttcc atgtcgtctg ccagttctgc 32400<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctctttctct tcacgggcga gctgctggta gtgacgcgcc cagctctgag cctcaagacg 32460<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> atcctgaatg taataagcgt tcatggctga actcctgaaa tagctgtgaa aatatcgccc 32520<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> gcgaaatgcc gggctgatta ggaaaacagg aaagggggtt agtgaatgct tttgcttgat 32580<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ctcagtttca gtattaatat ccatttttta taagcgtcga cggcttcacg aaacatcttt 32640<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tcatcgccaa taaaagtggc gatagtgaat ttagtctgga tagccataag tgtttgatcc 32700<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> attctttggg actcctggct gattaagtat gtcgataagg cgtttccatc cgtcacgtaa 32760<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> tttacgggtg attcgttcaa gtaaagattc ggaagggcag ccagcaacag gccaccctgc 32820<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aatggcatat tgcatggtgt gctccttatt tatacataac gaaaaacgcc tcgagtgaag 32880<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> cgttattggt atgcggtaaa accgcactca ggcggccttg atagtcatat catctgaatc 32940<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> aaatattcct gatgtatcga tatcggtaat tcttattcct tcgctaccat ccattggagg 33000<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> ccatccttcc tgaccatttc catcattcca gtcgaactca cacacaacac catatgcatt 33060 taagtcgctt gaaattgcta taagcagagc atgttgcgcc agcatgatta atacagcatt 33120 taatacagag ccgtgtttat tgagtcggta ttcagagtct gaccagaaat tattaatctg 33180 gtgaagtttt tcctctgtca ttacgtcatg gtcgatttca atttctattg atgctttcca 33240 gtcgtaatca atgatgtatt ttttgatgtt tgacatctgt tcatatcctc acagataaaa 33300 aatcgccctc acactggagg gcaaagaaga tttccaataa tcagaacaag tcggctcctg 33360 tttagttacg agcgacattg ctccgtgtat tcactcgttg gaatgaatac acagtgcagt 33420 gtttattctg ttatttatgc caaaaataaa ggccactatc aggcagcttt gttgttctgt 33480 ttaccaagtt ctctggcaat cattgccgtc gttcgtattg cccatttatc gacatatttc 33540 ccatcttcca ttacaggaaa catttcttca ggcttaacca tgcattccga ttgcagcttg 33600 catccattgc atcgcttgaa ttgtccacac cattgatttt tatcaatagt cgtagtcata 33660 cggatagtcc tggtattgtt ccatcacatc ctgaggatgc tcttcgaact cttcaaattc 33720 ttcttccata tatcacctta aatagtggat tgcggtagta aagattgtgc ctgtctttta 33780 accacatcag gctcggtggt tctcgtgtac ccctacagcg agaaatcgga taaactatta 33840 caacccctac agtttgatga gtatagaaat ggatccactc gttattctcg gacgagtgtt 33900 cagtaatgaa cctctggaga gaaccatgta tatgatcgtt atctgggttg gacttctgct 33960 tttaagccca gataactggc ctgaatatgt taatgagaga atcggtattc ctcatgtgtg 34020 gcatgttttc gtctttgctc ttgcattttc gctagcaatt aatgtgcatc gattatcagc 34080 tattgccagc gccagatata agcgatttaa gctaagaaaa cgcattaaga tgcaaaacga 34140 taaagtgcga tcagtaattc aaaaccttac agaagagcaa tctatggttt tgtgcgcagc 34200 ccttaatgaa ggcaggaagt atgtggttac atcaaaacaa ttcccataca ttagtgagtt 34260 gattgagctt ggtgtgttga acaaaacttt ttcccgatgg aatggaaagc atatattatt 34320 ccctattgag gatatttact ggactgaatt agttgccagc tatgatccat ataatattga 34380 gataaagcca aggccaatat ctaagtaact agataagagg aatcgatttt cccttaattt 34440 tctggcgtcc actgcatgtt atgccgcgtt cgccaggctt gctgtaccat gtgcgctgat 34500 tcttgcgctc aatacgttgc aggttgcttt caatctgttt gtggtattca gccagcactg 34560 taaggtctat cggatttagt gcgctttcta ctcgtgattt cggtttgcga ttcagcgaga 34620 gaatagggcg gttaactggt tttgcgctta ccccaaccaa caggggattt gctgctttcc 34680 attgagcctg tttctctgcg cgacgttcgc ggcggcgtgt ttgtgcatcc atctggattc 34740 tcctgtcagt tagctttggt ggtgtgtggc agttgtagtc ctgaacgaaa accccccgcg 34800 attggcacat tggcagctaa tccggaatcg cacttacggc caatgcttcg tttcgtatca 34860 cacaccccaa agccttctgc tttgaatgct gcccttcttc agggcttaat ttttaagagc 34920 gtcaccttca tggtggtcag tgcgtcctgc tgatgtgctc agtatcaccg ccagtggtat 34980 ttatgtcaac accgccagag ataatttatc accgcagatg gttatctgta tgttttttat 35040 atgaatttat tttttgcagg ggggcattgt ttggtaggtg agagatctga attgctatgt 35100 ttagtgagtt gtatctattt attttcaat aaatacaatt ggttatgtgt ttggggggcg 35160 atcgtgaggc aaagaaaacc cggcgctgag gccggtttat tcttgttctc tggtcaatt 35220 atatagttgg aaaacagga tgcatatatg atgaacgat gcagaggcaa tgccgatggc 35280 gatagtgggt atcatgtagc cgcttatgct ggaaagaagc ataacccgc agaaaaaaaaa 35340 agctccaagc tcaaaac taagggcata gataac accgatgtca tatacccata 35400 ctctctaatc ttggccagtc gggcgttct gcttccgatt agaaacgtca aggcagcaat 35460 caggattgca atcatggttc ctgcatatga tgacaatgtc gcccagac catctctatg 35520 agctgaaaaa gaacaccag gatgtagtg gcggaaagg agatagcaaa tgcttacgat 35580 aacgtaagga attackacta tgtaacacc aggcatgatt ctgttccgca attackccc 35640 tgataattaa tccttaactt tgcccacctg ccttttaaaa cattccagta tatcactttt 35700 cattcttgcg tagcaatg ccatctctc agctatctca gcattggtga ccttgttcag 35760 aggcgctgag agatggcctt tttctgatag atatgttct gttaaaatat ctccggccctc 35820 atcttttgcc cgcaggctaa tgtctgaaaa tgaggtgac gggttaaaa taatatcctt 35880 ggcaaccttt tttataccc ttttaattt tggcttaatg actatatcca atggtcaa 35940 aagctcccct tcaatctctg ttgccccta gacctttaat atcgcca atacaggtag 36000 cttggcttct accttcaccg ttgttcggcc gatgaatgc atatgcataa catcgtcttt 36060 ggtggttccc ctcatcagtg gcttactcg aacgcgctct ccactgctta atgacattcc 36120 ttcccgatt aaaaaatctg tcagatcgga tgtggtcggc ccgaaacag ttctggcaaa 36180 accaatggtg tcgccttcaaaaaaaaa agatgggaat cccaatgatt cgtcatctgc 36240 gaggctgttc ttatatctt caacgaagc tttagagcga ttatctct gaaccagact 36300 cttgtcattt gttttggtaa aggaaaagt ttttccatcg attttatgaa tatacaata 36360 attggagcca acctgcaggt gatgattatc agccagcaga gatttagga aacagacag 36420 gtttattgag cgcttatctt tccctttatt ttgctgcgg taagtcgcat aaaaaccatt 36480 cttcataatt caatccattt actatgttat gttctgaggg gagtgaaaat tcccctaatt 36540 cgatgaagat tcttgctcaa ttgttatcag ctatgcgccg accagaacac cttgccgatc 36600 agccaaacgt ctcttcaggc cactgactag cgataacttt ccccacaacg gaacaactct 36660 cattgcatgg gatcattggg tactgtgggt ttagtggttg taaaaacacc tgaccgctat 36720 ccctgatcag tttcttgaag gtaaactcat cacccccaag tctggctatg cagaaatcac 36780 ctggctcaac agcctgctca gggtcaacga gaattaacat tccgtcagga aagcttggct 36840 tggagcctgt tggtgcggtc atggaattac cttcaacctc aagccagaat gcagaatcac 36900 tggctttttt ggttgtgctt acccatctct ccgcatcacc tttggtaaag gttctaagct 36960 caggtgagaa catccctgcc tgaacatgag aaaaaacagg gtactcatac tcacttctaa 37020 gtgacggctg catactaacc gcttcataca tctcgtagat ttctctggcg attgaagggc 37080 taaattcttc aacgctaact ttgagaattt ttgcaagcaa tgcggcgtta taagcattta 37140 atgcattgat gccattaaat aaagcaccaa cgcctgactg ccccatcccc atcttgtctg 37200 cgacagattc ctgggataag ccaagttcat ttttcttttt ttcataaatt gctttaaggc 37260 gacgtgcgtc ctcaagctgc tcttgtgtta atggtttctt ttttgtgctc atacgttaaa 37320 37380 gtgataatgg ttgcatgtac taaggaggtt gtatggaaca acgcataacc ctgaaagatt 37440 atgcaatgcg ctttgggcaa accaagacag ctaaagatct cggcgtatat caaagcgcga 37500 tcaacaaggc cattcatgca ggccgaaaga tttttttaac tataaacgct gatggaagcg 37560 37620 cgctcttaca cattccagcc ctgaaaaagg gcatcaaatt aaaccacacc tatggtgtat 37680 gcatttattt gcatacattc aatcaattgt tatctaagga aatacttaca tatggttcgt 37740 gcaaacaaac gcaacgaggc tctacgaatc gagagtgcgt tgcttaacaa aatcgcaatg 37800 cttggaactg agaagacagc ggaagctgtg ggcgttgata agtcgcagat cagcaggtgg 37860 aagagggact ggattccaaa gttctcaatg ctgcttgctg ttcttgaatg gggggtcgtt 37920 gacgacgaca tggctcgatt ggcgcgacaa gttgctgcga ttctcaccaa taaaaaacgc 37980 ccggcggcaa ccgagcgttc tgaacaaatc cagatggagt tctgaggtca ttactggatc 38040 tatcaacagg agtcattatg aaaatacag caaaaatact caacttcggc agaggtaact 38100 ttgccggaca ggagcgtaat gtggcagatc tcgatgatgg ttacgccaga ctatcaaata 38160 tgctgcttga ggcttattcg ggcgcagatc tgaccaagcg acagtttaaa gtgctgcttg 38220 ccattctgcg taaaacctat gggtggataa aaccaatgga cagaatcacc gattctcaac 38280 ttagcgagat tacaaagtta cctgtcaaac ggtgcaatga agccaagtta gaactcgtca 38340 gaatgaatat tatcaagcag caaggcggca tgtttggacc aaataaaaac atctcagaat 38400 ggtgcatccc tcaaaacgag ggaaaatccc ctaaaacgag ggataaaaca tccctcaaat 38460 tgggggattg ctatccctca aaacaggggg acacaaaaga cactattaca aaagaaaaaa 38520 gaaaagatta ttcgtcagag aattctggcg aatcctctga ccagccagaa aacgaccttt 38580 ctgtggtgaa accggatgct gcaattcaga gcggcagcaa gtgggggaca gcagaagacc 38640 tgaccgccgc agagtggatg tttgacatgg tgaagactat cgcaccatca gccagaaaac 38700 cgaattttgc tgggtgggct aacgatatcc gcctgatgcg tgaacgtgac ggacgtaacc 38760 accgcga 38767 <210> 8 <211> 9107 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 8 catgtgtgtg ctgttccgct gggcatgcca ggacaacttc tggtccggta acgtgctgag 60 cccggccaaa ctccgcgata agtggaccca actcgaaatc aaccgtaaca agcaacaggc 120 aggcgtgaca gccagcaaac caaaactcga cctgacaaac acagactgga tttacggggt 180 ggatctatga aaaacatcgc cgcacagatg gttaactttg accgtgagca gatgcgtcgg 240 atcgccaaca acatgccgga acagtacgac gaaaagccgc aggtacagca ggtagcgcag 300 atcatcaacg gtgtgttcag ccagttactg gcaactttcc cggcgagcct ggctaaccgt 360 gaccagaacg aagtgaacga aatccgtcgc cagtgggttc tggcttttcg ggaaaacggg 420 atcaccacga tggaacaggt taacgcagga atgcgcgtag cccgtcggca gaatcgacca 480 tttctgccat cacccgggca gtttgttgca tggtgccggg aagaagcatc cgttaccgcc 540 ggactgccaa acgtcagcga gctggttgat atggtttacg agtattgccg gaagcgaggc 600 ctgtatccgg atgcggagtc ttatccgtgg aaatcaaacg cgcactactg gctggttacc 660 aacctgtatc agaacatgcg ggccaatgcg cttactgatg cggaattacg ccgtaaggcc 720 gcagatgagc ttgtccatat gactgcgaga attaaccgtg gtgaggcgat ccctgaacca 780 gtaaaacaac ttcctgtcat gggcggtaga cctctaaatc gtgcacaggc tctggcgaag 840 atcgcagaaa tcaaagctaa gttcggactg aaaggagcaa gtgtatgacg ggcaaagagg 900 caattattca ttacctgggg acgcataata gcttctgtgc gccggacgtt gccgcgctaa 960 caggcgcaac agtaaccagc ataaatcagg ccgcggctaa aatggcacgg gcaggtcttc 1020 tggttatcga aggtaaggtc tggcgaacgg tgtattaccg gtttgctacc agggaagaac 1080 gggaggaa gatgagcacg aacctggttt ttaaggagtg tcgccagagt gccgcgatga aacgggtatt ggcggtatat ggagttaaaa gatgaccatc tacattactg agctaataac aggcctgctg gtaatcgcag gcctttttat ttggggggaga gggaagtcat gaaaaaacta 1260. acctttgaaa ttcgatctcc agcacatcag caaaacgcta ttcacgcagt acagcaaatc cttccagacc caccaaacc aatcgtagta accattcagg aacgcaaccg cagcttagac caaaacagga agctatgggc ctgcttaggt gacgtctctc gtcaggttga atggcatggt cgctggctgg atgcagaaag ctggaagtgt gtgtttaccg cagcattaaa gcagcaggat gttgttccta accttgccgg gaatggcttt gtggtaatag gccagtcaac cagcaggatg cgtgtaggcg aatttgcgga gctattagg cttatacagg cattcggtac agagcgtggc gttaagtggt cagacgaagc gagactggct ctggagtgga aagcgagatg gggagacagg gctgcatgat aaatgtcgtt agtttctccg gtggcaggac gtcagcatat ttgctctggc 1740 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800. 1800 gtgaacatcc aatgacatat cggtttgtca gggaagttgt gaagttctgg gatataccgc tcaccgtatt gcaggttgat atcaacccgg agcttggaca gccaaatggt tatacggtat gggaccaaa ggatattcag acgcgaatgc ctgttctga gccatttatc gatatggtaa aaatatgg cactccatac gtcggcggcg cgttctgcac tgacagatta aaactcgttc ccttcacca atactgtgat gaccatttcg ggcgaggga ttacaccacg tggattggca tcagagctga tgaaccgaag cggctaaagc caaagcctgg aatcagatat cttgctgaac tgtcagactt tgagaagga gatatcctcg catggtgga gcaacaacca ttcgatttgc aaataccgga acatctcggt aactgcatat tctgcattaa aaaatcaacg caaaaaatcg gacttgcctg caaagatgag gagggattgc agcgtgtttt taatgaggtc atcacgggat cccatgtgcg tgacggacat cgggaaacgc caaaggagat tatgtaccga ggaagaatgt cgctggacgg tatcgcgaaa atgtattcag aaaatgatta tcaagccctg tatcaggaca tggtacgagc taaagattc gataccggct cttgttctga gtcatgcgaa atatttggag ggcagcttga tttcgacttc gggagggaag ctgcatgatg cgatgttatc ggtgcggtga 2580 atgcaaagaa gataaccgct tccgaccaaa tcaaccttac tggaatcgat ggtgtctccg 2640 gtgtgaaaga acaccaacag gggtgttacc actaccgcag gaaaaggagg acgtgtggcg 2700 agacagcgac gaagtatcac cgacataatc tgcgaaaact gcaaatacct tccaacgaaa 2760 cgcaccagaa ataaacccaa gccaatccca aaagaatctg acgtaaaaac cttcaactac 2820 acggctcacc tgtgggatat ccggtggcta agacgtcgtg cgaggaaaac aaggtgattg 2880 accaaaatcg aagttacgaa caagaaagcg tcgagcgagc tttaacgtgc gctaactgcg 2940 gtcagaagct gcatgtgctg gaagttcacg tgtgtgagca ctgctgcgca gaactgatga 3000 gcgatccgaa tagctcgatg cacgaggaag aagatgatgg ctaaaccagc gcgaagacga 3060 tgtaaaaacg atgaatgccg ggaatggttt caccctgcat tcgctaatca gtggtggtgc 3120 tctccagagt gtggaaccaa gatagcactc gaacgacgaa gtaaagaacg cgaaaaagcg 3180 gaaaaagcag cagagaagaa acgacgacga gaggagcaga aacagaaaga taaacttaag 3240 attcgaaac tcgccttaaa gccccgcagt tactggatta aacaagccca acaagccgta aacgccttca tcagagaaag agaccgcgac ttaccatgta tctcgtgcgg aacgctcacg tctgctcagt gggatgccgg acattaccgg acaactgctg cggcacctca actccgattt 3420 aatgaacgca atattcacaa gcaatgcgtg gtgtgcaacc agcacaaaag cggaaatctc gttccgtatc gcgtcgaact gattagccgc atcgggcagg aagcagtaga cgaaatcgaa tcaaaccata accgccatcg ctggactatc gaagagtgca aggcgatcaa ggcagagtac 3660. ttctcagtaa ccgcatgagg ccgcatgacg ttctcagtaa aaaccattcc agacatgctc gttgaagcat acggaatca gacagaagta gcacgcagac tgaaatgtag tcgcggtacg gtcagaaaat acgttgatga taaagacggg aaaatgcacg ccatcgtcaa cgacgttctc atggttcatc gcggatggag tgaaagagat gcgctattac 3840. gaaaaaattg atggcagcaa ataccgaat atttgggtag ttggcgatct gcacggatgc 3960. 3960. 3960. 3960. 100% atctcggtgg gcgatttggt tgatcgtggt gcagagaacg ttgaatgcct ggaattaatc 4020 acattccct ggttcagagc tgtacgtgga aacatgagc aaatgatgat tgatggctta 4080 tcagagcgtg gaaacgttaa tcactggctg cttaatggcg gtggctggtt ctttaatctc 4140 gattacgaca aagaaattct ggctaaagct cttgcccata aagcagatga acttccgtta 4200 atcatcgaac tggtgagcaa agataaaaaa tatgttatct gccacgccga ttatcccttt 4260 gacgaatacg agtttggaaa gccagttgat catcagcagg taatctggaa ccgcgaacga 4320 atcagcaact cacaaaacgg gatcgtgaaa gaaatcaaag gcgcggacac gttcatcttt 4380 ggtcatacgc cagcagtgaa accactcaag tttgccaacc aaatgtatat cgataccggc 4440 gcagtgttct gcggaaacct aacattgatt caggtacagg gagaaggcgc atgagactcg 4500 aaagcgtagc taaatttcat tcgccaaaaa gcccgatgat gagcgactca ccacgggcca 4560 cggcttctga ctctctttcc ggtactgatg tgatggctgc tatggggatg gcgcaatcac 4620 aagccggatt cggtatggct gcattctgcg gtaagcacga actcagccag aacgacaaac 4680 aaaaggctat caactatctg atgcaatttg cacaaaggt atcgggaaa taccgtggtg 4740 tggcaaagct tgaaggaat actaaggcaa aggtactgca agtgctcgca acattcgctt 4800 atgcggatta ttgccgtagt gccgcgacgc cggggcaag atgcagagat tgccatggta 4860 caggccgtgc ggttgatatt gccaaaacag agctgtgggg gagagttgtc gagaaagagt 4920 gcggaagatg caaggcgtc ggctattcaa ggatgccagc aagcgcagca tatcgcgctg 4980 tgacgatgct aatcccaaac cttacccaac ccacctggtc acgcactgtt aagccgctgt 5040 atgacgctct ggtggtgcaa tgccacaaag aagagtcaat cgcagacaac attttgaatg 5100 cggtcacacg ttagcagcat gattgccacg gatggcaaca tattaacggc atgatattga 5160 cttattgaat aaattgggt aaatttgact caacgatggg ttaattcgct cgttgtggta 5220 gtgagatgaa aagaggcggc gcttactacc gattccgcct agttggtcac ttcgacgtat 5280 cgtctggaac tccaaccatc gcaggcagag aggtctgcaa aatgcaatcc cgaaacagtt 5340 cgcaggtaat agttagagcc tgcataacgg tttcgggatt ttttatatct gcacaacagg 5400 taagagcatt gagtcgataa tcgtgaagag tcggcgagcc tggttagcca gtgctctttc 5460 cgttgtgctg aattaagcga ataccggaag cagaaccgga tcaccaaatg cgtacaggcg 5520 tcatcgccgc ccagcaacag cacaacccaa actgagccgt agccactgtc tgtcctgaat 5580 tcattagtaa tagttacgct gcggccttt acacatgacc ttcgtgaaag cgggtggcag 5640 gaggtcgcgc taacaacctc ctgccgtttt gccgtgcat atcggtcacg aaaaatctg 5700 attactaaac acagtagcct ggatttgttc tatcagtaat cgaccttatt cctaattaaa 5760 tagagcaaat ccccttattg ggggtaagac atgaagatgc cagaaaaaca tgacctgttg 5820 gccgccattc tcgcggcaaa ggaacaaggc atcggggcaa tccttgcgtt tgcaatggcg 5880 taccttcgcg gcagatataa tggcggtgcg tttacaaaaa cagtaatcga cgcaacgatg 5940 tgcgccatta tcgcctggtt cattcgtgac cttctcgact tcgccggact aagtagcaat 6000 ctcgcttata taacgagcgt gtttatcggc tacatcggta ctgactcgat tggttcgctt 6060 atcaaacgct tcgctgctaa aaaagccgga gtagaagatg gtagaaatca ataatcaacg 6120 taaggcgttc ctcgatatgc tggcgtggtc ggagggaact gataacggac gtcagaaaac 6180 cagaaatcat ggttatgacg tcattgtagg cggagagcta tttactgatt actccgatca 6240 ccctcgcaaa cttgtcacgc taaacccaaa actcaaatca acaggcgccg gacgctacca 6300 gcttctttcc cgttggtggg atgcctaccg caagcagctt ggcctgaaag acttctctcc 6360 gaaaagtcag gacgctgtgg cattgcagca gattaaggag cgtggcgctt tacctatgat 6420 tgatcgtggt gatatccgtc aggcaatcga ccgttgcagc aatatctggg cttcactgcc 6480 gggcgctggt tatggtcagt tcgagcataa ggctgacagc ctgattgcaa aattcaaaga 6540 agcgggcgga acggtcagag agattgatgt atgagcagag tcaccgcgat tatctccgct 6600 ctggttatct gcatcatcgt ctgcctgtca tgggctgtta atcattaccg tgataacgcc 6660 attack aagcccagcg cgacaaaaat gccagagaac tgaagctggc gaacgcggca 6720 attactgaca tgcagatgcg tcagcgtgat gttgctgcgc tcgatgcaaa atacacgaag 6780 gagttagctg atgctaaagc tgaaaatgat gctctgcgtg atgatgttgc cgctggtcgt 6840 cgtcggttgc acatcaaagc agtctgtcag tcagtgcgtg aagccaccac cgctcccggc 6900 gtggataatg cagcctcccc ccgactggca gacaccgctg aacgggatta tttcaccctc 6960 agagagaggc tgatcactat gcaaaaacaa ctggaaggaa cccagaagta tattaatgag 7020 cagtgcagat agagttgccc atatcgatgg gcaactcatg caattatgt gagcaataca 7080 cacgcgcttc cagcggagta taaatgccta aagtaataaa accgagcaat ccatttacga 7140 atgtttgctg ggtttctgtt ttaacaacat tttctgcgcc gccacaaatt ttggctgcat 7200 cgacagttt cttctgccca attccagaaa cgaagaaatg atgggtgatg gtttcctttg 7260 gtgctactgc tgccggtttg ttttgaacag taaacgtctg ttgagcacat cctgtaataa 7320 gcagggccag cgcagtagcg agtagcattt ttttcatggt gttatcccg atgctttttg 7380 aagttcgcag aatcgtatgt gtagaaaatt aaacaaaccc taaacaatga gttgaaattt 7440 catattgtta atatttatta atgtatgtca ggtgcgatga atcgtcattg tattcccgga 7500 ttaactatgt ccacagccct gacggggaac ttctctgcgg gagtgtccgg gaataattaa 7560 aacgatgcac acagggttta gcgcgtacac gtattgcatt atgccaacgc cccggtgctg 7620 acacggaaga aaccggacgt tatgatttag cgtggaaaga tttgtgtagt gttctgaatg 7680 ctctcagtaa atagtaatga attatcaaag gtatagtaat atcttttatg ttcatggata 7740 tttgtaaccc atcggaaaac tcctgcttta gcaagatttt ccctgtattg ctgaaatgtg 7800 atttctcttg atttcaacct atcataggac gtttctataa gatgcgtgtt tcttgagaat 7860 ttaacattta caaccttttt aagtcctttt attaacacgg tgttatcgtt ttctaacacg 7920 atgtgaatat tatctgtggc tagatagtaa atataatgtg agacgttgtg acgttttagt 7980 tcagaataaa acaattcaca gtctaaatct ttcgcactt gatcgaatat ttctttaaaa 8040 atggcaacct gagccattgg taaaaccttc catgtgatac gagggcgcgt agtttgcatt 8100 atcgttttta tcgtttcaat ctggtctgac ctccttgtgt tttgttgatg attatgtca 8160 aatattagga atgttttcac ttaatagtat tggttgcgta acaaagtgcg gtcctgctgg 8220 cattctggag ggaaatacaa ccgacagatg tatgtaaggc caacgtgctc aaatcttcat 8280 acagaaagat ttgaagtaat atttaaccg ctagatgaag agcaagcgca tggagcgaca 8340 aaatgaataa agaacaatct gctgatgatc cctccgtgga tctgattcgt gtaaaaaata 8400 tgcttaatag caccatttct atgagttacc ctgatgttgt aattgcatgt atagaacata 8460 aggtgtctct ggaagcattc agagcaattg aggcagcgtt ggtgaagcac gataataata 8520 tgaaggatta ttccctggtg gttgactgat caccataact gctaatcatt caaactattt 8580 agtctgtgac agagccaaca cgcagtctgt cactgtcagg aaagtggtaa aactgcaact 8640 caattactgc aatgccctcg taattaagtg aatttacaat atcgtcctgt tcggagggaa 8700 gaacgcggga tgttcattct tcatcacttt taattgatgt atatgctctc ttttctgacg 8760 ttagtctccg acggcaggct tcaatgaccc aggctgagaa attcccggac cctttttgct 8820 caagagcgat gttaatttgt tcaatcattt ggttaggaaa gcggatgttg cgggttgttg 8880 ttctgcgggt tctgttcttc gttgacatga ggttgccccg tattcagtgt cgctgatttg 8940 tattgtctga agttgttttt acgttaagtt gatgcagatc attaatacg atacctgcgt 9000 cataattgat tatttgacgt ggtttgatgg cctccacgca cgttgtgata ttagatgat 9060 aatcattatc actttacggg tcctttccgg tgatccgaca ggttacg 9107 <210> 9 <211> 19604 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 9 catgttgatt tcctgaaacg ggatatcatc aaagccatga acaaagcagc cgcgctggat 60 gaactgatac cggggttgct gagtgaatat atcgaacagt caggttaaca ggctgcggca 120 ttttgtccgc gccgggcttc gctcactgtt caggccggag ccacagaccg ccgttgaatg 180 ggcggatgct aattactatc tcccgaaaga atccgcatac caggaagggc gctgggaaac 240 actgcccttt cagcgggcca tcatgaatgc gatgggcagc gactacatcc gtgaggtgaa 300 tgtggtgaag tctgcccgtg tcggttattc caaaatgctg ctgggtgttt atgcctactt 360 tatagagcat aagcagcgca acacccttat ctggttgccg acggatggtg atgccgagaa 420 ctttatgaaa acccacgttg agccgactat tcgtgatatt ccgtcgctgc tggcgctggc 480 cccgtggtat ggcaaaaagc accgggataa cacgctcacc atgaagcgtt tcactaatgg 540 gcgtggcttc tggtgcctgg gcggtaaagc ggcaaaaaac taccgtgaaa agtcggtgga 600 tgtggcgggt tatgatgaac ttgctgcttt tgatgatgat attgaacagg aaggctctcc 660 gacgttcctg ggtgacaagc gtattgaagg ctcggtctgg ccaaagtcca tccgtggctc 720 cacgccaaaa gtgagaggca cctgtcagat tgagcgtgca gccagtgaat ccccgcattt 780 tatgcgtttt catgttgcct gcccgcattg cggggaggag cagtatctta aatttggcga 840 caaagagacg ccgtttggcc tcaaatggac gccggatgac ccctccagcg tgttttatct 900 ctgcgagcat aatgcctgcg tcatccgcca gcaggagctg gactttactg atgcccgtta 960 tatctgcgaa aagaccggga tctggacccg tgatggcatt ctctggtttt cgtcatccgg 1020 tgaagagatt gagccacctg acagtgtgac ctttcacatc tggacagcgt acagcccgtt 1080 caccacctgg gtgcagattg tcaaagactg gatgaaaacg aaaggggata cgggaaaacg 1140 taaaaccttc gtaaacacca cgctcggtga gacgtgggag gcgaaaattg gcgaacgtcc 1200 ggatgctgaa gtgatggcag agcggaaaga gcattattca gcgcccgttc ctgaccgtgt 1260 ggcttacctg accgccggta tcgactccca gctggaccgc tacgaaatgc gcgtatgggg 1320 atgggggccg ggtgaggaaa gctggctgat tgaccggcag attattatgg gccgccacga 1380 cgatgaacag acgctgctgc gtgtggatga ggccatcaat aaaacctata cccgccggaa 1440 tggtgcagaa atgtcgatat cccgtatctg ctgggatact ggcgggattg acccgaccat 1500 tgtgtatgaa cgctcgaaaa aacatgggct gttccgggtg atccccatta aaggggcatc 1560 cgtctacgga aagccggtgg ccagcatgcc acgtaagcga aacaaaaacg gggtttacct 1620 taccgaaatc ggtacggata ccgcgaaaga gcagatttat aaccgcttca cactgacgcc 1680 ggaaggggat gaaccgcttc ccggtgccgt tcacttcccg aataacccgg atatttttga 1740 tctgaccgaa gcgcagcagc tgactgctga agagcaggtc gaaaaatggg tggatggcag 1800 gaaaaaaata ctgtgggaca gcaaaaagcg acgcaatgag gcactcgact gcttcgttta 1860 tgcgctggcg gcgctgcgca tcagtatttc ccgctggcag ctggatctca gtgcgctgct 1920 ggcgagcctg caggaagagg atggtgcagc aaccaacaag aaaacactgg cagattacgc 1980 ccgtgcctta tccggagagg atgaatgacg cgacaggaag aacttgccgc tgcccgtgcg 2040 gcactgcatg acctgatgac aggtaaacgg gtggcaacag tacagaaaga cggacgaagg 2100 gtggagttta cggccacttc cgtgtctgac ctgaaaaaat atattgcaga gctggaagtg 2160 cagaccggca tgacacagcg acgcaggga cctgcaggat tttatgtatg aaaacgccca 2220 ccattcccac ccttctgggg ccggacggca tgacatcgct gcgcgaatat gccggttatc 2280 acggcggtgg cagcggattt ggagggcagt tgcggtcgtg gaacccaccg agtgaaagtg 2340 tggatgcagc cctgttgccc aactttaccc gtggcaatgc ccgcgcagac gatctggtac 2400 gcaataacgg ctatgccgcc aacgccatcc agctgcatca ggatcatatc gtcgggtctt 2460 ttttccggct cagtcatcgc ccaagctggc gctatctggg catcggggag gaaagccc 2520 gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct 2580 gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc 2640 acgcctttaa cggtgaactg ttcgttcagg ccacctgggga taccagttcg tcgcggcttt tccggacaca gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg acagccgga ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt acgtcagcga ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg agttacccgg cgggcgcgcc tcgttcattc acgtttttga acccgtggag gacgggcaga 2940. ctcgcggtgc aaatgtgttt tacagcgtga tggagcagat gaagatgctc gacacgctgc agaacacgca gctgcagagc gccattgtga aggcgatgta tgccgccacc attgagagtg 3060. agctggatac gcagtcagcg atggatttta ttctgggcgc gaacagtcag gagcagcggg 3180. aaaggctgac cggctggatt ggtgaaattg ccgcgtatta cgccgcagcg ccggtccggc tgggaggcgc aaaagtaccg cacctgatc cgggtgactc actgaacctg cagacggctc 3240 aggatacgga taacggctac tccgtgtttg agcagtcact gctgcggtat atcgctgccg ggctgggtgt ctcgtatgag cagctttccc ggaattacgc ccagatgagc tactccacgg 3360 cacgggccag tgcgaacgag tcgtgggcgt actttatggg gcggcgaaaa ttcgtcgcat 3420. cccgtcaggc gagccagatg tttctgtgct ggctggaga ggccatcgtt cgccgcgtgg 3480 3540. tgacgttacc ttcaaaagcg cgcttcagtt ttcaggaagc ccgcagtgcc tgggggaact gcgactggat aggctccggt cgtatggcca tcgatggtct gaaagaagtt caggaagcgg tgatgctgat agaagccgga ctgagtacct acgagaaaga gtgcgcaaaa cgcggtgacg 3660 actatcagga aatttttgcc cagcaggtcc gtgaaacgat ggagcgccgt gcagccggtc ttaaccgcc cgcctgggcg gctgcagcat ttgaatccgg gctgcgacaa tcaacagagg aggagagag tgacagcaga gctgcgtaat ctcccgcata ttgccagcat ggcctttaat gagccgctga tgcttgaacc cgcctatgcg cggggttttct tttgtgcgct tgcaggccag 3900 cttgggatca gcagcctgac ggatgcggtg tccggcgaca gcctgactgc ccaggaggca 3960. ctcgcgacgc tggcattatc cggtgatgat gacggaccac gacaggcccg cagttatcag gtcatgaacg gcatcgccgt gctgccggtg tccggcacgc tggtcagccg gacgcggggcg 4080. ctgcagccgt actcggggat gaccggttac aacggcatta tcgccccgtct gcaacaggct 4140 gccagcgatc cgatggtgga cggcattctg ctcgatatgg acacgccccgg cgggatggtg 4200 gcggggcat ttgactgcgc tgacatcatc gcccgtgtgc gtgacataaa accggtatgg 4260 gcgcttgcca acgacatgaa ctgcagtgca ggtcagttgc ttgccagtgc cgctcccgg 4320 cgtctggtca cgcagaccgc ccggacaggc tccatcggcg tcatgatggc tcacagtaat 4380 tacggtgctg cgctggagaa acagggtgtg gaatcacgc tgatttacag cggcagccat 4440 aaggtggatg gcaacccta cagccatctt ccggatgacg tccgggagac actgcagtcc 4500 cggatggacg caacccgcca gatgtttgcg cagaaggtgt cggcatatac cggcctgtcc 4560 gtgcaggttg tgctggatac cgaggctgca gtgtacagcg gtcaggaggc cattgatgcc 4620 ggactggctg atgaacttgt taacagcacc gatgcgatca ccgtcatgcg tgatgcactg 4680 gatgcacgta aatcccgtct ctcaggaggg cgaatgacca aagagactca atcaacaact 4740 gtttcagcca ctgcttcgca ggctgacgtt actgacgtgg tgccagcgac ggagggcgag 4800 aacgccagcg cggcgcagcc ggacgtgaac gcgcagatca ccgcagcggt tgcggcagaa 4860 aacagccgca ttatggggat cctcaactgt gaggaggctc acggacgcga agaacaggca 4920 cgcgtgctgg cagaaacccc cggtatgacc gtgaaaacgg cccgccgcat tctggccgca 4980 gcaccacaga gtgcacaggc gcgcagtgac actgcgctgg atcgtctgat gcagggggca 5040 ccggcaccgc tggctgcagg taacccggca tctgatgccg ttaacgattt gctgaacaca 5100 ccagtgtaag ggatgtttat gacgagcaaa gaaaccttta cccattacca gccgcagggc 5160 aacagtgacc cggctcatac cgcaaccgcg cccggcggat tgagtgcgaa agcgcctgca 5220 atgaccccgc tgatgctgga cacctccagc cgtaagctgg ttgcgtggga tggcaccacc 5280 gacggtgctg ccgttggcat tcttgcggtt gctgctgacc agaccagcac cacgctgacg 5340 ttctacaagt ccggcacgtt ccgttatgag gatgtgctct ggccggaggc tgccagcgac 5400 gagacgaaaa aacggaccgc gtttgccgga acggcaatca gcatcgttta actttaccct 5460 tcatcactaa aggccgcctg tgcggctttt tttacgggat ttttttatgt cgatgtacac 5520 aaccgcccaa ctgctggcgg caaatgagca gaaatttaag tttgatccgc tgtttctgcg 5580 tctctttttc cgtgagagct atcccttcac cacggagaaa gtctatctct cacaaattcc 5640 gggactggta aacatggcgc tgtacgtttc gccgattgtt tccggtgagg ttatccgttc 5700 ccgtggcggc tccacctctg aatttacgcc gggatatgtc aagccgaagc atgaagtgaa 5760 tccgcagatg accctgcgtc gcctgccgga tgaagatccg cagaatctgg cggacccggc 5820 ttaccgccgc cgtcgcatca tcatgcagaa catgcgtgac gaagagctgg ccattgctca 5880 ggtcgaagag atgcaggcag tttctgccgt gcttaagggc aaatacacca tgaccggtga 5940 agccttcgat ccggttgagg tggatatggg ccgcagtgag gagaataaca tcacgcagtc 6000 cggcggcacg gagtggagca agcgtgacaa gtccacgtat gacccgaccg acgatatcga 6060 agcctacgcg ctgaacgcca gcggtgtggt gaatatcatc gtgttcgatc cgaaaggctg 6120 ggcgctgttc cgttccttca aagccgtcaa ggagaagctg gatacccgtc gtggctctaa 6180 ttccgagctg gagacagcgg tgaaagacct gggcaaagcg gtgtcctata aggggatgta 6240 tggcgatgtg gccatcgtcg tgtattccgg acagtacgtg gaaaacggcg tcaaaaagaa 6300 cttcctgccg gacaacacga tggtgctggg gaacactcag gcacgcggtc tgcgcaccta 6360 tggctgcatt caggatgcgg acgcacagcg cgaaggcatt aacgcctctg cccgttaccc 6420 gaaaaactgg gtgaccaccg gcgatccggc gcgtgagttc accatgattc agtcagcacc 6480 gctgatgctg ctggctgacc ctgatgagtt cgtgtccgta caactggcgt aatcatggcc 6540 cttcggggcc attgtttctc tgtggaggag tccatgacga aagatgaact gattgcccgt 6600 ctccgctcgc tgggtgaaca actgaaccgt gatgtcagcc tgacggggac gaaagaagaa 6660 ctggcgctcc gtgtggcaga gctgaaagag gagcttgatg acacggatga aactgccggt 6720 caggacaccc ctctcagccg ggaaaatgtg ctgaccggac atgaaaatga ggtgggatca 6780 gcgcagccgg ataccgtgat tctggatacg tctgaactgg tcacggtcgt ggcactggtg 6840 aagctgcata ctgatgcact tcacgccacg cgggatgaac ctgtggcatt tgtgctgccg 6900 ggaacggcgt ttcgtgtctc tgccggtgtg gcagccgaaa tgacagagcg cggcctggcc 6960 agaatgcaat aacgggaggc gctgtggctg atttcgataa cctgttcgat gctgccattg 7020 cccgcgccga tgaaacgata cgcgggtaca tgggaacgtc agccaccatt acatccggtg 7080 agcagtcagg tgcggtgata cgtggtgttt ttgatgaccc tgaaaatatc agctatgccg 7140 gacagggcgt gcgcgttgaa ggctccagcc cgtccctgtt tgtccggact gatgaggtgc 7200 ggcagctgcg gcgtggagac acgctgacca tcggtgagga aaatttctgg gtagatcggg 7260 tttcgccgga tgatggcgga agttgtcatc tctggcttgg acggggcgta ccgcctgccg 7320 ttaaccgtcg ccgctgaaag ggggatgtat ggccataaaa ggtcttgagc aggccgttga 7380 aaacctcagc cgtatcagca aaacggcggt gcctggtgcc gccgcaatgg ccattaaccg 7440 cgttgcttca tccgcgatat cgcagtcggc gtcacaggtt gcccgtgaga caaaggtacg 7500 ccggaaactg gtaaaggaaa gggccaggct gaaaagggcc acggtcaaaa atccgcaggc 7560 cagaatcaaa gttaaccggg gggatttgcc cgtaatcaag ctgggtaatg cgcgggttgt 7620 cctttcgcgc cgcaggcgtc gtaaaaaggg gcagcgttca tccctgaaag gtggcggcag 7680 cgtgcttgtg gtgggtaacc gtcgtattcc cggcgcgttt attcagcaac tgaaaaatgg 7740 ccggtggcat gtcatgcagc gtgtggctgg gaaaaaccgt taccccattg atgtggtgaa 7800 aatcccgatg gcggtgccgc tgaccacggc gtttaaacaa aatattgagc ggatacggcg 7860 tgaacgtctt ccgaaagagc tgggctatgc gctgcagcat caactgagga tggtaataaa 7920 gcgatgaaac atactgaact ccgtgcagcc gtactggatg cactggagaa gcatgacacc 7980 ggggcgacgt tttttgatgg tcgccccgct gtttttgatg aggcggattt tccggcagtt 8040 gccgtttatc tcaccggcgc tgaatacacg ggcgaagagc tggacagcga tacctggcag 8100 gcggagctgc atatcgaagt tttcctgcct gctcaggtgc cggattcaga gctggatgcg 8160 tggatggagt cccggattta tccggtgatg agcgatatcc cggcactgtc agatttgatc 8220 accagtatgg tggccagcgg ctatgactac cggcgcgacg atgatgcggg cttgtggagt 8280 tcagccgatc tgacttatgt cattacctat gaaatgtgag gacgctatgc ctgtaccaaa 8340 tcctacaatg ccggtgaaag gtgccgggac caccctgtgg gtttataagg ggagcggtga 8400 cccttacgcg aatccgcttt cagacgttga ctggtcgcgt ctggcaaaag ttaaagacct 8460 gacgcccggc gaactgaccg ctgagtccta tgacgacagc tatctcgatg atgaagatgc 8520 agactggact gcgaccgggc aggggcagaa atctgccgga gataccagct tcacgctggc 8580 gtggatgccc ggagagcagg ggcagcaggc gctgctggcg tggtttaatg aaggcgatac 8640 ccgtgcctat aaaatccgct tcccgaacgg cacggtcgat gtgttccgtg gctgggtcag 8700 cagtatcggt aaggcggtga cggcgaagga agtgatcacc cgcacggtga aagtcaccaa 8760 tgtgggacgt ccgtcgatgg cagaagatcg cagcacggta acagcggcaa ccggcatgac 8820 cgtgacgcct gccagcacct cggtggtgaa agggcagagc accacgctga ccgtggcctt 8880 ccagccggag ggcgtaaccg acaagagctt tcgtgcggtg tctgcggata aaacaaaagc 8940 caccgtgtcg gtcagtggta tgaccatcac cgtgaacggc gttgctgcag gcaaggtcaa 9000 cattccggtt gtatccggta atggtgagtt tgctgcggtt gcagaaatta ccgtcaccgc 9060 cagttaatcc ggagagtcag cgatgttcct gaaaaccgaa tcatttgaac ataacggtgt 9120 gaccgtcacg ctttctgaac tgtcagccct gcagcgcatt gagcatctcg ccctgatgaa 9180 acggcaggca gaacaggcgg agtcagacag caaccggaag tttactgtgg aagacgccat 9240 cagaaccggc gcgtttctgg tggcgatgtc cctgtggcat aaccatccgc agaagacgca 9300 gatgccgtcc atgaatgaag ccgttaaaca gattgagcag gaagtgctta ccacctggcc 9360 cacggaggca atttctcatg ctgaaaacgt ggtgtaccgg ctgtctggta tgtatgagtt 9420 tgtggtgaat aatgcccctg aacagacaga ggacgccggg cccgcagagc ctgtttctgc 9480 gggaaagtgt tcgacggtga gctgagtttt gccctgaaac tggcgcgtga gatggggcga 9540 cccgactggc gtgccatgct tgccgggatg tcatccacgg agtatgccga ctggcaccgc 9600 ttttacagta cccattattt tcatgatgtt ctgctggata tgcacttttc cgggctgacg 9660 tacaccgtgc tcagcctgtt tttcagcgat ccggatatgc atccgctgga tttcagtctg 9720 ctgaaccggc gcgaggctga cgaagagcct gaagatgatg tgctgatgca gaaagcggca 9780 gggcttgccg gaggtgtccg ctttggcccg gacgggaatg aagttatccc cgcttcccg 9840 gatgtggcgg acatgacgga ggatgacgta atgctgatga cagtatcaga agggatcgca 9900 ggaggagtcc ggtatggctg aaccggtagg cgatctggtc gttgatttga gtctggatgc 9960 ggccagattt gacgagcaga tggccagagt caggcgtcat ttttctggta cggaaagtga 10020 tgcgaaaaaa acagcggcag tcgttgaaca gtcgctgagc cgacaggcgc tggctgcaca 10080 gaaagcgggg atttccgtcg ggcagtataa agccgccatg cgtatgctgc ctgcacagtt 10140 caccgacgtg gccacgcagc ttgcaggcgg gcaaagtccg tggctgatcc tgctgcaaca 10200 gggggggcag gtgaaggact ccttcggcgg gatgatcccc atgttcaggg ggcttgccgg 10260 tgcgatcacc ctgccgatgg tgggggccac ctcgctggcg gtggcgaccg gtgcgctggc 10320 gtatgcctgg tatcagggca actcaaccct gtccgatttc aacaaaacgc tggtcctttc 10380 cggcaatcag gcgggactga cggcagatcg tatgctggtc ctgtccagag ccgggcaggc 10440 ggcagggctg acgtttaacc agaccagcga gtcactcagc gcactggtta aggcgggggt 10500 aagcggtgag gctcagattg cgtccatcag ccagagtgtg gcgcgtttct cctctgcatc 10560 cggcgtggag gtggacaagg tcgctgaagc cttcgggaag ctgaccacag acccgacgtc ggggctgacg gcgatggctc gccagttcca taacgtgtcg gcggagcaga ttgcgtatgt 10680 tgctcagttg cagcgttccg gcgatgaagc cggggcattg caggcggcga acgaggccgc 10740 aacgaaaggg tttgatgacc agacccgccg cctgaaagag aacatgggca cgctggagac ctgggcagac aggactgcgc gggcattcaa atccatgtgg gatgcggtgc tggatattgg 10860 tcgtcctgat accgcgcagg agatgctgat taaggcagag gctgcgtata agaaagcaga 10920 cgacatctgg aatctgcgca aggatgatta ttttgttaac gatgaagcgc gggcgcgtta 10980. ctgggatgat cgtgaaaagg cccgtcttgc gcttgaagcc gcccgaaaga aggctgagca gcagactcaa caggacaaaa atgcgcagca gcagagcgat accgaagcgt cacggctgaa atataccga gaggcgcaga aggcttacga acggctgcag acgccgctgg agaaatatac cgcccgtcag gaagaactga acaaggcact gaaagacggg aaaatcctgc aggcggatta caacacgctg atggcggcgg cgaaaagga ttatgaagcg acgctgaaaa agccgaaaca gtccagcgtg aaggtgtctg cgggcgatcg tcaggaagac agtgctcatg ctgccctgct gacgcttcag gcagaactcc ggacgctgga gagcatgcc ggagcaaatg agaaaatcag ccagcagcgc cgggatttgt ggaggcgga gagtcagttc gcggtactgg aggaggcggc 11460 gcaacgtcgc cagctgtctg cacaggaga atccctgctg gcgcataaag atgagacgct ggagtacaaa cgccagctgg ctgcacttgg cgacaaggtt acgtatcagg agcgcctgaa cgcgctggcg cagcaggcgg ataattcgc acagcagcaa cgggcaaaac gggccgccat tgatgcgaaa agccgggggc tgactgaccg gcaggcagaa cgggaagcca cggaacagcg cctgaagga cagtatggcg fatherccgct ggcgctgaat aacgtcatgt cagagcagaa aaagacctgg gcggctgaag accagcttcg cgggaactgg atggcaggcc tgaagtccgg ctggagtgag tgggagaga gcgccacgga cagtatgtcg caggtaaaa gtgcagccac gcagaccttt gatggtattg cacagaatat ggcggcgatg ctgaccggca gtgagcagaa ctggcgcagc ttcaccccgtt ccgtgctgtc catgatgaca gaaattctgc ttaagcaggc 12000 aatggtgggg attgtcggga gtatcggcag cgccattggc ggggctggtg gtggcggcgc 12060 atccgcgtca ggcggtacag ccattcaggc cgctgcggcg aaattccatt ttgcaaccgg 12120 aggatttacg ggaaccggcg gcaatatga gccagcgggg attgttcacc gtggtgagtt 12180 tgtcttcacg aaagggcaa ccagccggat tggcgtgggg aatctttacc ggctgatgcg 12240 cggctatgcc accggcggtt atgtcggtac accgggcagc atggcagaca gccggtcgca 12300 ggcgtccggg acgtttgagc agaataacca tgtggtgatt aacaacgacg gcacgaacgg 12360 gcagataggt ccggctgctc tgaaggcggt gtatgacatg gcccgcaagg gtgcccgtga 12420 tgaaattcag acacagatgc gtgatggtgg cctgttctcc ggaggtggac gatgaagacc 12480 ttccgctgga aagtgaaacc cggtatggat gtggcttcgg tcccttctgt aagaaaggtg 12540 cgctttggtg atggctattc tcagcgagcg cctgccgggc tgaatgccaa cctgaaaacg 12600 tacagcgtga cgctttctgt cccccgtgag gaggccacgg tactggagtc gtttctggaa 12660 gagcacgggg gctggaaatc ctttctgtgg acgccgcctt atgagtggcg gcagataaag 12720 gtgacctgcg caaaatggtc gtcgcgggtc agtatgctgc gtgttgagtt cagcgcagag 12780 tttgaacagg tggtgaactg atgcaggata tccggcagga aacactgaat gaatgcaccc 12840 gtgcggagca gtcggccagc gtggtgctct gggaaatcga cctgacagag gtcggtggag 12900 aacgttattt tttctgtaat gagcagaacg aaaaaggtga gccggtcacc tggcaggggc 12960 gacagtatca gccgtatccc attcaggggga gcggttttga actgaatggc aaaggcacca 13020 gtacgcgccc cacgctgacg gtttctaacc tgtacggtat ggtcaccggg atggcggaag 13080 atatgcagag tctggtcggc ggaacggtgg tccggcgtaa ggtttacgcc cgttttctgg 13140 atgcggtgaa cttcgtcaac ggaaacagtt acgccgatcc ggagcaggag gtgatcagcc 13200 gctggcgcat tgagcagtgc agcgaactga gcgcggtgag tgcctccttt gtactgtcca 13260 cgccgacgga aacggatggc gctgtttttc cgggacgtat catgctggcc aacacctgca 13320 cctggaccta tcgcggtgac gagtgcggtt atagcggtcc ggctgtcgcg gatgaatatg 13380 accagccaac gtccgatatc acgaaggata aatgcagcaa atgcctgagc ggttgtaagt 13440 tccgcaataa cgtcggcaac tttggcggct tcctttccat taacaaactt tcgcagtaaa 13500 tcccatgaca cagacagaat cagcgattct ggcgcacgcc cggcgatgtg cgccagcgga 13560 gtcgtgcggc ttcgtggtaa gcacgccgga gggggaaaga tatttcccct gcgtgaatat 13620 ctccggtgag ccggaggcta tttccgtatg tcgccggaag actggctgca ggcagaaatg 13680 cagggtgaga ttgtggcgct ggtccacagc caccccggtg gtctgccctg gctgagtgag 13740 gccgaccggc ggctgcaggt gcagagtgat ttgccgtggt ggctggtctg ccgggggacg 13800 attcataagt tccgctgtgt gccgcatctc accgggcggc gctttgagca cggtgtgacg 13860 gactgttaca cactgttccg ggatgcttat catctggcgg ggattgagat gccggacttt 13920 catcgtgagg atgactggtg gcgtaacggc cagaatctct atctggataa tctggaggcg 13980 acggggctgt atcaggtgcc gttgtcagcg gcacagccgg gcgatgtgct gctgtgctgt 14040 tttggttcat cagtgccgaa tcacgccgca atttactgcg gcgacggcga gctgctgcac 14100 catattcctg aacaactgag caaacgagag aggtacaccg acaaatggca gcgacgcaca 14160 cactccctct ggcgtcaccg ggcatggcgc gcatctgcct ttacggggat ttacaacgat 14220 ttggtcgccg catcgacctt cgtgtgaaaa cgggggctga agccatccgg gcactggcca 14280 cacagctccc ggcgtttcgt cagaaactga gcgacggctg gtatcaggta cggattgccg 14340 ggcgggacgt cagcacgtcc gggttaacgg cgcagttaca tgagactctg cctgatggcg 14400 ctgtaattca tattgttccc agagtcgccg gggccaagtc aggtggcgta ttccagattg 14460 tcctgggggc tgccgccatt gccggatcat tctttaccgc cggagccacc cttgcagcat 14520 ggggggcagc cattggggcc ggtggtatga ccggcatcct gttttctctc ggtgccagta 14580 tggtgctcgg tggtgtggcg cagatgctgg caccgaaagc cagaactccc cgtatacaga 14640 caacggataa cggtaagcag aacacctatt tctcctcact ggataacatg gttgcccagg 14700 gcaatgttct gcctgttctg tacgggggaaa tgcgcgtggg gtcacgcgtg gtttctcagg 14760 agatcagcac ggcaggaa ggggacggtg gtcaggttgt ggtgattggt cgctgatgca 14820 aaatgtttta tgtgaaaccg cctgcgggcg gttttgtcat ttatggagcg tgaggaatgg 14880 gtaaaggaag cattaagggg cataccccgc gcgaagcgaa ggacaacctg aagtccacgc 14940 agttgctgag tgtgatcgat gccatcagcg aagggccgat tgaaggtccg gtggatggct 15000 taaaaagcgt gctgctgaac agtacgccgg tgctggacac tgagggaat accaacatat 15060 ccggtgtcac ggtggtgttc cgggctggtg agcaggagca gactccgccg gagggatttg 15120 15180 gcaccattac gtctgcaaac atcgaccgtc tgcgctttac cttcggtgta caggcactgg 15240 tggaaccac ctcaaagggt gacggaatc cgtcggaagt ccgcctgctg gttcagatac 15300 aacgtaacgg tggctgggtg acggaaaaag acatcaccat taagggcaaa accacctcgc 15360 agtatctggc ctcggtggtg atgggtaacc tgccgccgcg cccgtttaat atccggatgc 15420 gcaggatgac gccggacagc accacagacc agctgcagaa caaaacgctc tggtcgtcat 15480 acactgaaat catcgatgtg aaacagtgct acccgaacac ggcactggtc ggcgtgcagg 15540 tggactcgga gcagttcggc agccagcagg tgagccgtaa ttatcatctg cgcgggcgta 15600 ttctgcaggt gccgtcgaac tataacccgc agacgcggca atacagcggt atctgggacg 15660 gaacgtttaa accggcatac agcaacaaca tggcctggtg tctgtgggat atgctgaccc 15720 atccgcgcta cggcatgggg aaacgtcttg gtgcggcgga tgtggataaa tgggcgctgt 15780 atgtcatcgg ccagtactgc gaccagtcag tgccggacgg ctttggcggc acggagccgc 15840 gcatcacctg taatgcgtac ctgaccacac agcgtaaggc gtgggatgtg ctcagcgatt 15900 tctgctcggc gatgcgctgt atgccggtat ggaacgggca gacgctgacg ttcgtgcagg 15960 accgaccgtc ggataagacg tggacctata accgcagtaa tgtggtgatg ccggatgatg 16020 gcgcgccgtt ccgctacagc ttcagcgccc tgaaggaccg ccataatgcc gttgaggtga 16080 actggattga cccgaacaac ggctgggaga cggcgacaga gcttgttgaa gatacgcagg 16140 ccattgcccg ttacggtcgt aatgttacga agatggatgc ctttggctgt accagccggg 16200 ggcaggcaca ccgcgccggg ctgtggctga ttaaaacaga actgctggaa acgcagaccg 16260 tggatttcag cgtcggcgca gaagggcttc gccatgtacc gggcgatgtt attgaaatct 16320 gcgatgatga ctatgccggt atcagcaccg gtggtcgtgt gctggcggtg aacagccaga 16380 cccggacgct gacgctcgac cgtgaaatca cgctgccatc ctccggtacc gcgctgataa 16440 gcctggttga cggaagtggc aatccggtca gcgtggaggt tcagtccgtc accgacggcg 16500 tgaaggtaaa agtgagccgt gttcctgacg gtgttgctga atacagcgta tgggagctga 16560 agctgccgac gctgcgccag cgactgttcc gctgcgtgag tatccgtgag aacgacgacg 16620 gcacgtatgc catcaccgcc gtgcagcatg tgccggaaaa agaggccatc gtggataacg 16680 gggcgcactt tgacggcgaa cagagtggca cggtgaatgg tgtcacgccg ccagcggtgc 16740 agcacctgac cgcagaagtc actgcagaca gcgggaata tcaggtgctg gcgcgatggg 16800 acacaccgaa ggtggtgaag ggcgtgagtt tcctgctccg tctgaccgta acagcggacg 16860 acggcagtga gcggctggtc agcacggccc ggacgacgga aaccacatac cgcttcacgc 16920 aactggcgct ggggaactac aggctgacag tccggcggt aactgcgtgg gggcagcagg 16980 gcgatccggc gtcggtatcg ttccggattg ccgcaccggc agcaccgtcg aggattgagc 17040 tgacgccggg ctattttcag ataccgcca cgccgcatct tgccgtttat gacccgacgg 17100 tacagtttga gttctggttc tcggaaagc agattgcgga tatcagacag gttgaaacca 17160 gcacgcgtta tcttggtacg gcgctgtact ggatagccgc cagtatcaat atcaaccgg 17220 gccatgatta ttacttat atccgcagtg tgaacaccgt tggcaatcg gcattcgtgg 17280 aggccgtcgg tcggcgagc gatgatgcgg aaggttacct ggatttttc aaggcaaga 17340 taaccgaatc ccatctcggc aaggagctgc tggaaaagt cgagctgacg gaggataacg 17400 cagcagact ggaggatttt tcgaagagt ggaaggatgc cagtgataag tggaatgcca 17460 tgtgggctgt caaaattgag cagaccaaag acggcaaaca ttatgtcgcg ggtattggcc 17520 tcagcatgga ggacacggag gaaggcaaac tgagccagtt tctggttgcc gccaatcgta 17580 tcgcatttat tgacccggca aacgggaatg aaacgccgat gtttgtggcg cagggcaacc 17640 agatattcat gaacgacgtg ttcctgaagc gcctgacggc ccccaccatt accagcggcg 17700 gcaatcctcc ggccttttcc ctgacaccgg acggaaagct gaccgctaaa aatgcggata 17760 tcagtggcag tgtgaatgcg aactccggga cgctcagtaa tgtgacgata gctgaaaact 17820 gtacgataaa cggtacgctg agggcggaaa aaatcgtcgg ggacattgta aaggcggcga 17880 gcgcggcttt tccgcgccag cgtgaaagca gtgtggactg gccgtcaggt acccgtactg 17940 tcaccgtgac cgatgaccat ccttttgatc gccagatagt ggtgcttccg ctgacgtttc 18000 gcggaagtaa gcgtactgtc agcggcagga caacgtattc gatgtgttat ctgaaagtac 18060 tgatgaacgg tgcggtgatt tatgatggcg cggcgaacga ggcggtacag gtgttctccc 18120 gtattgttga catgccagcg ggtcggggaa acgtgatcct gacgttcacg cttacgtcca 18180 cacggcattc ggcagatatt ccgccgtata cgtttgccag cgatgtgcag gttatggtga 18240 ttaagaaaca ggcgctgggc atcagcgtgg tctgagtgtg ttacagaggt tcgtccggga 18300 acgggcgttt tattataaaa cagtgagagg tgaacgatgc gtaatgtgtg tattgccgtt 18360 gctgtctttg ccgcacttgc ggtgacagtc actccggccc gtgcggaagg tggacatggt 18420 acgtttacgg tgggctattt tcaagtgaaa ccgggtacat tgccgtcgtt gtcgggcggg 18480 gataccggtg tgagtcatct gaaagggatt aacgtgaagt accgttatga gctgacggac 18540 agtgtggggg tgatggcttc cctggggttc gccgcgtcga aaaagagcag cacagtgatg 18600 accggggagg atacgtttca ctatgagagc ctgcgtggac gttatgtgag cgtgatggcc 18660 ggaccggttt tacaaatcag taagcaggtc agtgcgtacg ccatggccgg agtggctcac 18720 agtcggtggt ccggcagtac aatggattac cgtaagacgg aaatcactcc cgggtatatg 18780 aaagagacga ccactgccag ggacgaaagt gcaatgcggc atacctcagt ggcgtggagt 18840 gcaggtatac agattaatcc ggcagcgtcc gtcgttgttg atattgctta tgaaggctcc 18900 ggcagtggcg actggcgtac tgacggattc atcgttgggg tcggttataa attctgatta 18960 gccaggtaac acagtgttat gacagcccgc cggaaccggt gggcttttt gtggggtgaa 19020 tatggcagta aagatttcag gagtcctgaa agacggcaca ggaaaaccgg tacagaactg 19080 caccattcag ctgaaagcca gacgtaacag caccacggtg gtggtgaaca cggtgggctc 19140 agagaatccg gatgaagccg ggcgttacag catggatgtg gagtacggtc agtacagtgt 19200 catcctgcag gttgacggtt ttccaccatc gcacgccggg accatcaccg tgtatgaaga 19260 ttcacaaccg gggacgctga atgattttct ctgtgccatg acggaggatg atgcccggcc 19320 ggaggtgctg cgtcgtcttg aactgatggt ggaagaggtg gcgcgtaacg cgtccgtggt 19380 ggcacagagt acggcagacg cgaagaaatc agccggcgat gccagtgcat cagctgctca 19440 ggtcgcggcc cttgtgactg atgcaactga ctcagcacgc gccgccagca cgtccgccgg 19500 acaggctgca tcgtcagctc aggaagcgtc ctccggcgca gaagcggcat cagcaaaggc 19560 cactgaagcg gaaaaaagtg ccgcagccgc agagtcctca aaaa 19604 <210> 10 <211> 10058 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 10 acgcggcggc caccagtgcc ggtgcggcga aaacgtcaga aacgaatgct gcagcgtcac 60 aacaatcagc cgccacgtct gcctccaccg cggccacgaa agcgtcagag gccgccactt 120 cagcacgaga tgcggtggcc tcaaaagagg cagcaaaatc atcagaaacg aacgcatcat 180 caagtgccgg tcgtgcagct tcctcggcaa cggcggcaga aaattctgcc agggcggcaa 240 aaacgtccga gacgaatgcc aggtcatctg aaacagcagc ggaacggagc gcctctgccg 300 cggcagacgc aaaaacagcg gcggcgggga gtgcgtcaac ggcatccacg aaggcgacag 360 aggctgcggg aagtgcggta tcagcatcgc agagcaaaag tgcggcagaa gcggcggcaa 420 tacgtgcaaa aaattcggca aaacgtgcag aagatatagc ttcagctgtc gcgcttgagg 480 atgcggacac aacgagaaag gggatagtgc agctcagcag tgcaaccaac agcacgtctg 540 aaacgcttgc tgcaacgcca aaggcggtta aggtggtaat ggatgaaacg aacagaaaag 600 cccactggac agtccggcac tgaccggaac gccaacagca ccaaccgcgc tcaggggaac 660 aaacaatacc cagattgcga acaccgcttt tgtactggcc gcgattgcag atgttatcga 720 cgcgtcacct gacgcactga atacgctgaa tgaactggcc gcagcgctcg ggaatgatcc 780 agattttgct accaccatga ctaacgcgct tgcgggtaaa caaccgaaga atgcgacact 840 gacggcgctg gcagggcttt ccacggcgaa aaataaatta ccgtattttg cggaaaatga 900 tgccgccagc ctgactgaac tgactcaggt tggcagggat attctggcaa aaaattccgt 960 tgcagatgtt cttgaatacc ttggggccgg tgagaattcg gcctttccgg caggtgcgcc 1020 gatcccgtgg ccatcagata tcgttccgtc tggctacgtc ctgatgcagg ggcaggcgtt 1080 tgacaaatca gcctacccaa aacttgctgt cgcgtatcca tcgggtgtgc ttcctgatat 1140 gcgaggctgg acaatcaagg ggaaacccgc cagcggtcgt gctgtattgt ctcaggaaca 1200 ggatggaatt aagtcgcaca cccacagtgc cagtgcatcc ggtacggatt tggggacgaa 1260 aaccacatcg tcgttgatt acgggacgaa aaaacaggc agtttcgatt acggcaccaa 1320 atcgacgaat aacacgggggg ctcatgctca cagtctgagc ggttcacag gggccgcggg 1380 tgctcatgcc cacacaagtg gtttaaggat gaacagttct ggctggagtc agtatggaac 1440 acaccatt acaggagtt tatccacagt acaccaggt acacacagg gtattgctta 1500 tttatcgaaa acggacagtc agggcagcca cagtcactca ttgtccggta cagccgtgag 1560 tgccggtgca catgcgcata cagttggtat tggtgcgcac cagcatccgg ttgttatcgg 1620 tgctcatgcc cattctttca gtattggttc acacggacac accatcaccg ttaacgctgc 1680 gggtaacgcg gaaaacaccg tcaaaacat tgcatttaac tatattgtga ggcttgcata 1740 atggcattca gatgagtga acaccacgg accataaaa tttataatct gctggccgga 1800 actaatgaat ttattggtga aggtgacgca tatattccgc ctcataccgg tctgcctgca 1860 aacagtaccg atattgcacc gccagatatt ccggctggct tgtggctgt ttcacagt 1920 gatgaggcat cgtggcatct cgttgaagac catcggggta aaaccgtcta tgacgtggct 1980 tccggcgacg cgttatttat ttctgaactc ggtccgttac cggaaaattt tacctggtta 2040 tcgccgggag gggaatatca gaagtggaac ggcacagcct gggtgaagga tacggaagca 2100 gaaaaactgt tccggatccg ggaggcggaa gaaacaaaaa aaagcctgat gcaggtagcc 2160 agtgagcata ttgcgccgct tcaggatgct gcagatctgg aaattgcaac gaaggaagaa 2220 acctcgttgc tggaagcctg gaagaagtat cgggtgttgc tgaaccgtgt tgatacatca 2280 actgcacctg atattgagtg gcctgctgtc cctgttatgg agtaatcgtt ttgtgatatg 2340 ccgcagaaac gttgtatgaa ataacgttct gcggttagtt agtatattgt aaagctgagt 2400 attggtttat ttggcgatta ttatcttcag gagaataatg gaagttctat gactcaattg 2460 ttcatagtgt ttacatcacc gccaattgct tttaagactg aacgcatgaa atatggtttt 2520 tcgtcatgtt ttgagtctgc tgttgatatt tctaaagtcg gttttttttc ttcgttttct 2580 ctaactattt tccatgaaat acatttttga ttattatttg aatcaattcc aattacctga 2640 agtctttcat ctataatttgg cattgtatgt attggtttat tggagtagat gcttgctttt 2700 ctgagccata gctctgatat ccaatgaag ccataggcat ttgttattt ggctctgtca 2760 gctgcataac gccaaaaaat atatttatct gcttgatctt caatgttgt attgattaa 2820 tcaatttggat ggaattgttt atcataaaaa attaatgttt gatgtgata accgtccttt 2880 aaaaaagtcg ttctgcaag ctggctgta tagtcaacta actctctgt cgaagtgata 2940 ttttaggct tattctaccag ttttaggacg tctttatat cttcaggaat tattttg 3000 tcatattgta tcatgctaaa tgacaatttg cttatggagt aatcttta ttttaaataa 3060 gttattctcc tggcttcatc aaaayagg tcgaatg tggcgaat cacatcgtca 3120 cccattggat tgtttatttg tatgccaaga gagttacagc agttacat tctgccatag 3180 attatagcta aggcatgtaa taattcgtaa tcttttagcg tattagcgac ccatcgtctt 3240 tctgatttaa taatagatga ttcagttaaa tatgaagta atttctttg tgcaagtctg 3300 actaactttt ttataccaat gtttaacata ctttcattg tataaactc aatgtcattt 3360 tcttcaatgt aagatgaat aagagtagcc tttgcctcgc tatacatttc taaatcgcct 3420 tgttttcta tcgtattgcg agaattttta gcccaagcca ttaatggatc atttttccat 3480 tttcataa cattattgtt ataccaatg tcatatccta taatctggtt ttgtttttt 3540 tgaataata atgttactgt tcttgcggtt tggagat gattcaaatt caagcgaat 3600 aattcagggt caaatatgt atcaatgcag catttgagca agtgcgataa atctttaagt 3660 cttctttccc atggttttt agtcataaaa ctctccattt tgataggttg catgctagat 3720 gctgatatat tttagaggtg aaaattaa ctgcttaact gtcaatgtaa tacaagttgt 3780 ttgatctttg caatgattct tatcagaac catatagtaa attagttaca caggaattt 3840 ttaatattat tattatcatt cattatgtat taaaattaga gttgtggctt ggctctgcta 3900 acacgttgct cataggagat atggtagagc cgcagacacg tcgtatgcag gaacgtgctg 3960 cggctggctg gtgaacttcc gatagtgcgg gtgttgaatg atttccagtt gctaccgatt 4020 ttacatattt ttgcatgag agaatttgta ccacctccca ccgaccatct atgactgtac 4080 gccactgtcc ctaggactgc tatgtgccgg agcggacatt acaaacgtcc ttctcggtgc 4140 atgccactgt tgccaatgac ctgcctagga attggttagc aagttactac cggattttgt 4200 aaaaacagcc ctcctcatat aaaaagtatt cgttcacttc cgataagcgt cgtaattttc 4260 tatctttcat catattctag atccctctga aaaaatcttc cgagtttgct aggcactgat 4320 acataactct tttccaataa ttggggaagt cattcaaatc tataataggt ttcagatttg 4380 cttcaataaa ttctgactgt agctgctgaa acgttgcggt tgaactatat ttccttataa 4440 cttttacgaa agagtttctt tgagtaatca cttcactcaa gtgcttccct gcctccaaac 4500 gatacctgtt agcaatattt aatagcttga aatgatgaag agctctgtgt ttgtcttcct 4560 gcctccagtt cgccgggcat tcaacataaa aactgatagc acccggagtt ccggaaacga 4620 aatttgcata tacccattgc tcacgaaaaa aaatgtcctt gtcgatatag ggatgaatcg 4680 cttggtgtac ctcatctact gcgaaaactt gacctttctc tcccatattg cagtcgcggc 4740 acgatggaac taaattaata ggcatcaccg aaaattcagg ataatgtgca ataggaagaa 4800 aatgatctat attttttgtc tgtcctatat caccacaaaa tggacatttt tcacctgatg aaacaagcat gtcatcgtaa tatgttctag cgggtttgtt tttatctcgg agattatttt cataaagctt ttctaattta acctttgtca ggttaccaac tactaaggtt gtaggctcaa gagggtgtgt cctgtcgtag gtaaataact gacctgtcga gcttaatt ctatattgtt gttctttctg caaaaaagtg gggaagtgag taatgaatt atttctaaca tttatctgca tcataccttc cgagcattta ttaagcattt cgctatagt tctcgctgga agaggtagtt ttttcattgt actttacctt catctctgtt cattatcatc gcttttaaaa cggttcgacc 5220 ttctaatcct atctgaccat fathertttt tagaatggtt tcataagaaa gctctgaatc 5340. aacggactgc gataataagt ggtggtatcc agaatttgtc acttcaagta aaaacacctc acgagttaa acacctaagt tctcaccgaa tgtctcaata tccggacgga taatatttat tgcttctctt gaccgtagga ctttccacat gcaggatttt ggaacctctt gcagtactac 5460 tggggaatga gttgcaatta ttgctacacc attgcgtgca tcgagtaagt cgcttaatgt tcgtaaaaaa gcagagagca aaggtggatg cagatgaacc tctggttcat cgaataaaac 5580 taatgacttt tcgccaacga catctactaa tcttgtgata gtaaataaaa caattgcatg 5640 tccagagctc attcgaagca gatatttctg gatattgtca taaaacaatt tagtgaattt 5700 atcatcgtcc acttgaatct gtggttcatt acgtcttaac tcttcatatt tagaaatgag 5760 gctgatgagt tccatatttg aaaagttttc atcactactt agttttttga tagcttcaag 5820 ccagagttgt ctttttctat ctactctcat acaaccaata aatgctgaaa tgaattctaa 5880 gcggagatcg cctagtgatt ttaaactatt gctggcagca ttcttgagtc caatataaaa 5940 gtattgtgta ccttttgctg ggtcaggttg ttctttagga ggagtaaaag gatcaaatgc 6000 actaaacgaa actgaaacaa gcgatcgaaa atatcccttt gggattcttg actcgataag 6060 tctattattt tcagagaaaa aatattcatt gttttctggg ttggtgattg caccaatcat 6120 tccattcaaa attgttgttt taccacaccc attccgcccg ataaaagcat gaatgttcgt 6180 gctgggcata gaattaaccg tcacctcaaa aggtatagtt aaatcactga atccgggagc 6240 actttttcta ttaaatgaaa agtggaaatc tgacaattct ggcaaaccat ttaacacacg 6300 tgcgaactgt ccatgaattt ctgaaagagt tacccctcta agtaatgagg tgttaaggac 6360 gctttcattt tcaatgtcgg ctaatcgatt tggccatact actaaatcct gaatagcttt 6420 aagaaggtta tgtttaaaac catcgcttaa tttgctgaga ttaacatagt agtcaatgct 6480 ttcacctaag gaaaaaaaca tttcagggag ttgactgaat tttttatcta ttaatgaata 6540 agtgcttact tcttcttttt gacctacaaa accaatttta acatttccga tatcgcattt 6600 ttcaccatgc tcatcaaaga cagtaagata aaacattgta acaaaggaat agtcattcca 6660 accatctgct cgtaggaatg ccttattttt ttctactgca ggaatatacc cgcctctttc 6720 aataacacta aactccaaca tatagtaacc cttaatttta ttaaaataac cgcaatttat 6780 ttggcggcaa cacaggatct ctcttttaag ttactctcta ttacatacgt tttccatcta 6840 aaaattagta gtattgaact taacggggca tcgtattgta gttttccata tttagctttc 6900 tgcttcctt tggataaccc actgttattc atgttgcatg gtgcactgtt tataccaacg 6960 atatagtcta ttaatgcata tatagtatcg ccgaacgatt agctcttcag gcttctgaag 7020 aagcgtttca agtactaata agccgataga tagccacgga cttcgtagcc atttttcata 7080 agtgttaact tccgctcctc gctcataaca gacattcact acagttatgg cggaaaggta 7140 tgcatgctgg gtgtggggaa gtcgtgaaag aaaagaagtc agctgcgtcg tttgacatca 7200 ctgctatctt cttactggtt atgcaggtcg tagtgggtgg cacacaaagc tttgcactgg 7260 attgcgaggc tttgtgcttc tctggagtgc gacaggtttg atgacaaaaa attagcgcaa 7320 gaagacaaaa atcaccttgc gctaatgctc tgttacaggt cactaatacc atctaagtag 7380 ttgattcata gtgactgcat atgttgtgtt ttacagtatt atgtagtctg ttttttatgc 7440 aaaatctaat ttaatatatt gatatttata tcattttacg tttctcgttc agctttttta 7500 tactaagttg gcattataaa aaagcattgc ttatcaattt gttgcaacga acaggtcact 7560 atcagtcaaa ataaaatcat tatttgattt caattttgtc ccactccctg cctctgtcat 7620 cacgatactg tgatgccatg gtgtccgact tatgcccgag aagatgttga gcaaacttat 7680 cgcttatctg cttctcatag agtcttgcag acaaactgcg caactcgtga aaggtaggcg 7740 gatccccttc gaaggaaaga cctgatgctt ttcgtgcgcg cataaaatac cttgatactg 7800 tgccggatga aagcggttcg cgacgagtag atgcaattat ggtttctccg ccaagaatct 7860 ctttgcattt atcaagtgtt tccttcattg atattccgag agcatcaata tgcaatgctg 7920 ttgggatggc aatttttacg cctgttttgc tttgctcgac ataaagatat ccatctacga 7980 tatcagacca cttcatttcg cataaatcac caactcgttg cccggtaaca acagccagtt 8040 ccattgcaag tctgagccaa catggtgatg attctgctgc ttgataaatt ttcaggtatt 8100 cgtcagccgt aagtcttgat ctccttacct ctgattttgc tgcgcgagtg gcagcgacat 8160 ggtttgttgt tatatggcct tcagctattg cctctcggaa tgcatcgctc agtgttgatc 8220 tgattaactt ggctgacgcc gccttgccct cgtctatgta tccattgagc attgccgcaa 8280 tttcttttgt ggtgatgtct tcaagtggag catcaggcag acccctcctt attgctttaa 8340 ttttgctcat gtaatttatg agtgtcttct gcttgattcc tctgctggcc aggatttttt 8400 cgtagcgatc aagccatgaa tgtaacgtaa cggaattatc actgttgatt ctcgctgtca 8460 gaggcttgtg tttgtgtcct gaaaataact caatgttggc ctgtatagct tcagtgattg 8520 cgattcgcct gtctctgcct aatccaaact ctttacccgt ccttgggtcc ctgtagcagt 8580 aatatccatt gtttcttata taaaggttag ggggtaaatc ccggcgctca tgacttcgcc 8640 ttcttcccat ttctgatcct cttcaaaagg ccacctgtta ctggtcgatt taagtcaacc 8700 tttaccgctg attcgtggaa cagatactct cttccatcct taaccggagg tgggaatatc 8760 ctgcattccc gaacccatcg acgaactgtt tcaaggcttc ttggacgtcg ctggcgtgcg 8820 ttccactcct gaagtgtcaa gtacatcgca aagtctccgc aattacacgc aagaaaaaac 8880 cgccatcagg cggcttggtg ttctttcagt tcttcaattc gaatattggt tacgtctgca 8940 tgtgctatct gcgcccatat catccagtgg tcgtagcagt cgttgatgtt ctccgcttcg 9000 ataactctgt tgaatggctc tccattccat tctcctgtga ctcggaagtg catttatcat 9060 ctccataaaa caaaacccgc cgtagcgagt tcagataaaa taaatccccg cgagtgcgag 9120 gattgttatg tatattggg tttaatcatc tatatgtttt gtacagagag ggcaagtatc 9180 gtttccaccg tactcgtgat aataattttg cacggtatca gtcatttctc gcacattgca 9240 gaatggggat ttgtcttcat tagacttata aaccttcatg gaatatttgt atgccgactc 9300 tatatctata ccttcatcta cataaacacc ttcgtgatgt ctgcatggag acaagacacc 9360 ggatctgcac aacattgata acgcccaatc tttttgctca gactctaact cattgatact 9420 catttataaa ctccttgcaa tgtatgtcgt ttcagctaaa cggtatcagc aatgttatg 9480 taagaaaca gtaagataat actcaacccg atgtttgagt acggtcatca tctgacacta 9540 cagactctgg catcgctgtg aagacgacgc gaaattcagc atttcacaa gcgttacttt 9600 ttacaaaacc gatctcactc tcctttgatg cgaatgccag cgtcagacat catatgcaga 9660 tactcacctg catcctgaac ccattgacct ccaaccccgt aatagcgatg cgtaatgatg 9720 tcgatagtta ctaacgggtc ttgttcgatt aactgccgca gaaactcttc caggtcacca 9780 gtgcagtgct tgataacagg agtcttccca ggatggcgaa caacaagaaa ctggtttccg 9840 tcttcacgga cttcgttgct ttccagttta gcaatacgct tactcccatc cgagataaca ccttcgtaat actcacgctg ctcgttgagt tttgattttg ctgtttcaag ctcaacacgc agtttcccta ctgttagcgc aatatcctcg ttctcctggt cgcggcgttt gatgtattgc 10020. tggtttcttt cccgttcatc cagcagttcc agcacaat 10058 <210> 11 <211> 9105 <212> DNA <213> artificial sequence <220> <223> Lambda DNA <400> 11 cgatggtgtt accaattcat ggaaaaggtc tgcgtcaaat ccccagtcgt catgcattgc ctgctctgcc gcttcacgca gtgcctgaga gttaattcg ctcacttcga acctctctgt 180. ttactgataa gttccagatc ctcctggcaa cttgcacaag tccgacaacc ctgaacgacc aggcgtcttc gttcatctat cggatcgcca cactcacaac aatgagtggc agatatagcc 240 tggtggttca ggcggcgcat ttttattgct gtgttgcgct gtaattcttc tatttctgat 300 gctgaatcaa tgatgtctgc catctttcat taatccctga actgttggtt aatacgcttg agggtgaatg cgaataataa aaaaggagcc tgtagctccc tgatgatttt gcttttcatg 420 ttcatcgttc cttaaagacg ccgtttaaca tgccgattgc caggcttaaa tgagtcggtg 480 tgaatcccat cagcgttacc gtttcgcggt gcttcttcag tacgctacgg caaatgtcat 540 cgacgttttt atccggaaac tgctgtctgg ctttttttga tttcagaatt agcctgacgg 600 gcaatgctgc gaagggcgtt ttcctgctga ggtgtcattg aacaagtccc atgtcggcaa 660 gcataagcac acagaatatg aagcccgctg ccagaaaaat gcattccgtg gttgtcatac 720 ctggtttctc tcatctgctt ctgctttcgc caccatcatt tccagctttt gtgaaaggga 780 tgcggctaac gtatgaaatt cttcgtctgt ttctactggt attggcacaa acctgattcc 840 aatttgagca aggctatgtg ccatctcgat actcgttctt aactcaacag aagatgcttt 900 gtgcatacag cccctcgttt attatttatc tcctcagcca gccgctgtgc tttcagtgga 960 tttcggataa cagaaaggcc gggaaatacc cagcctcgct ttgtaacgga gtagacgaaa 1020 gtgattgcgc ctacccggat attatcgtga ggatgcgtca tcgccattgc tccccaaata 1080 caaaaccaat ttcagccagt gcctcgtcca ttttttcgat gaactccggc acgatctcgt 1140 caaaactcgc catgtacttt tcatcccgct caatcacgac ataatgcagg ccttcacgct 1200 tcatacgcgg gtcatagttg gcaaagtacc aggcattttt tcgcgtcacc cacatgctgt 1260 actgcacctg ggccatgtaa gctgacttta tggcctcgaa accaccgagc cggaacttca 1320 tgaaatcccg ggaggtaaac gggcatttca gttcaaggcc gttgccgtca ctgcataaac 1380 catcgggaga gcaggcggta cgcatacttt cgtcgcgata gatgatcggg gattcagtaa 1440 cattcacgcc ggaagtgaat tcaaacaggg ttctggcgtc gttctcgtac tgttttcccc 1500 aggccagtgc tttagcgtta acttccggag ccacaccggt gcaaacctca gcaagcaggg 1560 tgtggaagta ggacattttc atgtcaggcc acttctttcc ggagcggggt tttgctatca 1620 cgttgtgaac ttctgaagcg gtgatgacgc cgagccgtaa tttgtgccac gcatcatccc 1680 cctgttcgac agctctcaca tcgatcccgg tacgctgcag gataatgtcc ggtgtcatgc 1740 tgccaccttc tgctctgcgg ctttctgttt caggaatcca agagctttta ctgcttcggc 1800 ctgtgtcagt tctgacgatg cacgaatgtc gcggcgaaat atctgggaac agagcggcaa 1860 taagtcgtca tcccatgttt tatccagggc gatcagcaga gtgttaatct cctgcatggt 1920 ttcatcgtta accggagtga tgtcgcgttc cggctgacgt tctgcagtgt atgcagtatt 1980 ttcgacaatg cgctcggctt catccttgtc atagatacca gcaaatccga aggccagacg 2040 ggcacactga atcatggctt tatgacgtaa catccgtttg ggatgcgact gccacggccc 2100 cgtgatttct ctgccttcgc gagttttgaa tggttcgcgg cggcattcat ccatccattc 2160 ggtaacgcag atcggatgat tacggtcctt gcggtaaatc cggcatgtac aggattcatt 2220 gtcctgctca aagtccatgc catcaaactg ctggttttca ttgatgatgc gggaccagcc 2280 atcaacgccc accaccggaa cgatgccatt ctgcttatca ggaaaggcgt aaatttcttt 2340 cgtccacgga ttaaggccgt actggttggc aacgatcagt aatgcgatga actgcgcatc 2400 gctggcatca cctttaaatg ccgtctggcg aagagtggtg atcagttcct gtgggtcgac 2460 agaatccatg ccgacacgtt cagccagctt cccagccagc gttgcgagtg cagtactcat 2520 tcgttttata cctctgaatc aatatcaacc tggtggtgag caatggtttc aaccatgtac cggatgtgtt ctgccatgcg ctcctgaaac tcaacatcgt catcaaacgc acgggtaatg gattttttgc tggccccgtg gcgttgcaaa tgatcgatgc atagcgattc aaacaggtgc 2700. tggggcaggc ctttttccat gtcgtctgcc agttctgcct ctttctcttc acggcgagc 2760 tgctggtagt gacgcgccca gctctgagcc tcaagacgat cctgaatgta ataagcgttc atggctgaac tcctgaaata gctgtgaaaa tatcgcccgc gaaatgccgg gctgattagg aaaacaggaa agggggttag tgaatgcttt tgcttgatct cagtttcagt attaatatcc attttttata agcgtcgacg gcttcacga acatcttttc atcgccaata aaagtggcga tagtgaattt agtctggata gccataagtg tttgatccat tctttggggac tcctggctga ttaagtatgt cgataaggcg tttccatccg tcacgtaatt tacgggtgat tcgttcaagt aaagattcgg aagggcagcc agcaacaggc caccctgcaa tggcatattg catggtgtgc tccttattta tacataacga aaaacgcctc gagtgaagcg ttattggtat gcggtaaaac cgcactcagg cggccttgat agtcatatca tctgaatcaa atattcctga tgtatcgata 3300 tcggtaattc ttattccttc gctaccatcc attggaggcc atccttcctg accatttcca 3360 tcattccagt cgaactcaca cacaacacca tatgcattta agtcgcttga aattgctata 3420 agcagagcat gttgcgccag catgattaat acagcattta atacagagcc gtgtttattg 3480 agtcggtatt cagagtctga ccagaaatta ttaatctggt gaagtttttc ctctgtcatt 3540 acgtcatggt cgatttcaat ttctattgat gctttccagt cgtaatcaat gatgtatttt 3600 ttgatgtttg acatctgttc atatcctcac agataaaaaa tcgccctcac actggagggc 3660 aaagaagatt tccaataatc agaacaagtc ggctcctgtt tagttacgag cgacattgct 3720 ccgtgtattc actcgttgga atgaatacac agtgcagtgt ttattctgtt atttatgcca 3780 aaaataaagg ccactatcag gcagctttgt tgttctgttt accaagttct ctggcaatca 3840 ttgccgtcgt tcgtattgcc catttatcga catatttccc atcttccatt acaggaaaca 3900 tttcttcagg cttaaccatg cattccgatt gcagcttgca tccattgcat cgcttgaatt 3960 gtccacacca ttgattttta tcaatagtcg tagtcatacg gatagtcctg gtattgttcc 4020 atcacatcct gaggatgctc ttcgaactct tcaaattctt cttccatata tcaccttaaa 4080 tagtggattg cggtagtaaa gattgtgcct gtcttttaac cacatcaggc tcggtggttc 4140 tcgtgtaccc ctacagcgag aaatcggata aactattaca acccctacag tttgatgagt 4200 atagaaatgg atccactcgt tattctcgga cgagtgttca gtaatgaacc tctggagaga 4260 accatgtata tgatcgttat ctgggttgga cttctgcttt taagcccaga taactggcct 4320 gaatatgtta atgagagaat cggtattcct catgtgtggc atgttttcgt ctttgctctt 4380 gcatttcgc tagcaattaa tgtgcatcga tttcagcta ttgccagcgc cagatataag 4440 cgatttaagc taagaaaacg cattaagatg caaaacgata aagtgcgatc agtaattcaa 4500 aaccttacag aagagcaatc tatggttttg tgcgcagccc ttaatgaagg caggaagtat 4560 gtggttacat caaaacaatt cccatacatt agtgagttga ttgagcttgg tgtgttgaac 4620 aaaacttttt cccgatggaa tggaaagcat atattattcc ctattgagga tatttactgg 4680 actgaattag ttgccagcta tgatccatat aatattgaga taaagccaag gccaatatct 4740 aagtaactag ataagaggaa tcgattttcc cttaattttc tggcgtccac tgcatgttat 4800 gccgcgttcg ccaggcttgc tgtaccatgt gcgctgattc ttgcgctcaa tacgttgcag 4860 gttgctttca atctgtttgt ggtattcagc cagcactgta aggtctatcg gatttagtgc 4920 gctttctact cgtgatttcg gtttgcgatt cagcgagaga atagggcggt taactggttt 4980 tgcgcttacc ccaaccaaca ggggatttgc tgctttccat tgagcctgtt tctctgcgcg 5040 acgttcgcgg cggcgtgttt gtgcatccat ctggattctc ctgtcagtta gctttggtgg 5100 tgtgtggcag ttgtagtcct gaacgaaaac cccccgcgat tggcacattg gcagctaatc 5160 cggaatcgca cttacggcca atgcttcgt...

Claims

1. A method for preparing adaptor-ligated target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps: a) providing a sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest; b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, both comprising a type II CRISPR nuclease capable of inducing a DSB, thereby generating a target nucleic acid fragment comprising a sequence of interest and two non-target nucleic acid fragments, wherein the sequence of interest is protected from exonuclease cleavage; c) contacting the cleaved nucleic acid molecule obtained in step b) with an exonuclease V and allowing the exonuclease V to digest the two non-target nucleic acid fragments, wherein the first and second gRNA-CAS complexes remain bound to the target nucleic acid fragments during step c); d) optionally, purifying the target nucleic acid fragments comprising the sequence of interest from the digest obtained in step c; and e) connecting the sequencing adapter to the target nucleic acid fragment, Wherein the type II CRISPR nuclease is a Cas9 protein.

2. The method of claim 1, wherein the method does not comprise a further step of protecting the target nucleic acid fragment or the ends of the target nucleic acid fragment prior to the exonuclease V digestion in step c). 3 . The method of claim 1 , wherein before ligating sequencing adapters in step e), a non-template extension reaction is performed on the target nucleic acid fragments. The method of claim 3 , wherein the non-templated extension reaction is a 3′-A addition.

5. The method of claim 1, wherein at least one of the following is satisfied: i) step b) is performed as follows: incubating the first and second gRNA-CAS complexes with the nucleic acid molecule at 10-90° C. for 1 minute to 18 hours; and ii) Step c) is performed by incubating the cleaved nucleic acid molecule with Exonuclease V at 10-90°C for 1 minute to 12 hours.

6. The method of claim 1, wherein at least one of the first and second gRNA-CAS complexes comprises an sgRNA.

7. The method of claim 1, wherein at least one of the first and second gRNA-CAS complexes comprises crRNA and tracrRNA as different molecules.

8. The method according to any one of the preceding claims, wherein in step e) a first sequencing adapter is ligated to the 5' end of the target nucleic acid fragment and a second adapter is ligated to the 3' end of the target nucleic acid fragment.

9. A method for sequencing target nucleic acid fragments from a sample comprising nucleic acid molecules, wherein the target nucleic acid fragments comprise a sequence of interest, and wherein the method comprises the following steps: a) providing a sample comprising the nucleic acid molecule, wherein the nucleic acid molecule comprises the sequence of interest; b) cleaving the nucleic acid molecule with at least a first and a second gRNA-CAS complex, both of which comprise a type II CRISPR nuclease capable of inducing DSBs, thereby generating a target nucleic acid fragment comprising the sequence of interest and two non-target nucleic acid fragments; c) contacting the cleaved nucleic acid molecule obtained in step b) with exonuclease V and allowing the exonuclease V to digest the two non-target nucleic acid fragments; d) optionally, purifying the target nucleic acid fragment containing the sequence of interest from the digest obtained in step c; e) optionally, ligating an adapter to the target nucleic acid fragment; and f) sequencing the at least one target nucleic acid fragment, Wherein the type II CRISPR nuclease is a Cas9 protein.

10. The method of claim 1 or 9, wherein the method is performed in parallel on multiple nucleic acid samples.

11. The method of claim 1 or 9, wherein the nucleic acid molecule is genomic DNA.

12. The method of claim 1 or 9, wherein the nucleic acid molecule is a nucleic acid molecule obtainable from a plant, an animal or a microorganism.

13. The method of claim 1 or 9, wherein the nucleic acid molecule is a nucleic acid molecule obtainable from a human.

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