CN110016481A - 一种pX335-xCas9n载体及其构建方法和应用 - Google Patents
一种pX335-xCas9n载体及其构建方法和应用 Download PDFInfo
- Publication number
- CN110016481A CN110016481A CN201910441500.1A CN201910441500A CN110016481A CN 110016481 A CN110016481 A CN 110016481A CN 201910441500 A CN201910441500 A CN 201910441500A CN 110016481 A CN110016481 A CN 110016481A
- Authority
- CN
- China
- Prior art keywords
- carrier
- xcas9n
- xcas9
- gene
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000010276 construction Methods 0.000 title claims abstract description 15
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 21
- 239000002773 nucleotide Substances 0.000 claims abstract description 15
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 15
- 238000012239 gene modification Methods 0.000 claims abstract description 13
- 108020004414 DNA Proteins 0.000 claims description 90
- 230000029087 digestion Effects 0.000 claims description 32
- 108091034117 Oligonucleotide Proteins 0.000 claims description 19
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 claims description 16
- 239000012634 fragment Substances 0.000 claims description 16
- 239000013612 plasmid Substances 0.000 claims description 15
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 13
- 241000124008 Mammalia Species 0.000 claims description 10
- 239000000969 carrier Substances 0.000 claims description 8
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 7
- 101150033433 Msh2 gene Proteins 0.000 claims description 6
- 101100205885 Mus musculus Tada1 gene Proteins 0.000 claims description 6
- 101150085542 relA gene Proteins 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 3
- 210000004962 mammalian cell Anatomy 0.000 claims description 3
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 2
- 238000005034 decoration Methods 0.000 claims 1
- 108091033409 CRISPR Proteins 0.000 abstract description 13
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 11
- 108091028043 Nucleic acid sequence Proteins 0.000 abstract description 9
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000012163 sequencing technique Methods 0.000 description 20
- 210000004027 cell Anatomy 0.000 description 13
- 238000012795 verification Methods 0.000 description 8
- 239000013600 plasmid vector Substances 0.000 description 7
- 238000000137 annealing Methods 0.000 description 6
- 238000013461 design Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 4
- 239000005089 Luciferase Substances 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 210000005260 human cell Anatomy 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 3
- 102100030091 Dickkopf-related protein 2 Human genes 0.000 description 3
- 101000864647 Homo sapiens Dickkopf-related protein 2 Proteins 0.000 description 3
- 108060001084 Luciferase Proteins 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 241001494479 Pecora Species 0.000 description 2
- 210000004618 arterial endothelial cell Anatomy 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000001890 transfection Methods 0.000 description 2
- 102220605874 Cytosolic arginine sensor for mTORC1 subunit 2_D10A_mutation Human genes 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 241000218636 Thuja Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 101150063416 add gene Proteins 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 230000005782 double-strand break Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/907—Stable introduction of foreign DNA into chromosome using homologous recombination in mammalian cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Cell Biology (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供了一种pX335‑xCas9n载体及其构建方法和应用，属于基因工程技术领域，所述pX335‑xCas9n载体的核苷酸序列如SEQ ID No.1所示。采用本发明提供的pX335‑xCas9n基因修饰效率较高，可以识别40bp的靶标DNA序列并开展特异性切割，产生特定基因的序列突变。普通的CRISPR/xCas9系统仅可识别20bp的靶标系列，本发明将识别序列长度提高了一倍，其特异性大大提高。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及一种pX335-xCas9n载体及其构建方法和应用。
背景技术
文献Hu,J.H.et al.Evolved Cas9variants with broad PAM compatibilityand high DNA specificity.Nature.556(7699),57-63,doi:10.1038/nature26155,(2018)中，公开了CRISPR/xCas9双链断裂基因敲除技术。传统的CRISPR/Cas9系统识别的PAM序列是3’-NGG，这限制了它的应用范围。而CRISPR/xCas9技术将识别范围扩展到了3’-NG(也就是说DNA序列当中只要有一个G就可以被识别)，显著扩展了基因敲除的靶标范围。
虽然CRISPR/xCas9技术相对于传统的CRISPR/Cas9系统特异性有所提高，但是由于它仅仅识别20nt左右的碱基，这就决定了它仍然会存在一定的潜在脱靶效应。
发明内容
有鉴于此，本发明的目的在于提供一种pX335-xCas9n载体及其构建方法和应用，采用本发明提供的pX335-xCas9n载体在基因修饰时，可识别40nt的靶标DNA序列。
为了实现上述发明目的，本发明提供了以下技术方案：
本发明提供了一种pX335-xCas9n载体，所述pX335-xCas9n载体的核苷酸序列如SEQ ID No.1所示。
本发明还提供了上述技术方案所述的pX335-xCas9n载体的构建方法，包括：将pX335载体和pX330-xCas9载体分别经BglⅡ与EcoRⅠ进行双酶切，将分别得到的酶切产物进行连接，得到pX335-xCas9n载体；
所述pX330-xCas9质粒的核苷酸序列如SEQ ID No.2所示。
优选的，所述pX330-xCas9载体的构建方法包括：将pX330质粒和xCas9-39K片段分别经BglⅡ与EcoRⅠ进行双酶切，将分别得到的酶切产物进行连接，得到pX330-xCas9载体；
所述xCas9-39K片段的核苷酸序列如SEQ ID No.3所示。
本发明还提供了上述技术方案所述的pX335-xCas9n载体在哺乳动物的基因修饰中的应用。
优选的，所述应用包括以下步骤：
1)根据哺乳动物的基因组上的基因设计两对单链DNA寡核苷酸，将所述两对单链DNA寡核苷酸分别退火，得到两个双链DNA片段；
2)将所述步骤1)得到的两个双链DNA片段分别插入到权利要求1所述的pX335-xCas9n载体的sgRNA表达框中，得到两个载体；
3)将所述步骤2)得到的两个载体共转染到哺乳动物细胞中，实现对哺乳动物的基因修饰。
优选的，所述两个载体的质量比为(1～5):(1～5)。
优选的，所述单链DNA寡核苷酸的长度为20～30nt。
优选的，所述基因包括人MSH2基因、小鼠Tada1基因和猪RELA基因。
本发明一种pX335-xCas9n载体及其构建方法和应用，所述pX335-xCas9n载体的核苷酸序列如SEQ ID No.1所示。采用本发明提供的pX335-xCas9n载体在基因修饰时，可识别40nt的靶标DNA序列。在本发明中，需要一对(2个)pX335-xCas9n靶标载体互相配合才能够发挥DNA双链断裂的功能，一个靶标载体识别20个碱基，一对靶标载体就识别40个的碱基序列。
本发明的有益效果：采用本发明提供的pX335-xCas9n基因修饰效率较高，可以识别40bp左右的靶标DNA序列并开展特异性切割，产生特定基因的序列突变。普通的CRISPR/xCas9系统仅可识别20bp的靶标系列，本发明将识别序列长度提高了一倍，其特异性大大提高。
附图说明
图1为双荧光素酶报告基因活性检测结果；
图2为菌落PCR检测结果；
图3为实施例3的6个单克隆的序列发生改变结果；
图4为实施例4的5个单克隆的序列发生改变结果；
图5为实施例5的6个单克隆的序列发生改变结果。
具体实施方式
本发明提供了一种pX335-xCas9n载体，所述pX335-xCas9n载体的核苷酸序列如SEQ ID No.1所示。
本发明还提供了上述技术方案所述的pX335-xCas9n载体的构建方法，包括：将pX335载体和pX330-xCas9载体分别经BglⅡ与EcoRⅠ进行双酶切，将分别得到的酶切产物进行连接，得到pX335-xCas9n载体；所述pX330-xCas9质粒的核苷酸序列如SEQ ID No.2所示。
本发明对所述pX335载体和pX330-xCas9载体分别经BglⅡ与EcoRⅠ进行双酶切的条件没有特殊限定，采用常规双酶切的条件即可。
本发明对所述酶切产物进行连接的条件没有特殊限定，采用常规酶切产物连接的条件即可。
在本发明中，所述pX335载体经BglⅡ与EcoRⅠ进行双酶切，酶切得到的片段大小为4538bp，序列如SEQ IDNo.4所示。
在本发明中，所述pX330-xCas9载体经BglⅡ与EcoRⅠ进行双酶切，酶切得到的片段大小为3911bp，序列如SEQ ID No.3所示。
在本发明中，所述pX330-xCas9质粒的核苷酸序列如SEQ ID No.2所示。
在本发明中，所述pX330-xCas9载体的构建方法优选包括：将pX330质粒和xCas9-39K片段分别经BglⅡ与EcoRⅠ进行双酶切，将分别得到的酶切产物进行连接，得到pX330-xCas9载体；所述xCas9-39K片段的核苷酸序列如SEQ ID No.3所示。
本发明对所述pX330质粒和xCas9-39K片段分别经BglⅡ与EcoRⅠ进行双酶切的条件没有特殊限定，采用常规双酶切的条件即可。本发明对所述酶切产物进行连接的条件没有特殊限定，采用常规即可。
在本发明中，所述xCas9-39K片段的核苷酸序列优选如SEQ ID No.3所示。
在本发明中，所述pX330-xCas9载体表达的xCas9蛋白具有DNA靶标切割活性。在本发明中，所述xCas9蛋白的表达框部分来自pX330载体(其中CMV启动子位于pX330-xCas9载体全序列SEQ ID No.2的440-725区段，xCas9基因的5’端编码区位于pX330-xCas9载体全序列SEQ ID No.2的1251-1619区段，转录终止信号bGH pA位于pX330-xCas9载体全序列SEQID No.2的5553-5760区段，这些元件均来自pX330，其在序列SEQ ID No.3的相应位置的数字均依次减去439，详述如下：CMV启动子位于SEQ ID No.3的1-286区段，xCas9基因的5’端编码区位于SEQ ID No.3的812-1180区段，转录终止信号bGH pA位于pX330-xCas9载体全序列SEQ ID No.3的5114-5321区段)，部分来自生物公司合成(即xCas9 39k片段，SEQ IDNo.3)，所述xCas9蛋白的表达框为pX330-xCas9载体全序列SEQ IDNo.2的440-5760片段。
本发明还提供了上述技术方案所述的pX335-xCas9n载体在哺乳动物的基因修饰中的应用。
在本发明中，所述基因优选包括人MSH2基因、小鼠Tada1基因和猪RELA基因。
在本发明中，所述应用优选包括以下步骤：
1)根据哺乳动物的基因组上的基因设计两对单链DNA寡核苷酸，将所述两对单链DNA寡核苷酸分别退火，得到两个双链DNA片段；
2)将所述步骤1)得到的两个双链DNA片段分别插入到上述技术方案所述的pX335-xCas9n载体的sgRNA表达框中，得到两个载体；
3)将所述步骤2)得到的两个载体共转染到哺乳动物细胞中，实现对哺乳动物的基因修饰。
本发明优选根据哺乳动物的基因组上的靶基因设计两对单链DNA寡核苷酸，将所述两对单链DNA寡核苷酸分别退火，得到两个双链DNA片段。本发明对所述设计单链DNA寡核苷酸的原则没有特殊限定，采用常规设计寡核苷酸的原则即可。
本发明优选将得到的两个双链DNA片段分别插入到上述技术方案所述的pX335-xCas9n载体的sgRNA表达框中，得到两个载体。
本发明对所述将双链DNA片段插入到pX335-xCas9n载体的sgRNA表达框中的方法没有特殊限定，采用常规方法即可。
在本发明中，所述两个载体的质量比优选为(1～5):(1～5)。在本发明中，所述单链DNA寡核苷酸的长度优选为20～30nt。
在本发明中，所述pX335-xCas9n载体中的xCas9n具有SEQ ID No.5所示的核苷酸序列。
在本发明中，所述xCas9n表达的蛋白具有造成靶序列区域单链DNA切口的作用。在本发明中，所述xCas9n表达的蛋白的表达框部分来自pX335载体(其中CMV启动子位于pX335-xCas9n全序列的区段440-725，xCas9n基因5’端编码区位于pX335-xCas9n全序列的1251-1566区段，bGH pA转录终止信号位于pX335-xCas9n全序列的5495-5702区段，这些元件均来自pX335,其在序列SEQ ID No.5的相应位置的数字均依次减去439，详述如下：CMV启动子位于SEQ ID No.5的区段1-286，xCas9n基因5’端编码区位于SEQ ID No.5的812-1127区段，bGH pA转录终止信号位于SEQ ID No.5的5056-5263区段)，部分来自pX330-xCas9载体(即BglⅡ、EcoRⅠ双酶切所得3.9kb片段)。在本发明中，xCas9n表达的蛋白的表达框来自两个载体，pX330-xCas9提供识别NGN PAM靶标的7个氨基酸突变，pX335载体提供D10A突变(此突变使得xCas9蛋白失去DNA双链切割的功能，但保留制造DNA单链切口的功能)，两个载体优势互补，共同提供在NGN PAM靶标处制造DNA单链切口的功能。在本发明中，所述xCas9n表达的蛋白的表达框的序列如SEQ ID No.6所示。
在本发明中，所述基因优选包括人MSH2基因、小鼠Tada1基因和猪RELA基因。
下面结合实施例对本发明提供的技术方案进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
质粒载体pX330-xCas9的构建及效率
1、质粒载体pX330-xCas9的构建
合成3911bp的DNA片段xCas9-39K(SEQ ID No.3)，通过BglⅡ与EcoRⅠ双酶切克隆到pX330载体(购自Addgene,货号42230)的多克隆位点，通过测序验证，获得质粒载体pX330-xCas9(SEQ ID No.2)。测序验证采用常规方法即可，测序验证使用的正向测序引物pX330-F(SEQ ID No.7)和反向测序引物pX330-R(SEQ ID No8)。
2、sgRNA靶标载体的构建
针对绵羊DKK2第一外显子序列(SEQ ID No.9)设计xCas9的sgRNA寡核苷酸。
合成6段DNA寡核苷酸，如下：
TX1-F(SEQ ID No.10)：
CACCGTGCCTGCTCCTACTGGCCGC(205-224)，205-224表示该靶标在绵羊DKK2第一外显子序列中的相应位置，以下也表明相应位置，不再解释；
TX1-R(SEQ ID No.11)：
AAACGCGGCCAGTAGGAGCAGGCAC
TX2-F(SEQ ID No.12):
CACCGATCAAGTCCTCTCTGGGCGG(280-299)
TX2-R(SEQ ID No.13):
AAACCCGCCCAGAGAGGACTTGATC
TX3-F(SEQ ID No.14):
caccgcccgcgagctgccgaactg(266-247)
TX3-R(SEQ ID No.15):
AAACCAGTTCGGCAGCTCGCGGGC
TX1-F与TX1-R、TX2-F与TX2-R、TX3-F与TX3-R两两退火(95℃5min，72℃10min，置冰上)，获得三条双链DNA寡核苷酸(TX1、TX2、TX3)，同时使用BbsⅠ内切酶切割pX330-xCas9载体并纯化，将TX1、TX2、TX3分别与切割后的pX330-xCas9载体连接并转化，经PCR检测(PCR检测采用常规方法即可)、测序获得xCas9-TX1、xCas9-TX2、xCas9-TX3载体。
PCR检测引物：TX1-F、TX2-F、TX3-F分别与测序引物bbsR配对，检测相应载体。
测序引物bbsR(SEQ ID No.16)：AAAGTCCCTATTGGCGTTAC。
空载体检测引物：
330F(SEQ ID No.17):5'CGGGTCTTCGAGAAGACCT 3'
bbsR(SEQ ID No.18)：5'AAAGTCCCTATTGGCGTTAC 3'
3、双荧光素酶报告基因活性检测
使用xCas9-TX1、xCas9-TX2和xCas9-TX3载体分别与双荧光素酶报告载体SSA-DKK2(SEQ ID No.19)以1:1的比例共转染猪血管内皮细胞系PIEC(购自中国科学院上海细胞库，目录号：GNO15)，48小时后使用试剂盒检测双荧光素酶报告基因活性(结果如图1，数值的高低反映的是海肾荧光素酶与萤火虫荧光素酶活性的比值，比值越高，说明基因编辑的效率越高)。结果说明，pX330-xCas9载体表达的xCas9蛋白确实具有DNA靶标切割活性。
实施例2
质粒载体pX335-xCas9n的构建
通过BglⅡ与EcoRⅠ对pX335质粒和实施例1获得的pX330-xCas9质粒实施双酶切，将前者的4.5kb片段(SEQ ID No.4)与后者的3.9kb片段(SEQ ID No.3)分别切胶回收并连接、转化、涂板、挑菌、摇菌，使用两对引物(F1和R1、F2和R2)进行PCR检测(PCR检测采用常规方法)，选取扩增片段分别为107bp、192bp的单菌落(如图2所示)送公司测序，获得pX335-xCas9n载体。
F1(SEQ ID No.20)、R1(SEQ ID No.21)、F2(SEQ ID No.22)和R2(SEQ ID No.23)。
实施例3
质粒载体pX335-xCas9n在人细胞系293T中的基因修饰效率
人细胞系293T，购自中国科学院上海细胞库，目录号：SCSP-502。人MSH2基因的第一外显子序列如SEQ IDNo.24所示。
选取人MSH2基因的第一外显子序列的120-190bp区段作为靶标，针对这一靶标设计并合成2对DNA寡核苷酸(如下所示)，
MSH2T1-F(SEQ ID No.25)、MSH2T1-R(SEQ ID No.26)、MSH2T2-F(SEQ ID No.27)和MSH2T2-R(SEQ ID No.28)。
其中MSH2T1-F与MSH2T1-R退火获得带粘性末端的双链DNA片段，经BbsⅠ酶切，连入pX335-xCas9n载体中，获得pX335-xCas9n-F1载体；MSH2T2-F与MSH2T2-R退火获得带粘性末端的双链DNA片段，经BbsⅠ酶切，连入pX335-xCas9n载体中，获得pX335-xCas9n-R1载体。两个质粒均送北京六合华大基因科技股份有限公司测序验证，测序引物bbsR的序列为(SEQID No.29)。
将pX335-xCas9n-F1与pX335-xCas9n-R1载体按照1:1的比例转染人细胞系293T，48小时后，提取细胞基因组DNA，使用引物对MSH2F、MSH2R进行PCR扩增(扩增条件：95℃3min；95℃30S，60℃30S，72℃30S，30个循环；72℃5min)(MSH2F序列SEQ ID No.30；MSH2R序列SEQ ID No.31，对获得的258bp的PCR产物进行克隆测序。共挑取20个单克隆菌落测序，其中有6个单克隆的序列发生改变(如图3所示)。该结果表明，pX335-xCas9n-MSH2TF、pX335-xCas9n-MSH2TR载体混合转染对人细胞系293T的敲除效率达到30％。这一结果说明，本发明构建的pX335-xCas9n载体效率较高，可以识别40bp左右的靶标DNA序列并开展特异性切割，产生特定基因的序列突变。而普通的CRISPR/xCas9系统仅可识别20bp的靶标系列，本发明将识别序列长度提高了一倍，其特异性大大提高。
实施例4
实施例2制备的质粒载体pX335-xCas9n在小鼠细胞系NIH/3T3中的基因修饰效率
小鼠细胞系NIH/3T3,购自中国科学院上海细胞库，目录号：SCSP-515。
小鼠Tada1基因的第一外显子序列SEQ ID No.32。
选取小鼠Tada1基因的第一外显子序列的200-280bp区段作为基因修饰的靶标，针对这一靶标设计并合成2对DNA寡核苷酸(如下)：
Tada1T1-F(SEQ ID No.33)、Tada1T1-R(SEQ ID No.34)、Tada1T2-F(SEQ IDNo.35)和Tada1T2-R(SEQ ID No.36)。
其中Tada1T1-F与Tada1T1-R退火获得带粘性末端的双链DNA片段，经BbsⅠ酶切，连入pX335-xCas9n载体中，获得pX335-xCas9n-F2载体；Tada1T2-F与Tada1T2-R退火获得带粘性末端的双链DNA片段，经BbsⅠ酶切，连入pX335-xCas9n载体中，获得pX335-xCas9n-R2载体。两个质粒均送北京六合华大基因科技股份有限公司测序验证，测序引物bbsR的序列为(SEQ ID No.37)。
测序验证方法(常规方法)：使用测序引物bbsR进行常规测序，测序结果与相应靶标的正向寡核苷酸Tada1T1-F、Tada1T2-F进行比对，同源性为100％者判定为正确的质粒。
将pX335-xCas9n-F2与pX335-xCas9n-R2载体按照1:1的比例转染小鼠细胞系NIH/3T3，48小时后，提取细胞基因组DNA，使用引物对Tada1F、Tada1R进行PCR扩增(Tada1F序列见序列SEQ IDNo.38；Tada1R序列SEQ ID No.39，扩增条件同实施例2)，对获得的157bp的PCR产物进行克隆测序。共挑取20个单克隆菌落测序，其中有5个单克隆的序列发生改变(如图4所示)。该结果表明，pX335-xCas9n-F2与pX335-xCas9n-R2载体混合转染对小鼠细胞系NIH/3T3的敲除效率达到25％。这一结果说明，本实验构建的pX335-xCas9n效率较高，可以识别40bp左右的靶标DNA序列并开展特异性切割，产生特定基因的序列突变。而普通的CRISPR/xCas9系统仅可识别20bp的靶标系列，本发明将识别序列长度提高了一倍，其特异性大大提高。
实施例5
实施例2制备的质粒载体pX335-xCas9n在猪细胞系PIEC中的基因修饰效率
猪血管内皮细胞系，购自中国科学院上海细胞库，目录号：GNO15。
猪RELA基因的第二外显子序列SEQ ID No.40。
选取猪RELA基因的第二外显子序列180-240bp区段作为基因修饰的靶标，针对这一靶标设计合成2对DNA寡核苷酸，如下：
RELAT1-F(SEQ ID No.41)、RELAT1-R(SEQ ID No.42)、RELAT2-F(SEQ ID No.43)和RELAT2-R(SEQ ID No.44)。
其中RELATF-F与RELATF-R退火获得带粘性末端的双链DNA片段，经BbsⅠ酶切，连入pX335-xCas9n载体中，获得pX335-xCas9n-F3载体；RELATR-F与RELATR-R退火获得带粘性末端的双链DNA片段，经BbsⅠ酶切，连入pX335-xCas9n载体中，获得pX335-xCas9n-R3载体。两个质粒均送北京六合华大基因科技股份有限公司测序验证，测序引物bbsR的序列为(SEQID No.45)。测序验证方法：使用测序引物bbsR进行常规测序，测序结果与相应靶标的正向寡核苷酸RELAT1-F、RELAT2-F进行比对，同源性为100％者判定为正确的质粒。
将pX335-xCas9n-F3与pX335-xCas9n-R3载体按照1:1的比例转染猪细胞系PIEC，48小时后，提取细胞基因组DNA，使用引物对RELAF、RELAR进行PCR扩增(RELAF序列(SEQ IDNo.46)；RELAR序列(SEQ ID No.47)，扩增条件同实施例2)，对获得的316bp的PCR产物进行克隆测序。共挑取20个单克隆菌落测序，其中有6个单克隆的序列发生改变(如图5所示)。该结果表明，pX335-xCas9n-F3与pX335-xCas9n-R3载体混合转染对猪细胞系PIEC的敲除效率达到30％。这一结果说明，本发明构建的pX335-xCas9n效率较高，可以识别40bp左右的靶标DNA序列并开展特异性切割，产生特定基因的序列突变。而普通的CRISPR/xCas9系统仅可识别20bp的靶标系列，本发明将识别序列长度提高了一倍，其特异性大大提高。
由以上实施例可以得出，采用本发明提供的pX335-xCas9n基因修饰效率较高，可以识别40bp左右的靶标DNA序列并开展特异性切割，产生特定基因的序列突变。普通的CRISPR/xCas9系统仅可识别20bp的靶标系列，本发明将识别序列长度提高了一倍，其特异性大大提高。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 青岛农业大学
<120> 一种pX335-xCas9n载体及其构建方法和应用
<160> 47
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8449
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360
agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420
aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480
ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900
gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960
ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1020
tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1080
accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1140
ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1200
ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atgtacccat 1260
acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg tccgacaaga 1320
agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg atcaccgacg 1380
agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg cacagcatca 1440
agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag gccacccggc 1500
tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc tatctgcaag 1560
agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 1620
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 1680
acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 1740
acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 1800
tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 1860
tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 1920
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 1980
atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 2040
tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gataccaaac 2100
tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 2160
accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 2220
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaagc 2280
tgtacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 2340
ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 2400
acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 2460
acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 2520
ccttcgacaa cggcatcatc ccccaccaga tccacctggg agagctgcac gccattctgc 2580
ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 2640
tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 2700
ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgagaag gtggtggaca 2760
agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 2820
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 2880
tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgacc 2940
agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 3000
tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 3060
aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 3120
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 3180
cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 3240
acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 3300
ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 3360
agtccgacgg cttcgccaac agaaacttca tccagctgat ccacgacgac agcctgacct 3420
ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 3480
ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 3540
tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 3600
ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 3660
tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 3720
cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 3780
tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 3840
agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 3900
ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 3960
ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 4020
agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 4080
cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 4140
acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 4200
ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 4260
cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 4320
tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 4380
agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 4440
actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 4500
agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 4560
ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 4620
gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 4680
aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 4740
tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 4800
tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 4860
ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 4920
tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgtgctg cagaagggaa 4980
acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 5040
agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 5100
actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 5160
acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 5220
agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 5280
tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 5340
acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 5400
agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 5460
agtaagaatt cctagagctc gctgatcagc ctcgactgtg ccttctagtt gccagccatc 5520
tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct 5580
ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg 5640
gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca ggcatgctgg 5700
ggagcggccg caggaacccc tagtgatgga gttggccact ccctctctgc gcgctcgctc 5760
gctcactgag gccgggcgac caaaggtcgc ccgacgcccg ggctttgccc gggcggcctc 5820
agtgagcgag cgagcgcgca gctgcctgca ggggcgcctg atgcggtatt ttctccttac 5880
gcatctgtgc ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc 5940
ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 6000
gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 6060
ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac 6120
ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc gccctgatag 6180
acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 6240
actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg gattttgccg 6300
atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac 6360
aaaatattaa cgtttacaat tttatggtgc actctcagta caatctgctc tgatgccgca 6420
tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 6480
ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg 6540
ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc tcgtgatacg cctattttta 6600
taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat 6660
gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 6720
agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa 6780
catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac 6840
ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac 6900
atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt 6960
ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc 7020
gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca 7080
ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc 7140
ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag 7200
gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa 7260
ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg 7320
gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa 7380
ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg 7440
gctggctggt ttattgctga taaatctgga gccggtgagc gtggaagccg cggtatcatt 7500
gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt 7560
caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag 7620
cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat 7680
ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct 7740
taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct 7800
tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca 7860
gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc 7920
agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc 7980
aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct 8040
gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag 8100
gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc 8160
tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg 8220
agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag 8280
cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt 8340
gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac 8400
gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgt 8449
<210> 2
<211> 8506
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag 60
ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga 120
aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat 180
atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga 240
cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag 300
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct 360
agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac 420
aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 480
ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc 540
aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 600
caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt 660
acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 720
ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac 780
ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg 840
ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga 900
gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc 960
ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc 1020
tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg 1080
accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag 1140
ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac 1200
ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata 1260
aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga 1320
tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt 1380
acagcatcgg cctggacatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt 1440
acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga 1500
agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga 1560
agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga 1620
tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct 1680
tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg 1740
aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca 1800
gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc 1860
ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt 1920
tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg 1980
gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc 2040
tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggaaacctg attgccctga 2100
gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat accaaactgc 2160
agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc 2220
agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca 2280
tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagctgt 2340
acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg 2400
agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg 2460
gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg 2520
gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct 2580
tcgacaacgg catcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc 2640
ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga 2700
ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga 2760
tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgagaaggtg gtggacaagg 2820
gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg 2880
agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga 2940
ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgaccaga 3000
aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga 3060
aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag 3120
atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg 3180
acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac 3240
tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg 3300
acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga 3360
agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt 3420
ccgacggctt cgccaacaga aacttcatcc agctgatcca cgacgacagc ctgaccttta 3480
aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg 3540
ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg 3600
acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca 3660
gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg 3720
aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc 3780
agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg 3840
accaggaact ggacatcaac cggctgtccg actacgatgt ggaccatatc gtgcctcaga 3900
gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg 3960
gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc 4020
agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga 4080
gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc 4140
ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg 4200
agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg 4260
atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc 4320
acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg 4380
aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga 4440
gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact 4500
ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga 4560
caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga 4620
aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct 4680
tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg 4740
actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg 4800
tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg 4860
ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca 4920
agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg 4980
agctggaaaa cggccggaag agaatgctgg cctctgccgg cgtgctgcag aagggaaacg 5040
aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc 5100
tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact 5160
acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg 5220
ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc 5280
aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca 5340
agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg 5400
ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc 5460
tgggaggcga caaaaggccg gcggccacga aaaaggccgg ccaggcaaaa aagaaaaagt 5520
aagaattcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt 5580
tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc 5640
ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg 5700
tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga 5760
gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct 5820
cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt 5880
gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca 5940
tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc 6000
gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc 6060
ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc 6120
cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc 6180
gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg 6240
gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact 6300
ggaacaacac tcaaccctat ctcgggctat tcttttgatt tataagggat tttgccgatt 6360
tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa 6420
atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag 6480
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc 6540
ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt 6600
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag 6660
gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg 6720
cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga 6780
caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat 6840
ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca 6900
gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc 6960
gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca 7020
atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg 7080
caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca 7140
gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata 7200
accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag 7260
ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg 7320
gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca 7380
acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta 7440
atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct 7500
ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca 7560
gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag 7620
gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat 7680
tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt 7740
taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa 7800
cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga 7860
gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg 7920
gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc 7980
agagcgcaga taccaaatac tgtccttcta gtgtagccgt agttaggcca ccacttcaag 8040
aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc 8100
agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg 8160
cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac 8220
accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga 8280
aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt 8340
ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag 8400
cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg 8460
gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt 8506
<210> 3
<211> 3911
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga ctggaagagt 60
ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc aacatcgtgg 120
acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag aaactggtgg 180
acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac atgatcaagt 240
tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac gtggacaagc 300
tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc atcaacgcca 360
gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga cggctggaaa 420
atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac ctgattgccc 480
tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag gataccaaac 540
tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc cagatcggcg 600
accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc ctgctgagcg 660
acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct atgatcaagc 720
tgtacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg cagcagctgc 780
ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc ggctacattg 840
acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg gaaaagatgg 900
acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg aagcagcgga 960
ccttcgacaa cggcatcatc ccccaccaga tccacctggg agagctgcac gccattctgc 1020
ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc gagaagatcc 1080
tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc agattcgcct 1140
ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgagaag gtggtggaca 1200
agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag aacctgccca 1260
acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg tataacgagc 1320
tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg agcggcgacc 1380
agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc gtgaagcagc 1440
tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc tccggcgtgg 1500
aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt atcaaggaca 1560
aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg ctgaccctga 1620
cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc cacctgttcg 1680
acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc aggctgagcc 1740
ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg gatttcctga 1800
agtccgacgg cttcgccaac agaaacttca tccagctgat ccacgacgac agcctgacct 1860
ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg cacgagcaca 1920
ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca gtgaaggtgg 1980
tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg atcgaaatgg 2040
ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga atgaagcgga 2100
tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc gtggaaaaca 2160
cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg gatatgtacg 2220
tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat atcgtgcctc 2280
agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc gacaagaacc 2340
ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag aactactggc 2400
ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg accaaggccg 2460
agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag ctggtggaaa 2520
cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac actaagtacg 2580
acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc aagctggtgt 2640
ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac taccaccacg 2700
cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag taccctaagc 2760
tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag atgatcgcca 2820
agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc aacatcatga 2880
actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg cctctgatcg 2940
agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt gccaccgtgc 3000
ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg cagacaggcg 3060
gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc gccagaaaga 3120
aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc tattctgtgc 3180
tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg aaagagctgc 3240
tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac tttctggaag 3300
ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag tactccctgt 3360
tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgtgctg cagaagggaa 3420
acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc cactatgaga 3480
agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa cagcacaagc 3540
actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg atcctggccg 3600
acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag cccatcagag 3660
agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc cctgccgcct 3720
tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa gaggtgctgg 3780
acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc gacctgtctc 3840
agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca aaaaagaaaa 3900
agtaagaatt c 3911
<210> 4
<211> 4538
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc 60
cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa 120
atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg 180
ggcaggacag caagggggag gattgggaag agaatagcag gcatgctggg gagcggccgc 240
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 300
ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc 360
gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg 420
gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag 480
cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc 540
cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc 600
tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa 660
aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg 720
ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac 780
actcaaccct atctcgggct attcttttga tttataaggg attttgccga tttcggccta 840
ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac 900
gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca 960
gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc 1020
cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc 1080
atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt 1140
catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac 1200
ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc 1260
ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt 1320
cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct 1380
ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga 1440
tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag 1500
cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca 1560
actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga 1620
aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag 1680
tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc 1740
ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa 1800
tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt 1860
gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg 1920
gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt 1980
tattgctgat aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg 2040
gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat 2100
ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact 2160
gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa 2220
aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt 2280
ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt 2340
ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg 2400
tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca 2460
gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt 2520
agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga 2580
taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc 2640
gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact 2700
gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga 2760
caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg 2820
aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt 2880
tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt 2940
acggttcctg gccttttgct ggccttttgc tcacatgtga gggcctattt cccatgattc 3000
cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg 3060
taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt 3120
ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt 3180
atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccggg tcttcgagaa 3240
gacctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa 3300
aaagtggcac cgagtcggtg cttttttgtt ttagagctag aaatagcaag ttaaaataag 3360
gctagtccgt ttttagcgcg tgcgccaatt ctgcagacaa atggctctag aggtacccgt 3420
tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc gcccattgac 3480
gtcaatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta 3540
aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt 3600
caatgacggt aaatggcccg cctggcattg tgcccagtac atgaccttat gggactttcc 3660
tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg tgagccccac 3720
gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt atttatttat 3780
tttttaatta ttttgtgcag cgatgggggc gggggggggg ggggggcgcg cgccaggcgg 3840
ggcggggcgg ggcgaggggc ggggcggggc gaggcggaga ggtgcggcgg cagccaatca 3900
gagcggcgcg ctccgaaagt ttccttttat ggcgaggcgg cggcggcggc ggccctataa 3960
aaagcgaagc gcgcggcggg cgggagtcgc tgcgacgctg ccttcgcccc gtgccccgct 4020
ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc ccacaggtga 4080
gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg taagggttta 4140
agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg cctgaaatca 4200
ctttttttca ggttggaccg gtgccaccat gtacccatac gatgttccag attacgcttc 4260
gccgaagaaa aagcgcaagg tcgaagcgtc cgacaagaag tacagcatcg gcctggccat 4320
cggcaccaac tctgtgggct gggccgtgat caccgacgag tacaaggtgc ccagcaagaa 4380
attcaaggtg ctgggcaaca ccgaccggca cagcatcaag aagaacctga tcggagccct 4440
gctgttcgac agcggcgaaa cagccgaggc cacccggctg aagagaaccg ccagaagaag 4500
atacaccaga cggaagaacc ggatctgcta tctgcaag 4538
<210> 5
<211> 4215
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgtacccat acgatgttcc agattacgct tcgccgaaga aaaagcgcaa ggtcgaagcg 60
tccgacaaga agtacagcat cggcctggcc atcggcacca actctgtggg ctgggccgtg 120
atcaccgacg agtacaaggt gcccagcaag aaattcaagg tgctgggcaa caccgaccgg 180
cacagcatca agaagaacct gatcggagcc ctgctgttcg acagcggcga aacagccgag 240
gccacccggc tgaagagaac cgccagaaga agatacacca gacggaagaa ccggatctgc 300
tatctgcaag agatcttcag caacgagatg gccaaggtgg acgacagctt cttccacaga 360
ctggaagagt ccttcctggt ggaagaggat aagaagcacg agcggcaccc catcttcggc 420
aacatcgtgg acgaggtggc ctaccacgag aagtacccca ccatctacca cctgagaaag 480
aaactggtgg acagcaccga caaggccgac ctgcggctga tctatctggc cctggcccac 540
atgatcaagt tccggggcca cttcctgatc gagggcgacc tgaaccccga caacagcgac 600
gtggacaagc tgttcatcca gctggtgcag acctacaacc agctgttcga ggaaaacccc 660
atcaacgcca gcggcgtgga cgccaaggcc atcctgtctg ccagactgag caagagcaga 720
cggctggaaa atctgatcgc ccagctgccc ggcgagaaga agaatggcct gttcggaaac 780
ctgattgccc tgagcctggg cctgaccccc aacttcaaga gcaacttcga cctggccgag 840
gataccaaac tgcagctgag caaggacacc tacgacgacg acctggacaa cctgctggcc 900
cagatcggcg accagtacgc cgacctgttt ctggccgcca agaacctgtc cgacgccatc 960
ctgctgagcg acatcctgag agtgaacacc gagatcacca aggcccccct gagcgcctct 1020
atgatcaagc tgtacgacga gcaccaccag gacctgaccc tgctgaaagc tctcgtgcgg 1080
cagcagctgc ctgagaagta caaagagatt ttcttcgacc agagcaagaa cggctacgcc 1140
ggctacattg acggcggagc cagccaggaa gagttctaca agttcatcaa gcccatcctg 1200
gaaaagatgg acggcaccga ggaactgctc gtgaagctga acagagagga cctgctgcgg 1260
aagcagcgga ccttcgacaa cggcatcatc ccccaccaga tccacctggg agagctgcac 1320
gccattctgc ggcggcagga agatttttac ccattcctga aggacaaccg ggaaaagatc 1380
gagaagatcc tgaccttccg catcccctac tacgtgggcc ctctggccag gggaaacagc 1440
agattcgcct ggatgaccag aaagagcgag gaaaccatca ccccctggaa cttcgagaag 1500
gtggtggaca agggcgcttc cgcccagagc ttcatcgagc ggatgaccaa cttcgataag 1560
aacctgccca acgagaaggt gctgcccaag cacagcctgc tgtacgagta cttcaccgtg 1620
tataacgagc tgaccaaagt gaaatacgtg accgagggaa tgagaaagcc cgccttcctg 1680
agcggcgacc agaaaaaggc catcgtggac ctgctgttca agaccaaccg gaaagtgacc 1740
gtgaagcagc tgaaagagga ctacttcaag aaaatcgagt gcttcgactc cgtggaaatc 1800
tccggcgtgg aagatcggtt caacgcctcc ctgggcacat accacgatct gctgaaaatt 1860
atcaaggaca aggacttcct ggacaatgag gaaaacgagg acattctgga agatatcgtg 1920
ctgaccctga cactgtttga ggacagagag atgatcgagg aacggctgaa aacctatgcc 1980
cacctgttcg acgacaaagt gatgaagcag ctgaagcggc ggagatacac cggctggggc 2040
aggctgagcc ggaagctgat caacggcatc cgggacaagc agtccggcaa gacaatcctg 2100
gatttcctga agtccgacgg cttcgccaac agaaacttca tccagctgat ccacgacgac 2160
agcctgacct ttaaagagga catccagaaa gcccaggtgt ccggccaggg cgatagcctg 2220
cacgagcaca ttgccaatct ggccggcagc cccgccatta agaagggcat cctgcagaca 2280
gtgaaggtgg tggacgagct cgtgaaagtg atgggccggc acaagcccga gaacatcgtg 2340
atcgaaatgg ccagagagaa ccagaccacc cagaagggac agaagaacag ccgcgagaga 2400
atgaagcgga tcgaagaggg catcaaagag ctgggcagcc agatcctgaa agaacacccc 2460
gtggaaaaca cccagctgca gaacgagaag ctgtacctgt actacctgca gaatgggcgg 2520
gatatgtacg tggaccagga actggacatc aaccggctgt ccgactacga tgtggaccat 2580
atcgtgcctc agagctttct gaaggacgac tccatcgaca acaaggtgct gaccagaagc 2640
gacaagaacc ggggcaagag cgacaacgtg ccctccgaag aggtcgtgaa gaagatgaag 2700
aactactggc ggcagctgct gaacgccaag ctgattaccc agagaaagtt cgacaatctg 2760
accaaggccg agagaggcgg cctgagcgaa ctggataagg ccggcttcat caagagacag 2820
ctggtggaaa cccggcagat cacaaagcac gtggcacaga tcctggactc ccggatgaac 2880
actaagtacg acgagaatga caagctgatc cgggaagtga aagtgatcac cctgaagtcc 2940
aagctggtgt ccgatttccg gaaggatttc cagttttaca aagtgcgcga gatcaacaac 3000
taccaccacg cccacgacgc ctacctgaac gccgtcgtgg gaaccgccct gatcaaaaag 3060
taccctaagc tggaaagcga gttcgtgtac ggcgactaca aggtgtacga cgtgcggaag 3120
atgatcgcca agagcgagca ggaaatcggc aaggctaccg ccaagtactt cttctacagc 3180
aacatcatga actttttcaa gaccgagatt accctggcca acggcgagat ccggaagcgg 3240
cctctgatcg agacaaacgg cgaaaccggg gagatcgtgt gggataaggg ccgggatttt 3300
gccaccgtgc ggaaagtgct gagcatgccc caagtgaata tcgtgaaaaa gaccgaggtg 3360
cagacaggcg gcttcagcaa agagtctatc ctgcccaaga ggaacagcga taagctgatc 3420
gccagaaaga aggactggga ccctaagaag tacggcggct tcgacagccc caccgtggcc 3480
tattctgtgc tggtggtggc caaagtggaa aagggcaagt ccaagaaact gaagagtgtg 3540
aaagagctgc tggggatcac catcatggaa agaagcagct tcgagaagaa tcccatcgac 3600
tttctggaag ccaagggcta caaagaagtg aaaaaggacc tgatcatcaa gctgcctaag 3660
tactccctgt tcgagctgga aaacggccgg aagagaatgc tggcctctgc cggcgtgctg 3720
cagaagggaa acgaactggc cctgccctcc aaatatgtga acttcctgta cctggccagc 3780
cactatgaga agctgaaggg ctcccccgag gataatgagc agaaacagct gtttgtggaa 3840
cagcacaagc actacctgga cgagatcatc gagcagatca gcgagttctc caagagagtg 3900
atcctggccg acgctaatct ggacaaagtg ctgtccgcct acaacaagca ccgggataag 3960
cccatcagag agcaggccga gaatatcatc cacctgttta ccctgaccaa tctgggagcc 4020
cctgccgcct tcaagtactt tgacaccacc atcgaccgga agaggtacac cagcaccaaa 4080
gaggtgctgg acgccaccct gatccaccag agcatcaccg gcctgtacga gacacggatc 4140
gacctgtctc agctgggagg cgacaaaagg ccggcggcca cgaaaaaggc cggccaggca 4200
aaaaagaaaa agtaa 4215
<210> 6
<211> 5264
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt 60
gacgtcaata gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg 120
gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga 180
cgtcaatgac ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt 240
tcctacttgg cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc 300
cacgttctgc ttcactctcc ccatctcccc cccctcccca cccccaattt tgtatttatt 360
tattttttaa ttattttgtg cagcgatggg ggcggggggg gggggggggc gcgcgccagg 420
cggggcgggg cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa 480
tcagagcggc gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta 540
taaaaagcga agcgcgcggc gggcgggagt cgctgcgacg ctgccttcgc cccgtgcccc 600
gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta ctcccacagg 660
tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag aggtaagggt 720
ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac ctgcctgaaa 780
tcactttttt tcaggttgga ccggtgccac catgtaccca tacgatgttc cagattacgc 840
ttcgccgaag aaaaagcgca aggtcgaagc gtccgacaag aagtacagca tcggcctggc 900
catcggcacc aactctgtgg gctgggccgt gatcaccgac gagtacaagg tgcccagcaa 960
gaaattcaag gtgctgggca acaccgaccg gcacagcatc aagaagaacc tgatcggagc 1020
cctgctgttc gacagcggcg aaacagccga ggccacccgg ctgaagagaa ccgccagaag 1080
aagatacacc agacggaaga accggatctg ctatctgcaa gagatcttca gcaacgagat 1140
ggccaaggtg gacgacagct tcttccacag actggaagag tccttcctgg tggaagagga 1200
taagaagcac gagcggcacc ccatcttcgg caacatcgtg gacgaggtgg cctaccacga 1260
gaagtacccc accatctacc acctgagaaa gaaactggtg gacagcaccg acaaggccga 1320
cctgcggctg atctatctgg ccctggccca catgatcaag ttccggggcc acttcctgat 1380
cgagggcgac ctgaaccccg acaacagcga cgtggacaag ctgttcatcc agctggtgca 1440
gacctacaac cagctgttcg aggaaaaccc catcaacgcc agcggcgtgg acgccaaggc 1500
catcctgtct gccagactga gcaagagcag acggctggaa aatctgatcg cccagctgcc 1560
cggcgagaag aagaatggcc tgttcggaaa cctgattgcc ctgagcctgg gcctgacccc 1620
caacttcaag agcaacttcg acctggccga ggataccaaa ctgcagctga gcaaggacac 1680
ctacgacgac gacctggaca acctgctggc ccagatcggc gaccagtacg ccgacctgtt 1740
tctggccgcc aagaacctgt ccgacgccat cctgctgagc gacatcctga gagtgaacac 1800
cgagatcacc aaggcccccc tgagcgcctc tatgatcaag ctgtacgacg agcaccacca 1860
ggacctgacc ctgctgaaag ctctcgtgcg gcagcagctg cctgagaagt acaaagagat 1920
tttcttcgac cagagcaaga acggctacgc cggctacatt gacggcggag ccagccagga 1980
agagttctac aagttcatca agcccatcct ggaaaagatg gacggcaccg aggaactgct 2040
cgtgaagctg aacagagagg acctgctgcg gaagcagcgg accttcgaca acggcatcat 2100
cccccaccag atccacctgg gagagctgca cgccattctg cggcggcagg aagattttta 2160
cccattcctg aaggacaacc gggaaaagat cgagaagatc ctgaccttcc gcatccccta 2220
ctacgtgggc cctctggcca ggggaaacag cagattcgcc tggatgacca gaaagagcga 2280
ggaaaccatc accccctgga acttcgagaa ggtggtggac aagggcgctt ccgcccagag 2340
cttcatcgag cggatgacca acttcgataa gaacctgccc aacgagaagg tgctgcccaa 2400
gcacagcctg ctgtacgagt acttcaccgt gtataacgag ctgaccaaag tgaaatacgt 2460
gaccgaggga atgagaaagc ccgccttcct gagcggcgac cagaaaaagg ccatcgtgga 2520
cctgctgttc aagaccaacc ggaaagtgac cgtgaagcag ctgaaagagg actacttcaa 2580
gaaaatcgag tgcttcgact ccgtggaaat ctccggcgtg gaagatcggt tcaacgcctc 2640
cctgggcaca taccacgatc tgctgaaaat tatcaaggac aaggacttcc tggacaatga 2700
ggaaaacgag gacattctgg aagatatcgt gctgaccctg acactgtttg aggacagaga 2760
gatgatcgag gaacggctga aaacctatgc ccacctgttc gacgacaaag tgatgaagca 2820
gctgaagcgg cggagataca ccggctgggg caggctgagc cggaagctga tcaacggcat 2880
ccgggacaag cagtccggca agacaatcct ggatttcctg aagtccgacg gcttcgccaa 2940
cagaaacttc atccagctga tccacgacga cagcctgacc tttaaagagg acatccagaa 3000
agcccaggtg tccggccagg gcgatagcct gcacgagcac attgccaatc tggccggcag 3060
ccccgccatt aagaagggca tcctgcagac agtgaaggtg gtggacgagc tcgtgaaagt 3120
gatgggccgg cacaagcccg agaacatcgt gatcgaaatg gccagagaga accagaccac 3180
ccagaaggga cagaagaaca gccgcgagag aatgaagcgg atcgaagagg gcatcaaaga 3240
gctgggcagc cagatcctga aagaacaccc cgtggaaaac acccagctgc agaacgagaa 3300
gctgtacctg tactacctgc agaatgggcg ggatatgtac gtggaccagg aactggacat 3360
caaccggctg tccgactacg atgtggacca tatcgtgcct cagagctttc tgaaggacga 3420
ctccatcgac aacaaggtgc tgaccagaag cgacaagaac cggggcaaga gcgacaacgt 3480
gccctccgaa gaggtcgtga agaagatgaa gaactactgg cggcagctgc tgaacgccaa 3540
gctgattacc cagagaaagt tcgacaatct gaccaaggcc gagagaggcg gcctgagcga 3600
actggataag gccggcttca tcaagagaca gctggtggaa acccggcaga tcacaaagca 3660
cgtggcacag atcctggact cccggatgaa cactaagtac gacgagaatg acaagctgat 3720
ccgggaagtg aaagtgatca ccctgaagtc caagctggtg tccgatttcc ggaaggattt 3780
ccagttttac aaagtgcgcg agatcaacaa ctaccaccac gcccacgacg cctacctgaa 3840
cgccgtcgtg ggaaccgccc tgatcaaaaa gtaccctaag ctggaaagcg agttcgtgta 3900
cggcgactac aaggtgtacg acgtgcggaa gatgatcgcc aagagcgagc aggaaatcgg 3960
caaggctacc gccaagtact tcttctacag caacatcatg aactttttca agaccgagat 4020
taccctggcc aacggcgaga tccggaagcg gcctctgatc gagacaaacg gcgaaaccgg 4080
ggagatcgtg tgggataagg gccgggattt tgccaccgtg cggaaagtgc tgagcatgcc 4140
ccaagtgaat atcgtgaaaa agaccgaggt gcagacaggc ggcttcagca aagagtctat 4200
cctgcccaag aggaacagcg ataagctgat cgccagaaag aaggactggg accctaagaa 4260
gtacggcggc ttcgacagcc ccaccgtggc ctattctgtg ctggtggtgg ccaaagtgga 4320
aaagggcaag tccaagaaac tgaagagtgt gaaagagctg ctggggatca ccatcatgga 4380
aagaagcagc ttcgagaaga atcccatcga ctttctggaa gccaagggct acaaagaagt 4440
gaaaaaggac ctgatcatca agctgcctaa gtactccctg ttcgagctgg aaaacggccg 4500
gaagagaatg ctggcctctg ccggcgtgct gcagaaggga aacgaactgg ccctgccctc 4560
caaatatgtg aacttcctgt acctggccag ccactatgag aagctgaagg gctcccccga 4620
ggataatgag cagaaacagc tgtttgtgga acagcacaag cactacctgg acgagatcat 4680
cgagcagatc agcgagttct ccaagagagt gatcctggcc gacgctaatc tggacaaagt 4740
gctgtccgcc tacaacaagc accgggataa gcccatcaga gagcaggccg agaatatcat 4800
ccacctgttt accctgacca atctgggagc ccctgccgcc ttcaagtact ttgacaccac 4860
catcgaccgg aagaggtaca ccagcaccaa agaggtgctg gacgccaccc tgatccacca 4920
gagcatcacc ggcctgtacg agacacggat cgacctgtct cagctgggag gcgacaaaag 4980
gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa aagtaagaat tcctagagct 5040
cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 5100
gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 5160
attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 5220
agcaaggggg aggattggga agagaatagc aggcatgctg ggga 5264
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
accgccagaa gaagatacac 20
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ctcagacaat gcgatgca 18
<210> 9
<211> 440
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agactgagtt cacacggtgc tgggccccca aagccaagtg gggttggggg aacagagtct 60
gcgagtcccg gcgccccgag tgcagggccc cgtgttgggg tcctccttcc catttgtatc 120
cgtatccttg cgggctttgc gcctccccgg gggacccctc gccgggagat ggccgcactg 180
atgcggggca aggactcctc ccgctgcctg ctcctactgg ccgcggtgct gatggtggag 240
agctcacagt tcggcagctc gcgggccaaa ctcaactcca tcaagtcctc tctgggcggg 300
gagacgcctg cccaggccgc caatcgatct gcgggcactt accaaggact ggctttcggc 360
ggcagtaaga agggcaaaaa cctggggcag gtaggaaaat acccccaata cactcttcaa 420
ccagaagagg tagggacccg 440
<210> 10
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
caccgtgcct gctcctactg gccgc 25
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
aaacgcggcc agtaggagca ggcac 25
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
caccgatcaa gtcctctctg ggcgg 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aaacccgccc agagaggact tgatc 25
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caccgcccgc gagctgccga actg 24
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
aaaccagttc ggcagctcgc gggc 24
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aaagtcccta ttggcgttac 20
<210> 17
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
cgggtcttcg agaagacct 19
<210> 18
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aaagtcccta ttggcgttac 20
<210> 19
<211> 7123
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
agatctgcgc agcaccatgg cctgaaataa cctctgaaag aggaacttgg ttaggtacct 60
tctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag 120
gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg 180
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 240
caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc 300
attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg 360
cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa 420
agcttgattc ttctgacaca acagtctcga acttaagctg cagaagttgg tcgtgaggca 480
ctgggcaggt aagtatcaag gttacaagac aggtttaagg agaccaatag aaactgggct 540
tgtcgagaca gagaagactc ttgcgtttct gataggcacc tattggtctt actgacatcc 600
actttgcctt tctctccaca ggtgtccact cccagttcaa ttacagctct taaggctaga 660
gtacttaata cgactcacta taggctagcc accatggctt ccaaggtgta cgaccccgag 720
caacgcaaac gcatgatcac tgggcctcag tggtgggctc gctgcaagca aatgaacgtg 780
ctggactcct tcatcaacta ctatgattcc gagaagcacg ccgagaacgc cgtgattttt 840
ctgcatggta acgctgcctc cagctacctg tggaggcacg tcgtgcctca catcgagccc 900
gtggctagat gcatcatccc tgatctgatc ggaatgggta agtccggcaa gagcgggaat 960
ggctcatatc gcctcctgga tcactacaag tacctcaccg cttggttcga gctgctgaac 1020
cttccaaaga aaatcatctt tgtgggccac gactgggggg cttgtctggc ctttcactac 1080
tcctacgagc accaagacaa gatcaaggcc atcgtccatg ctgagagtgt cgtggacgtg 1140
atcgagtcct gggacgagtg gcctgacatc gaggaggata tcgccctgat caagagcgaa 1200
gagggcgaga aaatggtgct tgagaataac ttcttcgtcg agaccatgct cccaagcaag 1260
atcatgcgga aactggagcc tgaggagttc gctgcctacc tggagccatt caaggagaag 1320
ggcgaggtta gacggcctac cctctcctgg cctcgcgaga tccctctcgt taagggaggc 1380
aagcccgacg tcaccggtaa aggcgcgcca gactgagttc acacggtgct gggcccccaa 1440
agccaagtgg ggttggggga acagagtctg cgagtcccgg cgccccgagt gcagggcccc 1500
gtgttggggt cctccttccc atttgtatcc gtatccttgc gggctttgcg cctccccggg 1560
ggacccctcg ccgggagatg gccgcactga tgcggggcaa ggactcctcc cgctgcctgc 1620
tcctactggc cgcggtgctg atggtggaga gctcacagtt cggcagctcg cgggccaaac 1680
tcaactccat caagtcctct ctgggcgggg agacgcctgc ccaggccgcc aatcgatctg 1740
cgggcactta ccaaggactg gctttcggcg gcagtaagaa gggcaaaaac ctggggcagg 1800
taggaaaata cccccaatac actcttcaac cagaagaggt agggacccgg tcgacaaacc 1860
tgcaggaaaa ctagtcctca ccgcttggtt cgagctgctg aaccttccaa agaaaatcat 1920
ctttgtgggc cacgactggg gggcttgtct ggcctttcac tactcctacg agcaccaaga 1980
caagatcaag gccatcgtcc atgctgagag tgtcgtggac gtgatcgagt cctgggacga 2040
gtggcctgac atcgaggagg atatcgccct gatcaagagc gaagagggcg agaaaatggt 2100
gcttgagaat aacttcttcg tcgagaccat gctcccaagc aagatcatgc ggaaactgga 2160
gcctgaggag ttcgctgcct acctggagcc attcaaggag aagggcgagg ttagacggcc 2220
taccctctcc tggcctcgcg agatccctct cgttaaggga ggcaagcccg acgtcgtcca 2280
gattgtccgc aactacaacg cctaccttcg ggccagcgac gatctgccta agatgttcat 2340
cgagtccgac cctgggttct tttccaacgc tattgtcgag ggagctaaga agttccctaa 2400
caccgagttc gtgaaggtga agggcctcca cttcagccag gaggacgctc cagatgaaat 2460
gggtaagtac atcaagagct tcgtggagcg cgtgctgaag aacgagcagt aattctaggc 2520
gatcgctcga gcccgggaat tcgtttaaac ctagagcggc cgctggccgc aataaaatat 2580
ctttattttc attacatctg tgtgttggtt ttttgtgtga ggatctaaat gagtcttcgg 2640
acctcgcggg ggccgcttaa gcggtggtta gggtttgtct gacgcggggg gagggggaag 2700
gaacgaaaca ctctcattcg gaggcggctc ggggtttggt cttggtggcc acgggcacgc 2760
agaagagcgc cgcgatcctc ttaagcaccc ccccgccctc cgtggaggcg ggggtttggt 2820
cggcgggtgg taactggcgg gccgctgact cgggcgggtc gcgcgcccca gagtgtgacc 2880
ttttcggtct gctcgcagac ccccgggcgg cgccgccgcg gcggcgacgg gctcgctggg 2940
tcctaggctc catggggacc gtatacgtgg acaggctctg gagcatccgc acgactgcgg 3000
tgatattacc ggagaccttc tgcgggacga gccgggtcac gcggctgacg cggagcgtcc 3060
gttgggcgac aaacaccagg acggggcaca ggtacactat cttgtcaccc ggaggcgcga 3120
gggactgcag gagcttcagg gagtggcgca gctgcttcat ccccgtggcc cgttgctcgc 3180
gtttgctggc ggtgtccccg gaagaaatat atttgcatgt ctttagttct atgatgacac 3240
aaaccccgcc cagcgtcttg tcattggcga attcgaacac gcagatgcag tcggggcggc 3300
gcggtcccag gtccacttcg catattaagg tgacgcgtgt ggcctcgaac accgagcgac 3360
cctgcagcga cccgcttaaa agcttggcat tccggtactg ttggtaaagc caccatggcc 3420
gatgctaaga acattaagaa gggccctgct cccttctacc ctctggagga tggcaccgct 3480
ggcgagcagc tgcacaaggc catgaagagg tatgccctgg tgcctggcac cattgccttc 3540
accgatgccc acattgaggt ggacatcacc tatgccgagt acttcgagat gtctgtgcgc 3600
ctggccgagg ccatgaagag gtacggcctg aacaccaacc accgcatcgt ggtgtgctct 3660
gagaactctc tgcagttctt catgccagtg ctgggcgccc tgttcatcgg agtggccgtg 3720
gcccctgcta acgacattta caacgagcgc gagctgctga acagcatggg catttctcag 3780
cctaccgtgg tgttcgtgtc taagaagggc ctgcagaaga tcctgaacgt gcagaagaag 3840
ctgcctatca tccagaagat catcatcatg gactctaaga ccgactacca gggcttccag 3900
agcatgtaca cattcgtgac atctcatctg cctcctggct tcaacgagta cgacttcgtg 3960
ccagagtctt tcgacaggga caaaaccatt gccctgatca tgaacagctc tgggtctacc 4020
ggcctgccta agggcgtggc cctgcctcat cgcaccgcct gtgtgcgctt ctctcacgcc 4080
cgcgacccta ttttcggcaa ccagatcatc cccgacaccg ctattctgag cgtggtgcca 4140
ttccaccacg gcttcggcat gttcaccacc ctgggctacc tgatttgcgg ctttcgggtg 4200
gtgctgatgt accgcttcga ggaggagctg ttcctgcgca gcctgcaaga ctacaaaatt 4260
cagtctgccc tgctggtgcc aaccctgttc agcttcttcg ctaagagcac cctgatcgac 4320
aagtacgacc tgtctaacct gcacgagatt gcctctggcg gcgccccact gtctaaggag 4380
gtgggcgaag ccgtggccaa gcgctttcat ctgccaggca tccgccaggg ctacggcctg 4440
accgagacaa ccagcgccat tctgattacc ccagagggcg acgacaagcc tggcgccgtg 4500
ggcaaggtgg tgccattctt cgaggccaag gtggtggacc tggacaccgg caagaccctg 4560
ggagtgaacc agcgcggcga gctgtgtgtg cgcggcccta tgattatgtc cggctacgtg 4620
aataaccctg aggccacaaa cgccctgatc gacaaggacg gctggctgca ctctggcgac 4680
attgcctact gggacgagga cgagcacttc ttcatcgtgg accgcctgaa gtctctgatc 4740
aagtacaagg gctaccaggt ggccccagcc gagctggagt ctatcctgct gcagcaccct 4800
aacattttcg acgccggagt ggccggcctg cccgacgacg atgccggcga gctgcctgcc 4860
gccgtcgtcg tgctggaaca cggcaagacc atgaccgaga aggagatcgt ggactatgtg 4920
gccagccagg tgacaaccgc caagaagctg cgcggcggag tggtgttcgt ggacgaggtg 4980
cccaagggcc tgaccggcaa gctggacgcc cgcaagatcc gcgagatcct gatcaaggct 5040
aagaaaggcg gcaagatcgc cgtgtaataa ttctagagtc ggggcggccg gccgcttcga 5100
gcagacatga taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa 5160
aaatgcttta tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc 5220
aataaacaag ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg 5280
tgggaggttt tttaaagcaa gtaaaacctc tacaaatgtg gtaaaatcga taaggatcca 5340
ggtggcactt ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat 5400
tcaaatatgt atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa 5460
aggaagagta tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt 5520
tgccttcctg tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag 5580
ttgggtgcac gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt 5640
tttcgccccg aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg 5700
gtattatccc gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag 5760
aatgacttgg ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta 5820
agagaattat gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg 5880
acaacgatcg gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta 5940
actcgccttg atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac 6000
accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt 6060
actctagctt cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca 6120
cttctgcgct cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag 6180
cgtgggtctc gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta 6240
gttatctaca cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag 6300
ataggtgcct cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt 6360
tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat 6420
aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta 6480
gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa 6540
acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt 6600
tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag 6660
ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta 6720
atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca 6780
agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag 6840
cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa 6900
agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga 6960
acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc 7020
gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc 7080
ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tgg 7123
<210> 20
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
cacggatcga cctgtctcag 20
<210> 21
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ctgatcagcg agctctagga a 21
<210> 22
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ctggagcacc tgcctgaaa 19
<210> 23
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
gcaccttgta ctcgtcggtg 20
<210> 24
<211> 336
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aagctgattg ggtgtggtcg ccgtggccgg acgccgctcg ggggacgtgg gaggggaggc 60
gggaaacagc ttagtgggtg tggggtcgcg cattttcttc aaccaggagg tgaggaggtt 120
tcgacatggc ggtgcagccg aaggagacgc tgcagttgga gagcgcggcc gaggtcggct 180
tcgtgcgctt ctttcagggc atgccggaga agccgaccac cacagtgcgc cttttcgacc 240
ggggcgactt ctatacggcg cacggcgagg acgcgctgct ggccgcccgg gaggtgttca 300
agacccaggg ggtgatcaag tacatggggc cggcag 336
<210> 25
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
caccgagcgc ggccgaggtc ggctt 25
<210> 26
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
aaacaagccg acctcggccg cgctc 25
<210> 27
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
caccgagcgt ctccttcggc tgcac 25
<210> 28
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
aaacgtgcag ccgaaggaga cgctc 25
<210> 29
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
aaagtcccta ttggcgttac 20
<210> 30
<211> 17
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ttgggtgtgg tcgccgt 17
<210> 31
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ccgtgcgccg tatagaagtc 20
<210> 32
<211> 310
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
agagccgagc cgagccgagc cgagcggagc cgagccgagc cgagcggagc cgagccgagc 60
cgagccgagc cgaaccgagc cgagccgagc cgaaccgagc cgagccgagc cgagtggaat 120
cgagtcgagt cgagcctcca gcgtccggcg cgcaggcctt ccgccgcgtt gatctttcgg 180
ttgctggtgg ccgtgggccg cgcggtctac ggtcgggctg aaagacgcgc gctgcaatgg 240
cgacctttgt gagcgagctg gaggcagcca agaagaactt gagcgaggcg ctgggggaca 300
acgtgaaaca 310
<210> 33
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
caccgtttgt gagcgagctg gaggc 25
<210> 34
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
aaacgcctcc agctcgctca caaac 25
<210> 35
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
caccgcgcgc gtctttcagc ccgac 25
<210> 36
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
aaacgtcggg ctgaaagacg cgcgc 25
<210> 37
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
aaagtcccta ttggcgttac 20
<210> 38
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
cgcgttgatc tttcggttgc 20
<210> 39
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
gcggctctta ctgtttcacg 20
<210> 40
<211> 151
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
gccggccccg gcctcgggcc cctatgtgga gatcatcgag cagcccaagc agcggggcat 60
gcgcttccgc tacaagtgcg agggccgctc agccggcagt atcccgggcg agaggagcac 120
ggataccacc aagacccacc ccaccatcaa g 151
<210> 41
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 41
caccgcgagg gccgctcagc cggc 24
<210> 42
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 42
aaacgccggc tgagcggccc tcgc 24
<210> 43
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 43
caccgtgtag cggaagcgca tgccc 25
<210> 44
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 44
aaacgggcat gcgcttccgc tacac 25
<210> 45
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 45
aaagtcccta ttggcgttac 20
<210> 46
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 46
ttcccctcgg gtaagttgga 20
<210> 47
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 47
tggggtttca cccctactga 20
Claims (8)
1.一种pX335-xCas9n载体，其特征在于，所述pX335-xCas9n载体的核苷酸序列如SEQID No.1所示。
2.权利要求1所述的pX335-xCas9n载体的构建方法，其特征在于，包括：将pX335载体和pX330-xCas9载体分别经BglⅡ与EcoRⅠ进行双酶切，将分别得到的酶切产物进行连接，得到pX335-xCas9n载体；
所述pX330-xCas9质粒的核苷酸序列如SEQ ID No.2所示。
3.根据权利要求2所述的构建方法，其特征在于，所述pX330-xCas9载体的构建方法包括：将pX330质粒和xCas9-39K片段分别经BglⅡ与EcoRⅠ进行双酶切，将分别得到的酶切产物进行连接，得到pX330-xCas9载体；
所述xCas9-39K片段的核苷酸序列如SEQ ID No.3所示。
4.权利要求1所述的pX335-xCas9n载体在哺乳动物的基因修饰中的应用。
5.根据权利要求4所述的应用，其特征在于，包括以下步骤：
1)根据哺乳动物的基因组上的基因设计两对单链DNA寡核苷酸，将所述两对单链DNA寡核苷酸分别退火，得到两个双链DNA片段；
2)将所述步骤1)得到的两个双链DNA片段分别插入到权利要求1所述的pX335-xCas9n载体的sgRNA表达框中，得到两个载体；
3)将所述步骤2)得到的两个载体共转染到哺乳动物细胞中，实现对哺乳动物的基因修饰。
6.根据权利要求5所述的应用，其特征在于，所述步骤3)两个载体的质量比为(1～5):(1～5)。
7.根据权利要求5所述的应用，其特征在于，所述步骤1)单链DNA寡核苷酸的长度为20～30nt。
8.根据权利要求5所述的应用，其特征在于，所述步骤1)基因包括人MSH2基因、小鼠Tada1基因和猪RELA基因。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910441500.1A CN110016481A (zh) | 2019-05-24 | 2019-05-24 | 一种pX335-xCas9n载体及其构建方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910441500.1A CN110016481A (zh) | 2019-05-24 | 2019-05-24 | 一种pX335-xCas9n载体及其构建方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110016481A true CN110016481A (zh) | 2019-07-16 |
Family
ID=67194392
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910441500.1A Pending CN110016481A (zh) | 2019-05-24 | 2019-05-24 | 一种pX335-xCas9n载体及其构建方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110016481A (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501169A (zh) * | 2020-11-30 | 2021-03-16 | 武汉爱博泰克生物科技有限公司 | 一种构建msh2基因敲除细胞系的方法 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293786A (zh) * | 2014-09-15 | 2015-01-21 | 青岛农业大学 | 一对靶向绵羊SFN基因的sgRNA |
| CN106399373A (zh) * | 2016-11-18 | 2017-02-15 | 青岛市畜牧兽医研究所 | 一种Cas9表达载体 |
-
2019
- 2019-05-24 CN CN201910441500.1A patent/CN110016481A/zh active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104293786A (zh) * | 2014-09-15 | 2015-01-21 | 青岛农业大学 | 一对靶向绵羊SFN基因的sgRNA |
| CN106399373A (zh) * | 2016-11-18 | 2017-02-15 | 青岛市畜牧兽医研究所 | 一种Cas9表达载体 |
Non-Patent Citations (3)
| Title |
|---|
| JOHNNY H. HU等: "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity compatibility and high DNA specificity", 《NATURE》 * |
| NCBI: "GenBank登录号：KU341332.1", 《NCBI GENBANK》 * |
| NCBI: "GenBank登录号：KU341333.1", 《NCBI GENBANK》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112501169A (zh) * | 2020-11-30 | 2021-03-16 | 武汉爱博泰克生物科技有限公司 | 一种构建msh2基因敲除细胞系的方法 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2019204429B2 (en) | Modified hematopoietic stem/progenitor and non-T effector cells, and uses thereof | |
| KR102451510B1 (ko) | Pd-1 호밍 엔도뉴클레아제 변이체, 조성물 및 사용 방법 | |
| KR102135239B1 (ko) | 세포 면역요법을 위한 방법 및 조성물 | |
| KR20210149060A (ko) | Tn7-유사 트랜스포존을 사용한 rna-유도된 dna 통합 | |
| DK2768848T3 (en) | METHODS AND PROCEDURES FOR EXPRESSION AND SECRETARY OF PEPTIDES AND PROTEINS | |
| US20200188531A1 (en) | Single-vector gene construct comprising insulin and glucokinase genes | |
| KR20240119152A (ko) | 재조합 glut1 아데노-관련 바이러스 벡터 작제물 및 glut1 발현을 회복하기 위한 관련된 방법 | |
| CN116083398B (zh) | 分离的Cas13蛋白及其应用 | |
| KR102409420B1 (ko) | 형질전환 생물체 선별용 마커 조성물, 형질전환 생물체 및 형질전환 방법 | |
| CN111849978B (zh) | 一种基于Type I-F CRISPR/Cas的染色质成像方法和染色质成像系统 | |
| CN101328486A (zh) | 一种可用于丙酮丁醇梭菌基因中断的重组质粒 | |
| CN110016481A (zh) | 一种pX335-xCas9n载体及其构建方法和应用 | |
| CN108714210B (zh) | 重组减毒李斯特菌在制备间皮素高表达癌症治疗性疫苗中的应用 | |
| US20200017917A1 (en) | Mapping a Functional Cancer Genome Atlas of Tumor Suppressors Using AAV-CRISPR Mediated Direct In Vivo Screening | |
| CN110241099B (zh) | 酿脓链球菌的CRISPR核酸酶SpCas9 的截短变异体及其应用 | |
| CN115867295A (zh) | 用于靶向hpv感染细胞的组合物和方法 | |
| CN112921007A (zh) | 用于预防猫感染新冠病毒的重组腺相关病毒及其构建方法和应用 | |
| KR20230012530A (ko) | 이소프레노이드의 생산을 위한 개선된 방법 | |
| RU2781083C2 (ru) | Варианты, композиции и методы применения хоминг-эндонуклеазы pd-1 | |
| CN106947775A (zh) | 一种在体实时长时间连续记录基因转录活性的方法和装置 | |
| RU2761660C1 (ru) | Штамм клеток Escherichia coli BL21(DE3)/pET32v11-Flpo, продуцирующих сайт-специфическую рекомбиназу Flpe | |
| CN115698332A (zh) | 在单细胞水平上评估载体转导效率和/或特异性的方法 | |
| TW202246316A (zh) | 連結cd40訊息傳遞與抗原結合之系統及方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20190716 |
|
| RJ01 | Rejection of invention patent application after publication |