CN114717207B - 一种酵母细胞同源重组酶系、dna体外组装试剂及其应用 - Google Patents

一种酵母细胞同源重组酶系、dna体外组装试剂及其应用 Download PDF

Info

Publication number
CN114717207B
CN114717207B CN202210436102.2A CN202210436102A CN114717207B CN 114717207 B CN114717207 B CN 114717207B CN 202210436102 A CN202210436102 A CN 202210436102A CN 114717207 B CN114717207 B CN 114717207B
Authority
CN
China
Prior art keywords
leu
dna
ser
yeast
ala
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.)
Active
Application number
CN202210436102.2A
Other languages
English (en)
Other versions
CN114717207A (zh
Inventor
柳伟强
杨平
付煜烨
许映冲
张斌
徐杰
齐金才
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Synbio Technologies
Original Assignee
Synbio Technologies
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Synbio Technologies filed Critical Synbio Technologies
Priority to CN202210436102.2A priority Critical patent/CN114717207B/zh
Publication of CN114717207A publication Critical patent/CN114717207A/zh
Application granted granted Critical
Publication of CN114717207B publication Critical patent/CN114717207B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1217—Phosphotransferases with a carboxyl group as acceptor (2.7.2)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1223—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • 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
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1241—Nucleotidyltransferases (2.7.7)
    • C12N9/1252—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • 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)
    • 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
    • C12N9/93—Ligases (6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates
    • C12P19/28—N-glycosides
    • C12P19/30—Nucleotides
    • C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0104—Pyruvate kinase (2.7.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/02—Phosphotransferases with a carboxy group as acceptor (2.7.2)
    • C12Y207/02001—Acetate kinase (2.7.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/03—Phosphotransferases with a nitrogenous group as acceptor (2.7.3)
    • C12Y207/03002—Creatine kinase (2.7.3.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04001—Polyphosphate kinase (2.7.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
    • C12Y207/04006—Nucleoside-diphosphate kinase (2.7.4.6)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/07—Nucleotidyltransferases (2.7.7)
    • C12Y207/07007—DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/11—Exodeoxyribonucleases producing 5'-phosphomonoesters (3.1.11)
    • C12Y301/11001—Exodeoxyribonuclease I (3.1.11.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y605/00—Ligases forming phosphoric ester bonds (6.5)
    • C12Y605/01—Ligases forming phosphoric ester bonds (6.5) forming phosphoric ester bonds (6.5.1)
    • C12Y605/01001—DNA ligase (ATP) (6.5.1.1)
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Microbiology (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明提供了一种酵母细胞同源重组酶系、DNA体外组装试剂及其应用，属于分子生物学与合成生物学技术领域。本发明提供利用酵母细胞同源重组酶系以及部分原核细胞表达重组酶制备DNA体外组装试剂，利用所述DNA体外组装试剂对两端带有同源臂的DNA片段进行体外组装，实现快速体外单链DNA与双链DNA组装构建。实验证明，本发明提供的方法在1h内单次可以实现4～40条单链引物或2～6个双链片段的无缝组装，具有高效快速特点，较Gibsonassembly组装具有明显优势。

Description

一种酵母细胞同源重组酶系、DNA体外组装试剂及其应用
技术领域
本发明属于分子生物学与合成生物学技术领域，具体涉及一种酵母细胞同源重组酶系、DNA体外组装试剂及其应用。
背景技术
酵母作为真核微生物，其同源重组酶系的活性高于噬菌体、细菌等原核生物的同源重组酶。酿酒酵母细胞凭借其强大的同源重组酶系可以将人工合成的数千碱基对的短基因片段组装成支原体、细菌等原核基因组与酵母、哺乳动物等真核基因组。但由于DNA片段转化入酵母细胞中较为困难的，转化效率很低，而且还涉及到酵母细胞原生质体制备、电转化等复杂操作。另外，酵母的生长周期也比较长，非常耗时。可见，使用酵母胞内同源重组酶系进行DNA片段的组装既耗时又费力。
已有的DNA体外组装试剂为Gibson Assembly。Gibson Assembly具体由T5外切酶、耐热DNA聚合和耐热DNA连接酶构成，所有酶组分使用噬菌体与细菌的酶，且片段之间的退火依赖随机的布朗运动而进行DNA片段组装，存在组装效率低的问题。
发明内容
有鉴于此，本发明的目的在于提供一种酵母细胞同源重组酶系、DNA体外组装试剂及其应用，实现多个单链或双链DNA片段的高效快速组装。
本发明提供的一种酵母细胞同源重组酶系，包括酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA。
优选的，所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52、酵母单链DNA结合蛋白RPA是经过原核表达***密码子优化获得；
优化后的酵母核酸外切酶ExoI的核苷酸序列如SEQ ID NO:1所示；
优化后的酵母重组酶RAD51的核苷酸序列如SEQ ID NO:2所示；
优化后的酵母重组酶RAD52的核苷酸序列如SEQ ID NO:3所示；
优化后的酵母单链DNA结合蛋白RPA的核苷酸序列如SEQ ID NO:4所示。
优选的，所述体外同源重组酶系还包括大肠杆菌DNA聚合酶I、大肠杆菌DNA连接酶和ATP再生酶；
所述ATP再生酶包括以下一种或几种激酶：肌酸激酶、丙酮酸激酶、乙酸激酶、聚磷酸激酶和核苷二磷酸激酶。
优选的，所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52、酵母单链DNA结合蛋白RPA、大肠杆菌DNA聚合酶I、大肠杆菌DNA连接酶和ATP再生酶的活性比为(0.001～0.1)：(10～100)：(10～100)：(10～100)：(0.1～1)：(0.1～1)：(1～100)。
本发明提供了一种DNA体外组装试剂，包括所述体外同源重组酶系和2.6×缓冲液；
所述2.6×缓冲液为包含1～50mM镁离子源、5～50mM碱金属离子源、质量百分含量2％～10％聚乙二醇、1～10mM NAD、2-20mM二硫苏糖醇、1～10mMATP再生酶底物、1～10mM脱氧核苷三磷酸的10～200mM Tris base缓冲液，pH值为7.0～8.0。
优选的，所述DNA体外组装试剂中酵母核酸外切酶ExoI的浓度为1～10U/ml，酵母重组酶RAD51的浓度为10～100nM，酵母重组酶RAD52的浓度为10～100nM，酵母单链DNA结合蛋白RPA的浓度为100～500nM，大肠杆菌DNA聚合酶I的浓度为10～100U/ml，大肠杆菌DNA连接酶的浓度为1000～5000U/ml，ATP再生酶的浓度为1～10mM。
本发明提供了所述体外同源重组酶系或所述DNA体外组装试剂在DNA片段体外组装中的应用。
优选的，所述应用包括两条及两条以上DNA片段组装；
所述DNA片段包括单链或双链的DNA片段；
所述DNA片段包括以下一种或几种：动物来源靶基因、病毒来源靶基因和抗体库DNA片段。
本发明提供了一种DNA片段体外组装的方法，包括以下步骤：
将待组装的DNA片段、线性载体与所述DNA体外组装试剂混合反应，得到直链或环状的DNA长片段；
所述待组装的DNA片段含有长度为15～200bp的碱基同源或互补区域。
优选的，所述混合反应后，将反应产物导入原核细胞中完成DNA片段的扩增，得到双链DNA分子。
本发明提供的一种酵母细胞同源重组酶系，包括酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA。
本发明提供的一种酵母细胞同源重组酶系，包括酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA。本发明提供的酵母核酸外切酶yExoI将两段双链DNA末端酶解成单链DNA；酵母重组酶yRAD51、酵母重组酶yRAD52、酵母单链DNA结合蛋白yRPA，相互协同作用，将同源的两个DNA片段的单链部分退火到一起，完成两端DNA的组装。由于酵母同源重组酶系本身的多酶协同反应与各种酶的高活性和精心的体外重组体系重构研发工作，使得本申请的DNA体外组装有更高的组装效率。
进一步的，本申请具体限定了酵母细胞同源重组酶系中各酶经过原核表达细胞密码子优化。本发明将酵母的同源重组酶系的核心同源重组酶基因进行了密码子优化经重组表达获得酵母同源重组酶的重组蛋白，进一步实现体外条件下多个单链或双链DNA片段的高效快速的无缝组装。
本发明提供的DNA片段体外组装的方法，将待组装的DNA片段与所述DNA体外组装试剂混合反应，得到直链或环状的DNA长片段。本发明提供的体外组装方法，能够高效地连接多个单链或双链DNA片段而得到直链状或环状的DNA，具有较高的连接准确性。采用本发明方法对EGFP基因、新冠病毒突变体Omicron S蛋白基因、酵母细胞表面展示纳米抗体库进行体外组装，结果表明，与Gibson assembly组装效率相比，本发明提供的方法阳性率有明显优势，阳性组装率为50％～95％。
附图说明
图1为酵母细胞同源重组酶系进行体外重组原理示意图；
图2为本发明实施例1中酵母核酸外切酶ExoI、酵母重组酶RAD51重组蛋白电泳图；
图3为本发明实施例1中酵母重组酶RAD52和酵母单链DNA结合蛋白RPA重组蛋白电泳图；
图4为EGFP基因组装菌落PCR验证图；
图5～图10为测序峰图与部分EGFP序列比对结果；
图11为Omicron S基因组装效率菌落PCR鉴定图(扩增部分序列)；
图12为纳米抗体库组装后菌落PCR扩增结果。
具体实施方式
本发明提供的一种酵母细胞同源重组酶系，包括酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA。
在本发明中，所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA优选是按照原核表达***为宿主进行密码子优化，经过重组表达获得。酵母细胞同源重组酶系优选根据原核表达***优化后的酵母核酸外切酶ExoI的核苷酸序列优选如SEQ ID NO:1所示。优化后的酵母重组酶RAD51的核苷酸序列优选如SEQ ID NO:2所示。优化后的酵母重组酶RAD52的核苷酸序列优选如SEQ ID NO:3所示。优化后的酵母单链DNA结合蛋白RPA的核苷酸序列优选如SEQ ID NO:4所示。本发明对所述重组表达的方法有没有特殊限制，采用本领域所熟知的原核表达***重组表达的方法即可。
在本发明中，所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA的工作原理示意图见图1，具体原理如下：H为两段DNA存在的同源序列区，即需要组装到一起的两段DNA片段，组装接头需要有15～200bp的同源序列。在酵母核酸外切酶yExoI的作用下，将两段双链DNA末端酶解成单链DNA，在酵母重组酶yRAD51、酵母重组酶yRAD52、酵母单链DNA结合蛋白yRPA相互协同作用，将带有两个单链DNA中两条单链发生退火，连接形成一条DNA片段，完成DNA片段的组装。
在本发明中，所述体外同源重组酶系作用后，还需补充重组后的DNA片段中缺口或孔隙，该过程可以体外反应或者进行原核细胞表达***中完成。当在体外条件下完成时，所述试剂盒还优选包括大肠杆菌DNA聚合酶I、大肠杆菌DNA连接酶和ATP再生酶。所述ATP再生酶包括以下一种或几种激酶：肌酸激酶、丙酮酸激酶、乙酸激酶、聚磷酸激酶和核苷二磷酸激酶。所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52、酵母单链DNA结合蛋白RPA、大肠杆菌DNA聚合酶I、大肠杆菌DNA连接酶和ATP再生酶的活性比比优选为(0.001～0.1)：(10～100)：(10～100)：(10～100)：(0.1～1)：(0.1～1)：(1～100)。
本发明提供了一种DNA体外组装试剂，包括所述体外同源重组酶系和2.6×缓冲液；所述2.6×缓冲液为包含1～50mM镁离子源、5～50mM碱金属离子源、质量百分含量2％～10％聚乙二醇、1～10mM NAD、2-20mM二硫苏糖醇、1～10mMATP再生酶底物、1～10mM脱氧核苷三磷酸的10～200mM Tris base缓冲液，pH值为7.0～8.0。
在本发明实施例中，所述镁离子源优选为MgAc2。所述碱金属离子源优选为磷酸肌酸钠。所述聚乙二醇优选为PEG-8000。当所述ATP再生酶为肌酸激酶，所述ATP再生酶底物为磷酸肌酸；当所述ATP再生酶为丙酮酸激酶时，所ATP再生酶底物为磷酸烯醇丙酮酸；当所述ATP再生酶为乙酸激酶，所述ATP再生酶底物为乙酰磷酸；当所述ATP再生酶为聚磷酸激酶，所述ATP再生酶底物为聚磷酸；所述ATP再生酶为核苷二磷酸激酶，所述ATP再生酶底物为核苷三磷酸。
在本发明中，所述DNA体外组装试剂中酵母核酸外切酶ExoI的浓度优选为1～10U/ml，更优选为2～8U/ml，进一步优选为4～7U/ml，最优选为5U/ml。酵母重组酶RAD51的浓度优选为10～100nM，更优选为20～90nM，进一步优选为40～70nM，最优选为50nM。酵母重组酶RAD52的浓度优选为10～100nM，更优选为为20～90nM，进一步优选为40～70nM，最优选为50nM。酵母单链DNA结合蛋白RPA的浓度优选为100～500nM，为200～400nM，进一步优选为250～350nM，最优选为300nM。大肠杆菌DNA聚合酶I的浓度优选为10～100U/ml，为20～90U/ml，进一步优选为40～70U/ml，最优选为50U/ml。大肠杆菌DNA连接酶的浓度优选为1000～5000U/ml，更优选为1500～4500U/ml，更优选为2000～4000U/ml，进一步优选为2500～3500U/ml，最优选为3000U/ml。ATP再生酶的浓度优选为1～10mM，更优选为2～8mM，进一步优选为4～7mM，最优选为5mM。
本发明提供了所述体外同源重组酶系或所述DNA体外组装试剂在DNA片段体外组装中的应用。
在本发明中，所述应用优选包括两条及两条以上DNA片段组装。在本发明实施例中分别进行了4～14条DNA片段的同时组装。所述DNA片段包括单链或双链的DNA片段。所述DNA片段体外组装包括动物来源靶基因的组装、病毒来源靶基因或基因组的组装和抗体库DNA片段的组装。
在本发明中，当进行靶基因短片段进行组装时，优选基于靶基因的DNA序列，设计待组装的引物，将所述引物连接接头后与所述DNA体外组装试剂混合反应完成组装。所述引物的设计优选采用DNAworks软件完成，设计时参数为Tm值为62℃，设计原则为65～70nt引物长度。所述接头的序列根据引物与引物之间的连接顺序，选择分别与上游引物的3’端序列互补或同源的序列和选择下游引物的5’端序列互补或同源的序列即可。靶基因的5’段引物的上游接头与待克隆的载体序列同源或互补即可。所述靶基因的3’段引物的下游接头与待克隆的载体序列同源或互补即可。所述接头的长度为15～200bp。
本发明提供了一种DNA片段体外组装的方法，包括以下步骤：
将待组装的DNA片段、线性载体与所述DNA体外组装试剂混合反应，得到直链或环状的DNA长片段；
所述待组装的DNA片段含有长度为15～200bp的碱基同源或互补区域。
在本发明中，不同的待组装的DNA片段混合时，优选按照等摩尔数混合。所述待组装的DNA片段的终浓度优选为10～500ng/μl，更优选为100～200ng/μl。所述线性载体的终浓度优选为10～500ng/μl，更优选为50～200ng/μl。所述混合反应的温度优选为20～27℃，更优选为25℃。所述混合反应的时间优选为1～1.5h。混合反应的体系优选为：10μM待组装引物各1μl、50～200ng/μl线性载体为6μl、所述DNA体外组装试剂20μl，用水补充至50μl。
在本发明中，所述混合反应后，将反应产物优选导入原核表达***中完成DNA片段的空隙或切口的修复和扩增，得到双链DNA分子。
得到双链DNA分子后，优选进行阳性组装率的检测。所述检测方法优选采用琼脂糖凝胶电泳和测序完成。将电泳图中目标长度的条带进行切胶回收纯化，送样测序，比对测序结果靶基因序列，序列完全一致表示成功得到组装后的序列。
下面结合实施例对本发明提供的一种酵母细胞同源重组酶系、DNA体外组装试剂及其应用进行详细的说明，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
一种酵母细胞同源重组酶核心酶组分的表达与纯化方法
1.酵母细胞同源重组酶系核心酶组分：酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母单链DNA退火蛋白RAD52、酵母单链DNA结合蛋白RPA，经过密码子优化，获得优化后的DNA序列，具体序列如序列表SEQ ID NO:1～SEQ ID NO:4所示，氨基酸序列如SEQ ID NO:5～SEQ ID NO:8所示。
2.根据4种酶基因的DNA序列人工合成含有酶切位点的DNA片段，通过NdeI与XhoI位点装入pET28a,测序正确后，按照下述流程进行诱导表达与重组蛋白纯化。
3.质粒的转化和摇瓶培养
1)将质粒1μl加入100μlArctic Expression(DE3)感受态细菌中，置于冰上20min；
2)42℃热激90sec，迅速置冰中3min；加入600μl LB培养液；
3)37℃，220rpm振摇培养1h，取200μl菌液涂布于含50μg/ml Kan的LB平板，37℃倒置培养过夜；
4)次日早晨挑取平板上的单克隆接种于3支含50μg/ml Kan的4ml LB培养液的试管中，37℃220rpm振摇培养至下午1点左右，OD约0.6；
5)按1：250比例，接种于3瓶含100μg/ml Kan的1L LB培养液中，在37℃、220rpm下振摇至菌体OD600为0.5～0.6(约4h)；
6)1L发酵培养基中加入诱导剂IPTG至终浓度为0.1mM，220rpm 16℃过夜培养；
7)5000rpm，5min离心去上清收集发酵菌体，-20℃保存待破碎纯化。
4.菌体的破碎和蛋白的纯化
1)超声破碎菌体
破碎条件：350W功率，破碎4s，间隔7s，共120循环，破菌缓冲液：20mM Tris，500mMNaCl，pH8.0。
2)Ni-IMAC纯化重组蛋白
破碎后的菌体12000rpm，离心20min，4℃，收集上清进行镍柱纯化，填料类型为Ni-IMAC。平衡液：20mM Tris,500mM NaCl,pH8.0，洗脱液：20mM Tris,500mM NaCl,500mMimidazole,pH8.0。分别使用含有20mM、50mM、250mM、500mM imidazole的4种洗脱液进行洗脱，根据吸收峰值收集洗脱液。
结果见图2～图3。由图可知，本发明成功重组表达得到酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母单链DNA退火蛋白RAD52、酵母单链DNA结合蛋白RPA。
实施例2
采用实施例1制备的酵母细胞同源重组酶系配制DNA体外组装试剂，具体见表1和表2：
表1 DNA体外组装用缓冲液
成分 浓度
Trisbase 200mM
MgAc2 20mM
DTT 20mM
NAD 2mM
磷酸肌酸钠 10mM
ATP钠盐 2mM
PEG-8000 10％
dNTP，10mM 0.2mM
ddH<sub>2</sub>O 补充到500ml
冰乙酸 调pH值至7.5
表2 DNA体外组装试剂
2.6×缓冲液 300ml
肌酸激酶，1mg/ml 10ml
酵母核酸外切酶ExoI，1mg/ml 0.5ml
酵母重组酶RAD51，1mg/ml 20ml
酵母单链退火蛋白RAD52，1mg/ml 20ml
酵母单链DNA结合蛋白RPA，1mg/ml 30ml
大肠杆菌聚合酶I，0.25mg/ml 10ml
大肠杆菌DNA连接酶，0.25mg/ml 10ml
实施例3
采用实施例2制备的DNA体外组装试剂进行EGFP基因的组装，具体方法如下：
EGFP基因序列和pUC57质粒载体序列如下，并通过EcoRI与HindIII位点克隆入pUC57质粒载体。
EGFP序列见SEQ ID NO:9。
pUC57质粒载体序列见SEQ ID NO:10。
其中，含下划线标记的pUC57质粒载体EcoRI与HindIII位点附近的同源序列)：
根据EGFP基因序列，使用DNAworks软件设计组装用引物见表3。
表3待组装的引物序列
Figure BDA0003612929390000061
Figure BDA0003612929390000071
在200μl PCR管中，加入用ddH2O稀释至10μM的上述14条组装EGFP的引物，各加入1μl，再加入EcoRI/HindIII酶切后的pUC57载体6μl(50～200ng)，再加入20μl本发明所述的2×DNA片段组装用混合液，混合均匀，并低速离心，保证所有液体沉至PCR管底部，25℃恒温反应1h。然后将反应后的组装产物转化至大肠杆菌感受态细胞，涂布LB固体培养基后，37摄氏度培养过夜，次日，用菌落PCR鉴定组装效率。
组装反应液转化大肠杆菌感受态方法如下：
1.大肠杆菌感受态细胞从-80℃拿出，迅速***冰中，5分钟后待菌块融化，加入组装后的反应液并用手拨打EP管底轻轻混匀，冰中静置30分钟。
2. 42℃水浴热激90秒，迅速放回冰上并静置2分钟。
3.向离心管中加入600μl不含抗生素的灭菌LB培养基，混匀后37℃，200rpm复苏60分钟。
4. 5000rpm离心1分钟收集菌体，留取100μl左右上清轻轻吹打重悬菌块并涂布到含Amp抗生素的LB培养基上。
5.将平板倒置放于37℃培养箱过夜培养。
菌落PCR鉴定方法如下：
1.单菌落挑取
把LB培养基倒入排枪槽中，然后用排枪加400μL LB培养基到96孔深孔板，用镊子拿已灭菌的牙签挑取平板上的单菌落并放到96孔深孔板中，37℃摇床上培养一个小时。
2.菌落PCR反应
配制好PCR反应的反应体系，把配好的反应液加到96孔PCR反应板中，用移液器加2.5μL的菌液到其中。把96孔反应板放在PCR仪器上盖好橡胶垫，按照PCR反应条件设置反应程序。
3.琼脂糖凝胶电泳
配制1.5％琼脂糖凝胶，在96孔反应板每个管中加5μL Loading buffer，震荡均匀，然后点样电泳，电泳好的琼脂糖凝胶拍照。
4.判断阳性克隆
根据琼脂糖凝胶电泳图条带来判断DNA组装成功的克隆。
琼脂糖凝胶电泳图见4。从图4可知，48个克隆中有42个克隆含有片段大小复核预期长度的组装成功的EGFP基因，阳性率为87.5％。
制备阳性克隆的质粒，进行测序验证。测序结果与预期需求比对，结果见图5～图10。
测序结果显示，所组装的EGFP DNA符合预期设计序列。
实施例4
新冠病毒突变体Omicron S蛋白基因组装，并通过EcoRI/ApaI装入哺乳动物细胞表达载体pMT7-CMV。
Omicron S蛋白氨基酸序列如SEQ ID NO:25所示。
Omicron S蛋白DNA序列如SEQ ID NO:26所示。
pMT7-CMV序列如SEQ ID NO:27所示。
将Omicron S蛋白的DNA序列分别设计A、B、C、D四段序列，并通过于实施例1类似的组装方法获得四个DNA片段，再用2X DNA组装混合液再次组装出Omicron S蛋白全长基因序列。
片段A见SEQ ID NO:28。
片段B见SEQ ID NO:29。
片段C见SEQ ID NO:30。
片段D见SEQ ID NO:31。
四个片段通过PCR扩增获得，经过琼脂糖凝胶电泳纯化与回收，回收后浓度50ng/μl。载体pMT7-CMV用EcoRI/ApaI酶切后，同样经过琼脂糖凝胶电泳纯化与回收，回收后浓度100ng/μl。同时采用Gibson assembly试剂进行组装。
组装反应见表4。
表4组装反应体系
pMT7-CMV(EcoRI/ApaI) 10μl
A片段 2.5μl
B片段 2.5μl
C片段 2.5μl
D片段 2.5μl
2×DNA组装混合液 20μl
合计 40μl
在25℃恒温反应1小时。然后按照实施例1相同的方法将反应后的组装产物转化至大肠杆菌感受态细胞，37℃培养过夜，次日，用菌落PCR鉴定组装效率。
结果见图11。从菌落PCR鉴定图来看，上边儿一排是用Gibson assembly(购自NEB公司)组装的鉴定结果，24个克隆中只有2个阳性克隆，阳性率8.3％。下边儿一排是用本发明的DNA组装混合液组装的鉴定结果，24个克隆中有13个阳性克隆，阳性率超过了50％。
实施例5
酵母细胞表面展示纳米抗体库的组装
纳米抗体库序列见SEQ ID NO:32。
氨基酸对应的密码子序列见表5。
表5氨基酸对应的密码子序列
Figure BDA0003612929390000081
Figure BDA0003612929390000091
通过EcoRI位点组装入酵母细胞表面展示载体pSBT-YD2见SEQ ID NO:33所示。
在1.5nl离心管中，加入如表6所示试剂：
表6反应体系
pMT7-CMV(EcoRI)，100ng/μl 125μl
文库组装各个片段混合液 125μl
2×DNA组装混合液 250μl
合计 500μl
25℃恒温反应3小时。然后用苯酚氯仿抽提，异丙醇沉淀的方法纯化组装产物，再将纯化后的组装产物电击转化至大肠杆菌感受态细胞，37℃培养过夜。次日，用菌落PCR鉴定组装效率。
组装产物纯化：
1.将组装产物500μl(不足500μl重组产物用水补齐)转移至1.5ml离心管中，加入500μl酚氯仿异戊醇(25:24:1)，震荡混匀。
2.室温，12000rpm离心5分钟；
3.取上层水相，加入500μl氯仿异戊醇，震荡混合。
4.室温，12000rpm离心5分钟；
5.取上层水相，加入10％3M NaAC(pH5.2)，颠倒混匀后加入等体积异丙醇，颠倒多次混匀，将其置于室温15min，然后-20℃放置2小时。
6. 4℃，12000rpm离心30分钟；
.7小心移去上清，避免接触到管底的沉淀物。
8.加入1ml室温70％的乙醇，轻轻颠倒几次洗涤沉淀。
9. 4℃，12000rpm离心10分钟。
10.小心移去上清，重复步骤8、9，再次漂洗沉淀。
11.小心移去上清，瞬时离心，去残留液体后将Eppendorf管置超净台中吹干5-10min直至无乙醇气味。
12.加入10-15μl ddH2O重新溶解沉淀，-20℃保存备用。
电转化
1.电转
取1μg纯化产物，加入电转感受态(冰水浴10min，转移至预冷的电转杯，电击转化，30℃恢复培养1.5h。
2.涂布平板
库容测定按照公式I计算。具体方法如下：取100μl 6.4.1产物，加入900μl LB培养基，混匀取100μl涂布平板，菌液即被稀释100倍，依次按10倍梯度稀释。剩余原始菌液涂布平板。30℃培养约20h或37℃培养过夜。
库容＝长斑数×稀释倍数公式I
按照实施例1的方法，挑取24个克隆进行菌落PCR，鉴定组装效率。
菌落PCR电泳图见图12。从图上可知，可以判断文库的组长效率很高，24个克隆中有22个克隆含有文库序列，阳性率91.6％。
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> 苏州泓迅生物科技股份有限公司
<120> 一种酵母细胞同源重组酶系、DNA体外组装试剂及其应用
<160> 33
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2109
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggtattc aaggtctgct gccgcaactg aaaccgattc agaatccggt ctctctgcgt 60
cgttatgaag gcgaagttct ggccattgac ggttatgctt ggctgcatcg cgcagcttgt 120
tcttgcgctt acgaactggc gatgggtaaa ccgaccgaca aatacctgca gttcttcatc 180
aaacgcttca gcctgctgaa aaccttcaaa gtcgaaccgt acctggtttt tgacggcgat 240
gcgattccgg tcaaaaaatc caccgaaagc aaacgccgcg acaaacgcaa agagaacaaa 300
gcaattgctg aacgtctgtg ggcttgcggc gagaaaaaga acgcgatgga ctacttccag 360
aaatgcgtcg atatcacccc ggaaatggcg aaatgcatca tctgctactg caaactgaac 420
ggcatccgtt acattgttgc gccgtttgaa gccgattcac agatggttta cctggaacag 480
aaaaacatcg tccagggcat catcagcgag gatagcgatc tgctggtttt tggttgccgt 540
cgtctgatca ccaaactgaa cgattacggc gagtgtctgg aaatctgccg cgataacttc 600
atcaaactgc cgaaaaaatt cccgctgggt agcctgacca acgaagaaat tatcaccatg 660
gtctgtctga gcggttgcga ttataccaac ggtattccga aagtcggtct gattaccgca 720
atgaaactgg tccgtcgctt caacaccatc gaacgtatca tcctgagcat tcagcgcgaa 780
ggtaaactga tgatcccgga cacctacatc aacgaatacg aagcagcggt tctggcgttt 840
caatttcagc gcgtattttg cccgattcgc aaaaagattg tcagcctgaa cgagattccg 900
ctgtacctga aagacaccga aagcaaacgc aaacgcctgt acgcttgcat tggctttgtc 960
atccaccgcg aaacccagaa aaaacagatc gtgcacttcg acgacgatat cgatcaccat 1020
ctgcacctga aaattgcaca gggcgatctg aacccgtacg attttcatca gccgctggca 1080
aatcgcgaac ataaactgca gctggcaagc aaaagcaaca tcgagttcgg caaaaccaac 1140
accaccaact ctgaagcgaa agtcaaaccg atcgagagct tcttccagaa aatgaccaaa 1200
ctggaccaca acccgaaagt tgcgaacaat atccacagcc tgcgtcaggc agaagataaa 1260
ctgaccatgg cgatcaaacg tcgcaaactg tctaacgcga acgttgttca ggaaaccctg 1320
aaagacaccc gcagcaaatt cttcaacaaa ccgagcatga ccgtcgtcga aaacttcaaa 1380
gagaaaggcg acagcatcca ggacttcaaa gaggatacca atagccagag cctggaagaa 1440
ccggtttctg aaagtcagct gagtacccaa attccgagca gctttatcac caccaacctg 1500
gaagacgacg ataacctgag cgaagaagtc agcgaagttg tcagcgatat cgaagaagat 1560
cgcaaaaaca gcgaaggtaa aaccatcggc aacgagatct ataataccga cgacgacggc 1620
gacggcgata ccagcgaaga ttatagcgaa accgcagaaa gtcgcgttcc gaccagtagt 1680
accaccagtt ttccgggtag tagtcaacgt agcattagcg gttgcaccaa agttctgcag 1740
aaattccgct atagcagcag ctttagcggc gttaacgcaa atcgtcaacc gctgtttccg 1800
cgtcacgtta atcagaaaag ccgcggtatg gtctacgtta accagaatcg cgacgacgat 1860
tgcgacgata acgacggcaa aaaccagatc acccaacgtc cgagtctgcg taaaagtctg 1920
attggcgcac gtagccagcg tattgtcatt gacatgaaaa gcgtcgacga acgcaaaagc 1980
ttcaatagca gcccgattct gcacgaagag agcaaaaaac gcgacatcga gaccaccaaa 2040
agcagtcaag cacgtccggc agttcgtagt attagcctgc tgagccagtt cgtctacaaa 2100
ggcaaataa 2109
<210> 2
<211> 1203
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgagccagg tgcaggagca gcatattagc gaaagccagc tgcagtatgg caacggcagc 60
ctgatgagca ccgtgccagc ggatctgagc cagtctgtgg ttgatggcaa cggcaatggc 120
agcagcgaag atattgaagc gaccaacggc agcggcgatg gcggtggctt acaggaacaa 180
gcagaagcgc aaggtgaaat ggaagatgaa gcgtatgatg aagcggcgct gggcagcttt 240
gtgccgattg aaaagctgca ggtgaacggc attaccatgg cggatgtgaa aaagctgcgc 300
gaaagcggcc tgcataccgc ggaagcagtt gcatatgcac cgcgcaaaga tctgctggaa 360
atcaaaggca ttagcgaagc gaaagcggat aaactgctga acgaagcggc gcgcctggtg 420
ccgatgggct ttgttactgc agcagatttt catatgcgcc gcagcgaact gatttgcctg 480
accaccggca gcaaaaacct ggataccctg ctgggcggcg gtgttgaaac cggcagcatt 540
accgagctgt ttggcgaatt tcgcaccggc aaaagccagc tgtgccatac cctggcggtg 600
acctgccaga ttccgctgga tattggcggc ggtgaaggta aatgcctgta tattgatacc 660
gaaggcacct ttcgcccggt gcgcctggtg agcattgcgc aacgctttgg tctggatcca 720
gatgatgcgc tgaacaacgt ggcgtatgcg cgcgcgtata acgcggatca tcagctgcgc 780
ttattagatg cggcggcgca gatgatgagc gaaagccgct ttagcctgat tgtggtggat 840
agcgtgatgg cgctgtatcg caccgatttt agcggccgcg gcgaactgtc tgcacgtcag 900
atgcatctgg cgaaatttat gcgcgcgctg cagcgcctgg cggatcagtt tggcgtggca 960
gttgttgtta ccaaccaggt ggtggcgcag gtggatggcg gcatggcgtt taatccggat 1020
ccgaaaaagc cgattggcgg caacattatg gcgcatagca gcaccacccg cctgggcttt 1080
aagaagggca aaggctgcca gcgcttatgc aaagtggtgg atagcccgtg cctgccggaa 1140
gcggaatgcg tgtttgcgat ttatgaagat ggcgtgggcg atccgcgcga agaagatgaa 1200
taa 1203
<210> 3
<211> 1416
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgaacgaaa ttatggacat ggatgaaaag aaaccggtgt ttggcaacca tagcgaagat 60
attcagacca aactggataa aaagctgggc ccggaatata ttagcaaacg cgtgggcttt 120
ggcaccagcc gcattgcgta tattgaaggc tggcgcgtga ttaacctggc gaaccagatt 180
tttggctata acggctggag caccgaagtg aaaagcgtgg tgattgattt tctggatgaa 240
cgccagggca aatttagcat tggctgcacc gcgattgtgc gcgtgaccct gaccagcggc 300
acttatcgtg aagacattgg ctatggcacc gtggaaaacg aacgccgcaa accggcggcg 360
tttgaacgcg cgaaaaagag cgcggtgact gatgcgctga aacgcagcct gcgtggcttt 420
ggtaacgcgc tgggcaactg cctgtatgat aaagactttc tggcgaaaat cgacaaagtg 480
aagtttgacc cgccggattt tgatgaaaac aacctgtttc gcccgaccga tgaaattagc 540
gaaagcagcc gcaccaacac cctgcatgaa aaccaggaac agcagcagta tccgaacaaa 600
cgccgccagc tgaccaaagt gaccaacacc aatccggata gcaccaagaa cctggtgaaa 660
attgaaaaca ccgtgagccg cggcactcca atgatggcag caccagcaga ggcaaacagc 720
aagaacagca gcaacaaaga taccgatctg aaaagcctgg atgcgagcaa acaggatcag 780
gacgatctgc tggatgatag cctgatgttt agcgatgatt ttcaggacga tgacctgatt 840
aacatgggca acaccaacag caacgtgctg accaccgaaa aagatccggt ggtggcgaaa 900
cagagcccga ccgcgagctc taatccggaa gcagaacaga ttacctttgt gaccgcgaaa 960
gcggcgacca gcgtgcagaa cgaacgctat atcggcgaag aaagcatttt tgatccgaaa 1020
tatcaggcgc agagcattcg ccataccgtg gatcagacca ccagcaaaca tattccggcg 1080
agcgtgctga aagataaaac catgaccacc gcgcgcgata gcgtgtatga aaaattcgcg 1140
ccgaaaggca aacagctgag catgaaaaac aacgataaag aactgggtcc gcatatgctg 1200
gaaggcgcgg gcaaccaggt tccacgcgaa accactccga ttaaaaccaa cgcgaccgcg 1260
tttccgccag cagcagcacc acgttttgca ccaccaagca aagtggtgca tccgaatggc 1320
aacggcgcgg tgccagcggt tccacaacaa cgctctactc gtcgtgaagt gggccgcccg 1380
aaaattaacc cgctgcatgc gcgcaaaccg acctaa 1416
<210> 4
<211> 1866
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgagttcag tacaactatc taggggagat ttccatagca tttttactaa caaacaacgt 60
tacgataacc caacgggtgg cgtgtaccag gtgtacaaca cccgtaaatc cgacggcgca 120
aacagcaacc gcaagaacct tataatgatt agcgatggta tttatcatat gaaagcgtta 180
ttgcgtaatc aagctgcatc gaagtttcag agtatggaac tgcagcgtgg tgatattatt 240
cgcgtgatta tcgcggaacc ggcgatcgtg cgtgaacgta agaaatacgt tctcttagtt 300
gacgacttcg agctggtgca gtctcgtgcc gacatggtga atcaaaccag cacctttctg 360
gataattact tcagcgagca cccgaatgaa accctgaagg acgaagacat caccgattcc 420
ggtaatgttg cgaaccagac caacgcttcc aacgcgggtg ttccggatat gctgcactcg 480
aactccaact tgaatgctaa cgagcgcaag ttcgcgaacg agaacccgaa ctcgcaaaaa 540
actcgtccca tctttgccat cgaacagctg tccccgtatc agaacgtgtg gaccatcaaa 600
gcgagagtct cctataaagg tgaaatcaaa acctggcata atcaacgcgg cgatggcaaa 660
ctgtttaacg tcaacttctt ggacaccagc ggtgaaatcc gtgcgaccgc atttaacgac 720
ttcgcaacta agttcaacga gatcctgcaa gaaggtaagg tttactacgt tagcaaggcg 780
aaactgcaac cggcgaaacc gcagtttacc aatctgacgc acccgtacga attgaatttg 840
gaccgcgata ccgtcatcga agagtgcttc gacgagagca atgtgccgaa aacgcacttt 900
aacttcatca agctggacgc aattcaaaac caagaagtga atagcaacgt ggacgttctg 960
ggcatcatcc agaccattaa cccgcatttt gaattgacgt cccgtgccgg caagaaattt 1020
gatcgtcgtg atattacgat cgttgatgat tctggcttca gcatttccgt tggtctgtgg 1080
aatcaacagg ctctggactt caacctgccg gagggcagcg tggccgcgat taagggtgtt 1140
cgtgtcaccg acttcggtgg caagagcctg tctatgggtt tttcaagcac cctgatcccg 1200
aacccagaga tcccggaggc atatgcatta aagggctggt atgactctaa ggggcgtaac 1260
gctaatttca tcaccttgaa gcaggagccg ggtatgggcg gtcagagcgc tgcatctctg 1320
acgaagttca tcgcgcaacg tattacgatc gcgcgtgcgc aggcggaaaa tttgggtcgt 1380
agcgagaaag gcgacttttt ctctgttaaa gctgccattt cctttttgaa ggttgacaac 1440
ttcgcgtacc cggcctgcag caacgagaac tgtaataaaa aagtacttga gcagccggat 1500
ggtacctggc gctgcgagaa atgtgatacc aataacgccc gccctaactg gcgttacatt 1560
ctgaccattt ctatcattga tgaaaccaac caactctggc tgaccctgtt cgacgaccag 1620
gctaaacagc tgttgggcgt ggacgctaat accttaatga gcctgaaaga agaagacccg 1680
aacgagttta ctaagatcac ccagagcatc caaatgaatg agtacgactt ccgcatccgc 1740
gcgagagagg acacatataa tgatcagagc cgtattcgct ataccgttgc gaatcttcac 1800
tccctgaact atcgtgctga ggcggactat ctggcagatg aactgagcaa ggcgctgctg 1860
gcgtaa 1866
<210> 5
<211> 702
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Gly Ile Gln Gly Leu Leu Pro Gln Leu Lys Pro Ile Gln Asn Pro
1 5 10 15
Val Ser Leu Arg Arg Tyr Glu Gly Glu Val Leu Ala Ile Asp Gly Tyr
20 25 30
Ala Trp Leu His Arg Ala Ala Cys Ser Cys Ala Tyr Glu Leu Ala Met
35 40 45
Gly Lys Pro Thr Asp Lys Tyr Leu Gln Phe Phe Ile Lys Arg Phe Ser
50 55 60
Leu Leu Lys Thr Phe Lys Val Glu Pro Tyr Leu Val Phe Asp Gly Asp
65 70 75 80
Ala Ile Pro Val Lys Lys Ser Thr Glu Ser Lys Arg Arg Asp Lys Arg
85 90 95
Lys Glu Asn Lys Ala Ile Ala Glu Arg Leu Trp Ala Cys Gly Glu Lys
100 105 110
Lys Asn Ala Met Asp Tyr Phe Gln Lys Cys Val Asp Ile Thr Pro Glu
115 120 125
Met Ala Lys Cys Ile Ile Cys Tyr Cys Lys Leu Asn Gly Ile Arg Tyr
130 135 140
Ile Val Ala Pro Phe Glu Ala Asp Ser Gln Met Val Tyr Leu Glu Gln
145 150 155 160
Lys Asn Ile Val Gln Gly Ile Ile Ser Glu Asp Ser Asp Leu Leu Val
165 170 175
Phe Gly Cys Arg Arg Leu Ile Thr Lys Leu Asn Asp Tyr Gly Glu Cys
180 185 190
Leu Glu Ile Cys Arg Asp Asn Phe Ile Lys Leu Pro Lys Lys Phe Pro
195 200 205
Leu Gly Ser Leu Thr Asn Glu Glu Ile Ile Thr Met Val Cys Leu Ser
210 215 220
Gly Cys Asp Tyr Thr Asn Gly Ile Pro Lys Val Gly Leu Ile Thr Ala
225 230 235 240
Met Lys Leu Val Arg Arg Phe Asn Thr Ile Glu Arg Ile Ile Leu Ser
245 250 255
Ile Gln Arg Glu Gly Lys Leu Met Ile Pro Asp Thr Tyr Ile Asn Glu
260 265 270
Tyr Glu Ala Ala Val Leu Ala Phe Gln Phe Gln Arg Val Phe Cys Pro
275 280 285
Ile Arg Lys Lys Ile Val Ser Leu Asn Glu Ile Pro Leu Tyr Leu Lys
290 295 300
Asp Thr Glu Ser Lys Arg Lys Arg Leu Tyr Ala Cys Ile Gly Phe Val
305 310 315 320
Ile His Arg Glu Thr Gln Lys Lys Gln Ile Val His Phe Asp Asp Asp
325 330 335
Ile Asp His His Leu His Leu Lys Ile Ala Gln Gly Asp Leu Asn Pro
340 345 350
Tyr Asp Phe His Gln Pro Leu Ala Asn Arg Glu His Lys Leu Gln Leu
355 360 365
Ala Ser Lys Ser Asn Ile Glu Phe Gly Lys Thr Asn Thr Thr Asn Ser
370 375 380
Glu Ala Lys Val Lys Pro Ile Glu Ser Phe Phe Gln Lys Met Thr Lys
385 390 395 400
Leu Asp His Asn Pro Lys Val Ala Asn Asn Ile His Ser Leu Arg Gln
405 410 415
Ala Glu Asp Lys Leu Thr Met Ala Ile Lys Arg Arg Lys Leu Ser Asn
420 425 430
Ala Asn Val Val Gln Glu Thr Leu Lys Asp Thr Arg Ser Lys Phe Phe
435 440 445
Asn Lys Pro Ser Met Thr Val Val Glu Asn Phe Lys Glu Lys Gly Asp
450 455 460
Ser Ile Gln Asp Phe Lys Glu Asp Thr Asn Ser Gln Ser Leu Glu Glu
465 470 475 480
Pro Val Ser Glu Ser Gln Leu Ser Thr Gln Ile Pro Ser Ser Phe Ile
485 490 495
Thr Thr Asn Leu Glu Asp Asp Asp Asn Leu Ser Glu Glu Val Ser Glu
500 505 510
Val Val Ser Asp Ile Glu Glu Asp Arg Lys Asn Ser Glu Gly Lys Thr
515 520 525
Ile Gly Asn Glu Ile Tyr Asn Thr Asp Asp Asp Gly Asp Gly Asp Thr
530 535 540
Ser Glu Asp Tyr Ser Glu Thr Ala Glu Ser Arg Val Pro Thr Ser Ser
545 550 555 560
Thr Thr Ser Phe Pro Gly Ser Ser Gln Arg Ser Ile Ser Gly Cys Thr
565 570 575
Lys Val Leu Gln Lys Phe Arg Tyr Ser Ser Ser Phe Ser Gly Val Asn
580 585 590
Ala Asn Arg Gln Pro Leu Phe Pro Arg His Val Asn Gln Lys Ser Arg
595 600 605
Gly Met Val Tyr Val Asn Gln Asn Arg Asp Asp Asp Cys Asp Asp Asn
610 615 620
Asp Gly Lys Asn Gln Ile Thr Gln Arg Pro Ser Leu Arg Lys Ser Leu
625 630 635 640
Ile Gly Ala Arg Ser Gln Arg Ile Val Ile Asp Met Lys Ser Val Asp
645 650 655
Glu Arg Lys Ser Phe Asn Ser Ser Pro Ile Leu His Glu Glu Ser Lys
660 665 670
Lys Arg Asp Ile Glu Thr Thr Lys Ser Ser Gln Ala Arg Pro Ala Val
675 680 685
Arg Ser Ile Ser Leu Leu Ser Gln Phe Val Tyr Lys Gly Lys
690 695 700
<210> 6
<211> 400
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 6
Met Ser Gln Val Gln Glu Gln His Ile Ser Glu Ser Gln Leu Gln Tyr
1 5 10 15
Gly Asn Gly Ser Leu Met Ser Thr Val Pro Ala Asp Leu Ser Gln Ser
20 25 30
Val Val Asp Gly Asn Gly Asn Gly Ser Ser Glu Asp Ile Glu Ala Thr
35 40 45
Asn Gly Ser Gly Asp Gly Gly Gly Leu Gln Glu Gln Ala Glu Ala Gln
50 55 60
Gly Glu Met Glu Asp Glu Ala Tyr Asp Glu Ala Ala Leu Gly Ser Phe
65 70 75 80
Val Pro Ile Glu Lys Leu Gln Val Asn Gly Ile Thr Met Ala Asp Val
85 90 95
Lys Lys Leu Arg Glu Ser Gly Leu His Thr Ala Glu Ala Val Ala Tyr
100 105 110
Ala Pro Arg Lys Asp Leu Leu Glu Ile Lys Gly Ile Ser Glu Ala Lys
115 120 125
Ala Asp Lys Leu Leu Asn Glu Ala Ala Arg Leu Val Pro Met Gly Phe
130 135 140
Val Thr Ala Ala Asp Phe His Met Arg Arg Ser Glu Leu Ile Cys Leu
145 150 155 160
Thr Thr Gly Ser Lys Asn Leu Asp Thr Leu Leu Gly Gly Gly Val Glu
165 170 175
Thr Gly Ser Ile Thr Glu Leu Phe Gly Glu Phe Arg Thr Gly Lys Ser
180 185 190
Gln Leu Cys His Thr Leu Ala Val Thr Cys Gln Ile Pro Leu Asp Ile
195 200 205
Gly Gly Gly Glu Gly Lys Cys Leu Tyr Ile Asp Thr Glu Gly Thr Phe
210 215 220
Arg Pro Val Arg Leu Val Ser Ile Ala Gln Arg Phe Gly Leu Asp Pro
225 230 235 240
Asp Asp Ala Leu Asn Asn Val Ala Tyr Ala Arg Ala Tyr Asn Ala Asp
245 250 255
His Gln Leu Arg Leu Leu Asp Ala Ala Ala Gln Met Met Ser Glu Ser
260 265 270
Arg Phe Ser Leu Ile Val Val Asp Ser Val Met Ala Leu Tyr Arg Thr
275 280 285
Asp Phe Ser Gly Arg Gly Glu Leu Ser Ala Arg Gln Met His Leu Ala
290 295 300
Lys Phe Met Arg Ala Leu Gln Arg Leu Ala Asp Gln Phe Gly Val Ala
305 310 315 320
Val Val Val Thr Asn Gln Val Val Ala Gln Val Asp Gly Gly Met Ala
325 330 335
Phe Asn Pro Asp Pro Lys Lys Pro Ile Gly Gly Asn Ile Met Ala His
340 345 350
Ser Ser Thr Thr Arg Leu Gly Phe Lys Lys Gly Lys Gly Cys Gln Arg
355 360 365
Leu Cys Lys Val Val Asp Ser Pro Cys Leu Pro Glu Ala Glu Cys Val
370 375 380
Phe Ala Ile Tyr Glu Asp Gly Val Gly Asp Pro Arg Glu Glu Asp Glu
385 390 395 400
<210> 7
<211> 471
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Met Asn Glu Ile Met Asp Met Asp Glu Lys Lys Pro Val Phe Gly Asn
1 5 10 15
His Ser Glu Asp Ile Gln Thr Lys Leu Asp Lys Lys Leu Gly Pro Glu
20 25 30
Tyr Ile Ser Lys Arg Val Gly Phe Gly Thr Ser Arg Ile Ala Tyr Ile
35 40 45
Glu Gly Trp Arg Val Ile Asn Leu Ala Asn Gln Ile Phe Gly Tyr Asn
50 55 60
Gly Trp Ser Thr Glu Val Lys Ser Val Val Ile Asp Phe Leu Asp Glu
65 70 75 80
Arg Gln Gly Lys Phe Ser Ile Gly Cys Thr Ala Ile Val Arg Val Thr
85 90 95
Leu Thr Ser Gly Thr Tyr Arg Glu Asp Ile Gly Tyr Gly Thr Val Glu
100 105 110
Asn Glu Arg Arg Lys Pro Ala Ala Phe Glu Arg Ala Lys Lys Ser Ala
115 120 125
Val Thr Asp Ala Leu Lys Arg Ser Leu Arg Gly Phe Gly Asn Ala Leu
130 135 140
Gly Asn Cys Leu Tyr Asp Lys Asp Phe Leu Ala Lys Ile Asp Lys Val
145 150 155 160
Lys Phe Asp Pro Pro Asp Phe Asp Glu Asn Asn Leu Phe Arg Pro Thr
165 170 175
Asp Glu Ile Ser Glu Ser Ser Arg Thr Asn Thr Leu His Glu Asn Gln
180 185 190
Glu Gln Gln Gln Tyr Pro Asn Lys Arg Arg Gln Leu Thr Lys Val Thr
195 200 205
Asn Thr Asn Pro Asp Ser Thr Lys Asn Leu Val Lys Ile Glu Asn Thr
210 215 220
Val Ser Arg Gly Thr Pro Met Met Ala Ala Pro Ala Glu Ala Asn Ser
225 230 235 240
Lys Asn Ser Ser Asn Lys Asp Thr Asp Leu Lys Ser Leu Asp Ala Ser
245 250 255
Lys Gln Asp Gln Asp Asp Leu Leu Asp Asp Ser Leu Met Phe Ser Asp
260 265 270
Asp Phe Gln Asp Asp Asp Leu Ile Asn Met Gly Asn Thr Asn Ser Asn
275 280 285
Val Leu Thr Thr Glu Lys Asp Pro Val Val Ala Lys Gln Ser Pro Thr
290 295 300
Ala Ser Ser Asn Pro Glu Ala Glu Gln Ile Thr Phe Val Thr Ala Lys
305 310 315 320
Ala Ala Thr Ser Val Gln Asn Glu Arg Tyr Ile Gly Glu Glu Ser Ile
325 330 335
Phe Asp Pro Lys Tyr Gln Ala Gln Ser Ile Arg His Thr Val Asp Gln
340 345 350
Thr Thr Ser Lys His Ile Pro Ala Ser Val Leu Lys Asp Lys Thr Met
355 360 365
Thr Thr Ala Arg Asp Ser Val Tyr Glu Lys Phe Ala Pro Lys Gly Lys
370 375 380
Gln Leu Ser Met Lys Asn Asn Asp Lys Glu Leu Gly Pro His Met Leu
385 390 395 400
Glu Gly Ala Gly Asn Gln Val Pro Arg Glu Thr Thr Pro Ile Lys Thr
405 410 415
Asn Ala Thr Ala Phe Pro Pro Ala Ala Ala Pro Arg Phe Ala Pro Pro
420 425 430
Ser Lys Val Val His Pro Asn Gly Asn Gly Ala Val Pro Ala Val Pro
435 440 445
Gln Gln Arg Ser Thr Arg Arg Glu Val Gly Arg Pro Lys Ile Asn Pro
450 455 460
Leu His Ala Arg Lys Pro Thr
465 470
<210> 8
<211> 621
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Met Ser Ser Val Gln Leu Ser Arg Gly Asp Phe His Ser Ile Phe Thr
1 5 10 15
Asn Lys Gln Arg Tyr Asp Asn Pro Thr Gly Gly Val Tyr Gln Val Tyr
20 25 30
Asn Thr Arg Lys Ser Asp Gly Ala Asn Ser Asn Arg Lys Asn Leu Ile
35 40 45
Met Ile Ser Asp Gly Ile Tyr His Met Lys Ala Leu Leu Arg Asn Gln
50 55 60
Ala Ala Ser Lys Phe Gln Ser Met Glu Leu Gln Arg Gly Asp Ile Ile
65 70 75 80
Arg Val Ile Ile Ala Glu Pro Ala Ile Val Arg Glu Arg Lys Lys Tyr
85 90 95
Val Leu Leu Val Asp Asp Phe Glu Leu Val Gln Ser Arg Ala Asp Met
100 105 110
Val Asn Gln Thr Ser Thr Phe Leu Asp Asn Tyr Phe Ser Glu His Pro
115 120 125
Asn Glu Thr Leu Lys Asp Glu Asp Ile Thr Asp Ser Gly Asn Val Ala
130 135 140
Asn Gln Thr Asn Ala Ser Asn Ala Gly Val Pro Asp Met Leu His Ser
145 150 155 160
Asn Ser Asn Leu Asn Ala Asn Glu Arg Lys Phe Ala Asn Glu Asn Pro
165 170 175
Asn Ser Gln Lys Thr Arg Pro Ile Phe Ala Ile Glu Gln Leu Ser Pro
180 185 190
Tyr Gln Asn Val Trp Thr Ile Lys Ala Arg Val Ser Tyr Lys Gly Glu
195 200 205
Ile Lys Thr Trp His Asn Gln Arg Gly Asp Gly Lys Leu Phe Asn Val
210 215 220
Asn Phe Leu Asp Thr Ser Gly Glu Ile Arg Ala Thr Ala Phe Asn Asp
225 230 235 240
Phe Ala Thr Lys Phe Asn Glu Ile Leu Gln Glu Gly Lys Val Tyr Tyr
245 250 255
Val Ser Lys Ala Lys Leu Gln Pro Ala Lys Pro Gln Phe Thr Asn Leu
260 265 270
Thr His Pro Tyr Glu Leu Asn Leu Asp Arg Asp Thr Val Ile Glu Glu
275 280 285
Cys Phe Asp Glu Ser Asn Val Pro Lys Thr His Phe Asn Phe Ile Lys
290 295 300
Leu Asp Ala Ile Gln Asn Gln Glu Val Asn Ser Asn Val Asp Val Leu
305 310 315 320
Gly Ile Ile Gln Thr Ile Asn Pro His Phe Glu Leu Thr Ser Arg Ala
325 330 335
Gly Lys Lys Phe Asp Arg Arg Asp Ile Thr Ile Val Asp Asp Ser Gly
340 345 350
Phe Ser Ile Ser Val Gly Leu Trp Asn Gln Gln Ala Leu Asp Phe Asn
355 360 365
Leu Pro Glu Gly Ser Val Ala Ala Ile Lys Gly Val Arg Val Thr Asp
370 375 380
Phe Gly Gly Lys Ser Leu Ser Met Gly Phe Ser Ser Thr Leu Ile Pro
385 390 395 400
Asn Pro Glu Ile Pro Glu Ala Tyr Ala Leu Lys Gly Trp Tyr Asp Ser
405 410 415
Lys Gly Arg Asn Ala Asn Phe Ile Thr Leu Lys Gln Glu Pro Gly Met
420 425 430
Gly Gly Gln Ser Ala Ala Ser Leu Thr Lys Phe Ile Ala Gln Arg Ile
435 440 445
Thr Ile Ala Arg Ala Gln Ala Glu Asn Leu Gly Arg Ser Glu Lys Gly
450 455 460
Asp Phe Phe Ser Val Lys Ala Ala Ile Ser Phe Leu Lys Val Asp Asn
465 470 475 480
Phe Ala Tyr Pro Ala Cys Ser Asn Glu Asn Cys Asn Lys Lys Val Leu
485 490 495
Glu Gln Pro Asp Gly Thr Trp Arg Cys Glu Lys Cys Asp Thr Asn Asn
500 505 510
Ala Arg Pro Asn Trp Arg Tyr Ile Leu Thr Ile Ser Ile Ile Asp Glu
515 520 525
Thr Asn Gln Leu Trp Leu Thr Leu Phe Asp Asp Gln Ala Lys Gln Leu
530 535 540
Leu Gly Val Asp Ala Asn Thr Leu Met Ser Leu Lys Glu Glu Asp Pro
545 550 555 560
Asn Glu Phe Thr Lys Ile Thr Gln Ser Ile Gln Met Asn Glu Tyr Asp
565 570 575
Phe Arg Ile Arg Ala Arg Glu Asp Thr Tyr Asn Asp Gln Ser Arg Ile
580 585 590
Arg Tyr Thr Val Ala Asn Leu His Ser Leu Asn Tyr Arg Ala Glu Ala
595 600 605
Asp Tyr Leu Ala Asp Glu Leu Ser Lys Ala Leu Leu Ala
610 615 620
<210> 9
<211> 772
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gttgtaaaac gacggccagt gaattcatgg tgagcaaggg cgaggagctg ttcaccgggg 60
tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg 120
gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg 180
gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 240
tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag 300
gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg 360
aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca 420
aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct 480
atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca 540
tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg 600
gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc 660
ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc 720
tcggcatgga cgagctgtac aagtaaaagc ttggcgtaat catggtcata gc 772
<210> 10
<211> 2710
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca 60
cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg 120
ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc 180
accagatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc 240
attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat 300
tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt 360
tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa 420
tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttggcg 480
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 540
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 600
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 660
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 720
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 780
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 840
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 900
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 960
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 1020
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 1080
tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 1140
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 1200
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 1260
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 1320
tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 1380
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 1440
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 1500
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 1560
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 1620
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 1680
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 1740
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg actcccacgc 1800
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 1860
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 1920
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 1980
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 2040
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 2100
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 2160
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 2220
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 2280
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 2340
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 2400
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 2460
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 2520
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 2580
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 2640
gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg 2700
ccctttcgtc 2710
<210> 11
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
gttgtaaaac gacggccag 19
<210> 12
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ccaggatggg caccaccccg gtgaacagct cctcgccctt gctcaccatg aattcactgg 60
ccgtcgtttt acaac 75
<210> 13
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc 60
ggcgagggcg agggc 75
<210> 14
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
gcacgggcag cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat 60
cgccctcgcc ctcgc 75
<210> 15
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct 60
tcagccgcta ccccg 75
<210> 16
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
gctcctggac gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt 60
cggggtagcg gctga 75
<210> 17
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 60
cgaggtgaag ttcga 75
<210> 18
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ccgtcctcct tgaagtcgat gcccttcagc tcgatgcggt tcaccagggt gtcgccctcg 60
aacttcacct cggcg 75
<210> 19
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca 60
caacgtctat atcat 75
<210> 20
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
tgttgtggcg gatcttgaag ttcaccttga tgccgttctt ctgcttgtcg gccatgatat 60
agacgttgtg gctgt 75
<210> 21
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag 60
aacaccccca tcggc 75
<210> 22
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac ggggccgtcg 60
ccgatggggg tgttc 75
<210> 23
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg 60
tgaccgccgc cggga 75
<210> 24
<211> 70
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
gctatgacca tgattacgcc aagcttttac ttgtacagct cgtccatgcc gagagtgatc 60
ccggcggcgg 70
<210> 25
<211> 1270
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 25
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Val Ile Ser Gly Thr Asn Gly Thr Lys Arg Phe Asp Asn Pro
65 70 75 80
Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Ile Glu Lys Ser
85 90 95
Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser Lys Thr
100 105 110
Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val
115 120 125
Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Asp His Lys Asn Asn
130 135 140
Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr Ser Ser Ala Asn Asn
145 150 155 160
Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu Met Asp Leu Glu Gly
165 170 175
Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe Val Phe Lys Asn Ile
180 185 190
Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr Pro Ile Ile Val Arg
195 200 205
Glu Pro Glu Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu Pro Leu Val
210 215 220
Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr Leu Leu Ala
225 230 235 240
Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser Gly Trp Thr
245 250 255
Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro Arg Thr Phe
260 265 270
Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala Val Asp Cys
275 280 285
Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys Ser Phe Thr
290 295 300
Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val Gln Pro Thr
305 310 315 320
Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys Pro Phe Asp
325 330 335
Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala Trp Asn Arg
340 345 350
Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu Tyr Asn Leu
355 360 365
Ala Pro Phe Phe Thr Phe Lys Cys Tyr Gly Val Ser Pro Thr Lys Leu
370 375 380
Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe Val Ile Arg
385 390 395 400
Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly Asn Ile Ala
405 410 415
Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys Val Ile Ala
420 425 430
Trp Asn Ser Asn Lys Leu Asp Ser Lys Val Ser Gly Asn Tyr Asn Tyr
435 440 445
Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe Glu Arg Asp
450 455 460
Ile Ser Thr Glu Ile Tyr Gln Ala Gly Asn Lys Pro Cys Asn Gly Val
465 470 475 480
Ala Gly Phe Asn Cys Tyr Phe Pro Leu Arg Ser Tyr Ser Phe Arg Pro
485 490 495
Thr Tyr Gly Val Gly His Gln Pro Tyr Arg Val Val Val Leu Ser Phe
500 505 510
Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys Lys Ser Thr
515 520 525
Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn Gly Leu Lys
530 535 540
Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu Pro Phe Gln
545 550 555 560
Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val Arg Asp Pro
565 570 575
Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe Gly Gly Val
580 585 590
Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val Ala Val Leu
595 600 605
Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile His Ala Asp
610 615 620
Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser Asn Val Phe
625 630 635 640
Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu Tyr Val Asn Asn Ser
645 650 655
Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala Ser Tyr Gln
660 665 670
Thr Gln Thr Lys Ser His Arg Arg Ala Arg Ser Val Ala Ser Gln Ser
675 680 685
Ile Ile Ala Tyr Thr Met Ser Leu Gly Ala Glu Asn Ser Val Ala Tyr
690 695 700
Ser Asn Asn Ser Ile Ala Ile Pro Thr Asn Phe Thr Ile Ser Val Thr
705 710 715 720
Thr Glu Ile Leu Pro Val Ser Met Thr Lys Thr Ser Val Asp Cys Thr
725 730 735
Met Tyr Ile Cys Gly Asp Ser Thr Glu Cys Ser Asn Leu Leu Leu Gln
740 745 750
Tyr Gly Ser Phe Cys Thr Gln Leu Lys Arg Ala Leu Thr Gly Ile Ala
755 760 765
Val Glu Gln Asp Lys Asn Thr Gln Glu Val Phe Ala Gln Val Lys Gln
770 775 780
Ile Tyr Lys Thr Pro Pro Ile Lys Tyr Phe Gly Gly Phe Asn Phe Ser
785 790 795 800
Gln Ile Leu Pro Asp Pro Ser Lys Pro Ser Lys Arg Ser Phe Ile Glu
805 810 815
Asp Leu Leu Phe Asn Lys Val Thr Leu Ala Asp Ala Gly Phe Ile Lys
820 825 830
Gln Tyr Gly Asp Cys Leu Gly Asp Ile Ala Ala Arg Asp Leu Ile Cys
835 840 845
Ala Gln Lys Phe Lys Gly Leu Thr Val Leu Pro Pro Leu Leu Thr Asp
850 855 860
Glu Met Ile Ala Gln Tyr Thr Ser Ala Leu Leu Ala Gly Thr Ile Thr
865 870 875 880
Ser Gly Trp Thr Phe Gly Ala Gly Ala Ala Leu Gln Ile Pro Phe Ala
885 890 895
Met Gln Met Ala Tyr Arg Phe Asn Gly Ile Gly Val Thr Gln Asn Val
900 905 910
Leu Tyr Glu Asn Gln Lys Leu Ile Ala Asn Gln Phe Asn Ser Ala Ile
915 920 925
Gly Lys Ile Gln Asp Ser Leu Ser Ser Thr Ala Ser Ala Leu Gly Lys
930 935 940
Leu Gln Asp Val Val Asn His Asn Ala Gln Ala Leu Asn Thr Leu Val
945 950 955 960
Lys Gln Leu Ser Ser Lys Phe Gly Ala Ile Ser Ser Val Leu Asn Asp
965 970 975
Ile Phe Ser Arg Leu Asp Lys Val Glu Ala Glu Val Gln Ile Asp Arg
980 985 990
Leu Ile Thr Gly Arg Leu Gln Ser Leu Gln Thr Tyr Val Thr Gln Gln
995 1000 1005
Leu Ile Arg Ala Ala Glu Ile Arg Ala Ser Ala Asn Leu Ala Ala Thr
1010 1015 1020
Lys Met Ser Glu Cys Val Leu Gly Gln Ser Lys Arg Val Asp Phe Cys
1025 1030 1035 1040
Gly Lys Gly Tyr His Leu Met Ser Phe Pro Gln Ser Ala Pro His Gly
1045 1050 1055
Val Val Phe Leu His Val Thr Tyr Val Pro Ala Gln Glu Lys Asn Phe
1060 1065 1070
Thr Thr Ala Pro Ala Ile Cys His Asp Gly Lys Ala His Phe Pro Arg
1075 1080 1085
Glu Gly Val Phe Val Ser Asn Gly Thr His Trp Phe Val Thr Gln Arg
1090 1095 1100
Asn Phe Tyr Glu Pro Gln Ile Ile Thr Thr Asp Asn Thr Phe Val Ser
1105 1110 1115 1120
Gly Asn Cys Asp Val Val Ile Gly Ile Val Asn Asn Thr Val Tyr Asp
1125 1130 1135
Pro Leu Gln Pro Glu Leu Asp Ser Phe Lys Glu Glu Leu Asp Lys Tyr
1140 1145 1150
Phe Lys Asn His Thr Ser Pro Asp Val Asp Leu Gly Asp Ile Ser Gly
1155 1160 1165
Ile Asn Ala Ser Val Val Asn Ile Gln Lys Glu Ile Asp Arg Leu Asn
1170 1175 1180
Glu Val Ala Lys Asn Leu Asn Glu Ser Leu Ile Asp Leu Gln Glu Leu
1185 1190 1195 1200
Gly Lys Tyr Glu Gln Tyr Ile Lys Trp Pro Trp Tyr Ile Trp Leu Gly
1205 1210 1215
Phe Ile Ala Gly Leu Ile Ala Ile Val Met Val Thr Ile Met Leu Cys
1220 1225 1230
Cys Met Thr Ser Cys Cys Ser Cys Leu Lys Gly Cys Cys Ser Cys Gly
1235 1240 1245
Ser Cys Cys Lys Phe Asp Glu Asp Asp Ser Glu Pro Val Leu Lys Gly
1250 1255 1260
Val Lys Leu His Tyr Thr
1265 1270
<210> 26
<211> 3862
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
tccagcctcc ggactctaga gaattcatgt ttgtttttct tgttttattg ccactagtct 60
ctagtcagtg tgttaatctt acaaccagaa ctcaattacc ccctgcatac actaattctt 120
tcacacgtgg tgtttattac cctgacaaag ttttcagatc ctcagtttta cattcaactc 180
aggacttgtt cttacctttc ttttccaatg ttacttggtt ccatgttatc tctgggacca 240
atggtactaa gaggtttgat aaccctgtcc taccatttaa tgatggtgtt tattttgctt 300
ccattgagaa gtctaacata ataagaggct ggatttttgg tactacttta gattcgaaga 360
cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc tgtgaatttc 420
aattttgtaa tgatccattt ttggaccaca aaaacaacaa aagttggatg gaaagtgagt 480
tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag ccttttctta 540
tggaccttga aggaaaacag ggtaatttca aaaatcttag ggaatttgtg tttaagaata 600
ttgatggtta ttttaaaata tattctaagc acacgcctat tatagtgcgt gagccagaag 660
atctccctca gggtttttcg gctttagaac cattggtaga tttgccaata ggtattaaca 720
tcactaggtt tcaaacttta cttgctttac atagaagtta tttgactcct ggtgattctt 780
cttcaggttg gacagctggt gctgcagctt attatgtggg ttatcttcaa cctaggactt 840
ttctattaaa atataatgaa aatggaacca ttacagatgc tgtagactgt gcacttgacc 900
ctctctcaga aacaaagtgt acgttgaaat ccttcactgt agaaaaagga atctatcaaa 960
cttctaactt tagagtccaa ccaacagaat ctattgttag atttcctaat attacaaact 1020
tgtgcccttt tgatgaagtt tttaacgcca ccagatttgc atctgtttat gcttggaaca 1080
ggaagagaat cagcaactgt gttgctgatt attctgtcct atataatctc gcaccatttt 1140
tcacttttaa gtgttatgga gtgtctccta ctaaattaaa tgatctctgc tttactaatg 1200
tctatgcaga ttcatttgta attagaggtg atgaagtcag acaaatcgct ccagggcaaa 1260
ctggaaatat tgctgattat aattataaat taccagatga ttttacaggc tgcgttatag 1320
cttggaattc taacaagctt gattctaagg ttagtggtaa ttataattac ctgtatagat 1380
tgtttaggaa gtctaatctc aaaccttttg agagagatat ttcaactgaa atctatcagg 1440
ccggtaacaa accttgtaat ggtgttgcag gttttaattg ttactttcct ttacgatcat 1500
atagtttccg acccacttat ggtgttggtc accaaccata cagagtagta gtactttctt 1560
ttgaacttct acatgcacca gcaactgttt gtggacctaa aaagtctact aatttggtta 1620
aaaacaaatg tgtcaatttc aacttcaatg gtttaaaagg cacaggtgtt cttactgagt 1680
ctaacaaaaa gtttctgcct ttccaacaat ttggcagaga cattgctgac actactgatg 1740
ctgtccgtga tccacagaca cttgagattc ttgacattac accatgttct tttggtggtg 1800
tcagtgttat aacaccagga acaaatactt ctaaccaggt tgctgttctt tatcagggtg 1860
ttaactgcac agaagtccct gttgctattc atgcagatca acttactcct acttggcgtg 1920
tttattctac aggttctaat gtttttcaaa cacgtgcagg ctgtttaata ggggctgaat 1980
atgtcaacaa ctcatatgag tgtgacatac ccattggtgc aggtatatgc gctagttatc 2040
agactcagac taagtctcat cggcgggcac gtagtgtagc tagtcaatcc atcattgcct 2100
acactatgtc acttggtgca gaaaattcag ttgcttactc taataactct attgccatac 2160
ccacaaattt tactattagt gttaccacag aaattctacc agtgtctatg accaagacat 2220
cagtagattg tacaatgtac atttgtggtg attcaactga atgcagcaat cttttgttgc 2280
aatatggcag tttttgtaca caattaaaac gtgctttaac tggaatagct gttgaacaag 2340
acaaaaacac ccaagaagtt tttgcacaag tcaaacaaat ttacaaaaca ccaccaatta 2400
aatattttgg tggttttaat ttttcacaaa tattaccaga tccatcaaaa ccaagcaaga 2460
ggtcatttat tgaagatcta cttttcaaca aagtgacact tgcagatgct ggcttcatca 2520
aacaatatgg tgattgcctt ggtgatattg ctgctagaga cctcatttgt gcacaaaagt 2580
ttaaaggcct tactgttttg ccacctttgc tcacagatga aatgattgct caatacactt 2640
ctgcactgtt agcgggtaca atcacttctg gttggacctt tggtgcaggt gctgcattac 2700
aaataccatt tgctatgcaa atggcttata ggtttaatgg tattggagtt acacagaatg 2760
ttctctatga gaaccaaaaa ttgattgcca accaatttaa tagtgctatt ggcaaaattc 2820
aagactcact ttcttccaca gcaagtgcac ttggaaaact tcaagatgtg gtcaaccata 2880
atgcacaagc tttaaacacg cttgttaaac aacttagctc caaatttggt gcaatttcaa 2940
gtgttttaaa tgatatcttt tcacgtcttg acaaagttga ggctgaagtg caaattgata 3000
ggttgatcac aggcagactt caaagtttgc agacatatgt gactcaacaa ttaattagag 3060
ctgcagaaat cagagcttct gctaatcttg ctgctactaa aatgtcagag tgtgtacttg 3120
gacaatcaaa aagagttgat ttttgtggaa agggctatca tcttatgtcc ttccctcagt 3180
cagcacctca tggtgtagtc ttcttgcatg tgacttatgt ccctgcacaa gaaaagaact 3240
tcacaactgc tcctgccatt tgtcatgatg gaaaagcaca ctttcctcgt gaaggtgtct 3300
ttgtttcaaa tggcacacac tggtttgtaa cacaaaggaa tttttatgaa ccacaaatca 3360
ttactacaga caacacattt gtgtctggta actgtgatgt tgtaatagga attgtcaaca 3420
acacagttta tgatcctttg caacctgaat tagattcatt caaggaggag ttagataaat 3480
attttaagaa tcatacatca ccagatgttg atttaggtga catctctggc attaatgctt 3540
cagttgtaaa cattcaaaaa gaaattgacc gcctcaatga ggttgccaag aatttaaatg 3600
aatctctcat cgatctccaa gaacttggaa agtatgagca gtatataaaa tggccatggt 3660
acatttggct aggttttata gctggcttga ttgccatagt aatggtgaca attatgcttt 3720
gctgtatgac cagttgctgt agttgtctca agggctgttg ttcttgtgga tcctgctgca 3780
aatttgatga agacgactct gagccagtgc tcaaaggagt caaattacat tacacagggc 3840
ccggttcttc tgaaaacctg ta 3862
<210> 27
<211> 7451
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atgggcagag cgcacatcgc 60
ccacagtccc cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt 120
ggcgcggggt aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg 180
ggggagaacc gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg 240
ccgccagaac acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc 300
ctacctgagg ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg 360
cctcctgaac tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt 420
gtccggcgct cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc 480
tgcttgctca actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc 540
tgtgaccggc gcctacacgc gtgccaccat ggtgagcaag ggcgaggagc tgttcaccgg 600
ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt tcagcgtgtc 660
cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca tctgcaccac 720
cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg 780
cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg ccatgcccga 840
aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca agacccgcgc 900
cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg gcatcgactt 960
caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca gccacaacgt 1020
ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga tccgccacaa 1080
catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga 1140
cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc tgagcaaaga 1200
ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg ccgggatcac 1260
tctcggcatg gacgagctgt acaagtaacc gcgctagcgc ctcgactgtg ccttctagtt 1320
gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc 1380
ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt 1440
ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa gagaatagca 1500
ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct 1560
ctagggggta tcccctgaca ttgattattg actagttatt aatagtaatc aattacgggg 1620
tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg 1680
cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata 1740
gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc 1800
cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac 1860
ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg 1920
cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc 1980
aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc 2040
aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc 2100
gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct 2160
cgtttagtga accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga 2220
agacaccggg accgatccag cctccggact ctagagaatt cgagctcggt acctcgcgaa 2280
tgcatctaga tatcggatcc cgggcccggt tcttctgaaa acctgtactt ccaatccaaa 2340
tcttctcacc atcaccatca ccatcaccat caccattagt gataaaccgg ttagtaatga 2400
gtttgatatc tcgacaatca acctctggat tacaaaattt gtgaaagatt gactggtatt 2460
cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc tttgtatcat 2520
gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg gttgctgtct 2580
ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac tgtgtttgct 2640
gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc cgggactttc 2700
gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc ccgctgctgg 2760
acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa gctgacgtcc 2820
tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc cttctgctac 2880
gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc ggctctgcgg 2940
cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg ggccgcctcc 3000
ccgcctggaa acgggggagg ctaactgaaa cacggaagga gacaataccg gaaggaaccc 3060
gcgctatgac ggcaataaaa agacagaata aaacgcacgg gtgttgggtc gtttgttcat 3120
aaacgcgggg ttcggtccca gggctggcac tctgtcgata ccccaccgag accccattgg 3180
ggccaatacg cccgcgtttc ttccttttcc ccaccccacc ccccaagttc gggtgaaggc 3240
ccagggctcg cagccaacgt cggggcggca ggccctgcca tagcagatct gcgcagctgg 3300
ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg 3360
gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc 3420
ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcggggcatc 3480
cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt 3540
gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag 3600
tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg 3660
gtctattctt ttgatttata agggattttg gggatttcgg cctattggtt aaaaaatgag 3720
ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg 3780
gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag 3840
caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc 3900
tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc 3960
ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg 4020
aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag 4080
gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat caagagacag 4140
gatgaggatc gtttcgcatg atgaccgagt acaagcccac ggtgcgcctc gccacccgcg 4200
acgacgtccc ccgggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc 4260
gccacaccgt cgacccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc 4320
tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg 4380
cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc 4440
gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg 4500
cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc 4560
accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg 4620
ccggggtgcc cgccttcctg gagacctccg cgccccgcaa cctccccttc tacgagcggc 4680
tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga 4740
cccgcaagcc cggtgcctag tgagcgggac tctggggttc gcgaaatgac cgaccaagcg 4800
acgcccaacc tgccatcacg agatttcgat tccaccgccg ccttctatga aaggttgggc 4860
ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg 4920
gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat 4980
agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 5040
aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg 5100
taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac 5160
atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca 5220
ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat 5280
taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc 5340
tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca 5400
aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca 5460
aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg 5520
ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg 5580
acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt 5640
ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt 5700
tctcaatgct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc 5760
tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt 5820
gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt 5880
agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc 5940
tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa 6000
agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt 6060
tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct 6120
acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta 6180
tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa 6240
agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc 6300
tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact 6360
acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc 6420
tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt 6480
ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta 6540
agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg 6600
tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt 6660
acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc 6720
agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt 6780
actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc 6840
tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc 6900
gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa 6960
ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac 7020
tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa 7080
aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt 7140
tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa 7200
tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct 7260
gacgtcgacg gatcgggaga tctcccgatc ccctatggtc gactctcagt acaatctgct 7320
ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt 7380
agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga 7440
atctgcttag g 7451
<210> 28
<211> 1040
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tccagcctcc ggactctaga gaattcatgt ttgtttttct tgttttattg ccactagtct 60
ctagtcagtg tgttaatctt acaaccagaa ctcaattacc ccctgcatac actaattctt 120
tcacacgtgg tgtttattac cctgacaaag ttttcagatc ctcagtttta cattcaactc 180
aggacttgtt cttacctttc ttttccaatg ttacttggtt ccatgttatc tctgggacca 240
atggtactaa gaggtttgat aaccctgtcc taccatttaa tgatggtgtt tattttgctt 300
ccattgagaa gtctaacata ataagaggct ggatttttgg tactacttta gattcgaaga 360
cccagtccct acttattgtt aataacgcta ctaatgttgt tattaaagtc tgtgaatttc 420
aattttgtaa tgatccattt ttggaccaca aaaacaacaa aagttggatg gaaagtgagt 480
tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag ccttttctta 540
tggaccttga aggaaaacag ggtaatttca aaaatcttag ggaatttgtg tttaagaata 600
ttgatggtta ttttaaaata tattctaagc acacgcctat tatagtgcgt gagccagaag 660
atctccctca gggtttttcg gctttagaac cattggtaga tttgccaata ggtattaaca 720
tcactaggtt tcaaacttta cttgctttac atagaagtta tttgactcct ggtgattctt 780
cttcaggttg gacagctggt gctgcagctt attatgtggg ttatcttcaa cctaggactt 840
ttctattaaa atataatgaa aatggaacca ttacagatgc tgtagactgt gcacttgacc 900
ctctctcaga aacaaagtgt acgttgaaat ccttcactgt agaaaaagga atctatcaaa 960
cttctaactt tagagtccaa ccaacagaat ctattgttag atttcctaat attacaaact 1020
tgtgcccttt tgatgaagtt 1040
<210> 29
<211> 1040
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tgtgcccttt tgatgaagtt tttaacgcca ccagatttgc atctgtttat gcttggaaca 60
ggaagagaat cagcaactgt gttgctgatt attctgtcct atataatctc gcaccatttt 120
tcacttttaa gtgttatgga gtgtctccta ctaaattaaa tgatctctgc tttactaatg 180
tctatgcaga ttcatttgta attagaggtg atgaagtcag acaaatcgct ccagggcaaa 240
ctggaaatat tgctgattat aattataaat taccagatga ttttacaggc tgcgttatag 300
cttggaattc taacaagctt gattctaagg ttagtggtaa ttataattac ctgtatagat 360
tgtttaggaa gtctaatctc aaaccttttg agagagatat ttcaactgaa atctatcagg 420
ccggtaacaa accttgtaat ggtgttgcag gttttaattg ttactttcct ttacgatcat 480
atagtttccg acccacttat ggtgttggtc accaaccata cagagtagta gtactttctt 540
ttgaacttct acatgcacca gcaactgttt gtggacctaa aaagtctact aatttggtta 600
aaaacaaatg tgtcaatttc aacttcaatg gtttaaaagg cacaggtgtt cttactgagt 660
ctaacaaaaa gtttctgcct ttccaacaat ttggcagaga cattgctgac actactgatg 720
ctgtccgtga tccacagaca cttgagattc ttgacattac accatgttct tttggtggtg 780
tcagtgttat aacaccagga acaaatactt ctaaccaggt tgctgttctt tatcagggtg 840
ttaactgcac agaagtccct gttgctattc atgcagatca acttactcct acttggcgtg 900
tttattctac aggttctaat gtttttcaaa cacgtgcagg ctgtttaata ggggctgaat 960
atgtcaacaa ctcatatgag tgtgacatac ccattggtgc aggtatatgc gctagttatc 1020
agactcagac taagtctcat 1040
<210> 30
<211> 1040
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
agactcagac taagtctcat cggcgggcac gtagtgtagc tagtcaatcc atcattgcct 60
acactatgtc acttggtgca gaaaattcag ttgcttactc taataactct attgccatac 120
ccacaaattt tactattagt gttaccacag aaattctacc agtgtctatg accaagacat 180
cagtagattg tacaatgtac atttgtggtg attcaactga atgcagcaat cttttgttgc 240
aatatggcag tttttgtaca caattaaaac gtgctttaac tggaatagct gttgaacaag 300
acaaaaacac ccaagaagtt tttgcacaag tcaaacaaat ttacaaaaca ccaccaatta 360
aatattttgg tggttttaat ttttcacaaa tattaccaga tccatcaaaa ccaagcaaga 420
ggtcatttat tgaagatcta cttttcaaca aagtgacact tgcagatgct ggcttcatca 480
aacaatatgg tgattgcctt ggtgatattg ctgctagaga cctcatttgt gcacaaaagt 540
ttaaaggcct tactgttttg ccacctttgc tcacagatga aatgattgct caatacactt 600
ctgcactgtt agcgggtaca atcacttctg gttggacctt tggtgcaggt gctgcattac 660
aaataccatt tgctatgcaa atggcttata ggtttaatgg tattggagtt acacagaatg 720
ttctctatga gaaccaaaaa ttgattgcca accaatttaa tagtgctatt ggcaaaattc 780
aagactcact ttcttccaca gcaagtgcac ttggaaaact tcaagatgtg gtcaaccata 840
atgcacaagc tttaaacacg cttgttaaac aacttagctc caaatttggt gcaatttcaa 900
gtgttttaaa tgatatcttt tcacgtcttg acaaagttga ggctgaagtg caaattgata 960
ggttgatcac aggcagactt caaagtttgc agacatatgt gactcaacaa ttaattagag 1020
ctgcagaaat cagagcttct 1040
<210> 31
<211> 802
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
ctgcagaaat cagagcttct gctaatcttg ctgctactaa aatgtcagag tgtgtacttg 60
gacaatcaaa aagagttgat ttttgtggaa agggctatca tcttatgtcc ttccctcagt 120
cagcacctca tggtgtagtc ttcttgcatg tgacttatgt ccctgcacaa gaaaagaact 180
tcacaactgc tcctgccatt tgtcatgatg gaaaagcaca ctttcctcgt gaaggtgtct 240
ttgtttcaaa tggcacacac tggtttgtaa cacaaaggaa tttttatgaa ccacaaatca 300
ttactacaga caacacattt gtgtctggta actgtgatgt tgtaatagga attgtcaaca 360
acacagttta tgatcctttg caacctgaat tagattcatt caaggaggag ttagataaat 420
attttaagaa tcatacatca ccagatgttg atttaggtga catctctggc attaatgctt 480
cagttgtaaa cattcaaaaa gaaattgacc gcctcaatga ggttgccaag aatttaaatg 540
aatctctcat cgatctccaa gaacttggaa agtatgagca gtatataaaa tggccatggt 600
acatttggct aggttttata gctggcttga ttgccatagt aatggtgaca attatgcttt 660
gctgtatgac cagttgctgt agttgtctca agggctgttg ttcttgtgga tcctgctgca 720
aatttgatga agacgactct gagccagtgc tcaaaggagt caaattacat tacacagggc 780
ccggttcttc tgaaaacctg ta 802
<210> 32
<211> 355
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (99)..(99)
<223> N=Y，R，S，T，F，G，AorD；
<220>
<221> misc_feature
<222> (100)..(100)
<223> n=Y，S，T，F，G，TorT (T的比例是其他的三倍)
<220>
<221> misc_feature
<222> (101)..(101)
<223> n=Y，S，S，S，F or W (S的比例是其他的三倍)
<220>
<221> misc_feature
<222> (102)..(102)
<223> n=Y，R，S，T，F，G，A，W，D，E，K or N
<220>
<221> misc_feature
<222> (103)..(103)
<223> n=S，T，F，G，A，W，D，E，N，I，H，R，Q or L
<220>
<221> misc_feature
<222> (104)..(104)
<223> n=S，T，Y，D or E
<220>
<221> misc_feature
<222> (105)..(105)
<223> n=S，T，G，A，D，E，N，I or V
<220>
<221> misc_feature
<222> (154)..(154)
<223> n=R，S，F，G，A，W，D，E or Y
<220>
<221> misc_feature
<222> (155)..(155)
<223> n=S，T，F，G，A，W，D，E，N，H，R，Q，L or Y
<220>
<221> misc_feature
<222> (156)..(156)
<223> n=S，T，F，G，A，W，D，E，N，H，Q or P
<220>
<221> misc_feature
<222> (157)..(157)
<223> n=G，S，T，N or D
<220>
<221> misc_feature
<222> (158)..(158)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P，V，W，K or M
<220>
<221> misc_feature
<222> (159)..(159)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P，V，W or K
<220>
<221> misc_feature
<222> (160)..(160)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P or V
<220>
<221> misc_feature
<222> (278)..(293)
<223> n=S，T，F，G，A，Y，D，E，N，I，H，R，Q，L，P，V，W，K or M
<400> 32
acaagagaga agctgaagct caagttcaac tggttgaatc tggcggcggt ctggttcaac 60
cgggcggtag tctgcgtctg agttgcgcag catctggtnn nnnnnatggg ttggtttcgt 120
caagcaccgg gtcaaggtct ggaagcagtt gcannnnnnn tactacgcgg atagcgtcaa 180
aggtcgcttt accatcagcc gcgataacag caaaaacacc ctgtacctgc agatgaattc 240
tctgcgcgca gaagataccg cggtttatta ttgcgcgnnn nnnnnnnnnn nnntattggg 300
gtcaaggtac gctggttacc gttagtagtg aattcggtaa gcctatccct aaccc 355
<210> 33
<211> 8165
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag 60
catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac 120
caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc 180
ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt 240
aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc 300
gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga 360
cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta 420
ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta 480
tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga 540
tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg 600
cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag 660
tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc 720
tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact 780
tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt 840
cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta 900
ccatctggcc ccagtgctgc aatgataccg cgactcccac gctcaccggc tccagattta 960
tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc 1020
gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat 1080
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt 1140
atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg 1200
tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca 1260
gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta 1320
agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg 1380
cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact 1440
ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg 1500
ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt 1560
actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga 1620
ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc 1680
atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa 1740
caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgaacg aagcatctgt 1800
gcttcatttt gtagaacaaa aatgcaacgc gagagcgcta atttttcaaa caaagaatct 1860
gagctgcatt tttacagaac agaaatgcaa cgcgaaagcg ctattttacc aacgaagaat 1920
ctgtgcttca tttttgtaaa acaaaaatgc aacgcgacga gagcgctaat ttttcaaaca 1980
aagaatctga gctgcatttt tacagaacag aaatgcaacg cgagagcgct attttaccaa 2040
caaagaatct atacttcttt tttgttctac aaaaatgcat cccgagagcg ctatttttct 2100
aacaaagcat cttagattac tttttttctc ctttgtgcgc tctataatgc agtctcttga 2160
taactttttg cactgtaggt ccgttaaggt tagaagaagg ctactttggt gtctattttc 2220
tcttccataa aaaaagcctg actccacttc ccgcgtttac tgattactag cgaagctgcg 2280
ggtgcatttt ttcaagataa aggcatcccc gattatattc tataccgatg tggattgcgc 2340
atactttgtg aacagaaagt gatagcgttg atgattcttc attggtcaga aaattatgaa 2400
cggtttcttc tattttgtct ctatatacta cgtataggaa atgtttacat tttcgtattg 2460
ttttcgattc actctatgaa tagttcttac tacaattttt ttgtctaaag agtaatacta 2520
gagataaaca taaaaaatgt agaggtcgag tttagatgca agttcaagga gcgaaaggtg 2580
gatgggtagg ttatataggg atatagcaca gagatatata gcaaagagat acttttgagc 2640
aatgtttgtg gaagcggtat tcgcaatgat gtgctgcaag gcgattaagt tgggtaacgc 2700
cagggttttc ccagtcacga cgttgtaaaa cgacggccag tgccgcaaat taaagccttc 2760
gagcgtccca aaaccttctc aagcaaggtt ttcagtataa tgttacatgc gtacacgcgt 2820
ctgtacagaa aaaaaagaaa aatttgaaat ataaataacg ttcttaatac taacataact 2880
ataaaaaaat aaatagggac ctagacttca ggttgtctaa ctccttcctt ttcggttaga 2940
gcggatgtgg ggggagggcg tgaatgtaag cgtgacataa ctaattacat gactttaact 3000
gaaaattaca ttgcaagcaa ctgccatgat ggcgaagacc aactttccta atggcaaacg 3060
cgtttgcgat ccactgccca tatattggct aattgttacc attgatgaag tttggtattg 3120
tatggagatt gaagtagtgg aaacttctgt agtggtaaca gatgtttgta gagaagaggg 3180
agcagatgat gatgagagga aaatagaaga taccgtcgga gtcaatgatg aagattcaat 3240
agtcgattcc acagaggtag atggtgtaga gatagatgtt gcggaaagtg atgatgtaga 3300
ggcagatatt gtagcaatag ctgtggaaga agtgggttgt aaaatctctg atgttgaaga 3360
aatttgaagt gtggtaactg gtgactgttc gacacttagt gatgatacag aactgaatag 3420
caaaatagta ctgtcagagg ataatgtagc agacaaggag gaggatgaaa ggaccgatgt 3480
cgataataag tgtgatgatt gttcttgaga ttctgagtca atgcttgatg tactcgtggc 3540
ttcacttaca ctagtcgaaa tggttgaggt acctgaagat gttacatgat acttaccggt 3600
agcgtgctta ttgaaatctt catccgaagt tgtcattgaa cttacccaac tctttgtgga 3660
tgtggtctgc tgtgttacag aacttgatga agctgtttgc acggaagaat acgagtgtga 3720
cgtttcagca catgtggagc atgcatatga ggattcaacc gttgaggtgc ttacagaagt 3780
tgcgacgttt ttagtggaca tttttataga agatgtaatc aaacttgttg tctgcgaggg 3840
catgcaagac tcgtgagtgc agactgtagc gtctgttgta accgtttcat gcatactggt 3900
taatgtgcta gtctcagatt cagagctata aggacaccat gtcgtataca ttgtagtgac 3960
tccactaaca gtggtgaaat atgtagacat ggaaaatgat gggatagtag tagaaattga 4020
ggatttggtg atatattcag acgaactttg tgaactaaca gttgtttcaa cagttgaaga 4080
agtcatagaa ggagttgcaa catttgacga agtcgaaggg acggagtaaa caggtgtgga 4140
tgggctgtat aatgacacag acgttgatga agatgctata gaggagccaa gggatgaagt 4200
tgaatcagta aaagtagagc taatagatgt tgaacttgga gaagtggaag ccaaagttgg 4260
agatgaagaa gtcaaacttg gggatgtaga tgttgaatat gaagaagtgg aagtggagct 4320
tgcagaagta gatttagaac ttggagatgt agatgttgaa cttgatgagg tagatgtaga 4380
gcttggagat gtagatgtcg aacttgatga ggtagatgta gaactttggg atgtagatgt 4440
agaacttgaa gatgtggatg tcaaacttga agatgtggat gtcgaacttg gagatgtaga 4500
tgtcgaactt gatgaggtag atgtcgaact tgatgaggta gatgtcgaac ttgatgaggt 4560
agatgtagag cttggagatg tagatgtaga gcttggagat gtagatgtcg aacttaggga 4620
ggtagtagtt ggattggacg atgttgtact cgaaagtgta gaggtgacag gtgagattat 4680
agaggcactt gatggttcaa tggcgctgga tgataccacg gttgtagttc ctacttcgga 4740
taatgagctg atagcggttg tgtgacaagt agggcaaata tatgaagtga acttggaagt 4800
cacagataag gtggtggttg gcaacgtcga tgacgaagga catgcctcat gggagcagac 4860
ttctgtagat aatgtcaaag tactaaatct ggatagggta gtagcgtacg atattgaagg 4920
acttatttcg gcagcagatg aagtggatac cgtcaatgga caccaagtcg tatacaatgt 4980
cgtagtgcca gcttgaacga tagtggatgt ggagactagc gcgggtgaaa ctgtagttgt 5040
aacaacccca tttgcatcgt ttgtcttggt tatcgtcact agaatcgttt ctggatcaga 5100
tgccaaggca atattagtta atcccaacaa tattgtgaac aggtaggtaa aatgagcgaa 5160
agataatgtc atggtggcgg cgtcgactta tattgaattt tcaaaaattc ttactttttt 5220
tttggatgga cgcaaagaag tttaataatc atattacatg gcattaccac catatacata 5280
tccatataca tatccatatc taatcttact tatatgttgt ggaaatgtaa agagccccat 5340
tatcttagcc taaaaaaacc ttctctttgg aactttcagt aatacgctta actgctcatt 5400
gctatattga agtacggatt agaagccgcc gagcgggtga cagccctccg aaggaagact 5460
ctcctccgtg cgtcctcgtc ttcaccggtc gcgttcctga aacgcagatg tgcctcgcgc 5520
cgcactgctc cgaacaataa agattctaca atactagctt ttatggttat gaagaggaaa 5580
aattggcagt aacctggccc cacaaacctt caaatgaacg aatcaaatta acaaccatag 5640
gatgataatg cgattagttt tttagcctta tttctggggt aattaatcag cgaagcgatg 5700
atttttgatc tattaacaga tatataaatg caaaaactgc ataaccactt taactaatac 5760
tttcaacatt ttcggtttgt attacttctt attcaaatgt aataaaagta tcaacaaaaa 5820
attgttaata tacctctata ctttaacgtc aaggagaaaa aaccccggat cggactacta 5880
gcagctgtaa tacgactcac tatagggaat attaagctaa ttccctactt catacatttt 5940
caattaagat gagattccca tctatcttca ccgctgtttt atttgctgct tcttctgcct 6000
tggccgctcc agctaacaca actactgaag atgaaaccgc tcaaatccca gctgaagccg 6060
tcattgacta ctcagatttg gaaggtgact tcgatgctgc tgctttgcca ctctctaact 6120
ccaccaacaa cggtttatcc tctactaata ccaccattgc ttctattgcc gctaaggaag 6180
aaggtgttca attggacaag agagaagctg aagctgaatt ctattggggt caaggtacgc 6240
tggttaccgt tagtagtgaa ttcggtaagc ctatccctaa ccctctcctc ggtctcgatt 6300
ctacgggtgg tggtggttct ggtggtggtg gttctggtgg tggtggttct caggaactga 6360
caactatatg cgagcaaatc ccctcaccaa ctttagaatc gacgccgtac tctttgtcaa 6420
cgactactat tttggccaac gggaaggcaa tgcaaggagt ttttgaatat tacaaatcag 6480
taacgtttgt cagtaattgc ggttctcacc cctcaacgac tagcaaaggc agccccataa 6540
acacacagta tgtttttaag cttctgcagg ctagttgata ataggtttaa acccgctgat 6600
ctgataacaa cagtgtagat gtaacaaaat cgactttgtt cccactgtac ttttagctcg 6660
tacaaaatac aatatacttt tcatttctcc gtaaacaaca tgttttccca tgtaatatcc 6720
ttttctattt ttcgttccgt taccaacttt acacatactt tatatagcta ttcacttcta 6780
tacactaaaa aactaagaca attttaattt tgctgcctgc catatttcaa tttgttataa 6840
attcctataa tttatcctat tagtagctaa aaaaagatga atgtgaatcg aatcctaaga 6900
gaattggcaa gtgcacaaac aatacttaaa taaatactac tcagtaataa ctgcagaggc 6960
ctgcatgcaa gcttggctcg agcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 7020
ctcacaattc cacacaacat acgagccgga agcataacta gtacactcta tattttttta 7080
tgcctcggta atgattttca tttttttttt tccacctagc ggatgactct ttttttttct 7140
tagcgattgg cattatcaca taatgaatta tacattatat aaagtaatgt gatttcttcg 7200
aagaatatac taaaaaatga gcaggcaaga taaacgaagg caaagatgac agagcagaaa 7260
gccctagtaa agcgtattac aaatgaaacc aagattcaga ttgcgatctc tttaaagggt 7320
ggtcccctag cgatagagca ctcgatcttc ccagaaaaag aggcagaagc agtagcagaa 7380
caggccacac aatcgcaagt gattaacgtc cacacaggta tagggtttct ggaccatatg 7440
atacatgctc tggccaagca ttccggctgg tcgctaatcg ttgagtgcat tggtgactta 7500
cacatagacg accatcacac cactgaagac tgcgggattg ctctcggtca agcttttaaa 7560
gaggccctac tggcgcgtgg agtaaaaagg tttggatcag gatttgcgcc tttggatgag 7620
gcactttcca gagcggtggt agatctttcg aacaggccgt acgcagttgt cgaacttggt 7680
ttgcaaaggg agaaagtagg agatctctct tgcgagatga tcccgcattt tcttgaaagc 7740
tttgcagagg ctagcagaat taccctccac gttgattgtc tgcgaggcaa gaatgatcat 7800
caccgtagtg agagtgcgtt caaggctctt gcggttgcca taagagaagc cacctcgccc 7860
aatggtacca acgatgttcc ctccaccaaa ggtgttctta tgtagtctct tctcgagtca 7920
tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct ctaaccgaaa 7980
aggaaggagt tagacaacct gaagtctagg tccctattta tttttttata gttatgttag 8040
tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg 8100
catgtaacat tatactgaaa accttgcttg agaaggtttt gggacgctcg aaggctttaa 8160
tttgc 8165

Claims (9)

1.一种酵母细胞同源重组酶系，其特征在于，包括酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52和酵母单链DNA结合蛋白RPA；
所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52、酵母单链DNA结合蛋白RPA是经过原核表达***密码子优化获得；
优化后的酵母核酸外切酶ExoI的核苷酸序列如SEQ ID NO:1所示；
优化后的酵母重组酶RAD51的核苷酸序列如SEQ ID NO:2所示；
优化后的酵母重组酶RAD52的核苷酸序列如SEQ ID NO:3所示；
优化后的酵母单链DNA结合蛋白RPA的核苷酸序列如SEQ ID NO:4所示。
2.根据权利要求1所述同源重组酶系，其特征在于，所述同源重组酶系还包括大肠杆菌DNA聚合酶I、大肠杆菌DNA连接酶和ATP再生酶；
所述ATP再生酶包括以下一种或几种激酶：肌酸激酶、丙酮酸激酶、乙酸激酶、聚磷酸激酶和核苷二磷酸激酶。
3.根据权利要求2所述同源重组酶系，其特征在于，所述酵母核酸外切酶ExoI、酵母重组酶RAD51、酵母重组酶RAD52、酵母单链DNA结合蛋白RPA、大肠杆菌DNA聚合酶I、大肠杆菌DNA连接酶和ATP再生酶的活性比为（0.001~0.1）：（10~100）：（10~100）：（10~100）：（0.1~1）：（0.1~1）：（1~100）。
4.一种DNA体外组装试剂，其特征在于，包括权利要求1~3任意一项所述同源重组酶系和2.6×缓冲液；
所述2.6×缓冲液为包含1~50mM镁离子源、5~50mM碱金属离子源、质量百分含量2%~10%聚乙二醇、1~10mM NAD、2-20mM二硫苏糖醇、1~10mM ATP再生酶底物、1~10mM脱氧核苷三磷酸的10~200mM Tris base缓冲液，pH值为7.0~8.0。
5.根据权利要求4所述DNA体外组装试剂，其特征在于，所述DNA体外组装试剂中酵母核酸外切酶ExoI的浓度为1~10 U/ml，酵母重组酶RAD51的浓度为10~100nM，酵母重组酶RAD52的浓度为10~100nM，酵母单链DNA结合蛋白RPA的浓度为100~500nM，大肠杆菌DNA聚合酶I的浓度为10~100U/ml，大肠杆菌DNA连接酶的浓度为1000~5000U/ml，ATP再生酶的浓度为1~10mM。
6.权利要求1~3任意一项所述同源重组酶系或权利要求4或5所述DNA体外组装试剂在DNA片段体外组装中的应用。
7.根据权利要求6所述应用，其特征在于，所述应用包括两条及两条以上DNA片段组装；
所述DNA片段包括单链或双链的DNA片段；
所述DNA片段体外组装包括以下一种或几种应用：动物来源靶基因的组装、病毒来源靶基因或基因组的组装和抗体库DNA片段的组装。
8.一种DNA片段体外组装的方法，其特征在于，包括以下步骤：
将待组装的DNA片段、线性载体与权利要求4或5所述DNA体外组装试剂混合反应，得到直链或环状的DNA长片段；
所述待组装的DNA片段含有长度为10~200bp的碱基同源或互补区域。
9.根据权利要求8所述方法，其特征在于，所述混合反应后，将反应产物导入原核细胞中完成DNA片段的扩增，得到双链DNA分子。
CN202210436102.2A 2022-04-25 2022-04-25 一种酵母细胞同源重组酶系、dna体外组装试剂及其应用 Active CN114717207B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210436102.2A CN114717207B (zh) 2022-04-25 2022-04-25 一种酵母细胞同源重组酶系、dna体外组装试剂及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210436102.2A CN114717207B (zh) 2022-04-25 2022-04-25 一种酵母细胞同源重组酶系、dna体外组装试剂及其应用

Publications (2)

Publication Number Publication Date
CN114717207A CN114717207A (zh) 2022-07-08
CN114717207B true CN114717207B (zh) 2023-03-07

Family

ID=82246465

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210436102.2A Active CN114717207B (zh) 2022-04-25 2022-04-25 一种酵母细胞同源重组酶系、dna体外组装试剂及其应用

Country Status (1)

Country Link
CN (1) CN114717207B (zh)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107699550A (zh) * 2017-08-11 2018-02-16 山东大学 一组伯克氏菌同源重组酶及其表达载体与应用
CN110691848A (zh) * 2017-04-10 2020-01-14 马克斯·普朗克科学促进学会 用于提高基因组编辑效率的化合物
CN111630175A (zh) * 2018-01-22 2020-09-04 庆尚大学校产学协力团 在植物体中基于同源重组提高基因编辑效率的方法
WO2021178898A1 (en) * 2020-03-05 2021-09-10 Flagship Pioneering Innovations Vi, Llc Host defense suppressing methods and compositions for modulating a genome

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110691848A (zh) * 2017-04-10 2020-01-14 马克斯·普朗克科学促进学会 用于提高基因组编辑效率的化合物
CN107699550A (zh) * 2017-08-11 2018-02-16 山东大学 一组伯克氏菌同源重组酶及其表达载体与应用
CN111630175A (zh) * 2018-01-22 2020-09-04 庆尚大学校产学协力团 在植物体中基于同源重组提高基因编辑效率的方法
WO2021178898A1 (en) * 2020-03-05 2021-09-10 Flagship Pioneering Innovations Vi, Llc Host defense suppressing methods and compositions for modulating a genome

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Pathways and assays for DNA double-strand break repair by homologous recombination;Jinbao Li等;《Acta Biochim Biophys Sin (Shanghai)》;20190906;第51卷(第9期);第879-889页 *
酵母细胞中构建HL-60细胞的酵母双杂交cDNA文库;黄功华等;《广东医学院学报》;20041009(第1期);第1-3页 *

Also Published As

Publication number Publication date
CN114717207A (zh) 2022-07-08

Similar Documents

Publication Publication Date Title
US20020025561A1 (en) Vectors for gene-self-assembly
CN113481327B (zh) 基于RAA扩增和CRISPR-Cas12a的新型冠状病毒ORF1ab基因检测方法
CN113549618B (zh) 基于RAA扩增和CRISPR-Cas13a***的SARS-CoV-2核酸检测方法
CN108395996B (zh) 一种猪瘟病毒亚单位疫苗及其制备方法和用途
CN108531471B (zh) 一种长基因合成方法
CN109609579B (zh) 一种产β-胡萝卜素的基因工程菌及其构建方法
CN108285886A (zh) 重组枯草芽孢杆菌全细胞转化生产n-乙酰神经氨酸的方法
CN114901816A (zh) 脂肪酶修饰的菌株
CN108531495A (zh) 一种光控基因表达***及其应用
CN114717207B (zh) 一种酵母细胞同源重组酶系、dna体外组装试剂及其应用
CN113604505A (zh) pSFV-p32病毒样颗粒及其制备方法和应用
CN109652352B (zh) 一株用于屎肠球菌谷氨酸脱羧酶高效固定化的基因工程菌及固定化方法
CN114292864B (zh) 高产表面活性素的贝莱斯芽孢杆菌突变株及其构建方法和应用
CN111979134B (zh) 一种合成胭脂红酸的重组酿酒酵母的构建及其应用
CN113584223B (zh) 基于CRISPR-Cas12a的SARS-CoV-2中D614G突变鉴定方法
CN112626116B (zh) 定点整合大片段外源dna的方法
CN111321163B (zh) 一种枯草芽孢杆菌线性质粒***的构建与应用
CN109456930A (zh) 一株用于快速纯化制备屎肠球菌谷氨酸脱羧酶天然酶纯酶的工程菌及纯化方法
CN107661496A (zh) 一种猪细小病毒免疫组合物及其制备方法与应用
CN112322706A (zh) 一种特异性人源基因片段及其引物探针和应用
CN111378718A (zh) 一种基因测序文库的构建方法
CN114540345B (zh) 一种发夹结构的标签荧光探针和荧光检测方法
CN114214347B (zh) 示踪肝前体细胞的质粒***及应用
CN110607380B (zh) 桑树植原体ltrA基因及其在桑树植原体分子检测中的应用
CN114214346B (zh) 靶向肝前体细胞的质粒***及应用

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
GR01 Patent grant
GR01 Patent grant