CN113355340A - 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用 - Google Patents

一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用 Download PDF

Info

Publication number
CN113355340A
CN113355340A CN202010157998.1A CN202010157998A CN113355340A CN 113355340 A CN113355340 A CN 113355340A CN 202010157998 A CN202010157998 A CN 202010157998A CN 113355340 A CN113355340 A CN 113355340A
Authority
CN
China
Prior art keywords
ala
leu
gly
ser
val
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.)
Granted
Application number
CN202010157998.1A
Other languages
English (en)
Other versions
CN113355340B (zh
Inventor
杜德尧
白超弦
张倩
李腾
张浩千
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.)
Shenzhen Blue Crystal Biotechnology Co ltd
Original Assignee
Shenzhen Blue Crystal Biotechnology Co ltd
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 Shenzhen Blue Crystal Biotechnology Co ltd filed Critical Shenzhen Blue Crystal Biotechnology Co ltd
Priority to CN202010157998.1A priority Critical patent/CN113355340B/zh
Publication of CN113355340A publication Critical patent/CN113355340A/zh
Application granted granted Critical
Publication of CN113355340B publication Critical patent/CN113355340B/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
    • 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
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1025—Acyltransferases (2.3)
    • 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.)
    • C12N9/1025—Acyltransferases (2.3)
    • C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • 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)
    • C12N9/1229—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
    • C12N9/88—Lyases (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
    • C12N9/90—Isomerases (5.)
    • 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
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/24—Preparation of oxygen-containing organic compounds containing a carbonyl group
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
    • C12Y101/01088—Hydroxymethylglutaryl-CoA reductase (1.1.1.88)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
    • C12Y101/03038—Vanillyl-alcohol oxidase (1.1.3.38)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/01—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
    • C12Y203/01009—Acetyl-CoA C-acetyltransferase (2.3.1.9)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/03—Acyl groups converted into alkyl on transfer (2.3.3)
    • C12Y203/0301—Hydroxymethylglutaryl-CoA synthase (2.3.3.10)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01001—Dimethylallyltranstransferase (2.5.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/01029—Geranylgeranyl diphosphate synthase (2.5.1.29)
    • 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)
    • C12Y207/01036—Mevalonate kinase (2.7.1.36)
    • 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)
    • C12Y207/04002—Phosphomevalonate kinase (2.7.4.2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01033—Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/03—Intramolecular oxidoreductases (5.3) transposing C=C bonds (5.3.3)
    • C12Y503/03002—Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)

Abstract

本发明涉及一种构建生产柠檬醛的重组菌的方法，及由其构建的重组菌及其应用。在本发明中，构建了两个质粒，其中一个质粒包含用于表达生成IPP和DMAPP的各种酶的编码基因：atoB、HMGS、HMGR、MK、PMK、PMD和idi，另一个质粒包含用于以IPP和DMAPP为前体合成柠檬醛的酶的编码基因：GPPS、GES、geoA和idi。用上述两质粒共转染常规的BW25113菌可得到重组菌，该重组菌可在经典LB培养基中以高产量发酵生产柠檬醛。

Description

一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及 其应用
技术领域
本发明属于生物工程领域，具体地，本发明涉及一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用。
背景技术
柠檬醛(citral)是开链单萜中最重要的代表之一，分子式为C10H16O，无色或淡黄色透明液体，其通常由互为异构体的香叶醛(反式柠檬醛，柠檬醛a，geranial，结构式如下)和橙花醛(顺式柠檬醛，柠檬醛b，neral，结构式如下)组成。
Figure BDA0002404765100000011
柠檬醛的应用包括：a)食用香料：其可直接用于烘烤食品、糖果、冷饮、口香糖和软饮料，同时也可用于配制草莓、苹果、杏、甜橙、柠檬等水果型的食用香精，尤其是在柑桔香味的香精中更是不可缺。因为柠檬醛对于柑桔香精，不仅发挥了其本身的柠檬香气，而且还对香精的整体气味起到增香作用。b)在日常生活中，其广泛用于餐具的洗涂液、肥皂和花露水的加香剂，以及人造柠檬油和柑橘油的调制。c)香料工业：主要用于合成高档香料紫罗兰酮、鸢尾酮系列产品。d)制药工业：其可用于合成维生素A、维生素E、β-类胡萝卜素、叶绿醇、异植物醇等。同时其具有驱避、杀虫、抗菌抗氧化、治疗心血管疾病、平喘镇咳、抗过敏等生物活性，可用于研发驱蚊剂、治疗皮肤类感染病、心血管疾病和抗白血病等药物。
柠檬醛现有的生产方法包括：a)天然获取：天然柠檬醛主要存在于山苍子油、柠檬艾油、柑桔油、柠檬罗勒油、桉叶油、柠檬草油、青草油、马鞭草油以及姜油等植物的提取精油中。b)化学合成：主要包括丙酮法、异戊二烯法、醇和醛缩合法、氮氧化物法、香叶醇气相氧化法和脱氨芳棒醇直接重排法。植物提取法存在的缺点是，原料受季节和地缘的影响，限制了天然柠檬醛的产能，而化学合成法存在环境污染和条件苛刻等问题。
柠檬醛是萜类化合物的重要代表之一，萜类化合物的生物合成是以IPP(异戊二烯焦磷酸)和DMAPP(二甲基烯丙基焦磷酸)作为单元结构进行合成。IPP和DMAPP的合成途径有明确路线，分为MVA途径和MEP途径。MVA途径：甲羟戊酸途径(mevalonate pathway)，缩写为MEV途径，该途径可以描述为3个乙酰辅酶A(acetyl-CoA)形成一个C6化合物甲羟戊酸(mevalonate)，再连接两个磷酸基团后，脱掉一个CO2，形成C5的IPP，IPP和DMAPP之间可以通过异构酶相互转化(VranováE,Coman D,Gruissem W.,2013,Network analysis of theMVA and MEP pathways for isoprenoid synthesis,Annu Rev Plant Biol.2013；64:665-700.)。具体地，AtoB以乙酰辅酶A为底物合成乙酰乙酰辅酶A(acetoacetyl-CoA)，HMGS以乙酰乙酰辅酶A为底物合成3-羟基-3-甲基戊二酰-CoA(3-Hydroxy-3-methylglutaryl-CoA，HMG-CoA)，HMGR(3-羟基-3-甲基戊二酰-CoA还原酶)以HMG-CoA为底物合成甲羟戊酸(MVA)，MK(MVA激酶)以MVA为底物合成mevalonate-5-phosphate，PMK(磷酸MVA激酶)以mevalonate-5-phosphate为底物合成mevalonate-5-diphosphate，PMD(MVA二磷酸脱羧酶)以mevalonate-5-diphosphate为底物合成isopentenyl diphosphate(IPP)，IPPI(IPP异构酶)可以将IPP转化为DMAPP。
一分子IPP和一分子DMAPP形成单萜前体GPP(香叶酯二磷酸，geranyldiphosphate)，催化该反应的酶是GPP合成酶，(GPP synthase，GPPS)。GPP经过磷酸酶(GES)水解生成单萜香叶醇(geraniol)。香叶醇经过氧化酶(GeoA)氧化生成柠檬醛，该合成过程可参见图1。
发明内容
本发明的技术目的是提供一种构建用于生产柠檬醛的重组菌的方法，以及由该方法构建的重组菌以及该重组菌用于生产柠檬醛的应用。
因此，一方面，本发明提供一种构建用于生产柠檬醛的重组菌的方法，其包括以下步骤：
1)构建用于生成IPP和DMAPP的表达载体，用以表达乙酰乙酰CoA硫解酶(优选SEQID NO:38)、3-羟基-3-甲基戊二酰-CoA合酶(优选SEQ ID NO:39)、3-羟基-3-甲基戊二酰-CoA还原酶(优选SEQ ID NO:40)、MVA激酶(优选SEQ ID NO:41)、磷酸MVA激酶(优选SEQ IDNO:42)、MVA焦磷酸脱羧酶(优选SEQ ID NO:43)以及IPP异构酶(优选SEQ ID NO:44)；
2)构建包含GPPS编码基因(优选SEQ ID NO:14)、GES编码基因(优选SEQ ID NO:15)、geoA编码基因(优选SEQ ID NO:16)和idi编码基因(优选SEQ ID NO:7)的表达载体，用以表达香叶酯二磷酸合成酶(优选SEQ ID NO:45)、香叶醇合成酶(优选SEQ ID NO:46)、香叶醇氧化酶(优选SEQ ID NO:47)以及IPP异构酶(优选SEQ ID NO:44)；
3)将步骤1)和步骤2)中构建的表达载体共转入出发菌株中，得到重组菌。
在一个具体实施方式中，所述出发菌株为大肠杆菌BW25113。
在一个具体实施方式中，所述用于生成IPP和DMAPP的表达载体为用于使重组菌完成MVA途径而得到IPP和DMAPP的表达载体。
在一个具体实施方式中，所述用于生成IPP和DMAPP的表达载体包括合成IPP和DMAPP的各个酶的编码序列：编码乙酰乙酰辅酶A硫解酶(acetoacetyl-CoA thiolase)的基因、编码3-羟基-3-甲基戊二酰-CoA合酶(3-Hydroxy-3-methylglutaryl-CoA synthase)的基因、编码3-羟基-3-甲基戊二酰-CoA还原酶(3-Hydroxy-3-methylglutaryl-CoAreductase)的基因、编码MVA激酶(MVA kinase)的基因、编码磷酸MVA激酶(phospho MVAkinase)的基因、编码MVA焦磷酸脱羧酶(MVA diphosphate decarboxylase)的基因和编码IPP异构酶的基因。
在一个具体实施方式中，所述用于生成IPP和DMAPP的表达载体包括：atoB(优选SEQ ID NO:1)、HMGS(优选SEQ ID NO:2)、HMGR(优选SEQ ID NO:3)、MK(优选SEQ ID NO:4)、PMK(优选SEQ ID NO:5)、PMD(优选SEQ ID NO:6)和idi(优选SEQ ID NO:7)。
其中atoB可为改造自大肠杆菌BW25113的atoB基因，编码乙酰乙酰辅酶A硫解酶(acetoacetyl-CoA thiolase)；HMGS可为改造自Enterococcus的HMGS，用于编码3-羟基-3-甲基戊二酰-CoA合酶(3-Hydroxy-3-methylglutaryl-CoA synthase)；HMGR可为改造自Enterococcus的HMGR，用于编码3-羟基-3-甲基戊二酰-CoA还原酶(3-Hydroxy-3-methylglutaryl-CoA reductase)；MK可为改造自Methanosarcina mazei Tuc01的MK基因，用于编码MVA激酶(MVA kinase)；PMK可为改造自Saccharomyces cerevisiae S288C的PMK基因，用于编码磷酸MVA激酶(phospho MVA kinase)；PMD可为改造自Saccharomycescerevisiae S288C的PMD基因，用于编码MVA焦磷酸脱羧酶(MVA diphosphatedecarboxylase)；idi可为改造自大肠杆菌的idi基因，用于编码IPP异构酶。
在一个具体实施方式中，atoB、HMGS、HMGR共用一套调控元件；MK、PMK、PMD和idi共用一套调控元件。
在一个具体实施方式中，所述表达载体为p15A-MVA质粒，其包括合成IPP和DMAPP的各个酶的编码序列：atoB、HMGS、HMGR、MK、PMK、PMD和idi，其中atoB、HMGS、HMGR共用一套调控元件，受诱导型启动子(例如，lac UV5启动子(SEQ ID NO:31))调控；MK、PMK、PMD和idi共用一套调控元件，受诱导型启动子(例如，pTac启动子(SEQ ID NO:32))调控。
在可选的实施方式中，上述启动子lac UV5、pTac可以被其它启动子替代，如pRha启动子(SEQ ID NO:33)、pTet启动子(SEQ ID NO:34)、pBAD启动子(SEQ ID NO:35)。
对于lac UV5启动子和pTac启动子，可采用IPTG作为诱导剂；对于pRha启动子，可采用鼠李糖作为诱导剂；对于pTet启动子，可采用四环素作为诱导剂；对于pBAD启动子，可采用阿拉伯糖作为诱导剂。
在一个具体实施方式中，所述包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体为用于使重组菌从IPP和DMAPP合成柠檬醛的表达质粒。
在一个具体实施方式中，所述包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体包括以IPP和DMAPP为前体合成柠檬醛的酶的编码基因：编码香叶酯二磷酸合成酶(GPPS)的基因、编码香叶醇合成酶(GES)的基因、编码香叶醇氧化酶(geoA)的基因和编码IPP异构酶的基因，
其中，GPPS编码基因可为改造自Abies grandis的GPPS基因，用于编码香叶酯二磷酸合成酶(SEQ ID NO:45)；GES编码基因可为改造自Ocimum basilicum的GES基因，用于编码香叶醇合成酶(SEQ ID NO:46)；geoA编码基因可为改造自Castellaniella defragrans的geoA基因，用于编码香叶醇氧化酶(SEQ ID NO:47)；idi编码基因可为改造自大肠杆菌的idi基因，用于编码IPP异构酶(SEQ ID NO:44)，这些编码基因共用一套调控元件，受组成型启动子(例如，启动子为sp2(SEQ ID NO:36))调控，启动子和核糖体结合位点之间插入有绝缘子功能RiboJ的序列(SEQ ID NO:37)。
所述组成型启动子也可被诱导性启动子替代，如pRha启动子(SEQ ID NO:33)、pTet启动子(SEQ ID NO:34)、pBAD启动子(SEQ ID NO:35)。
在一个具体实施方式中，所述包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体包含表达GPPS-GES-geoA-idi的基因盒序列，所述基因盒包括六个部分：组成型启动子(例如，sp2启动子(SEQ ID NO:36))，绝缘子RiboJ(SEQ ID NO:37)，带有核糖体结合序列的GPPS编码序列(SEQ ID NO:9)，带有rbs1的GES序列(SEQ ID NO:10)，带有rbs2的geoA序列(SEQ ID NO:11)，以及带有rbs3的idi序列(SEQ ID NO:12)。
在一个具体实施方式中，所述包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体为pTALE-GPPS-GES-geoA-idi质粒(SEQ ID NO:13)。
另一方面，本发明提供一种表达载体，其包含序列SEQ ID No：18。
再一方面，本发明提供一种表达载体，其序列如SEQ ID NO:13所示。
再一方面，本发明提供一种由上述方法构建的重组菌。
再一方面，本发明提供上述重组菌用于发酵生产柠檬醛的用途。
再一方面，本发明提供一种生产柠檬醛的方法，所述方法包括使用上述重组菌发酵生产柠檬醛。
在具体实施方式中，在所述方法中，当构建重组菌使用lac UV5启动子和pTac启动子时，使用异丙基-β-D-硫代半乳糖苷(IPTG)进行诱导发酵。
在具体实施方式中，使用LB培养基进行发酵。
在具体实施方式中，所述IPTG的浓度为50-500μM，优选为200μM。
在具体实施方式中，发酵时间为20-48小时，优选40小时。
在具体实施方式中，在所述方法中，OD600值为0.6-1.1，优选为1.0。
有益效果
本发明中构建了两个质粒，其中一个质粒包含用于表达生成IPP和DMAPP的各种酶的编码基因，如atoB、HMGS、HMGR、MK、PMK、PMD和idi，另一个质粒包含用于以IPP和DMAPP为前体合成柠檬醛的酶的编码基因，如GPPS、GES、geoA和idi。用上述两质粒共转染常规的BW25113菌得到的重组菌可在经典LB培养基中发酵生产柠檬醛，培养过程中通过IPTG诱导，获得高产量的柠檬醛(约0.1g/L)，表达过程中，杂质少，产物包括Neral和Geranial两种构象。由此表明，本发明中构建的重组菌具有强大的柠檬醛工业化生产的潜力。
附图说明
图1为IPP和DMAPP合成柠檬醛的生物合成过程。
图2示意性显示本发明中构建的p15A-MVA质粒。
图3示意性显示本发明中构建的pTALE-GPPS-GES-geoA-idi质粒。
具体实施方式
在下文中，将通过实施例详细描述本发明。然而，在此提供的实施例仅用于说明目的，并不用于限制本发明。
下述实施例所使用的实验方法如无特殊说明，均为常规方法。
下述实施例所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
所用酶试剂采购自ThermoFisher公司和New England Biolabs(NEB)公司，提取质粒所用的试剂盒购自天根生化科技(北京)有限公司，回收DNA片段的试剂盒购自美国omega公司，相应的操作步骤严格按照产品说明书进行，所有培养基如无特殊说明均用去离子水配制。基因合成由华大基因研究院完成。
实施例1：构建MVA表达载体
p15A-MVA质粒的构建：该质粒包括合成IPP和DMAPP的各个酶的编码序列，其中atoB、HMGS、HMGR共用一套调控元件，受lac UV5启动子(SEQ ID NO:31)调控；MK、PMK、PMD和idi共用一套调控元件，受pTac启动子(SEQ ID NO:32)调控。
其中atoB(SEQ ID NO:1)为acetoacetyl-CoA thiolase(SEQ ID NO:38)编码序列，HMGS(SEQ ID NO:2)编码3-Hydroxy-3-methylglutaryl-CoA synthase(3-羟基-3-甲基戊二酰-CoA合酶)(SEQ ID NO:39)，HMGR(SEQ ID NO:3)编码3-Hydroxy-3-methylglutaryl-CoA reductase(3-羟基-3-甲基戊二酰-CoA还原酶)(SEQ ID NO:40)，MK(SEQ ID NO:4)编码MVA kinase(MVA激酶)(SEQ ID NO:41)，PMK(SEQ ID NO:5)编码phospho MVA kinase(磷酸MVA激酶)(SEQ ID NO:42)，PMD(SEQ ID NO:6)编码MVAdiphosphate decarboxylase(MVA焦磷酸脱羧酶)(SEQ ID NO:43)，idi(SEQ ID NO:7)编码IPP异构酶(SEQ ID NO:44)。
用引物(PKMVA 1F和PK1-R)以pJBEI6409(JorgeAlonso-Gutierrez,RossanaChan,Tanveer S.Batth,Paul D.Adams,Jay D.Keasling,Christopher J.Petzold,TaekSoon Lee,2013.Metabolic engineering of Escherichia coli for limonene andperillyl alcohol production.Metab.Eng.,19,4,33-41.)为模板扩增得到p15A载体骨架片段；
用引物(PK2-F和PK2-R)以pJBEI6409为模板扩增得到DNA片段，该片段包括atoB、HMGS和HMGR三个基因的编码序列，atoB序列前包含lac UV5启动子片段；
用引物(PK3-F和PK3-R)以pJBEI6409为模板扩增得到DNA片段，该片段包括MK和PMK两个基因的序列，MK序列前包含pTac启动子片段；
用引物(PK4-F和PKMVA-3R)以pJBEI6409为模板扩增得到DNA片段，该片段包含PMD和idi序列片段。上述引物序列及模板参见以下表1。
表1
Figure BDA0002404765100000071
Figure BDA0002404765100000081
通过如下Gibson Assembly方法连接四个片段，即得到质粒p15A-MVA(图2)，其序列为SEQ ID NO:8，其中atoB、HMGS、HMGR共用一套调控元件，受lac UV5启动子调控；MK、PMK、PMD和idi共用一套调控元件，受pTac启动子调控。上述质粒能够促使表达菌完成MVA途径，得到IPP和DMAPP。
Gibson Assembly反应体系如下表2所示：
表2
Figure BDA0002404765100000082
操作步骤
(1)取含有Gibson Assembly Mix 15μL的PCR管，标好编号；
(2)分别加入各片段，ddH2O补至20μL；
Gibson Assembly反应条件(1h)如下表3中所示：
表3
步骤 温度 时间
消化和连接 50℃ 60min
保持 12℃ ∞
反应产物转化DH5a，长出克隆验证包含正确质粒的克隆；
提质粒待用。
实施例2：构建包含用于以IPP和DMAPP为前体合成柠檬醛的酶的编码基因的表达载体
pTALE-GPPS-GES-geoA-idi质粒的构建：以IPP和DMAPP为前体合成柠檬醛的四个酶的编码基因，GPPS、GES、geoA和idi共用一套调控元件，启动子和核糖体结合位点之间插入有绝缘子功能的序列，其中GPPS为香叶酯二磷酸合成酶基因、GES为甜罗勒香叶醇合成酶基因，idi为异戊烯焦磷酸异构酶基因。
选择pTALE为质粒载体，sp2为启动子序列，该启动子为组成型转录启动子，启动子序列与核糖体结合位点序列之间加入绝缘子功能RiboJ(排除其他调控元件的作用)的功能序列，四个酶的编码序列的排列顺序为GPPS-GES-geoA-idi(上述顺序仅是示例性的，本发明并不限于此)。GES、geoA和idi序列前分别加入核糖体结合位点功能的序列rbs1、rbs2和rbs3。
通过合成载体序列pTALE(SEQ ID NO:17)，合成功能序列GPPS-GES-geoA-idi(SEQID NO:18)，通过Gibson Assembly方法连接2个片段，即得到质粒pTALE-GPPS-GES-geoA-idi(SEQ ID NO:13)(参见图3)。上述质粒能够促使表达菌从IPP和DMAPP合成柠檬醛。
所得到的sp2启动子表达GPPS-GES-geoA-idi的基因盒序列为：基因盒包括六个部分：sp2启动子部分(SEQ ID NO:36)，绝缘子RiboJ部分(SEQ ID NO:37)，带有核糖体结合序列的GPPS编码序列(SEQ ID NO:9)，带有rbs1的GES序列(SEQ ID NO:10)，带有rbs2的geoA序列(SEQ ID NO:11)，以及带有rbs3的idi序列(SEQ ID NO:12)。
其中，带有核糖体结合位点序列的GPPS编码序列(SEQ ID NO:9)，下划线标记的是核糖体结合位点序列，其后为GPPS编码序列：
gatgattgcgatagaaattccaacggaggggtaaatggaatttgacttcaacaaatacatggactccaaagcgatgacggtaaatgaagcactgaacaaagcgatccctctgcgttatccgcagaaaatctacgaaagcatgcgttacagcctgctggcaggcggcaagcgtgttcgtccggttctgtgtattgccgcatgtgaactggtaggtggtaccgaagaactggcgatcccgaccgcgtgcgcaattgaaatgatccacacgatgtccctgatgcacgatgatctgccgtgtatcgacaacgacgatctgcgtcgcggtatggaatttgaaaaccgactaaccacaaaattttcggtgaggataccgcagtgactgctggtaacgcactgcactcttacgccttcgagcatatcgcggtttctacttctaaaaccgttggtgctgaccgcatcctgcgtatggtgtccgagctgggtcgtgctactggctctgaaggtgttatgggtggtcagatggtagacatcgcatccgaaggcgatccgtctatcgacctgcagaccctggaatggattcacatccacaaaaccgcaatgctgctggaatgctccgttgtttgcggtgcaatcattggcggtgccagcgaaatcgtaatcgaacgtgcccgtcgctacgcccgctgtgttggtctgctgttccaggtagttgatgacattctggacgtaactaaaagcagcgacgaactgggtaagactgcgggcaaggacctgatctctgataaagccacctacccaaagctgatgggtctggaaaaggccaaggagttctccgatgaactgctgaaccgtgcgaagggtgaactgtcctgcttcgacccagttaaagccgctccgctgctgggcctggcagactacgtggcatttcgtcagaattaa
带有rbs1的GES序列(SEQ ID NO:10)，下划线标记的是rbs1，其后为GES编码序列：
cagaaaatagtaaggaggttttcgatgagctgcgcgcgtatcaccgtgaccctgccgtatcgtagcgcgaaaaccagcatccagcgtggtattacccactatccggcgctgatccgtccgcgtttcagcgcgtgcaccccgctggcgagcgcgatgccgctgagcagcaccccgctgattaacggcgataacagccagcgtaagaacacccgtcaacacatggaggaaagcagcagcaaacgtcgtgaatatctgctggaggaaaccacccgtaagctgcaacgtaacgataccgaaagcgtggagaagctgaaactgatcgacaacattcagcaactgggtatcggctactatttcgaggatgcgattaacgcggttctgcgtagcccgtttagcaccggcgaggaagacctgttcaccgcggcgctgcgttttcgtctgctgcgtcacaacggcatcgaaattagcccggagatcttcctgaagtttaaagacgaacgtggtaaattcgatgagagcgacaccctgggcctgctgagcctgtacgaagcgagcaacctgggtgtggcgggcgaggaaattctggaggaagcgatggaatttgcggaggcgcgtctgcgtcgtagcctgagcgaaccggcggcgccgctgcatggtgaggtggcgcaggcgctggatgttccgcgtcacctgcgtatggcgcgtctggaagcgcgtcgttttatcgagcagtatggcaagcaaagcgaccacgatggcgacctgctggagctggcgattctggactacaaccaggtgcaagcgcagcaccaaagcgaactgaccgagatcattcgttggtggaaggaactgggtctggttgataaactgagcttcggccgtgaccgtccgctggagtgctttctgtggaccgtgggtctgctgccggaaccgaagtatagcagcgttcgtatcgagctggcgaaagcgatcagcattctgctggttatcgacgatattttcgacacctacggtgaaatggacgatctgatcctgtttaccgatgcgattcgtcgttgggacctggaagcgatggagggcctgccggaatatatgaaaatctgctacatggcgctgtataacaccaccaacgaggtgtgctacaaagttctgcgtgataccggtcgtattgtgctgctgaacctgaagagcacctggatcgacatgattgagggcttcatggaggaagcgaagtggtttaacggtggcagcgcgccgaaactggaggaatatatcgaaaacggtgttagcaccgcgggcgcgtacatggcgtttgcgcacatcttctttctgattggcgagggcgtgacccaccagaacagccaactgttcacccagaagccgtatccgaaagtttttagcgcggcgggtcgtatcctgcgtctgtgggacgatctgggtaccgcgaaagaggaacaggaacgtggcgatctggcgagctgcgttcaactgtttatgaaggagaaaagcctgaccgaggaagaggcgcgtagccgtatcctggaagagattaagggtctgtggcgtgacctgaacggcgagctggtgtacaacaagaacctgccgctgagcatcattaaagttgcgctgaacatggcgcgtgcgagccaggtggtttacaagcacgatcaagacacctatttcagcagcgtggataactacgttgacgcgctgttctttacccaataa
带有rbs2的geoA序列(SEQ ID NO:11)，下划线标记的是rbs2序列，其后为geoA序列：
attcccgcagtcgataccgttgccatgaacgatacccaagatttcatttctgctcaggccgcagtactgcgccaggtgggtggtccgctggctgtggaaccagtacgtatttctatgccgaagggcgacgaggttctgattcgcatcgcaggcgttggtgtgtgccacactgatctggtgtgtcgtgatggtttcccggtaccgctgccaatcgttctgggccatgagggctctggcactgttgaggcggttggtgaacaggtgcgcaccctgaagccaggtgatcgtgtagtgctgtctttcaactcctgcggtcactgtggcaactgtcacgacggccacccgtctaactgtctgcagatgctgccgctgaacttcggcggtgcccaacgcgtagatggcggccaggtactggacggcgcaggtcacccggtgcagtccatgttctttggtcagagcagctttggtactcacgcggttgctcgcgaaattaacgcagtgaaagtaggtgatgatctgccgctggagctgctgggtccgctgggctgtggcatccaaactggtgcaggtgcagcgatcaactctctgggtattggtccgggtcaaagcctggcgatcttcggcggtggtggcgttggcctgagcgctctgctgggcgctcgtgcagtgggtgctgatcgtgttgtagtaatcgagccgaacgcagctcgtcgtgcactggcgctggagctgggtgcatcccacgcactggacccgcacgcggagggtgatctggttgcggcgattaaagcagcgaccggcggtggtgcgactcattccctggacactactggcctgccaccggtaattggctccgcaattgcctgtaccctgccaggcggtaccgttggcatggtaggcctgccggcaccggatgcaccggtaccggcaactctgctggacctgctgtccaaatctgttactctgcgtccaattaccgaaggtgacgccgatccgcagcgtttcatcccgcgtatgctggacttccatcgtgcgggcaaattcccatttgaccgcctgatcactcgctatcgttttgatcagatcaacgaagccctgcatgctactgagaaaggtgaagctattaagccggttctggtgttctaa
带有rbs3的idi序列(SEQ ID NO:12)，下划线标记的是rbs3序列，其后为idi序列：
ttgctaaagaaagaaggcctgctcatgcaaacggaacacgtcattttattgaatgcacagggagttcccacgggtacgctggaaaagtatgccgcacacacggcagacacccgcttacatctcgcgttctccagttggctgtttaatgccaaaggacaattattagttacccgccgcgcactgagcaaaaaagcatggcctggcgtgtggactaactcggtttgtgggcacccacaactgggagaaagcaacgaagacgcagtgatccgccgttgccgttatgagcttggcgtggaaattacgcctcctgaatctatctatcctgactttcgctaccgcgccaccgatccgagtggcattgtggaaaatgaagtgtgtccggtatttgccgcacgcaccactagtgcgttacagatcaatgatgatgaagtgatggattatcaatggtgtgatttagcagatgtattacacggtattgatgccacgccgtgggcgttcagtccgtggatggtgatgcaggcgacaaatcgcgaagccagaaaacgattatctgcatttacccagcttaaataa
实施例3：质粒的转化
将实施例1中构建的质粒p15A-MVA和实施例2中构建的质粒pTALE-GPPS-GES-geoA-idi共转化转入大肠杆菌BW25113(ATCC编号：ATCC12435购自美国菌种保藏中心American Type Culture Collection，BW25113菌株来源于E.coli K-12W1485，是K12的衍生菌株，与MG1655类似，也是一种经过较少改造，比较接近于“野生型”的大肠杆菌工程菌株。)，经如下筛选过程获得产柠檬醛重组菌。
筛选过程：
(1)取50μL(100μL)感受态细胞，置于冰上缓慢解冻；
(2)加入p15A-MVA和pTALE-GPPS-GES-geoA-idi各100ng，轻轻吹打混匀，冰浴放置20min；
(3)42℃(金属浴)热激60s后，迅速冰浴2min，该过程不要摇动离心管；
(4)加入不含抗生素的LB液体培养基150μL(300μL)，37℃复苏培养60min；
(5)将培养后细胞重悬，全部涂布于含有25ug/mL氯霉素、50ug/mL卡纳霉素的LB培养基平板上，37℃倒置培养16-24h；
(6)挑单克隆进行后续生产。
实施例4：重组菌发酵生产柠檬醛
LB培养基：5g/L酵母提取物(购自英国OXID公司，产品目录号LP0021)，10g/L蛋白胨(购自英国OXID公司，产品目录号LP0042),10g/L NaCl，其余为水。调pH值至7.0-7.2，高压蒸汽灭菌。
将实施例3中得到的重组菌在LB培养基中以200rpm，37℃培养12小时后按5％接种至50ml LB培养基，200rmp，30℃，培养48小时。按5％接种至3L LB发酵培养基中，接种4小时后添加不同浓度的IPTG调控基因表达，紫外分光光计测定培养液的OD变化，描绘其生长曲线。
发酵结束后，取40ml发酵液，加入10ml正十二烷，混匀后离心。取上清检测发酵产物。检测方法和条件如下：采用GC分析柠檬醛含量，使用岛津公司的GC-2014型气相色谱仪。色谱仪的配置为：HP-5型毛细管色谱柱，氢火焰离子化检测器FID，SPL分流进样口；高纯氮气作为载气，氢气为燃气，空气为助燃气；使用AOC-20S型自动进样器，丙酮为洗涤液。GC分析程序的设置为：进样口温度240℃，检测器温度250℃，柱温起始温度为80℃，维持1.5分钟；以30℃/分钟的速率升至140℃并维持0分钟；以40℃/分钟的速率升至240℃并维持2分钟；总计时间为8分钟。GC结果采用内标归一法根据峰面积进行定量计算柠檬醛含量。
分别考察了在OD600 1.0时，诱导剂IPTG浓度对柠檬醛产量的影响，在IPTG为200μM时，OD值对于柠檬醛产量的影响，以及在IPTG200μM，OD600 1.0的诱导条件下，发酵时间对于柠檬醛产量的影响，结果分别示于下表4-6中。
表4 IPTG浓度柠檬醛产量的影响(mg/L)
IPTG(μM) 产量(mg/L) SD
0 3.14 0.29
25 44.77 0.27
50 42.99 1.03
100 50.00 0.32
200 55.24 6.67
500 37.65 4.29
表5不同OD600下，IPTG诱导后的柠檬醛含量/mg/L(IPTG诱导48h，30℃)
OD600 柠檬醛(mg/L)
0.6 55.02
0.7 70.56
0.8 75.83
0.9 76.67
1.0 89.96
1.1 75.12
表6不同发酵时间柠檬醛产量的变化
Figure BDA0002404765100000131
Figure BDA0002404765100000141
由以上表4-6中的数据可以看出，在IPTG终浓度为200μM，OD600为1.0，发酵时间为40小时的条件下，获得了最高的柠檬醛产量，约为91.32mg/L，这远高于CN 104195063A所公布的2320μg/L。
综上，本发明开发出一种可以高产量生产柠檬醛的工程菌，能够通过常规的BW菌和经典LB培养基即可进行发酵培养，培养过程中通过IPTG诱导，获得高产量的柠檬醛表达，约0.1g/L，表达过程中，杂质少，发酵产物包括Neral和Geranial两种构象，具有强大的柠檬醛工业化生产潜力。
序列表
<110> 深圳蓝晶生物科技有限公司
<120> 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用
<130> DI20-0010-XC03
<160> 47
<170> PatentIn version 3.5
<210> 1
<211> 1185
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> atoB
<400> 1
atgaaaaatt gtgtcatcgt cagtgcggta cgtactgcta tcggtagttt taacggttca 60
ctcgcttcca ccagcgccat cgacctgggg gcgacagtaa ttaaagccgc cattgaacgt 120
gcaaaaatcg attcacaaca cgttgatgaa gtgattatgg gtaacgtgtt acaagccggg 180
ctggggcaaa atccggcgcg tcaggcactg ttaaaaagcg ggctggcaga aacggtgtgc 240
ggattcacgg tcaataaagt atgtggttcg ggtcttaaaa gtgtggcgct tgccgcccag 300
gccattcagg caggtcaggc gcagagcatt gtggcggggg gtatggaaaa tatgagttta 360
gccccctact tactcgatgc aaaagcacgc tctggttatc gtcttggaga cggacaggtt 420
tatgacgtaa tcctgcgcga tggcctgatg tgcgccaccc atggttatca tatggggatt 480
accgccgaaa acgtggctaa agagtacgga attacccgtg aaatgcagga tgaactggcg 540
ctacattcac agcgtaaagc ggcagccgca attgagtccg gtgcttttac agccgaaatc 600
gtcccggtaa atgttgtcac tcgaaagaaa accttcgtct tcagtcaaga cgagttcccg 660
aaagcgaact caacggctga agcgttaggt gcattgcgcc cggccttcga taaagcagga 720
acagtcaccg ctgggaacgc gtctggtatt aacgacggtg ctgccgctct ggtgattatg 780
gaagaatctg cggcgctggc agcaggcctt acccccctgg ctcgcattaa aagttatgcc 840
agcggtggcg tgccccccgc attgatgggt atggggccag tacctgccac gcaaaaagcg 900
ttacaactgg cggggctgca actggcggat attgatctca ttgaggctaa tgaagcattt 960
gctgcacagt tccttgccgt tgggaaaaac ctgggctttg attctgagaa agtgaatgtc 1020
aacggcgggg ccatcgcgct cgggcatcct atcggtgcca gtggtgctcg tattctggtc 1080
acactattac atgccatgca ggcacgcgat aaaacgctgg ggctggcaac actgtgcatt 1140
ggcggcggtc agggaattgc gatggtgatt gaacggttga attga 1185
<210> 2
<211> 1167
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> HMGS
<400> 2
atgacaatag gtatcgataa aataaacttt tacgttccaa agtactatgt agacatggct 60
aaattagcag aagcacgcca agtagaccca aacaaatttt taattggaat tggtcaaact 120
gaaatggctg ttagtcctgt aaaccaagac atcgtttcaa tgggcgctaa cgctgctaag 180
gacattataa cagacgaaga caaaaagaaa attggtatgg taattgtggc aactgaatca 240
gcagttgatg ctgctaaagc agccgctgtt caaattcaca acttattagg tattcaacct 300
tttgcacgct gctttgaaat gaaagaagct tgttatgctg caacaccagc aattcaatta 360
gctaaagatt atttagcaac tagaccgaat gaaaaagtat tagttattgc tacagataca 420
gcacgttatg gattgaactc aggcggcgag ccaacacaag gtgctggcgc agttgcgatg 480
gttattgcac ataatccaag cattttggca ttaaatgaag atgctgttgc ttacactgaa 540
gacgtttatg atttctggcg tccaactgga cataaatatc cattagttga tggtgcatta 600
tctaaagatg cttatatccg ctcattccaa caaagctgga atgaatacgc aaaacgtcaa 660
ggtaagtcgc tagctgactt cgcatctcta tgcttccatg ttccatttac aaaaatgggt 720
aaaaaggcat tagagtcaat cattgataac gctgatgaaa caactcaaga gcgtttacgt 780
tcaggatatg aagatgctgt agattataac cgttatgtcg gtaatattta tactggatca 840
ttatatttaa gcctaatatc attacttgaa aatcgagatt tacaagctgg tgaaacaatc 900
ggtttattca gttatggctc aggttcagtt ggtgaatttt atagtgcgac attagttgaa 960
ggctacaaag atcatttaga tcaagctgca cataaagcat tattaaataa ccgtactgaa 1020
gtatctgttg atgcatatga aacattcttc aaacgttttg atgacgttga atttgacgaa 1080
gaacaagatg ctgttcatga agatcgtcat attttctact tatcaaatat tgaaaataac 1140
gttcgcgaat atcacagacc agagtaa 1167
<210> 3
<211> 1284
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> HMGR
<400> 3
atgtccatgc aaagtttaga taagaatttt cgacatttat ctcgtaaaga aaagttacaa 60
caattggttg ataagcaatg gttatcagaa gaacaattcg acattttact gaatcatcca 120
ttaatcgatg aagaagtagc caatagttta attgaaaatg tcatcgcgca aggtgcatta 180
cccgttggat tattaccgaa tatcattgtg gacgataagg catatgttgt acctatgatg 240
gtggaagagc cttcagttgt cgctgcagct agttatggtg caaagctagt gaatcagact 300
ggcggattta aaacggtatc ttctgaacgt attatgatag gtcaaatcgt ctttgatggc 360
gttgacgata ctgaaaaatt atcagcagac attaaagctt tagaaaagca aattcataaa 420
attgcggatg aggcatatcc ttctattaaa gcgcgtggtg gtggttacca acgtatagcg 480
attgatacat ttcctgagca acagttacta tctttaaaag tatttgttga tacgaaagat 540
gctatgggcg ctaatatgct taatacgatt ttagaggcca taactgcatt tttaaaaaat 600
gaatttccgc aaagcgacat tttaatgagt attttatcca atcatgcaac agcgtccgtt 660
gttaaagttc aaggcgaaat tgatgttaaa gatttagcaa ggggcgagag aactggagaa 720
gaggttgcca aacgaatgga acgtgcttct gtattggccc aagtagatat tcatcgtgca 780
gcaacacata ataaaggtgt tatgaatggc atacatgctg ttgttttagc aacaggaaat 840
gatacgcgtg gtgcagaagc aagtgcgcat gcatacgcga gtcgtgacgg acagtatcgt 900
ggtattgcta catggcgtta cgatcaagat cgtcaacgat tgattggtac aattgaagtg 960
cctatgacat tggcaatcgt tggcggtggt acaaaagtat taccaattgc taaagcttca 1020
ttagagctac taaatgtaga gtcagcacaa gaattaggtc atgtagttgc tgccgttggt 1080
ttagcgcaaa actttgcagc atgtcgcgcg cttgtgtcag aaggtattca acaaggtcat 1140
atgagtttac aatataaatc attagctatc gttgtagggg caaaaggtga tgaaattgct 1200
aaagtagctg aagctttgaa aaaagaaccc cgtgcaaata cacaagcagc ggaacatatt 1260
ttacaagaaa ttagacaaca ataa 1284
<210> 4
<211> 1332
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> MK
<400> 4
atgtctctgc cattcctgac gtctgcgcca ggtaaggtga tcatcttcgg cgagcactct 60
gcggtgtaca ataagccggc cgtcgccgcc tctgtgtctg cgttacgcac ctacctgctg 120
atcagcgaat cttctgcacc ggacacgatc gagctggact ttccggacat cagcttcaac 180
cacaagtgga gcatcaacga cttcaacgcg atcacggagg accaggtgaa cagccaaaag 240
ctggccaaag cccagcaagc aaccgacggt ctgtctcagg agctggtgtc tctgctggac 300
ccgctgttag cgcagttaag cgagagcttc cattaccacg ccgcgttctg cttcctgtac 360
atgttcgttt gcctgtgccc gcacgcaaag aacatcaagt tcagcctgaa gagcacgctg 420
ccgattggcg caggcttagg ctctagcgca tctatcagcg tgagcctggc gctggcgatg 480
gcctatctgg gtggcctgat tggcagcaac gacctggaga aactgagcga aaacgacaag 540
cacatcgtga accagtgggc ctttatcggc gagaagtgca ttcatggcac cccgagcggc 600
attgacaacg cagttgccac gtatggcaac gccctgctgt tcgagaaaga cagccacaac 660
ggcacgatca acacgaacaa cttcaagttc ctggacgact tcccggcgat cccgatgatt 720
ctgacctaca cccgtatccc acgcagcacc aaggatttag tcgcccgcgt gcgtgtttta 780
gtcaccgaaa agttcccgga ggtgatgaag ccgatcctgg acgcgatggg cgagtgcgcg 840
ctgcagggtc tggagatcat gaccaagctg agcaagtgca agggcaccga cgatgaggcg 900
gtggagacca acaatgagct gtacgagcag ctgctggagc tgatccgtat caatcacggc 960
ctgctggtct ctatcggtgt gtctcacccg ggcctggaac tgatcaaaaa cctgagcgac 1020
gacctgcgca ttggctctac gaaattaacg ggtgcaggtg gcggtggctg ctctttaacg 1080
ctgctgcgcc gtgacattac gcaggagcaa atcgacagct tcaagaagaa gctgcaggac 1140
gacttcagct acgagacgtt cgagacggac ctgggcggca cgggctgttg cctgctgagc 1200
gccaaaaatc tgaacaagga cctgaagatc aaaagcctgg tgttccagct gttcgaaaac 1260
aagacgacca cgaagcagca gatcgacgac ctgttactgc cgggtaacac caatctgccg 1320
tggacgtctt aa 1332
<210> 5
<211> 1356
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PMK
<400> 5
atgagcgaat tacgtgcatt cagcgcgcca ggtaaggcac tgctggccgg tggctacctg 60
gtgttagaca ccaagtacga ggcgttcgtc gtcggcttat ctgcccgtat gcatgcagtt 120
gcccacccgt atggtagcct gcagggctct gacaagttcg aagtgcgtgt gaagagcaag 180
cagttcaagg acggcgagtg gctgtaccac attagcccaa agagcggctt catcccggtt 240
agcattggtg gcagcaagaa cccatttatc gagaaggtca ttgccaacgt cttcagctac 300
ttcaagccga atatggacga ttactgcaac cgcaacctgt tcgtcatcga cattttcagc 360
gacgacgcgt accacagcca agaggactct gttacggagc atcgtggtaa ccgccgcctg 420
agcttccaca gccatcgcat tgaggaggtg ccgaagacgg gtctgggttc tagcgccggt 480
ttagttaccg tcttaacgac ggcgttagcg agcttcttcg tgagcgacct ggagaacaac 540
gtggacaagt accgcgaagt gattcataac ctggcgcagg tggcacattg tcaggcccaa 600
ggtaagattg gctctggttt tgatgtggca gcggccgcct atggctctat ccgctatcgc 660
cgctttccgc cggccctgat cagcaatctg ccggacatcg gctctgcgac gtatggtagc 720
aaactggcgc atctggtgga cgaagaagac tggaacatca ccattaagtc taatcacctg 780
ccgagcggct taacgttatg gatgggcgat atcaagaacg gcagcgaaac ggttaagctg 840
gtgcagaaag tgaaaaactg gtacgacagc cacatgccgg aaagcctgaa gatttacacg 900
gagctggacc acgccaatag ccgtttcatg gatggtctga gcaagctgga ccgcctgcac 960
gaaacccacg acgactacag cgaccaaatc ttcgagagcc tggagcgcaa tgactgcacc 1020
tgccagaagt acccggagat cacggaggtc cgcgatgccg tggcaacgat tcgccgtagc 1080
ttccgcaaaa ttacgaagga gagcggcgcg gatatcgaac caccggtcca gacgtctctg 1140
ctggacgact gtcaaacctt aaagggcgtg ttaacgtgcc tgattccggg cgcgggtggt 1200
tacgacgcca ttgccgtcat cacgaaacag gacgtcgatc tgcgcgcaca aacggccaac 1260
gacaaacgtt tcagcaaagt ccaatggctg gatgttacgc aggccgactg gggtgttcgc 1320
aaggagaagg acccggaaac gtatctggat aagtga 1356
<210> 6
<211> 1191
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PMD
<400> 6
atgaccgttt acacagcatc cgttaccgca cccgtcaaca tcgcaaccct taagtattgg 60
gggaaaaggg acacgaagtt gaatctgccc accaattcgt ccatatcagt gactttatcg 120
caagatgacc tcagaacgtt gacctctgcg gctactgcac ctgagtttga acgcgacact 180
ttgtggttaa atggagaacc acacagcatc gacaatgaaa gaactcaaaa ttgtctgcgc 240
gacctacgcc aattaagaaa ggaaatggaa tcgaaggacg cctcattgcc cacattatct 300
caatggaaac tccacattgt ctccgaaaat aactttccta cagcagctgg tttagcttcc 360
tccgctgctg gctttgctgc attggtctct gcaattgcta agttatacca attaccacag 420
tcaacttcag aaatatctag aatagcaaga aaggggtctg gttcagcttg tagatcgttg 480
tttggcggat acgtggcctg ggaaatggga aaagctgaag atggtcatga ttccatggca 540
gtacaaatcg cagacagctc tgactggcct cagatgaaag cttgtgtcct agttgtcagc 600
gatattaaaa aggatgtgag ttccactcag ggtatgcaat tgaccgtggc aacctccgaa 660
ctatttaaag aaagaattga acatgtcgta ccaaagagat ttgaagtcat gcgtaaagcc 720
attgttgaaa aagatttcgc cacctttgca aaggaaacaa tgatggattc caactctttc 780
catgccacat gtttggactc tttccctcca atattctaca tgaatgacac ttccaagcgt 840
atcatcagtt ggtgccacac cattaatcag ttttacggag aaacaatcgt tgcatacacg 900
tttgatgcag gtccaaatgc tgtgttgtac tacttagctg aaaatgagtc gaaactcttt 960
gcatttatct ataaattgtt tggctctgtt cctggatggg acaagaaatt tactactgag 1020
cagcttgagg ctttcaacca tcaatttgaa tcatctaact ttactgcacg tgaattggat 1080
cttgagttgc aaaaggatgt tgccagagtg attttaactc aagtcggttc aggcccacaa 1140
gaaacaaacg aatctttgat tgacgcaaag actggtctac caaaggaata a 1191
<210> 7
<211> 555
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> Idi
<400> 7
atgataatgc aaacggaaca cgtcatttta ttgaatgcac agggagttcc cacgggtacg 60
ctggaaaagt atgccgcaca cacggcagac acccgcttac atctcgcgtt ctccagttgg 120
ctgtttaatg ccaaaggaca attattagtt acccgccgcg cactgagcaa aaaagcatgg 180
cctggcgtgt ggactaactc ggtttgtggg cacccacaac tgggagaaag caacgaagac 240
gcagtgatcc gccgttgccg ttatgagctt ggcgtggaaa ttacgcctcc tgaatctatc 300
tatcctgact ttcgctaccg cgccaccgat ccgagtggca ttgtggaaaa tgaagtgtgt 360
ccggtatttg ccgcacgcac cactagtgcg ttacagatca atgatgatga agtgatggat 420
tatcaatggt gtgatttagc agatgtatta cacggtattg atgccacgcc gtgggcgttc 480
agtccgtgga tggtgatgca ggcgacaaat cgcgaagcca gaaaacgatt atctgcattt 540
acccagctta aataa 555
<210> 8
<211> 12103
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> p15A-MVA
<400> 8
gacgtcggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct 60
ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga 120
ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag 180
ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg 240
ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc 300
ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc cggactcggt 360
aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac 420
gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc 480
ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag 540
acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc 600
caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact 660
gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc 720
ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg 780
ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat 840
cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg 900
cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc 960
cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac 1020
tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg 1080
ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac 1140
cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcgccattc 1200
gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg aagcagccca 1260
gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg 1320
cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa caagcgctca 1380
tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata taggcgccag 1440
caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga 1500
tcgtttaggc accccaggct ttacacttta tgcttccggc tcgtataatg tgtggaattg 1560
tgagcggata acaatttcag aattcaaaag atcttaggag gaatataaaa tgaaaaattg 1620
tgtcatcgtc agtgcggtac gtactgctat cggtagtttt aacggttcac tcgcttccac 1680
cagcgccatc gacctggggg cgacagtaat taaagccgcc attgaacgtg caaaaatcga 1740
ttcacaacac gttgatgaag tgattatggg taacgtgtta caagccgggc tggggcaaaa 1800
tccggcgcgt caggcactgt taaaaagcgg gctggcagaa acggtgtgcg gattcacggt 1860
caataaagta tgtggttcgg gtcttaaaag tgtggcgctt gccgcccagg ccattcaggc 1920
aggtcaggcg cagagcattg tggcgggggg tatggaaaat atgagtttag ccccctactt 1980
actcgatgca aaagcacgct ctggttatcg tcttggagac ggacaggttt atgacgtaat 2040
cctgcgcgat ggcctgatgt gcgccaccca tggttatcat atggggatta ccgccgaaaa 2100
cgtggctaaa gagtacggaa ttacccgtga aatgcaggat gaactggcgc tacattcaca 2160
gcgtaaagcg gcagccgcaa ttgagtccgg tgcttttaca gccgaaatcg tcccggtaaa 2220
tgttgtcact cgaaagaaaa ccttcgtctt cagtcaagac gagttcccga aagcgaactc 2280
aacggctgaa gcgttaggtg cattgcgccc ggccttcgat aaagcaggaa cagtcaccgc 2340
tgggaacgcg tctggtatta acgacggtgc tgccgctctg gtgattatgg aagaatctgc 2400
ggcgctggca gcaggcctta cccccctggc tcgcattaaa agttatgcca gcggtggcgt 2460
gccccccgca ttgatgggta tggggccagt acctgccacg caaaaagcgt tacaactggc 2520
ggggctgcaa ctggcggata ttgatctcat tgaggctaat gaagcatttg ctgcacagtt 2580
ccttgccgtt gggaaaaacc tgggctttga ttctgagaaa gtgaatgtca acggcggggc 2640
catcgcgctc gggcatccta tcggtgccag tggtgctcgt attctggtca cactattaca 2700
tgccatgcag gcacgcgata aaacgctggg gctggcaaca ctgtgcattg gcggcggtca 2760
gggaattgcg atggtgattg aacggttgaa ttgaggatct tgaattaagg aggacagcta 2820
aatgacaata ggtatcgata aaataaactt ttacgttcca aagtactatg tagacatggc 2880
taaattagca gaagcacgcc aagtagaccc aaacaaattt ttaattggaa ttggtcaaac 2940
tgaaatggct gttagtcctg taaaccaaga catcgtttca atgggcgcta acgctgctaa 3000
ggacattata acagacgaag acaaaaagaa aattggtatg gtaattgtgg caactgaatc 3060
agcagttgat gctgctaaag cagccgctgt tcaaattcac aacttattag gtattcaacc 3120
ttttgcacgc tgctttgaaa tgaaagaagc ttgttatgct gcaacaccag caattcaatt 3180
agctaaagat tatttagcaa ctagaccgaa tgaaaaagta ttagttattg ctacagatac 3240
agcacgttat ggattgaact caggcggcga gccaacacaa ggtgctggcg cagttgcgat 3300
ggttattgca cataatccaa gcattttggc attaaatgaa gatgctgttg cttacactga 3360
agacgtttat gatttctggc gtccaactgg acataaatat ccattagttg atggtgcatt 3420
atctaaagat gcttatatcc gctcattcca acaaagctgg aatgaatacg caaaacgtca 3480
aggtaagtcg ctagctgact tcgcatctct atgcttccat gttccattta caaaaatggg 3540
taaaaaggca ttagagtcaa tcattgataa cgctgatgaa acaactcaag agcgtttacg 3600
ttcaggatat gaagatgctg tagattataa ccgttatgtc ggtaatattt atactggatc 3660
attatattta agcctaatat cattacttga aaatcgagat ttacaagctg gtgaaacaat 3720
cggtttattc agttatggct caggttcagt tggtgaattt tatagtgcga cattagttga 3780
aggctacaaa gatcatttag atcaagctgc acataaagca ttattaaata accgtactga 3840
agtatctgtt gatgcatatg aaacattctt caaacgtttt gatgacgttg aatttgacga 3900
agaacaagat gctgttcatg aagatcgtca tattttctac ttatcaaata ttgaaaataa 3960
cgttcgcgaa tatcacagac cagagtaatt aggatctatt caggaaacag accatgtcca 4020
tgcaaagttt agataagaat tttcgacatt tatctcgtaa agaaaagtta caacaattgg 4080
ttgataagca atggttatca gaagaacaat tcgacatttt actgaatcat ccattaatcg 4140
atgaagaagt agccaatagt ttaattgaaa atgtcatcgc gcaaggtgca ttacccgttg 4200
gattattacc gaatatcatt gtggacgata aggcatatgt tgtacctatg atggtggaag 4260
agccttcagt tgtcgctgca gctagttatg gtgcaaagct agtgaatcag actggcggat 4320
ttaaaacggt atcttctgaa cgtattatga taggtcaaat cgtctttgat ggcgttgacg 4380
atactgaaaa attatcagca gacattaaag ctttagaaaa gcaaattcat aaaattgcgg 4440
atgaggcata tccttctatt aaagcgcgtg gtggtggtta ccaacgtata gcgattgata 4500
catttcctga gcaacagtta ctatctttaa aagtatttgt tgatacgaaa gatgctatgg 4560
gcgctaatat gcttaatacg attttagagg ccataactgc atttttaaaa aatgaatttc 4620
cgcaaagcga cattttaatg agtattttat ccaatcatgc aacagcgtcc gttgttaaag 4680
ttcaaggcga aattgatgtt aaagatttag caaggggcga gagaactgga gaagaggttg 4740
ccaaacgaat ggaacgtgct tctgtattgg cccaagtaga tattcatcgt gcagcaacac 4800
ataataaagg tgttatgaat ggcatacatg ctgttgtttt agcaacagga aatgatacgc 4860
gtggtgcaga agcaagtgcg catgcatacg cgagtcgtga cggacagtat cgtggtattg 4920
ctacatggcg ttacgatcaa gatcgtcaac gattgattgg tacaattgaa gtgcctatga 4980
cattggcaat cgttggcggt ggtacaaaag tattaccaat tgctaaagct tcattagagc 5040
tactaaatgt agagtcagca caagaattag gtcatgtagt tgctgccgtt ggtttagcgc 5100
aaaactttgc agcatgtcgc gcgcttgtgt cagaaggtat tcaacaaggt catatgagtt 5160
tacaatataa atcattagct atcgttgtag gggcaaaagg tgatgaaatt gctaaagtag 5220
ctgaagcttt gaaaaaagaa ccccgtgcaa atacacaagc agcggaacat attttacaag 5280
aaattagaca acaataagga tctttttaag gatctccagg catcaaataa aacgaaaggc 5340
tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta 5400
gagtcacact ggctcacctt cgggtgggcc tttctgcgtt tatagcgaat tgatctggtt 5460
tgacagctta tcatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga 5520
agctgtggta tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca 5580
ctcccgttct ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga 5640
aatgagctgt tgacaattaa tcatccggct cgtataatgt gtggaattgt gagcggataa 5700
caatttcagg atctaggagg aaataaccat gtctctgcca ttcctgacgt ctgcgccagg 5760
taaggtgatc atcttcggcg agcactctgc ggtgtacaat aagccggccg tcgccgcctc 5820
tgtgtctgcg ttacgcacct acctgctgat cagcgaatct tctgcaccgg acacgatcga 5880
gctggacttt ccggacatca gcttcaacca caagtggagc atcaacgact tcaacgcgat 5940
cacggaggac caggtgaaca gccaaaagct ggccaaagcc cagcaagcaa ccgacggtct 6000
gtctcaggag ctggtgtctc tgctggaccc gctgttagcg cagttaagcg agagcttcca 6060
ttaccacgcc gcgttctgct tcctgtacat gttcgtttgc ctgtgcccgc acgcaaagaa 6120
catcaagttc agcctgaaga gcacgctgcc gattggcgca ggcttaggct ctagcgcatc 6180
tatcagcgtg agcctggcgc tggcgatggc ctatctgggt ggcctgattg gcagcaacga 6240
cctggagaaa ctgagcgaaa acgacaagca catcgtgaac cagtgggcct ttatcggcga 6300
gaagtgcatt catggcaccc cgagcggcat tgacaacgca gttgccacgt atggcaacgc 6360
cctgctgttc gagaaagaca gccacaacgg cacgatcaac acgaacaact tcaagttcct 6420
ggacgacttc ccggcgatcc cgatgattct gacctacacc cgtatcccac gcagcaccaa 6480
ggatttagtc gcccgcgtgc gtgttttagt caccgaaaag ttcccggagg tgatgaagcc 6540
gatcctggac gcgatgggcg agtgcgcgct gcagggtctg gagatcatga ccaagctgag 6600
caagtgcaag ggcaccgacg atgaggcggt ggagaccaac aatgagctgt acgagcagct 6660
gctggagctg atccgtatca atcacggcct gctggtctct atcggtgtgt ctcacccggg 6720
cctggaactg atcaaaaacc tgagcgacga cctgcgcatt ggctctacga aattaacggg 6780
tgcaggtggc ggtggctgct ctttaacgct gctgcgccgt gacattacgc aggagcaaat 6840
cgacagcttc aagaagaagc tgcaggacga cttcagctac gagacgttcg agacggacct 6900
gggcggcacg ggctgttgcc tgctgagcgc caaaaatctg aacaaggacc tgaagatcaa 6960
aagcctggtg ttccagctgt tcgaaaacaa gacgaccacg aagcagcaga tcgacgacct 7020
gttactgccg ggtaacacca atctgccgtg gacgtcttaa ggatctagga gggagatcat 7080
atgagcgaat tacgtgcatt cagcgcgcca ggtaaggcac tgctggccgg tggctacctg 7140
gtgttagaca ccaagtacga ggcgttcgtc gtcggcttat ctgcccgtat gcatgcagtt 7200
gcccacccgt atggtagcct gcagggctct gacaagttcg aagtgcgtgt gaagagcaag 7260
cagttcaagg acggcgagtg gctgtaccac attagcccaa agagcggctt catcccggtt 7320
agcattggtg gcagcaagaa cccatttatc gagaaggtca ttgccaacgt cttcagctac 7380
ttcaagccga atatggacga ttactgcaac cgcaacctgt tcgtcatcga cattttcagc 7440
gacgacgcgt accacagcca agaggactct gttacggagc atcgtggtaa ccgccgcctg 7500
agcttccaca gccatcgcat tgaggaggtg ccgaagacgg gtctgggttc tagcgccggt 7560
ttagttaccg tcttaacgac ggcgttagcg agcttcttcg tgagcgacct ggagaacaac 7620
gtggacaagt accgcgaagt gattcataac ctggcgcagg tggcacattg tcaggcccaa 7680
ggtaagattg gctctggttt tgatgtggca gcggccgcct atggctctat ccgctatcgc 7740
cgctttccgc cggccctgat cagcaatctg ccggacatcg gctctgcgac gtatggtagc 7800
aaactggcgc atctggtgga cgaagaagac tggaacatca ccattaagtc taatcacctg 7860
ccgagcggct taacgttatg gatgggcgat atcaagaacg gcagcgaaac ggttaagctg 7920
gtgcagaaag tgaaaaactg gtacgacagc cacatgccgg aaagcctgaa gatttacacg 7980
gagctggacc acgccaatag ccgtttcatg gatggtctga gcaagctgga ccgcctgcac 8040
gaaacccacg acgactacag cgaccaaatc ttcgagagcc tggagcgcaa tgactgcacc 8100
tgccagaagt acccggagat cacggaggtc cgcgatgccg tggcaacgat tcgccgtagc 8160
ttccgcaaaa ttacgaagga gagcggcgcg gatatcgaac caccggtcca gacgtctctg 8220
ctggacgact gtcaaacctt aaagggcgtg ttaacgtgcc tgattccggg cgcgggtggt 8280
tacgacgcca ttgccgtcat cacgaaacag gacgtcgatc tgcgcgcaca aacggccaac 8340
gacaaacgtt tcagcaaagt ccaatggctg gatgttacgc aggccgactg gggtgttcgc 8400
aaggagaagg acccggaaac gtatctggat aagtgaggat ctaggaggat tatgagatga 8460
ccgtttacac agcatccgtt accgcacccg tcaacatcgc aacccttaag tattggggga 8520
aaagggacac gaagttgaat ctgcccacca attcgtccat atcagtgact ttatcgcaag 8580
atgacctcag aacgttgacc tctgcggcta ctgcacctga gtttgaacgc gacactttgt 8640
ggttaaatgg agaaccacac agcatcgaca atgaaagaac tcaaaattgt ctgcgcgacc 8700
tacgccaatt aagaaaggaa atggaatcga aggacgcctc attgcccaca ttatctcaat 8760
ggaaactcca cattgtctcc gaaaataact ttcctacagc agctggttta gcttcctccg 8820
ctgctggctt tgctgcattg gtctctgcaa ttgctaagtt ataccaatta ccacagtcaa 8880
cttcagaaat atctagaata gcaagaaagg ggtctggttc agcttgtaga tcgttgtttg 8940
gcggatacgt ggcctgggaa atgggaaaag ctgaagatgg tcatgattcc atggcagtac 9000
aaatcgcaga cagctctgac tggcctcaga tgaaagcttg tgtcctagtt gtcagcgata 9060
ttaaaaagga tgtgagttcc actcagggta tgcaattgac cgtggcaacc tccgaactat 9120
ttaaagaaag aattgaacat gtcgtaccaa agagatttga agtcatgcgt aaagccattg 9180
ttgaaaaaga tttcgccacc tttgcaaagg aaacaatgat ggattccaac tctttccatg 9240
ccacatgttt ggactctttc cctccaatat tctacatgaa tgacacttcc aagcgtatca 9300
tcagttggtg ccacaccatt aatcagtttt acggagaaac aatcgttgca tacacgtttg 9360
atgcaggtcc aaatgctgtg ttgtactact tagctgaaaa tgagtcgaaa ctctttgcat 9420
ttatctataa attgtttggc tctgttcctg gatgggacaa gaaatttact actgagcagc 9480
ttgaggcttt caaccatcaa tttgaatcat ctaactttac tgcacgtgaa ttggatcttg 9540
agttgcaaaa ggatgttgcc agagtgattt taactcaagt cggttcaggc ccacaagaaa 9600
caaacgaatc tttgattgac gcaaagactg gtctaccaaa ggaataagga tctaggaggt 9660
aatgataatg caaacggaac acgtcatttt attgaatgca cagggagttc ccacgggtac 9720
gctggaaaag tatgccgcac acacggcaga cacccgctta catctcgcgt tctccagttg 9780
gctgtttaat gccaaaggac aattattagt tacccgccgc gcactgagca aaaaagcatg 9840
gcctggcgtg tggactaact cggtttgtgg gcacccacaa ctgggagaaa gcaacgaaga 9900
cgcagtgatc cgccgttgcc gttatgagct tggcgtggaa attacgcctc ctgaatctat 9960
ctatcctgac tttcgctacc gcgccaccga tccgagtggc attgtggaaa atgaagtgtg 10020
tccggtattt gccgcacgca ccactagtgc gttacagatc aatgatgatg aagtgatgga 10080
ttatcaatgg tgtgatttag cagatgtatt acacggtatt gatgccacgc cgtgggcgtt 10140
cagtccgtgg atggtgatgc aggcgacaaa tcgcgaagcc agaaaacgat tatctgcatt 10200
tacccagctt aaataaggat ctcgcaaaaa accccggatc caaactcgag taaggatctc 10260
caggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg 10320
tttgtcggtg aacgctctct actagagtca cactggctca ccttcgggtg ggcctttctg 10380
cgtttatacc tagggatata ttccgcttcc tcgctcactg actcgctacg ctcggtcgtt 10440
cgactgcggc gagcggaaat ggcttacgaa cggggcggag atttcctgga agatgccagg 10500
aagatactta acagggaagt gagagggccg cggcaaagcc gtttttccat aggctccgcc 10560
cccctgacaa gcatcacgaa atctgacgct caaatcagtg gtggcgaaac ccgacaggac 10620
tataaagata ccaggcgttt ccccctggcg gctccctcgt gcgctctcct gttcctgcct 10680
ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt gtctcattcc acgcctgaca 10740
ctcagttccg ggtaggcagt tcgctccaag ctggactgta tgcacgaacc ccccgttcag 10800
tccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccgga aagacatgca 10860
aaagcaccac tggcagcagc cactggtaat tgatttagag gagttagtct tgaagtcatg 10920
cgccggttaa ggctaaactg aaaggacaag ttttggtgac tgcgctcctc caagccagtt 10980
acctcggttc aaagagttgg tagctcagag aaccttcgaa aaaccgccct gcaaggcggt 11040
tttttcgttt tcagagcaag agattacgcg cagaccaaaa cgatctcaag aagatcatct 11100
tattaatcag ataaaatatt tctagatttc agtgcaattt atctcttcaa atgtagcacc 11160
tgaagtcagc cccatacgat ataagttgtt actagtgctt ggattctcac caataaaaaa 11220
cgcccggcgg caaccgagcg ttctgaacaa atccagatgg agttctgagg tcattactgg 11280
atctatcaac aggagtccaa gcgagctcga tatcaaatta cgccccgccc tgccactcat 11340
cgcagtactg ttgtaattca ttaagcattc tgccgacatg gaagccatca caaacggcat 11400
gatgaacctg aatcgccagc ggcatcagca ccttgtcgcc ttgcgtataa tatttgccca 11460
tggtgaaaac gggggcgaag aagttgtcca tattggccac gtttaaatca aaactggtga 11520
aactcaccca gggattggct gagacgaaaa acatattctc aataaaccct ttagggaaat 11580
aggccaggtt ttcaccgtaa cacgccacat cttgcgaata tatgtgtaga aactgccgga 11640
aatcgtcgtg gtattcactc cagagcgatg aaaacgtttc agtttgctca tggaaaacgg 11700
tgtaacaagg gtgaacacta tcccatatca ccagctcacc gtctttcatt gccatacgaa 11760
attccggatg agcattcatc aggcgggcaa gaatgtgaat aaaggccgga taaaacttgt 11820
gcttattttt ctttacggtc tttaaaaagg ccgtaatatc cagctgaacg gtctggttat 11880
aggtacattg agcaactgac tgaaatgcct caaaatgttc tttacgatgc cattgggata 11940
tatcaacggt ggtatatcca gtgatttttt tctccatttt agcttcctta gctcctgaaa 12000
atctcgataa ctcaaaaaat acgcccggta gtgatcttat ttcattatgg tgaaagttgg 12060
aacctcttac gtgccgatca acgtctcatt ttcgccagat atc 12103
<210> 9
<211> 939
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 带有核糖体结合位点序列的GPPS
<400> 9
gatgattgcg atagaaattc caacggaggg gtaaatggaa tttgacttca acaaatacat 60
ggactccaaa gcgatgacgg taaatgaagc actgaacaaa gcgatccctc tgcgttatcc 120
gcagaaaatc tacgaaagca tgcgttacag cctgctggca ggcggcaagc gtgttcgtcc 180
ggttctgtgt attgccgcat gtgaactggt aggtggtacc gaagaactgg cgatcccgac 240
cgcgtgcgca attgaaatga tccacacgat gtccctgatg cacgatgatc tgccgtgtat 300
cgacaacgac gatctgcgtc gcggtatgga atttgaaaac cgactaacca caaaattttc 360
ggtgaggata ccgcagtgac tgctggtaac gcactgcact cttacgcctt cgagcatatc 420
gcggtttcta cttctaaaac cgttggtgct gaccgcatcc tgcgtatggt gtccgagctg 480
ggtcgtgcta ctggctctga aggtgttatg ggtggtcaga tggtagacat cgcatccgaa 540
ggcgatccgt ctatcgacct gcagaccctg gaatggattc acatccacaa aaccgcaatg 600
ctgctggaat gctccgttgt ttgcggtgca atcattggcg gtgccagcga aatcgtaatc 660
gaacgtgccc gtcgctacgc ccgctgtgtt ggtctgctgt tccaggtagt tgatgacatt 720
ctggacgtaa ctaaaagcag cgacgaactg ggtaagactg cgggcaagga cctgatctct 780
gataaagcca cctacccaaa gctgatgggt ctggaaaagg ccaaggagtt ctccgatgaa 840
ctgctgaacc gtgcgaaggg tgaactgtcc tgcttcgacc cagttaaagc cgctccgctg 900
ctgggcctgg cagactacgt ggcatttcgt cagaattaa 939
<210> 10
<211> 1728
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 带有rbs1的GES
<400> 10
cagaaaatag taaggaggtt ttcgatgagc tgcgcgcgta tcaccgtgac cctgccgtat 60
cgtagcgcga aaaccagcat ccagcgtggt attacccact atccggcgct gatccgtccg 120
cgtttcagcg cgtgcacccc gctggcgagc gcgatgccgc tgagcagcac cccgctgatt 180
aacggcgata acagccagcg taagaacacc cgtcaacaca tggaggaaag cagcagcaaa 240
cgtcgtgaat atctgctgga ggaaaccacc cgtaagctgc aacgtaacga taccgaaagc 300
gtggagaagc tgaaactgat cgacaacatt cagcaactgg gtatcggcta ctatttcgag 360
gatgcgatta acgcggttct gcgtagcccg tttagcaccg gcgaggaaga cctgttcacc 420
gcggcgctgc gttttcgtct gctgcgtcac aacggcatcg aaattagccc ggagatcttc 480
ctgaagttta aagacgaacg tggtaaattc gatgagagcg acaccctggg cctgctgagc 540
ctgtacgaag cgagcaacct gggtgtggcg ggcgaggaaa ttctggagga agcgatggaa 600
tttgcggagg cgcgtctgcg tcgtagcctg agcgaaccgg cggcgccgct gcatggtgag 660
gtggcgcagg cgctggatgt tccgcgtcac ctgcgtatgg cgcgtctgga agcgcgtcgt 720
tttatcgagc agtatggcaa gcaaagcgac cacgatggcg acctgctgga gctggcgatt 780
ctggactaca accaggtgca agcgcagcac caaagcgaac tgaccgagat cattcgttgg 840
tggaaggaac tgggtctggt tgataaactg agcttcggcc gtgaccgtcc gctggagtgc 900
tttctgtgga ccgtgggtct gctgccggaa ccgaagtata gcagcgttcg tatcgagctg 960
gcgaaagcga tcagcattct gctggttatc gacgatattt tcgacaccta cggtgaaatg 1020
gacgatctga tcctgtttac cgatgcgatt cgtcgttggg acctggaagc gatggagggc 1080
ctgccggaat atatgaaaat ctgctacatg gcgctgtata acaccaccaa cgaggtgtgc 1140
tacaaagttc tgcgtgatac cggtcgtatt gtgctgctga acctgaagag cacctggatc 1200
gacatgattg agggcttcat ggaggaagcg aagtggttta acggtggcag cgcgccgaaa 1260
ctggaggaat atatcgaaaa cggtgttagc accgcgggcg cgtacatggc gtttgcgcac 1320
atcttctttc tgattggcga gggcgtgacc caccagaaca gccaactgtt cacccagaag 1380
ccgtatccga aagtttttag cgcggcgggt cgtatcctgc gtctgtggga cgatctgggt 1440
accgcgaaag aggaacagga acgtggcgat ctggcgagct gcgttcaact gtttatgaag 1500
gagaaaagcc tgaccgagga agaggcgcgt agccgtatcc tggaagagat taagggtctg 1560
tggcgtgacc tgaacggcga gctggtgtac aacaagaacc tgccgctgag catcattaaa 1620
gttgcgctga acatggcgcg tgcgagccag gtggtttaca agcacgatca agacacctat 1680
ttcagcagcg tggataacta cgttgacgcg ctgttcttta cccaataa 1728
<210> 11
<211> 1146
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 带有rbs2的geoA
<400> 11
attcccgcag tcgataccgt tgccatgaac gatacccaag atttcatttc tgctcaggcc 60
gcagtactgc gccaggtggg tggtccgctg gctgtggaac cagtacgtat ttctatgccg 120
aagggcgacg aggttctgat tcgcatcgca ggcgttggtg tgtgccacac tgatctggtg 180
tgtcgtgatg gtttcccggt accgctgcca atcgttctgg gccatgaggg ctctggcact 240
gttgaggcgg ttggtgaaca ggtgcgcacc ctgaagccag gtgatcgtgt agtgctgtct 300
ttcaactcct gcggtcactg tggcaactgt cacgacggcc acccgtctaa ctgtctgcag 360
atgctgccgc tgaacttcgg cggtgcccaa cgcgtagatg gcggccaggt actggacggc 420
gcaggtcacc cggtgcagtc catgttcttt ggtcagagca gctttggtac tcacgcggtt 480
gctcgcgaaa ttaacgcagt gaaagtaggt gatgatctgc cgctggagct gctgggtccg 540
ctgggctgtg gcatccaaac tggtgcaggt gcagcgatca actctctggg tattggtccg 600
ggtcaaagcc tggcgatctt cggcggtggt ggcgttggcc tgagcgctct gctgggcgct 660
cgtgcagtgg gtgctgatcg tgttgtagta atcgagccga acgcagctcg tcgtgcactg 720
gcgctggagc tgggtgcatc ccacgcactg gacccgcacg cggagggtga tctggttgcg 780
gcgattaaag cagcgaccgg cggtggtgcg actcattccc tggacactac tggcctgcca 840
ccggtaattg gctccgcaat tgcctgtacc ctgccaggcg gtaccgttgg catggtaggc 900
ctgccggcac cggatgcacc ggtaccggca actctgctgg acctgctgtc caaatctgtt 960
actctgcgtc caattaccga aggtgacgcc gatccgcagc gtttcatccc gcgtatgctg 1020
gacttccatc gtgcgggcaa attcccattt gaccgcctga tcactcgcta tcgttttgat 1080
cagatcaacg aagccctgca tgctactgag aaaggtgaag ctattaagcc ggttctggtg 1140
ttctaa 1146
<210> 12
<211> 573
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 带有rbs3的idi
<400> 12
ttgctaaaga aagaaggcct gctcatgcaa acggaacacg tcattttatt gaatgcacag 60
ggagttccca cgggtacgct ggaaaagtat gccgcacaca cggcagacac ccgcttacat 120
ctcgcgttct ccagttggct gtttaatgcc aaaggacaat tattagttac ccgccgcgca 180
ctgagcaaaa aagcatggcc tggcgtgtgg actaactcgg tttgtgggca cccacaactg 240
ggagaaagca acgaagacgc agtgatccgc cgttgccgtt atgagcttgg cgtggaaatt 300
acgcctcctg aatctatcta tcctgacttt cgctaccgcg ccaccgatcc gagtggcatt 360
gtggaaaatg aagtgtgtcc ggtatttgcc gcacgcacca ctagtgcgtt acagatcaat 420
gatgatgaag tgatggatta tcaatggtgt gatttagcag atgtattaca cggtattgat 480
gccacgccgt gggcgttcag tccgtggatg gtgatgcagg cgacaaatcg cgaagccaga 540
aaacgattat ctgcatttac ccagcttaaa taa 573
<210> 13
<211> 10918
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pTALE-GPPS-GES-geoA-idi
<400> 13
ctacatggct ctgctgtagt gagtgggttg cgctccggca gcggtcctga tcccccgcag 60
aaaaaaagga tctcaagaag atcctttgat cttttctacg gcgcgcccag ctgtctaggg 120
cggcggattt gtcctactca ggagagcgtt caccgacaaa caacagataa aacgaaaggc 180
ccagtctttc gactgagcct ttcgttttat ttgatgcctt taattaaagc ggataacaat 240
ttcacacagg atcgcggccg cttctagagc tcggtaccaa attccagaaa agaggcctcc 300
cgaaaggggg gccttttttc gttttggtcc tactggcgcg cctcagtcag agtattgact 360
taaagtctaa cctataggag atctacagcc atcgagagct gcgagactgt cgccggatgt 420
gtatccgacc tgacgatggc ccaaaagggc cgaaacagtc ctctacaaat aattttgttt 480
aaatcaattc atcgacgtga aaatggtaga tttaagaact ttaggatatt cacagcagca 540
acaggaaaag atcaagccca aagttaggtc gacagtcgcg cagcatcacg aagcgctggt 600
tggtcatggg tttacacatg cccacatcgt agccttatcg cagcaccctg ccgcccttgg 660
cacggtcgcc gtcaagtacc aggacatgat tgcggcgttg ccggaagcca cacatgaggc 720
gatcgtcggt gtggggaaac agtggagcgg agcccgagcg cttgaggccc tgttgacggt 780
cgcgggagag ctgagagggc ctccccttca gctggacacg ggccagttgc tgaagatcgc 840
gaagcgggga ggagtcacgg cggtcgaggc ggtgcacgcg tggcgcaatg cgctcacggg 900
agcacccctc aacctgaccc cggaccaggt agtcgcgatt gcttcacatg acgggggtaa 960
acaagcgctg gaaacggtgc agcgtctgct accggtgtta tgtcaggatc atgggctcac 1020
gccggaacag gtagtggcaa ttgcgagtca tgacggtggc aaacaggccc tggaaaccgt 1080
acagcggctg ctaccggtgc tgtgtcaagc gcatggcctg actccggacc aagttgtagc 1140
cattgcctcg aacgggggcg gcaagcaggc gctggagact gttcaacgtc tgctccccgt 1200
tctgtgtcag gcgcatggcc tgacgcctgc gcaggtcgtg gcgatcgctt caaacatcgg 1260
tgggaagcaa gccctggaga ctgtccaaag actgttgcca gtgttgtgtc aagatcatgg 1320
cttaacccca gatcaggtgg ttgcgattgc atcaaatgga ggtggtaaac aggcgctgga 1380
gactgtgcag cgcctgttgc cggttctgtg ccaagatcat gggctgactc cggaacaggt 1440
tgtggctatc gcaagcaata ttggtggcaa gcaggccctg gaaacagtac agcgcctgct 1500
gcctgtattg tgtcaagccc acggtcttac ccccgatcag gtagtcgcca tagcatcgca 1560
cgacggcggg aagcaggccc ttgagactgt acaacgcctc ctgccggttt tgtgccaggc 1620
gcacggcctg acgccagccc aggtggttgc gatagccagt aataacggcg gtaagcaagc 1680
ccttgaaacc gttcaacgtt tgctgccagt gctgtgccag gatcacggcc tgaccccgga 1740
tcaggtagtc gccattgcta gcaacattgg tggcaaacaa gcactggaga cagttcaacg 1800
cttactgccc gtgctttgcc aggatcatgg actgacccca gagcaagtgg tcgcgattgc 1860
ctcgcatgac ggaggtaaac aggcccttga gactgtccag cgtctgctgc cggtcctttg 1920
ccaggctcat gggctgacgc ccgaccaggt ggtagcaatc gcttcgaacg gcggaggcaa 1980
acaggcgtta gaaaccgttc aacgtctgtt accggtgctg tgccaagctc atggcttaac 2040
cccggcccag gtagtcgcaa tcgctagtca tgatggtggg aaacaggcat tagaaacagt 2100
acaacgtctg ctcccggtcc tctgtcagga tcacggtctg accccggatc aggttgtagc 2160
aatcgctagc aatattggtg gtaaacaggc gctggaaaca gtgcaaagat tattaccagt 2220
tctgtgtcag gaccatggcc tgacacctga gcaggtcgta gcgatcgcaa gtaatattgg 2280
aggtaaacag gccctggaaa ccgtgcagcg tctgctgcct gtgctctgtc aagcgcatgg 2340
tctgactccg gatcaggttg tcgcgatcgc cagtaacggg ggagggaaac aggcactcga 2400
gactgtgcag agattgctgc cggtcctgtg tcaggcgcat ggtctgaccc cagcgcaggt 2460
cgtggcaatc gcttccaaca taggtggtaa acaggccctc gaaactgtcc aacggctgtt 2520
accggtactg tgccaggatc atggtctgac ccctgagcag gtagtggcta ttgcatccaa 2580
cggagggggc agacccgcac tggagtcaat cgtggcccag ctttcgaggc cggaccccgc 2640
gctggccgca ctcactaatg atcatcttgt agcgctggcc tgcctcggcg gacgacccgc 2700
cttggatgcg gtgaagaagg ggctcccgca cgcgcctgca ttgattaagc ggaccaacag 2760
aaggattccc gagaggacat cacatcgagt ggcagatcac gcgcaagtgg tccgcgtgct 2820
cggattcttc cagtgtcact cccaccccgc acaagcgttc gatgacgcca tgactcaatt 2880
tggtatgtcg agacacggac tgcttcagct ctttcgtaga gtcggtgtca cagaactcga 2940
ggcccgctcg ggcacactgc ctcccgcctc ccagcggtgg gacaggattc tccaagcgag 3000
cggtatgaaa cgcgcgaagc cttcacctac gtcaactcag acacctgacc aggcgagcct 3060
tcatgcgttc gcagactcgc tggagaggga tttggacgcg ccctcgccca tgcatgaagg 3120
ggaccaaact cgcgcgtcat aataggtttc agccaaaaaa cttaagaccg ccggtcttgt 3180
ccactacctt gcagtaatgc ggtggacagg atcggcggtt ttcttttctc ttctcaagct 3240
tatcccgaaa atttatcaaa aagagtattg acttatattg agtcgtatag gatacttaca 3300
gccatcgaga gctgcgagct gtcaccggat gtgctttccg gtctgatgag tccgtgagga 3360
cgaaacagcc tctacaaata attttgttta agatgattgc gatagaaatt ccaacggagg 3420
ggtaaatgga atttgacttc aacaaataca tggactccaa agcgatgacg gtaaatgaag 3480
cactgaacaa agcgatccct ctgcgttatc cgcagaaaat ctacgaaagc atgcgttaca 3540
gcctgctggc aggcggcaag cgtgttcgtc cggttctgtg tattgccgca tgtgaactgg 3600
taggtggtac cgaagaactg gcgatcccga ccgcgtgcgc aattgaaatg atccacacga 3660
tgtccctgat gcacgatgat ctgccgtgta tcgacaacga cgatctgcgt cgcggtaaac 3720
cgactaacca caaaattttc ggtgaggata ccgcagtgac tgctggtaac gcactgcact 3780
cttacgcctt cgagcatatc gcggtttcta cttctaaaac cgttggtgct gaccgcatcc 3840
tgcgtatggt gtccgagctg ggtcgtgcta ctggctctga aggtgttatg ggtggtcaga 3900
tggtagacat cgcatccgaa ggcgatccgt ctatcgacct gcagaccctg gaatggattc 3960
acatccacaa aaccgcaatg ctgctggaat gctccgttgt ttgcggtgca atcattggcg 4020
gtgccagcga aatcgtaatc gaacgtgccc gtcgctacgc ccgctgtgtt ggtctgctgt 4080
tccaggtagt tgatgacatt ctggacgtaa ctaaaagcag cgacgaactg ggtaagactg 4140
cgggcaagga cctgatctct gataaagcca cctacccaaa gctgatgggt ctggaaaagg 4200
ccaaggagtt ctccgatgaa ctgctgaacc gtgcgaaggg tgaactgtcc tgcttcgacc 4260
cagttaaagc cgctccgctg ctgggcctgg cagactacgt ggcatttcgt cagaattaac 4320
agaaaatagt aaggaggttt tcgatgagct gcgcgcgtat caccgtgacc ctgccgtatc 4380
gtagcgcgaa aaccagcatc cagcgtggta ttacccacta tccggcgctg atccgtccgc 4440
gtttcagcgc gtgcaccccg ctggcgagcg cgatgccgct gagcagcacc ccgctgatta 4500
acggcgataa cagccagcgt aagaacaccc gtcaacacat ggaggaaagc agcagcaaac 4560
gtcgtgaata tctgctggag gaaaccaccc gtaagctgca acgtaacgat accgaaagcg 4620
tggagaagct gaaactgatc gacaacattc agcaactggg tatcggctac tatttcgagg 4680
atgcgattaa cgcggttctg cgtagcccgt ttagcaccgg cgaggaagac ctgttcaccg 4740
cggcgctgcg ttttcgtctg ctgcgtcaca acggcatcga aattagcccg gagatcttcc 4800
tgaagtttaa agacgaacgt ggtaaattcg atgagagcga caccctgggc ctgctgagcc 4860
tgtacgaagc gagcaacctg ggtgtggcgg gcgaggaaat tctggaggaa gcgatggaat 4920
ttgcggaggc gcgtctgcgt cgtagcctga gcgaaccggc ggcgccgctg catggtgagg 4980
tggcgcaggc gctggatgtt ccgcgtcacc tgcgtatggc gcgtctggaa gcgcgtcgtt 5040
ttatcgagca gtatggcaag caaagcgacc acgatggcga cctgctggag ctggcgattc 5100
tggactacaa ccaggtgcaa gcgcagcacc aaagcgaact gaccgagatc attcgttggt 5160
ggaaggaact gggtctggtt gataaactga gcttcggccg tgaccgtccg ctggagtgct 5220
ttctgtggac cgtgggtctg ctgccggaac cgaagtatag cagcgttcgt atcgagctgg 5280
cgaaagcgat cagcattctg ctggttatcg acgatatttt cgacacctac ggtgaaatgg 5340
acgatctgat cctgtttacc gatgcgattc gtcgttggga cctggaagcg atggagggcc 5400
tgccggaata tatgaaaatc tgctacatgg cgctgtataa caccaccaac gaggtgtgct 5460
acaaagttct gcgtgatacc ggtcgtattg tgctgctgaa cctgaagagc acctggatcg 5520
acatgattga gggcttcatg gaggaagcga agtggtttaa cggtggcagc gcgccgaaac 5580
tggaggaata tatcgaaaac ggtgttagca ccgcgggcgc gtacatggcg tttgcgcaca 5640
tcttctttct gattggcgag ggcgtgaccc accagaacag ccaactgttc acccagaagc 5700
cgtatccgaa agtttttagc gcggcgggtc gtatcctgcg tctgtgggac gatctgggta 5760
ccgcgaaaga ggaacaggaa cgtggcgatc tggcgagctg cgttcaactg tttatgaagg 5820
agaaaagcct gaccgaggaa gaggcgcgta gccgtatcct ggaagagatt aagggtctgt 5880
ggcgtgacct gaacggcgag ctggtgtaca acaagaacct gccgctgagc atcattaaag 5940
ttgcgctgaa catggcgcgt gcgagccagg tggtttacaa gcacgatcaa gacacctatt 6000
tcagcagcgt ggataactac gttgacgcgc tgttctttac ccaataaatt cccgcagtcg 6060
ataccgttgc catgaacgat acccaagatt tcatttctgc tcaggccgca gtactgcgcc 6120
aggtgggtgg tccgctggct gtggaaccag tacgtatttc tatgccgaag ggcgacgagg 6180
ttctgattcg catcgcaggc gttggtgtgt gccacactga tctggtgtgt cgtgatggtt 6240
tcccggtacc gctgccaatc gttctgggcc atgagggctc tggcactgtt gaggcggttg 6300
gtgaacaggt gcgcaccctg aagccaggtg atcgtgtagt gctgtctttc aactcctgcg 6360
gtcactgtgg caactgtcac gacggccacc cgtctaactg tctgcagatg ctgccgctga 6420
acttcggcgg tgcccaacgc gtagatggcg gccaggtact ggacggcgca ggtcacccgg 6480
tgcagtccat gttctttggt cagagcagct ttggtactca cgcggttgct cgcgaaatta 6540
acgcagtgaa agtaggtgat gatctgccgc tggagctgct gggtccgctg ggctgtggca 6600
tccaaactgg tgcaggtgca gcgatcaact ctctgggtat tggtccgggt caaagcctgg 6660
cgatcttcgg cggtggtggc gttggcctga gcgctctgct gggcgctcgt gcagtgggtg 6720
ctgatcgtgt tgtagtaatc gagccgaacg cagctcgtcg tgcactggcg ctggagctgg 6780
gtgcatccca cgcactggac ccgcacgcgg agggtgatct ggttgcggcg attaaagcag 6840
cgaccggcgg tggtgcgact cattccctgg acactactgg cctgccaccg gtaattggct 6900
ccgcaattgc ctgtaccctg ccaggcggta ccgttggcat ggtaggcctg ccggcaccgg 6960
atgcaccggt accggcaact ctgctggacc tgctgtccaa atctgttact ctgcgtccaa 7020
ttaccgaagg tgacgccgat ccgcagcgtt tcatcccgcg tatgctggac ttccatcgtg 7080
cgggcaaatt cccatttgac cgcctgatca ctcgctatcg ttttgatcag atcaacgaag 7140
ccctgcatgc tactgagaaa ggtgaagcta ttaagccggt tctggtgttc taattgctaa 7200
agaaagaagg cctgctcatg caaacggaac acgtcatttt attgaatgca cagggagttc 7260
ccacgggtac gctggaaaag tatgccgcac acacggcaga cacccgctta catctcgcgt 7320
tctccagttg gctgtttaat gccaaaggac aattattagt tacccgccgc gcactgagca 7380
aaaaagcatg gcctggcgtg tggactaact cggtttgtgg gcacccacaa ctgggagaaa 7440
gcaacgaaga cgcagtgatc cgccgttgcc gttatgagct tggcgtggaa attacgcctc 7500
ctgaatctat ctatcctgac tttcgctacc gcgccaccga tccgagtggc attgtggaaa 7560
atgaagtgtg tccggtattt gccgcacgca ccactagtgc gttacagatc aatgatgatg 7620
aagtgatgga ttatcaatgg tgtgatttag cagatgtatt acacggtatt gatgccacgc 7680
cgtgggcgtt cagtccgtgg atggtgatgc aggcgacaaa tcgcgaagcc agaaaacgat 7740
tatctgcatt tacccagctt aaataagaga gcagaggttg ataagttttc tccaggcatc 7800
aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg 7860
tgaacgctct ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata 7920
tactagagct gctaacaaag cccgaaagga agctgagttg gctgctgcca ccgctgagca 7980
ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt tgctgaaagg 8040
aggaactata tccggattac tagaggtcat gcttgccatc tgttttcttg caagatgctc 8100
actcaaaggc ggtaatgggt cgatgaagag caaaagctct tcactcgtcg tgactgggaa 8160
aaccctggcg actagtcttg gactcctgtt gatagatcca gtaatgacct cagaactcca 8220
tctggatttg ttcagaacgc tcggttgccg ccgggcgttt tttattggtg agaatccagg 8280
ggtccccaat aattacgatt taaattggcg aaaatgagac gtggatgaat gtcagctact 8340
gggctatctg gacaagggaa aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc 8400
ttacatggcg atagctagac tgggcggttt tatggacagc aagcgaaccg gaattgccag 8460
ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttcttgc 8520
cgccaaggat ctgatggcgc aggggatcaa gatctgatca agagacagga tgaggatcgt 8580
ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 8640
tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 8700
tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 8760
aactgcagga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 8820
ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 8880
ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 8940
caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 9000
atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 9060
acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgcgcatgc 9120
ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 9180
aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 9240
aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 9300
gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 9360
ttcttgacga gttcttctga tttgactttt gtccttttcc gctgcataac cctgcttcgg 9420
ggtcattata gcgatttttt cggtatatcc atcctttttc gcacgatata caggattttg 9480
ccaaagggtt cgtgtagact ttccttggtg tatccaacgg cgtcagccgg gcaggatagg 9540
tgaagtaggc ccacccgcga gcgggtgttc cttcttcact gtcccttatt cgcacctggc 9600
ggtgctcaac gggaatcctg ctctgcgagg ctggccgtag gccggccgcg atgcaggtgg 9660
ctgctgaacc cccagccgga actgacccca caaggcctca gatccttccg tatttagcca 9720
gtatgttctc tagtgtggtt cgttgttttt gcgtgagcca tgagaacgaa ccattgagat 9780
catacttact ttgcatgtca ctcaaaaatt ttgcctcaaa actggtgagc tgaatttttg 9840
cagttaaagc atcgtgtagt gtttttctta gtccgttatg taggtaggaa tctgatgtaa 9900
tggttgttgg tattttgtca ccattcattt ttatctggtt gttctcaagt tcggttacga 9960
gatccatttg tctatctagt tcaacttgga aaatcaacgt atcagtcggg cggcctcgct 10020
tatcaaccac caatttcata ttgctgtaag tgtttaaatc tttacttatt ggtttcaaaa 10080
cccattggtt aagcctttta aactcatggt agttattttc aagcattaac atgaacttaa 10140
attcatcaag gctaatctct atatttgcct tgtgagtttt cttttgtgtt agttctttta 10200
ataaccactc ataaatcctc atagagtatt tgttttcaaa agacttaaca tgttccagat 10260
tatattttat gaattttttt aactggaaaa gataaggcaa tatctcttca ctaaaaacta 10320
attctaattt ttcgcttgag aacttggcat agtttgtcca ctggaaaatc tcaaagcctt 10380
taaccaaagg attcctgatt tccacagttc tcgtcatcag ctctctggtt gctttagcta 10440
atacaccata agcattttcc ctactgatgt tcatcatctg agcgtattgg ttataagtga 10500
acgataccgt ccgttctttc cttgtagggt tttcaatcgt ggggttgagt agtgccacac 10560
agcataaaat tagcttggtt tcatgctccg ttaagtcata gcgactaatc gctagttcat 10620
ttgctttgaa aacaactaat tcagacatac atctcaattg gtctaggtga ttttaatcac 10680
tataccaatt gagatgggct agtcaatgat aattacatgt ccttttcctt tgagttgtgg 10740
gtatctgtaa attctgctag acctttgctg gaaaacttgt aaattctgct agaccctctg 10800
taaattccgc tagacctttg tgtgtttttt ttgtttatat tcaagtggtt ataatttata 10860
gaataaagaa agaataaaaa aagataaaaa gaatagatcc cagccctgtg tataactc 10918
<210> 14
<211> 894
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> GPPS
<400> 14
atggaatttg acttcaacaa atacatggac tccaaagcga tgacggtaaa tgaagcactg 60
aacaaagcga tccctctgcg ttatccgcag aaaatctacg aaagcatgcg ttacagcctg 120
ctggcaggcg gcaagcgtgt tcgtccggtt ctgtgtattg ccgcatgtga actggtaggt 180
ggtaccgaag aactggcgat cccgaccgcg tgcgcaattg aaatgatcca cacgatgtcc 240
ctgatgcacg atgatctgcc gtgtatcgac aacgacgatc tgcgtcgcgg taaaccgact 300
aaccacaaaa ttttcggtga ggataccgca gtgactgctg gtaacgcact gcactcttac 360
gccttcgagc atatcgcggt ttctacttct aaaaccgttg gtgctgaccg catcctgcgt 420
atggtgtccg agctgggtcg tgctactggc tctgaaggtg ttatgggtgg tcagatggta 480
gacatcgcat ccgaaggcga tccgtctatc gacctgcaga ccctggaatg gattcacatc 540
cacaaaaccg caatgctgct ggaatgctcc gttgtttgcg gtgcaatcat tggcggtgcc 600
agcgaaatcg taatcgaacg tgcccgtcgc tacgcccgct gtgttggtct gctgttccag 660
gtagttgatg acattctgga cgtaactaaa agcagcgacg aactgggtaa gactgcgggc 720
aaggacctga tctctgataa agccacctac ccaaagctga tgggtctgga aaaggccaag 780
gagttctccg atgaactgct gaaccgtgcg aagggtgaac tgtcctgctt cgacccagtt 840
aaagccgctc cgctgctggg cctggcagac tacgtggcat ttcgtcagaa ttaa 894
<210> 15
<211> 1704
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> GES
<400> 15
atgagctgcg cgcgtatcac cgtgaccctg ccgtatcgta gcgcgaaaac cagcatccag 60
cgtggtatta cccactatcc ggcgctgatc cgtccgcgtt tcagcgcgtg caccccgctg 120
gcgagcgcga tgccgctgag cagcaccccg ctgattaacg gcgataacag ccagcgtaag 180
aacacccgtc aacacatgga ggaaagcagc agcaaacgtc gtgaatatct gctggaggaa 240
accacccgta agctgcaacg taacgatacc gaaagcgtgg agaagctgaa actgatcgac 300
aacattcagc aactgggtat cggctactat ttcgaggatg cgattaacgc ggttctgcgt 360
agcccgttta gcaccggcga ggaagacctg ttcaccgcgg cgctgcgttt tcgtctgctg 420
cgtcacaacg gcatcgaaat tagcccggag atcttcctga agtttaaaga cgaacgtggt 480
aaattcgatg agagcgacac cctgggcctg ctgagcctgt acgaagcgag caacctgggt 540
gtggcgggcg aggaaattct ggaggaagcg atggaatttg cggaggcgcg tctgcgtcgt 600
agcctgagcg aaccggcggc gccgctgcat ggtgaggtgg cgcaggcgct ggatgttccg 660
cgtcacctgc gtatggcgcg tctggaagcg cgtcgtttta tcgagcagta tggcaagcaa 720
agcgaccacg atggcgacct gctggagctg gcgattctgg actacaacca ggtgcaagcg 780
cagcaccaaa gcgaactgac cgagatcatt cgttggtgga aggaactggg tctggttgat 840
aaactgagct tcggccgtga ccgtccgctg gagtgctttc tgtggaccgt gggtctgctg 900
ccggaaccga agtatagcag cgttcgtatc gagctggcga aagcgatcag cattctgctg 960
gttatcgacg atattttcga cacctacggt gaaatggacg atctgatcct gtttaccgat 1020
gcgattcgtc gttgggacct ggaagcgatg gagggcctgc cggaatatat gaaaatctgc 1080
tacatggcgc tgtataacac caccaacgag gtgtgctaca aagttctgcg tgataccggt 1140
cgtattgtgc tgctgaacct gaagagcacc tggatcgaca tgattgaggg cttcatggag 1200
gaagcgaagt ggtttaacgg tggcagcgcg ccgaaactgg aggaatatat cgaaaacggt 1260
gttagcaccg cgggcgcgta catggcgttt gcgcacatct tctttctgat tggcgagggc 1320
gtgacccacc agaacagcca actgttcacc cagaagccgt atccgaaagt ttttagcgcg 1380
gcgggtcgta tcctgcgtct gtgggacgat ctgggtaccg cgaaagagga acaggaacgt 1440
ggcgatctgg cgagctgcgt tcaactgttt atgaaggaga aaagcctgac cgaggaagag 1500
gcgcgtagcc gtatcctgga agagattaag ggtctgtggc gtgacctgaa cggcgagctg 1560
gtgtacaaca agaacctgcc gctgagcatc attaaagttg cgctgaacat ggcgcgtgcg 1620
agccaggtgg tttacaagca cgatcaagac acctatttca gcagcgtgga taactacgtt 1680
gacgcgctgt tctttaccca ataa 1704
<210> 16
<211> 1122
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> geoA
<400> 16
atgaacgata cccaagattt catttctgct caggccgcag tactgcgcca ggtgggtggt 60
ccgctggctg tggaaccagt acgtatttct atgccgaagg gcgacgaggt tctgattcgc 120
atcgcaggcg ttggtgtgtg ccacactgat ctggtgtgtc gtgatggttt cccggtaccg 180
ctgccaatcg ttctgggcca tgagggctct ggcactgttg aggcggttgg tgaacaggtg 240
cgcaccctga agccaggtga tcgtgtagtg ctgtctttca actcctgcgg tcactgtggc 300
aactgtcacg acggccaccc gtctaactgt ctgcagatgc tgccgctgaa cttcggcggt 360
gcccaacgcg tagatggcgg ccaggtactg gacggcgcag gtcacccggt gcagtccatg 420
ttctttggtc agagcagctt tggtactcac gcggttgctc gcgaaattaa cgcagtgaaa 480
gtaggtgatg atctgccgct ggagctgctg ggtccgctgg gctgtggcat ccaaactggt 540
gcaggtgcag cgatcaactc tctgggtatt ggtccgggtc aaagcctggc gatcttcggc 600
ggtggtggcg ttggcctgag cgctctgctg ggcgctcgtg cagtgggtgc tgatcgtgtt 660
gtagtaatcg agccgaacgc agctcgtcgt gcactggcgc tggagctggg tgcatcccac 720
gcactggacc cgcacgcgga gggtgatctg gttgcggcga ttaaagcagc gaccggcggt 780
ggtgcgactc attccctgga cactactggc ctgccaccgg taattggctc cgcaattgcc 840
tgtaccctgc caggcggtac cgttggcatg gtaggcctgc cggcaccgga tgcaccggta 900
ccggcaactc tgctggacct gctgtccaaa tctgttactc tgcgtccaat taccgaaggt 960
gacgccgatc cgcagcgttt catcccgcgt atgctggact tccatcgtgc gggcaaattc 1020
ccatttgacc gcctgatcac tcgctatcgt tttgatcaga tcaacgaagc cctgcatgct 1080
actgagaaag gtgaagctat taagccggtt ctggtgttct aa 1122
<210> 17
<211> 6577
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pTALE
<400> 17
gagagcagag gttgataagt tttctccagg catcaaataa aacgaaaggc tcagtcgaaa 60
gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctctacta gagtcacact 120
ggctcacctt cgggtgggcc tttctgcgtt tatatactag agctgctaac aaagcccgaa 180
aggaagctga gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct 240
ctaaacgggt cttgaggggt tttttgctga aaggaggaac tatatccgga ttactagagg 300
tcatgcttgc catctgtttt cttgcaagat gctcactcaa aggcggtaat gggtcgatga 360
agagcaaaag ctcttcactc gtcgtgactg ggaaaaccct ggcgactagt cttggactcc 420
tgttgataga tccagtaatg acctcagaac tccatctgga tttgttcaga acgctcggtt 480
gccgccgggc gttttttatt ggtgagaatc caggggtccc caataattac gatttaaatt 540
ggcgaaaatg agacgtggat gaatgtcagc tactgggcta tctggacaag ggaaaacgca 600
agcgcaaaga gaaagcaggt agcttgcagt gggcttacat ggcgatagct agactgggcg 660
gttttatgga cagcaagcga accggaattg ccagctgggg cgccctctgg taaggttggg 720
aagccctgca aagtaaactg gatggctttc ttgccgccaa ggatctgatg gcgcagggga 780
tcaagatctg atcaagagac aggatgagga tcgtttcgca tgattgaaca agatggattg 840
cacgcaggtt ctccggccgc ttgggtggag aggctattcg gctatgactg ggcacaacag 900
acaatcggct gctctgatgc cgccgtgttc cggctgtcag cgcaggggcg cccggttctt 960
tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc aggacgaggc agcgcggcta 1020
tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg 1080
ggaagggact ggctgctatt gggcgaagtg ccggggcagg atctcctgtc atctcacctt 1140
gctcctgccg agaaagtatc catcatggct gatgcaatgc ggcggctgca tacgcttgat 1200
ccggctacct gcccattcga ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg 1260
atggaagccg gtcttgtcga tcaggatgat ctggacgaag agcatcaggg gctcgcgcca 1320
gccgaactgt tcgccaggct caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc 1380
catggcgatg cctgcttgcc gaatatcatg gtggaaaatg gccgcttttc tggattcatc 1440
gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca tagcgttggc tacccgtgat 1500
attgctgaag agcttggcgg cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc 1560
gctcccgatt cgcagcgcat cgccttctat cgccttcttg acgagttctt ctgatttgac 1620
ttttgtcctt ttccgctgca taaccctgct tcggggtcat tatagcgatt ttttcggtat 1680
atccatcctt tttcgcacga tatacaggat tttgccaaag ggttcgtgta gactttcctt 1740
ggtgtatcca acggcgtcag ccgggcagga taggtgaagt aggcccaccc gcgagcgggt 1800
gttccttctt cactgtccct tattcgcacc tggcggtgct caacgggaat cctgctctgc 1860
gaggctggcc gtaggccggc cgcgatgcag gtggctgctg aacccccagc cggaactgac 1920
cccacaaggc ctcagatcct tccgtattta gccagtatgt tctctagtgt ggttcgttgt 1980
ttttgcgtga gccatgagaa cgaaccattg agatcatact tactttgcat gtcactcaaa 2040
aattttgcct caaaactggt gagctgaatt tttgcagtta aagcatcgtg tagtgttttt 2100
cttagtccgt tatgtaggta ggaatctgat gtaatggttg ttggtatttt gtcaccattc 2160
atttttatct ggttgttctc aagttcggtt acgagatcca tttgtctatc tagttcaact 2220
tggaaaatca acgtatcagt cgggcggcct cgcttatcaa ccaccaattt catattgctg 2280
taagtgttta aatctttact tattggtttc aaaacccatt ggttaagcct tttaaactca 2340
tggtagttat tttcaagcat taacatgaac ttaaattcat caaggctaat ctctatattt 2400
gccttgtgag ttttcttttg tgttagttct tttaataacc actcataaat cctcatagag 2460
tatttgtttt caaaagactt aacatgttcc agattatatt ttatgaattt ttttaactgg 2520
aaaagataag gcaatatctc ttcactaaaa actaattcta atttttcgct tgagaacttg 2580
gcatagtttg tccactggaa aatctcaaag cctttaacca aaggattcct gatttccaca 2640
gttctcgtca tcagctctct ggttgcttta gctaatacac cataagcatt ttccctactg 2700
atgttcatca tctgagcgta ttggttataa gtgaacgata ccgtccgttc tttccttgta 2760
gggttttcaa tcgtggggtt gagtagtgcc acacagcata aaattagctt ggtttcatgc 2820
tccgttaagt catagcgact aatcgctagt tcatttgctt tgaaaacaac taattcagac 2880
atacatctca attggtctag gtgattttaa tcactatacc aattgagatg ggctagtcaa 2940
tgataattac atgtcctttt cctttgagtt gtgggtatct gtaaattctg ctagaccttt 3000
gctggaaaac ttgtaaattc tgctagaccc tctgtaaatt ccgctagacc tttgtgtgtt 3060
ttttttgttt atattcaagt ggttataatt tatagaataa agaaagaata aaaaaagata 3120
aaaagaatag atcccagccc tgtgtataac tcctacatgg ctctgctgta gtgagtgggt 3180
tgcgctccgg cagcggtcct gatcccccgc agaaaaaaag gatctcaaga agatcctttg 3240
atcttttcta cggcgcgccc agctgtctag ggcggcggat ttgtcctact caggagagcg 3300
ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt 3360
atttgatgcc tttaattaaa gcggataaca atttcacaca ggatcgcggc cgcttctaga 3420
gctcggtacc aaattccaga aaagaggcct cccgaaaggg gggccttttt tcgttttggt 3480
cctactggcg cgcctcagtc agagtattga cttaaagtct aacctatagg agatctacag 3540
ccatcgagag ctgcgagact gtcgccggat gtgtatccga cctgacgatg gcccaaaagg 3600
gccgaaacag tcctctacaa ataattttgt ttaaatcaat tcatcgacgt gaaaatggta 3660
gatttaagaa ctttaggata ttcacagcag caacaggaaa agatcaagcc caaagttagg 3720
tcgacagtcg cgcagcatca cgaagcgctg gttggtcatg ggtttacaca tgcccacatc 3780
gtagccttat cgcagcaccc tgccgccctt ggcacggtcg ccgtcaagta ccaggacatg 3840
attgcggcgt tgccggaagc cacacatgag gcgatcgtcg gtgtggggaa acagtggagc 3900
ggagcccgag cgcttgaggc cctgttgacg gtcgcgggag agctgagagg gcctcccctt 3960
cagctggaca cgggccagtt gctgaagatc gcgaagcggg gaggagtcac ggcggtcgag 4020
gcggtgcacg cgtggcgcaa tgcgctcacg ggagcacccc tcaacctgac cccggaccag 4080
gtagtcgcga ttgcttcaca tgacgggggt aaacaagcgc tggaaacggt gcagcgtctg 4140
ctaccggtgt tatgtcagga tcatgggctc acgccggaac aggtagtggc aattgcgagt 4200
catgacggtg gcaaacaggc cctggaaacc gtacagcggc tgctaccggt gctgtgtcaa 4260
gcgcatggcc tgactccgga ccaagttgta gccattgcct cgaacggggg cggcaagcag 4320
gcgctggaga ctgttcaacg tctgctcccc gttctgtgtc aggcgcatgg cctgacgcct 4380
gcgcaggtcg tggcgatcgc ttcaaacatc ggtgggaagc aagccctgga gactgtccaa 4440
agactgttgc cagtgttgtg tcaagatcat ggcttaaccc cagatcaggt ggttgcgatt 4500
gcatcaaatg gaggtggtaa acaggcgctg gagactgtgc agcgcctgtt gccggttctg 4560
tgccaagatc atgggctgac tccggaacag gttgtggcta tcgcaagcaa tattggtggc 4620
aagcaggccc tggaaacagt acagcgcctg ctgcctgtat tgtgtcaagc ccacggtctt 4680
acccccgatc aggtagtcgc catagcatcg cacgacggcg ggaagcaggc ccttgagact 4740
gtacaacgcc tcctgccggt tttgtgccag gcgcacggcc tgacgccagc ccaggtggtt 4800
gcgatagcca gtaataacgg cggtaagcaa gcccttgaaa ccgttcaacg tttgctgcca 4860
gtgctgtgcc aggatcacgg cctgaccccg gatcaggtag tcgccattgc tagcaacatt 4920
ggtggcaaac aagcactgga gacagttcaa cgcttactgc ccgtgctttg ccaggatcat 4980
ggactgaccc cagagcaagt ggtcgcgatt gcctcgcatg acggaggtaa acaggccctt 5040
gagactgtcc agcgtctgct gccggtcctt tgccaggctc atgggctgac gcccgaccag 5100
gtggtagcaa tcgcttcgaa cggcggaggc aaacaggcgt tagaaaccgt tcaacgtctg 5160
ttaccggtgc tgtgccaagc tcatggctta accccggccc aggtagtcgc aatcgctagt 5220
catgatggtg ggaaacaggc attagaaaca gtacaacgtc tgctcccggt cctctgtcag 5280
gatcacggtc tgaccccgga tcaggttgta gcaatcgcta gcaatattgg tggtaaacag 5340
gcgctggaaa cagtgcaaag attattacca gttctgtgtc aggaccatgg cctgacacct 5400
gagcaggtcg tagcgatcgc aagtaatatt ggaggtaaac aggccctgga aaccgtgcag 5460
cgtctgctgc ctgtgctctg tcaagcgcat ggtctgactc cggatcaggt tgtcgcgatc 5520
gccagtaacg ggggagggaa acaggcactc gagactgtgc agagattgct gccggtcctg 5580
tgtcaggcgc atggtctgac cccagcgcag gtcgtggcaa tcgcttccaa cataggtggt 5640
aaacaggccc tcgaaactgt ccaacggctg ttaccggtac tgtgccagga tcatggtctg 5700
acccctgagc aggtagtggc tattgcatcc aacggagggg gcagacccgc actggagtca 5760
atcgtggccc agctttcgag gccggacccc gcgctggccg cactcactaa tgatcatctt 5820
gtagcgctgg cctgcctcgg cggacgaccc gccttggatg cggtgaagaa ggggctcccg 5880
cacgcgcctg cattgattaa gcggaccaac agaaggattc ccgagaggac atcacatcga 5940
gtggcagatc acgcgcaagt ggtccgcgtg ctcggattct tccagtgtca ctcccacccc 6000
gcacaagcgt tcgatgacgc catgactcaa tttggtatgt cgagacacgg actgcttcag 6060
ctctttcgta gagtcggtgt cacagaactc gaggcccgct cgggcacact gcctcccgcc 6120
tcccagcggt gggacaggat tctccaagcg agcggtatga aacgcgcgaa gccttcacct 6180
acgtcaactc agacacctga ccaggcgagc cttcatgcgt tcgcagactc gctggagagg 6240
gatttggacg cgccctcgcc catgcatgaa ggggaccaaa ctcgcgcgtc ataataggtt 6300
tcagccaaaa aacttaagac cgccggtctt gtccactacc ttgcagtaat gcggtggaca 6360
ggatcggcgg ttttcttttc tcttctcaag cttatcccga aaatttatca aaaagagtat 6420
tgacttatat tgagtcgtat aggatactta cagccatcga gagctgcgag ctgtcaccgg 6480
atgtgctttc cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt 6540
taagatgatt gcgatagaaa ttccaacgga ggggtaa 6577
<210> 18
<211> 4391
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> GPPS-GES-geoA-idi
<400> 18
gatagaaatt ccaacggagg ggtaaatgga atttgacttc aacaaataca tggactccaa 60
agcgatgacg gtaaatgaag cactgaacaa agcgatccct ctgcgttatc cgcagaaaat 120
ctacgaaagc atgcgttaca gcctgctggc aggcggcaag cgtgttcgtc cggttctgtg 180
tattgccgca tgtgaactgg taggtggtac cgaagaactg gcgatcccga ccgcgtgcgc 240
aattgaaatg atccacacga tgtccctgat gcacgatgat ctgccgtgta tcgacaacga 300
cgatctgcgt cgcggtaaac cgactaacca caaaattttc ggtgaggata ccgcagtgac 360
tgctggtaac gcactgcact cttacgcctt cgagcatatc gcggtttcta cttctaaaac 420
cgttggtgct gaccgcatcc tgcgtatggt gtccgagctg ggtcgtgcta ctggctctga 480
aggtgttatg ggtggtcaga tggtagacat cgcatccgaa ggcgatccgt ctatcgacct 540
gcagaccctg gaatggattc acatccacaa aaccgcaatg ctgctggaat gctccgttgt 600
ttgcggtgca atcattggcg gtgccagcga aatcgtaatc gaacgtgccc gtcgctacgc 660
ccgctgtgtt ggtctgctgt tccaggtagt tgatgacatt ctggacgtaa ctaaaagcag 720
cgacgaactg ggtaagactg cgggcaagga cctgatctct gataaagcca cctacccaaa 780
gctgatgggt ctggaaaagg ccaaggagtt ctccgatgaa ctgctgaacc gtgcgaaggg 840
tgaactgtcc tgcttcgacc cagttaaagc cgctccgctg ctgggcctgg cagactacgt 900
ggcatttcgt cagaattaac agaaaatagt aaggaggttt tcgatgagct gcgcgcgtat 960
caccgtgacc ctgccgtatc gtagcgcgaa aaccagcatc cagcgtggta ttacccacta 1020
tccggcgctg atccgtccgc gtttcagcgc gtgcaccccg ctggcgagcg cgatgccgct 1080
gagcagcacc ccgctgatta acggcgataa cagccagcgt aagaacaccc gtcaacacat 1140
ggaggaaagc agcagcaaac gtcgtgaata tctgctggag gaaaccaccc gtaagctgca 1200
acgtaacgat accgaaagcg tggagaagct gaaactgatc gacaacattc agcaactggg 1260
tatcggctac tatttcgagg atgcgattaa cgcggttctg cgtagcccgt ttagcaccgg 1320
cgaggaagac ctgttcaccg cggcgctgcg ttttcgtctg ctgcgtcaca acggcatcga 1380
aattagcccg gagatcttcc tgaagtttaa agacgaacgt ggtaaattcg atgagagcga 1440
caccctgggc ctgctgagcc tgtacgaagc gagcaacctg ggtgtggcgg gcgaggaaat 1500
tctggaggaa gcgatggaat ttgcggaggc gcgtctgcgt cgtagcctga gcgaaccggc 1560
ggcgccgctg catggtgagg tggcgcaggc gctggatgtt ccgcgtcacc tgcgtatggc 1620
gcgtctggaa gcgcgtcgtt ttatcgagca gtatggcaag caaagcgacc acgatggcga 1680
cctgctggag ctggcgattc tggactacaa ccaggtgcaa gcgcagcacc aaagcgaact 1740
gaccgagatc attcgttggt ggaaggaact gggtctggtt gataaactga gcttcggccg 1800
tgaccgtccg ctggagtgct ttctgtggac cgtgggtctg ctgccggaac cgaagtatag 1860
cagcgttcgt atcgagctgg cgaaagcgat cagcattctg ctggttatcg acgatatttt 1920
cgacacctac ggtgaaatgg acgatctgat cctgtttacc gatgcgattc gtcgttggga 1980
cctggaagcg atggagggcc tgccggaata tatgaaaatc tgctacatgg cgctgtataa 2040
caccaccaac gaggtgtgct acaaagttct gcgtgatacc ggtcgtattg tgctgctgaa 2100
cctgaagagc acctggatcg acatgattga gggcttcatg gaggaagcga agtggtttaa 2160
cggtggcagc gcgccgaaac tggaggaata tatcgaaaac ggtgttagca ccgcgggcgc 2220
gtacatggcg tttgcgcaca tcttctttct gattggcgag ggcgtgaccc accagaacag 2280
ccaactgttc acccagaagc cgtatccgaa agtttttagc gcggcgggtc gtatcctgcg 2340
tctgtgggac gatctgggta ccgcgaaaga ggaacaggaa cgtggcgatc tggcgagctg 2400
cgttcaactg tttatgaagg agaaaagcct gaccgaggaa gaggcgcgta gccgtatcct 2460
ggaagagatt aagggtctgt ggcgtgacct gaacggcgag ctggtgtaca acaagaacct 2520
gccgctgagc atcattaaag ttgcgctgaa catggcgcgt gcgagccagg tggtttacaa 2580
gcacgatcaa gacacctatt tcagcagcgt ggataactac gttgacgcgc tgttctttac 2640
ccaataaatt cccgcagtcg ataccgttgc catgaacgat acccaagatt tcatttctgc 2700
tcaggccgca gtactgcgcc aggtgggtgg tccgctggct gtggaaccag tacgtatttc 2760
tatgccgaag ggcgacgagg ttctgattcg catcgcaggc gttggtgtgt gccacactga 2820
tctggtgtgt cgtgatggtt tcccggtacc gctgccaatc gttctgggcc atgagggctc 2880
tggcactgtt gaggcggttg gtgaacaggt gcgcaccctg aagccaggtg atcgtgtagt 2940
gctgtctttc aactcctgcg gtcactgtgg caactgtcac gacggccacc cgtctaactg 3000
tctgcagatg ctgccgctga acttcggcgg tgcccaacgc gtagatggcg gccaggtact 3060
ggacggcgca ggtcacccgg tgcagtccat gttctttggt cagagcagct ttggtactca 3120
cgcggttgct cgcgaaatta acgcagtgaa agtaggtgat gatctgccgc tggagctgct 3180
gggtccgctg ggctgtggca tccaaactgg tgcaggtgca gcgatcaact ctctgggtat 3240
tggtccgggt caaagcctgg cgatcttcgg cggtggtggc gttggcctga gcgctctgct 3300
gggcgctcgt gcagtgggtg ctgatcgtgt tgtagtaatc gagccgaacg cagctcgtcg 3360
tgcactggcg ctggagctgg gtgcatccca cgcactggac ccgcacgcgg agggtgatct 3420
ggttgcggcg attaaagcag cgaccggcgg tggtgcgact cattccctgg acactactgg 3480
cctgccaccg gtaattggct ccgcaattgc ctgtaccctg ccaggcggta ccgttggcat 3540
ggtaggcctg ccggcaccgg atgcaccggt accggcaact ctgctggacc tgctgtccaa 3600
atctgttact ctgcgtccaa ttaccgaagg tgacgccgat ccgcagcgtt tcatcccgcg 3660
tatgctggac ttccatcgtg cgggcaaatt cccatttgac cgcctgatca ctcgctatcg 3720
ttttgatcag atcaacgaag ccctgcatgc tactgagaaa ggtgaagcta ttaagccggt 3780
tctggtgttc taattgctaa agaaagaagg cctgctcatg caaacggaac acgtcatttt 3840
attgaatgca cagggagttc ccacgggtac gctggaaaag tatgccgcac acacggcaga 3900
cacccgctta catctcgcgt tctccagttg gctgtttaat gccaaaggac aattattagt 3960
tacccgccgc gcactgagca aaaaagcatg gcctggcgtg tggactaact cggtttgtgg 4020
gcacccacaa ctgggagaaa gcaacgaaga cgcagtgatc cgccgttgcc gttatgagct 4080
tggcgtggaa attacgcctc ctgaatctat ctatcctgac tttcgctacc gcgccaccga 4140
tccgagtggc attgtggaaa atgaagtgtg tccggtattt gccgcacgca ccactagtgc 4200
gttacagatc aatgatgatg aagtgatgga ttatcaatgg tgtgatttag cagatgtatt 4260
acacggtatt gatgccacgc cgtgggcgtt cagtccgtgg atggtgatgc aggcgacaaa 4320
tcgcgaagcc agaaaacgat tatctgcatt tacccagctt aaataagaga gcagaggttg 4380
ataagttttc t 4391
<210> 19
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PKMVA-1F
<400> 19
cgcaaaaaac cccggatcca aactcgagta aggatctcc 39
<210> 20
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK1-R
<400> 20
cctaaacgat ctcgatcctc tacgc 25
<210> 21
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK2-F
<400> 21
gtagaggatc gagatcgttt aggcac 26
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK2-R
<400> 22
caacggcagc aactacatga cc 22
<210> 23
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK3-F
<400> 23
tcatgtagtt gctgccgttg g 21
<210> 24
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK3-R
<400> 24
ctctccttcg taattttgcg gaagct 26
<210> 25
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK4-F
<400> 25
cgcaaaatta cgaaggagag cgg 23
<210> 26
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PKMVA-3R
<400> 26
cgagtttgga tccggggttt tttgcgagat ccttatttaa gc 42
<210> 27
<211> 3390
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PKMVA-1F和PK1-R的PCR产物
<400> 27
cgcaaaaaac cccggatcca aactcgagta aggatctcca ggcatcaaat aaaacgaaag 60
gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctctac 120
tagagtcaca ctggctcacc ttcgggtggg cctttctgcg tttataccta gggatatatt 180
ccgcttcctc gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg 240
cttacgaacg gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga 300
gagggccgcg gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat 360
ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 420
ccctggcggc tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc 480
gctgttatgg ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc 540
gctccaagct ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg 600
gtaactatcg tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca 660
ctggtaattg atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa 720
aggacaagtt ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta 780
gctcagagaa ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag 840
attacgcgca gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc 900
tagatttcag tgcaatttat ctcttcaaat gtagcacctg aagtcagccc catacgatat 960
aagttgttac tagtgcttgg attctcacca ataaaaaacg cccggcggca accgagcgtt 1020
ctgaacaaat ccagatggag ttctgaggtc attactggat ctatcaacag gagtccaagc 1080
gagctcgata tcaaattacg ccccgccctg ccactcatcg cagtactgtt gtaattcatt 1140
aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa tcgccagcgg 1200
catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg gggcgaagaa 1260
gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg gattggctga 1320
gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt caccgtaaca 1380
cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt attcactcca 1440
gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt gaacactatc 1500
ccatatcacc agctcaccgt ctttcattgc catacgaaat tccggatgag cattcatcag 1560
gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct ttacggtctt 1620
taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag caactgactg 1680
aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg tatatccagt 1740
gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact caaaaaatac 1800
gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt gccgatcaac 1860
gtctcatttt cgccagatat cgacgtcggt gcctaatgag tgagctaact tacattaatt 1920
gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga 1980
atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt 2040
caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag 2100
caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg 2160
cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tgtccgcacc 2220
aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc 2280
aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc 2340
ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag 2400
atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa 2460
cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc 2520
ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc 2580
cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt 2640
aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc 2700
gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga 2760
tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc 2820
aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag 2880
ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt 2940
caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt 3000
tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc 3060
gcgaaaggtt ttgcgccatt cgatggtgtc cgggatctcg acgctctccc ttatgcgact 3120
cctgcattag gaagcagccc agtagtaggt tgaggccgtt gagcaccgcc gccgcaagga 3180
atggtgcatg caaggagatg gcgcccaaca gtcccccggc cacggggcct gccaccatac 3240
ccacgccgaa acaagcgctc atgagcccga agtggcgagc ccgatcttcc ccatcggtga 3300
tgtcggcgat ataggcgcca gcaaccgcac ctgtggcgcc ggtgatgccg gccacgatgc 3360
gtccggcgta gaggatcgag atcgtttagg 3390
<210> 28
<211> 3605
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK2-F和PK2-R的PCR产物
<400> 28
gtagaggatc gagatcgttt aggcacccca ggctttacac tttatgcttc cggctcgtat 60
aatgtgtgga attgtgagcg gataacaatt tcagaattca aaagatctta ggaggaatat 120
aaaatgaaaa attgtgtcat cgtcagtgcg gtacgtactg ctatcggtag ttttaacggt 180
tcactcgctt ccaccagcgc catcgacctg ggggcgacag taattaaagc cgccattgaa 240
cgtgcaaaaa tcgattcaca acacgttgat gaagtgatta tgggtaacgt gttacaagcc 300
gggctggggc aaaatccggc gcgtcaggca ctgttaaaaa gcgggctggc agaaacggtg 360
tgcggattca cggtcaataa agtatgtggt tcgggtctta aaagtgtggc gcttgccgcc 420
caggccattc aggcaggtca ggcgcagagc attgtggcgg ggggtatgga aaatatgagt 480
ttagccccct acttactcga tgcaaaagca cgctctggtt atcgtcttgg agacggacag 540
gtttatgacg taatcctgcg cgatggcctg atgtgcgcca cccatggtta tcatatgggg 600
attaccgccg aaaacgtggc taaagagtac ggaattaccc gtgaaatgca ggatgaactg 660
gcgctacatt cacagcgtaa agcggcagcc gcaattgagt ccggtgcttt tacagccgaa 720
atcgtcccgg taaatgttgt cactcgaaag aaaaccttcg tcttcagtca agacgagttc 780
ccgaaagcga actcaacggc tgaagcgtta ggtgcattgc gcccggcctt cgataaagca 840
ggaacagtca ccgctgggaa cgcgtctggt attaacgacg gtgctgccgc tctggtgatt 900
atggaagaat ctgcggcgct ggcagcaggc cttacccccc tggctcgcat taaaagttat 960
gccagcggtg gcgtgccccc cgcattgatg ggtatggggc cagtacctgc cacgcaaaaa 1020
gcgttacaac tggcggggct gcaactggcg gatattgatc tcattgaggc taatgaagca 1080
tttgctgcac agttccttgc cgttgggaaa aacctgggct ttgattctga gaaagtgaat 1140
gtcaacggcg gggccatcgc gctcgggcat cctatcggtg ccagtggtgc tcgtattctg 1200
gtcacactat tacatgccat gcaggcacgc gataaaacgc tggggctggc aacactgtgc 1260
attggcggcg gtcagggaat tgcgatggtg attgaacggt tgaattgagg atcttgaatt 1320
aaggaggaca gctaaatgac aataggtatc gataaaataa acttttacgt tccaaagtac 1380
tatgtagaca tggctaaatt agcagaagca cgccaagtag acccaaacaa atttttaatt 1440
ggaattggtc aaactgaaat ggctgttagt cctgtaaacc aagacatcgt ttcaatgggc 1500
gctaacgctg ctaaggacat tataacagac gaagacaaaa agaaaattgg tatggtaatt 1560
gtggcaactg aatcagcagt tgatgctgct aaagcagccg ctgttcaaat tcacaactta 1620
ttaggtattc aaccttttgc acgctgcttt gaaatgaaag aagcttgtta tgctgcaaca 1680
ccagcaattc aattagctaa agattattta gcaactagac cgaatgaaaa agtattagtt 1740
attgctacag atacagcacg ttatggattg aactcaggcg gcgagccaac acaaggtgct 1800
ggcgcagttg cgatggttat tgcacataat ccaagcattt tggcattaaa tgaagatgct 1860
gttgcttaca ctgaagacgt ttatgatttc tggcgtccaa ctggacataa atatccatta 1920
gttgatggtg cattatctaa agatgcttat atccgctcat tccaacaaag ctggaatgaa 1980
tacgcaaaac gtcaaggtaa gtcgctagct gacttcgcat ctctatgctt ccatgttcca 2040
tttacaaaaa tgggtaaaaa ggcattagag tcaatcattg ataacgctga tgaaacaact 2100
caagagcgtt tacgttcagg atatgaagat gctgtagatt ataaccgtta tgtcggtaat 2160
atttatactg gatcattata tttaagccta atatcattac ttgaaaatcg agatttacaa 2220
gctggtgaaa caatcggttt attcagttat ggctcaggtt cagttggtga attttatagt 2280
gcgacattag ttgaaggcta caaagatcat ttagatcaag ctgcacataa agcattatta 2340
aataaccgta ctgaagtatc tgttgatgca tatgaaacat tcttcaaacg ttttgatgac 2400
gttgaatttg acgaagaaca agatgctgtt catgaagatc gtcatatttt ctacttatca 2460
aatattgaaa ataacgttcg cgaatatcac agaccagagt aattaggatc tattcaggaa 2520
acagaccatg tccatgcaaa gtttagataa gaattttcga catttatctc gtaaagaaaa 2580
gttacaacaa ttggttgata agcaatggtt atcagaagaa caattcgaca ttttactgaa 2640
tcatccatta atcgatgaag aagtagccaa tagtttaatt gaaaatgtca tcgcgcaagg 2700
tgcattaccc gttggattat taccgaatat cattgtggac gataaggcat atgttgtacc 2760
tatgatggtg gaagagcctt cagttgtcgc tgcagctagt tatggtgcaa agctagtgaa 2820
tcagactggc ggatttaaaa cggtatcttc tgaacgtatt atgataggtc aaatcgtctt 2880
tgatggcgtt gacgatactg aaaaattatc agcagacatt aaagctttag aaaagcaaat 2940
tcataaaatt gcggatgagg catatccttc tattaaagcg cgtggtggtg gttaccaacg 3000
tatagcgatt gatacatttc ctgagcaaca gttactatct ttaaaagtat ttgttgatac 3060
gaaagatgct atgggcgcta atatgcttaa tacgatttta gaggccataa ctgcattttt 3120
aaaaaatgaa tttccgcaaa gcgacatttt aatgagtatt ttatccaatc atgcaacagc 3180
gtccgttgtt aaagttcaag gcgaaattga tgttaaagat ttagcaaggg gcgagagaac 3240
tggagaagag gttgccaaac gaatggaacg tgcttctgta ttggcccaag tagatattca 3300
tcgtgcagca acacataata aaggtgttat gaatggcata catgctgttg ttttagcaac 3360
aggaaatgat acgcgtggtg cagaagcaag tgcgcatgca tacgcgagtc gtgacggaca 3420
gtatcgtggt attgctacat ggcgttacga tcaagatcgt caacgattga ttggtacaat 3480
tgaagtgcct atgacattgg caatcgttgg cggtggtaca aaagtattac caattgctaa 3540
agcttcatta gagctactaa atgtagagtc agcacaagaa ttaggtcatg tagttgctgc 3600
cgttg 3605
<210> 29
<211> 3112
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK3-F和PK3-R的PCR产物
<400> 29
tcatgtagtt gctgccgttg gtttagcgca aaactttgca gcatgtcgcg cgcttgtgtc 60
agaaggtatt caacaaggtc atatgagttt acaatataaa tcattagcta tcgttgtagg 120
ggcaaaaggt gatgaaattg ctaaagtagc tgaagctttg aaaaaagaac cccgtgcaaa 180
tacacaagca gcggaacata ttttacaaga aattagacaa caataaggat ctttttaagg 240
atctccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc 300
tgttgtttgt cggtgaacgc tctctactag agtcacactg gctcaccttc gggtgggcct 360
ttctgcgttt atagcgaatt gatctggttt gacagcttat catcgactgc acggtgcacc 420
aatgcttctg gcgtcaggca gccatcggaa gctgtggtat ggctgtgcag gtcgtaaatc 480
actgcataat tcgtgtcgct caaggcgcac tcccgttctg gataatgttt tttgcgccga 540
catcataacg gttctggcaa atattctgaa atgagctgtt gacaattaat catccggctc 600
gtataatgtg tggaattgtg agcggataac aatttcagga tctaggagga aataaccatg 660
tctctgccat tcctgacgtc tgcgccaggt aaggtgatca tcttcggcga gcactctgcg 720
gtgtacaata agccggccgt cgccgcctct gtgtctgcgt tacgcaccta cctgctgatc 780
agcgaatctt ctgcaccgga cacgatcgag ctggactttc cggacatcag cttcaaccac 840
aagtggagca tcaacgactt caacgcgatc acggaggacc aggtgaacag ccaaaagctg 900
gccaaagccc agcaagcaac cgacggtctg tctcaggagc tggtgtctct gctggacccg 960
ctgttagcgc agttaagcga gagcttccat taccacgccg cgttctgctt cctgtacatg 1020
ttcgtttgcc tgtgcccgca cgcaaagaac atcaagttca gcctgaagag cacgctgccg 1080
attggcgcag gcttaggctc tagcgcatct atcagcgtga gcctggcgct ggcgatggcc 1140
tatctgggtg gcctgattgg cagcaacgac ctggagaaac tgagcgaaaa cgacaagcac 1200
atcgtgaacc agtgggcctt tatcggcgag aagtgcattc atggcacccc gagcggcatt 1260
gacaacgcag ttgccacgta tggcaacgcc ctgctgttcg agaaagacag ccacaacggc 1320
acgatcaaca cgaacaactt caagttcctg gacgacttcc cggcgatccc gatgattctg 1380
acctacaccc gtatcccacg cagcaccaag gatttagtcg cccgcgtgcg tgttttagtc 1440
accgaaaagt tcccggaggt gatgaagccg atcctggacg cgatgggcga gtgcgcgctg 1500
cagggtctgg agatcatgac caagctgagc aagtgcaagg gcaccgacga tgaggcggtg 1560
gagaccaaca atgagctgta cgagcagctg ctggagctga tccgtatcaa tcacggcctg 1620
ctggtctcta tcggtgtgtc tcacccgggc ctggaactga tcaaaaacct gagcgacgac 1680
ctgcgcattg gctctacgaa attaacgggt gcaggtggcg gtggctgctc tttaacgctg 1740
ctgcgccgtg acattacgca ggagcaaatc gacagcttca agaagaagct gcaggacgac 1800
ttcagctacg agacgttcga gacggacctg ggcggcacgg gctgttgcct gctgagcgcc 1860
aaaaatctga acaaggacct gaagatcaaa agcctggtgt tccagctgtt cgaaaacaag 1920
acgaccacga agcagcagat cgacgacctg ttactgccgg gtaacaccaa tctgccgtgg 1980
acgtcttaag gatctaggag ggagatcata tgagcgaatt acgtgcattc agcgcgccag 2040
gtaaggcact gctggccggt ggctacctgg tgttagacac caagtacgag gcgttcgtcg 2100
tcggcttatc tgcccgtatg catgcagttg cccacccgta tggtagcctg cagggctctg 2160
acaagttcga agtgcgtgtg aagagcaagc agttcaagga cggcgagtgg ctgtaccaca 2220
ttagcccaaa gagcggcttc atcccggtta gcattggtgg cagcaagaac ccatttatcg 2280
agaaggtcat tgccaacgtc ttcagctact tcaagccgaa tatggacgat tactgcaacc 2340
gcaacctgtt cgtcatcgac attttcagcg acgacgcgta ccacagccaa gaggactctg 2400
ttacggagca tcgtggtaac cgccgcctga gcttccacag ccatcgcatt gaggaggtgc 2460
cgaagacggg tctgggttct agcgccggtt tagttaccgt cttaacgacg gcgttagcga 2520
gcttcttcgt gagcgacctg gagaacaacg tggacaagta ccgcgaagtg attcataacc 2580
tggcgcaggt ggcacattgt caggcccaag gtaagattgg ctctggtttt gatgtggcag 2640
cggccgccta tggctctatc cgctatcgcc gctttccgcc ggccctgatc agcaatctgc 2700
cggacatcgg ctctgcgacg tatggtagca aactggcgca tctggtggac gaagaagact 2760
ggaacatcac cattaagtct aatcacctgc cgagcggctt aacgttatgg atgggcgata 2820
tcaagaacgg cagcgaaacg gttaagctgg tgcagaaagt gaaaaactgg tacgacagcc 2880
acatgccgga aagcctgaag atttacacgg agctggacca cgccaatagc cgtttcatgg 2940
atggtctgag caagctggac cgcctgcacg aaacccacga cgactacagc gaccaaatct 3000
tcgagagcct ggagcgcaat gactgcacct gccagaagta cccggagatc acggaggtcc 3060
gcgatgccgt ggcaacgatt cgccgtagct tccgcaaaat tacgaaggag ag 3112
<210> 30
<211> 2085
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> PK4-F和PKMVA-3R的PCR产物
<400> 30
cgcaaaatta cgaaggagag cggcgcggat atcgaaccac cggtccagac gtctctgctg 60
gacgactgtc aaaccttaaa gggcgtgtta acgtgcctga ttccgggcgc gggtggttac 120
gacgccattg ccgtcatcac gaaacaggac gtcgatctgc gcgcacaaac ggccaacgac 180
aaacgtttca gcaaagtcca atggctggat gttacgcagg ccgactgggg tgttcgcaag 240
gagaaggacc cggaaacgta tctggataag tgaggatcta ggaggattat gagatgaccg 300
tttacacagc atccgttacc gcacccgtca acatcgcaac ccttaagtat tgggggaaaa 360
gggacacgaa gttgaatctg cccaccaatt cgtccatatc agtgacttta tcgcaagatg 420
acctcagaac gttgacctct gcggctactg cacctgagtt tgaacgcgac actttgtggt 480
taaatggaga accacacagc atcgacaatg aaagaactca aaattgtctg cgcgacctac 540
gccaattaag aaaggaaatg gaatcgaagg acgcctcatt gcccacatta tctcaatgga 600
aactccacat tgtctccgaa aataactttc ctacagcagc tggtttagct tcctccgctg 660
ctggctttgc tgcattggtc tctgcaattg ctaagttata ccaattacca cagtcaactt 720
cagaaatatc tagaatagca agaaaggggt ctggttcagc ttgtagatcg ttgtttggcg 780
gatacgtggc ctgggaaatg ggaaaagctg aagatggtca tgattccatg gcagtacaaa 840
tcgcagacag ctctgactgg cctcagatga aagcttgtgt cctagttgtc agcgatatta 900
aaaaggatgt gagttccact cagggtatgc aattgaccgt ggcaacctcc gaactattta 960
aagaaagaat tgaacatgtc gtaccaaaga gatttgaagt catgcgtaaa gccattgttg 1020
aaaaagattt cgccaccttt gcaaaggaaa caatgatgga ttccaactct ttccatgcca 1080
catgtttgga ctctttccct ccaatattct acatgaatga cacttccaag cgtatcatca 1140
gttggtgcca caccattaat cagttttacg gagaaacaat cgttgcatac acgtttgatg 1200
caggtccaaa tgctgtgttg tactacttag ctgaaaatga gtcgaaactc tttgcattta 1260
tctataaatt gtttggctct gttcctggat gggacaagaa atttactact gagcagcttg 1320
aggctttcaa ccatcaattt gaatcatcta actttactgc acgtgaattg gatcttgagt 1380
tgcaaaagga tgttgccaga gtgattttaa ctcaagtcgg ttcaggccca caagaaacaa 1440
acgaatcttt gattgacgca aagactggtc taccaaagga ataaggatct aggaggtaat 1500
gataatgcaa acggaacacg tcattttatt gaatgcacag ggagttccca cgggtacgct 1560
ggaaaagtat gccgcacaca cggcagacac ccgcttacat ctcgcgttct ccagttggct 1620
gtttaatgcc aaaggacaat tattagttac ccgccgcgca ctgagcaaaa aagcatggcc 1680
tggcgtgtgg actaactcgg tttgtgggca cccacaactg ggagaaagca acgaagacgc 1740
agtgatccgc cgttgccgtt atgagcttgg cgtggaaatt acgcctcctg aatctatcta 1800
tcctgacttt cgctaccgcg ccaccgatcc gagtggcatt gtggaaaatg aagtgtgtcc 1860
ggtatttgcc gcacgcacca ctagtgcgtt acagatcaat gatgatgaag tgatggatta 1920
tcaatggtgt gatttagcag atgtattaca cggtattgat gccacgccgt gggcgttcag 1980
tccgtggatg gtgatgcagg cgacaaatcg cgaagccaga aaacgattat ctgcatttac 2040
ccagcttaaa taaggatctc gcaaaaaacc ccggatccaa actcg 2085
<210> 31
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> lac UV5 启动子
<400> 31
ttgacaatta atcatccggc tcgtataatg tgtggaattg tgagcggata acaa 54
<210> 32
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pTac 启动子
<400> 32
tgttgacaat taatcatcgg ctcgtataat gtgtggaatt gtgagcgctc acaatt 56
<210> 33
<211> 62
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pRha 启动子
<400> 33
aggtcgcgaa ttcaggcgct ttttagactg gtcgtaatga aattcaacta gtgctctgca 60
gg 62
<210> 34
<211> 59
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pTet 启动子
<400> 34
ttttttccct atcagtgata gagattgaca tccctatcag tgatagagat aatgagcac 59
<210> 35
<211> 81
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> pBAD 启动子
<400> 35
ttgctatgcc atagcatttt tatccataag attagcggat cctacctgac gctttttatc 60
gcaactctct actgtttctc c 81
<210> 36
<211> 54
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> sp2
<400> 36
tcaaaaagag tattgactta tattgagtcg tataggatac ttacagccat cgag 54
<210> 37
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<223> 绝缘子RiboJ部分
<400> 37
agctgtcacc ggatgtgctt tccggtctga tgagtccgtg aggacgaaac agcctctaca 60
aataattttg tttaa 75
<210> 38
<211> 394
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> atoB编码的氨基酸序列
<400> 38
Met Lys Asn Cys Val Ile Val Ser Ala Val Arg Thr Ala Ile Gly Ser
1 5 10 15
Phe Asn Gly Ser Leu Ala Ser Thr Ser Ala Ile Asp Leu Gly Ala Thr
20 25 30
Val Ile Lys Ala Ala Ile Glu Arg Ala Lys Ile Asp Ser Gln His Val
35 40 45
Asp Glu Val Ile Met Gly Asn Val Leu Gln Ala Gly Leu Gly Gln Asn
50 55 60
Pro Ala Arg Gln Ala Leu Leu Lys Ser Gly Leu Ala Glu Thr Val Cys
65 70 75 80
Gly Phe Thr Val Asn Lys Val Cys Gly Ser Gly Leu Lys Ser Val Ala
85 90 95
Leu Ala Ala Gln Ala Ile Gln Ala Gly Gln Ala Gln Ser Ile Val Ala
100 105 110
Gly Gly Met Glu Asn Met Ser Leu Ala Pro Tyr Leu Leu Asp Ala Lys
115 120 125
Ala Arg Ser Gly Tyr Arg Leu Gly Asp Gly Gln Val Tyr Asp Val Ile
130 135 140
Leu Arg Asp Gly Leu Met Cys Ala Thr His Gly Tyr His Met Gly Ile
145 150 155 160
Thr Ala Glu Asn Val Ala Lys Glu Tyr Gly Ile Thr Arg Glu Met Gln
165 170 175
Asp Glu Leu Ala Leu His Ser Gln Arg Lys Ala Ala Ala Ala Ile Glu
180 185 190
Ser Gly Ala Phe Thr Ala Glu Ile Val Pro Val Asn Val Val Thr Arg
195 200 205
Lys Lys Thr Phe Val Phe Ser Gln Asp Glu Phe Pro Lys Ala Asn Ser
210 215 220
Thr Ala Glu Ala Leu Gly Ala Leu Arg Pro Ala Phe Asp Lys Ala Gly
225 230 235 240
Thr Val Thr Ala Gly Asn Ala Ser Gly Ile Asn Asp Gly Ala Ala Ala
245 250 255
Leu Val Ile Met Glu Glu Ser Ala Ala Leu Ala Ala Gly Leu Thr Pro
260 265 270
Leu Ala Arg Ile Lys Ser Tyr Ala Ser Gly Gly Val Pro Pro Ala Leu
275 280 285
Met Gly Met Gly Pro Val Pro Ala Thr Gln Lys Ala Leu Gln Leu Ala
290 295 300
Gly Leu Gln Leu Ala Asp Ile Asp Leu Ile Glu Ala Asn Glu Ala Phe
305 310 315 320
Ala Ala Gln Phe Leu Ala Val Gly Lys Asn Leu Gly Phe Asp Ser Glu
325 330 335
Lys Val Asn Val Asn Gly Gly Ala Ile Ala Leu Gly His Pro Ile Gly
340 345 350
Ala Ser Gly Ala Arg Ile Leu Val Thr Leu Leu His Ala Met Gln Ala
355 360 365
Arg Asp Lys Thr Leu Gly Leu Ala Thr Leu Cys Ile Gly Gly Gly Gln
370 375 380
Gly Ile Ala Met Val Ile Glu Arg Leu Asn
385 390
<210> 39
<211> 388
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> HMGS编码的氨基酸序列
<400> 39
Met Thr Ile Gly Ile Asp Lys Ile Asn Phe Tyr Val Pro Lys Tyr Tyr
1 5 10 15
Val Asp Met Ala Lys Leu Ala Glu Ala Arg Gln Val Asp Pro Asn Lys
20 25 30
Phe Leu Ile Gly Ile Gly Gln Thr Glu Met Ala Val Ser Pro Val Asn
35 40 45
Gln Asp Ile Val Ser Met Gly Ala Asn Ala Ala Lys Asp Ile Ile Thr
50 55 60
Asp Glu Asp Lys Lys Lys Ile Gly Met Val Ile Val Ala Thr Glu Ser
65 70 75 80
Ala Val Asp Ala Ala Lys Ala Ala Ala Val Gln Ile His Asn Leu Leu
85 90 95
Gly Ile Gln Pro Phe Ala Arg Cys Phe Glu Met Lys Glu Ala Cys Tyr
100 105 110
Ala Ala Thr Pro Ala Ile Gln Leu Ala Lys Asp Tyr Leu Ala Thr Arg
115 120 125
Pro Asn Glu Lys Val Leu Val Ile Ala Thr Asp Thr Ala Arg Tyr Gly
130 135 140
Leu Asn Ser Gly Gly Glu Pro Thr Gln Gly Ala Gly Ala Val Ala Met
145 150 155 160
Val Ile Ala His Asn Pro Ser Ile Leu Ala Leu Asn Glu Asp Ala Val
165 170 175
Ala Tyr Thr Glu Asp Val Tyr Asp Phe Trp Arg Pro Thr Gly His Lys
180 185 190
Tyr Pro Leu Val Asp Gly Ala Leu Ser Lys Asp Ala Tyr Ile Arg Ser
195 200 205
Phe Gln Gln Ser Trp Asn Glu Tyr Ala Lys Arg Gln Gly Lys Ser Leu
210 215 220
Ala Asp Phe Ala Ser Leu Cys Phe His Val Pro Phe Thr Lys Met Gly
225 230 235 240
Lys Lys Ala Leu Glu Ser Ile Ile Asp Asn Ala Asp Glu Thr Thr Gln
245 250 255
Glu Arg Leu Arg Ser Gly Tyr Glu Asp Ala Val Asp Tyr Asn Arg Tyr
260 265 270
Val Gly Asn Ile Tyr Thr Gly Ser Leu Tyr Leu Ser Leu Ile Ser Leu
275 280 285
Leu Glu Asn Arg Asp Leu Gln Ala Gly Glu Thr Ile Gly Leu Phe Ser
290 295 300
Tyr Gly Ser Gly Ser Val Gly Glu Phe Tyr Ser Ala Thr Leu Val Glu
305 310 315 320
Gly Tyr Lys Asp His Leu Asp Gln Ala Ala His Lys Ala Leu Leu Asn
325 330 335
Asn Arg Thr Glu Val Ser Val Asp Ala Tyr Glu Thr Phe Phe Lys Arg
340 345 350
Phe Asp Asp Val Glu Phe Asp Glu Glu Gln Asp Ala Val His Glu Asp
355 360 365
Arg His Ile Phe Tyr Leu Ser Asn Ile Glu Asn Asn Val Arg Glu Tyr
370 375 380
His Arg Pro Glu
385
<210> 40
<211> 427
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> HMGR编码的氨基酸序列
<400> 40
Met Ser Met Gln Ser Leu Asp Lys Asn Phe Arg His Leu Ser Arg Lys
1 5 10 15
Glu Lys Leu Gln Gln Leu Val Asp Lys Gln Trp Leu Ser Glu Glu Gln
20 25 30
Phe Asp Ile Leu Leu Asn His Pro Leu Ile Asp Glu Glu Val Ala Asn
35 40 45
Ser Leu Ile Glu Asn Val Ile Ala Gln Gly Ala Leu Pro Val Gly Leu
50 55 60
Leu Pro Asn Ile Ile Val Asp Asp Lys Ala Tyr Val Val Pro Met Met
65 70 75 80
Val Glu Glu Pro Ser Val Val Ala Ala Ala Ser Tyr Gly Ala Lys Leu
85 90 95
Val Asn Gln Thr Gly Gly Phe Lys Thr Val Ser Ser Glu Arg Ile Met
100 105 110
Ile Gly Gln Ile Val Phe Asp Gly Val Asp Asp Thr Glu Lys Leu Ser
115 120 125
Ala Asp Ile Lys Ala Leu Glu Lys Gln Ile His Lys Ile Ala Asp Glu
130 135 140
Ala Tyr Pro Ser Ile Lys Ala Arg Gly Gly Gly Tyr Gln Arg Ile Ala
145 150 155 160
Ile Asp Thr Phe Pro Glu Gln Gln Leu Leu Ser Leu Lys Val Phe Val
165 170 175
Asp Thr Lys Asp Ala Met Gly Ala Asn Met Leu Asn Thr Ile Leu Glu
180 185 190
Ala Ile Thr Ala Phe Leu Lys Asn Glu Phe Pro Gln Ser Asp Ile Leu
195 200 205
Met Ser Ile Leu Ser Asn His Ala Thr Ala Ser Val Val Lys Val Gln
210 215 220
Gly Glu Ile Asp Val Lys Asp Leu Ala Arg Gly Glu Arg Thr Gly Glu
225 230 235 240
Glu Val Ala Lys Arg Met Glu Arg Ala Ser Val Leu Ala Gln Val Asp
245 250 255
Ile His Arg Ala Ala Thr His Asn Lys Gly Val Met Asn Gly Ile His
260 265 270
Ala Val Val Leu Ala Thr Gly Asn Asp Thr Arg Gly Ala Glu Ala Ser
275 280 285
Ala His Ala Tyr Ala Ser Arg Asp Gly Gln Tyr Arg Gly Ile Ala Thr
290 295 300
Trp Arg Tyr Asp Gln Asp Arg Gln Arg Leu Ile Gly Thr Ile Glu Val
305 310 315 320
Pro Met Thr Leu Ala Ile Val Gly Gly Gly Thr Lys Val Leu Pro Ile
325 330 335
Ala Lys Ala Ser Leu Glu Leu Leu Asn Val Glu Ser Ala Gln Glu Leu
340 345 350
Gly His Val Val Ala Ala Val Gly Leu Ala Gln Asn Phe Ala Ala Cys
355 360 365
Arg Ala Leu Val Ser Glu Gly Ile Gln Gln Gly His Met Ser Leu Gln
370 375 380
Tyr Lys Ser Leu Ala Ile Val Val Gly Ala Lys Gly Asp Glu Ile Ala
385 390 395 400
Lys Val Ala Glu Ala Leu Lys Lys Glu Pro Arg Ala Asn Thr Gln Ala
405 410 415
Ala Glu His Ile Leu Gln Glu Ile Arg Gln Gln
420 425
<210> 41
<211> 443
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> MK编码的氨基酸序列
<400> 41
Met Ser Leu Pro Phe Leu Thr Ser Ala Pro Gly Lys Val Ile Ile Phe
1 5 10 15
Gly Glu His Ser Ala Val Tyr Asn Lys Pro Ala Val Ala Ala Ser Val
20 25 30
Ser Ala Leu Arg Thr Tyr Leu Leu Ile Ser Glu Ser Ser Ala Pro Asp
35 40 45
Thr Ile Glu Leu Asp Phe Pro Asp Ile Ser Phe Asn His Lys Trp Ser
50 55 60
Ile Asn Asp Phe Asn Ala Ile Thr Glu Asp Gln Val Asn Ser Gln Lys
65 70 75 80
Leu Ala Lys Ala Gln Gln Ala Thr Asp Gly Leu Ser Gln Glu Leu Val
85 90 95
Ser Leu Leu Asp Pro Leu Leu Ala Gln Leu Ser Glu Ser Phe His Tyr
100 105 110
His Ala Ala Phe Cys Phe Leu Tyr Met Phe Val Cys Leu Cys Pro His
115 120 125
Ala Lys Asn Ile Lys Phe Ser Leu Lys Ser Thr Leu Pro Ile Gly Ala
130 135 140
Gly Leu Gly Ser Ser Ala Ser Ile Ser Val Ser Leu Ala Leu Ala Met
145 150 155 160
Ala Tyr Leu Gly Gly Leu Ile Gly Ser Asn Asp Leu Glu Lys Leu Ser
165 170 175
Glu Asn Asp Lys His Ile Val Asn Gln Trp Ala Phe Ile Gly Glu Lys
180 185 190
Cys Ile His Gly Thr Pro Ser Gly Ile Asp Asn Ala Val Ala Thr Tyr
195 200 205
Gly Asn Ala Leu Leu Phe Glu Lys Asp Ser His Asn Gly Thr Ile Asn
210 215 220
Thr Asn Asn Phe Lys Phe Leu Asp Asp Phe Pro Ala Ile Pro Met Ile
225 230 235 240
Leu Thr Tyr Thr Arg Ile Pro Arg Ser Thr Lys Asp Leu Val Ala Arg
245 250 255
Val Arg Val Leu Val Thr Glu Lys Phe Pro Glu Val Met Lys Pro Ile
260 265 270
Leu Asp Ala Met Gly Glu Cys Ala Leu Gln Gly Leu Glu Ile Met Thr
275 280 285
Lys Leu Ser Lys Cys Lys Gly Thr Asp Asp Glu Ala Val Glu Thr Asn
290 295 300
Asn Glu Leu Tyr Glu Gln Leu Leu Glu Leu Ile Arg Ile Asn His Gly
305 310 315 320
Leu Leu Val Ser Ile Gly Val Ser His Pro Gly Leu Glu Leu Ile Lys
325 330 335
Asn Leu Ser Asp Asp Leu Arg Ile Gly Ser Thr Lys Leu Thr Gly Ala
340 345 350
Gly Gly Gly Gly Cys Ser Leu Thr Leu Leu Arg Arg Asp Ile Thr Gln
355 360 365
Glu Gln Ile Asp Ser Phe Lys Lys Lys Leu Gln Asp Asp Phe Ser Tyr
370 375 380
Glu Thr Phe Glu Thr Asp Leu Gly Gly Thr Gly Cys Cys Leu Leu Ser
385 390 395 400
Ala Lys Asn Leu Asn Lys Asp Leu Lys Ile Lys Ser Leu Val Phe Gln
405 410 415
Leu Phe Glu Asn Lys Thr Thr Thr Lys Gln Gln Ile Asp Asp Leu Leu
420 425 430
Leu Pro Gly Asn Thr Asn Leu Pro Trp Thr Ser
435 440
<210> 42
<211> 451
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> PMK编码的氨基酸序列
<400> 42
Met Ser Glu Leu Arg Ala Phe Ser Ala Pro Gly Lys Ala Leu Leu Ala
1 5 10 15
Gly Gly Tyr Leu Val Leu Asp Thr Lys Tyr Glu Ala Phe Val Val Gly
20 25 30
Leu Ser Ala Arg Met His Ala Val Ala His Pro Tyr Gly Ser Leu Gln
35 40 45
Gly Ser Asp Lys Phe Glu Val Arg Val Lys Ser Lys Gln Phe Lys Asp
50 55 60
Gly Glu Trp Leu Tyr His Ile Ser Pro Lys Ser Gly Phe Ile Pro Val
65 70 75 80
Ser Ile Gly Gly Ser Lys Asn Pro Phe Ile Glu Lys Val Ile Ala Asn
85 90 95
Val Phe Ser Tyr Phe Lys Pro Asn Met Asp Asp Tyr Cys Asn Arg Asn
100 105 110
Leu Phe Val Ile Asp Ile Phe Ser Asp Asp Ala Tyr His Ser Gln Glu
115 120 125
Asp Ser Val Thr Glu His Arg Gly Asn Arg Arg Leu Ser Phe His Ser
130 135 140
His Arg Ile Glu Glu Val Pro Lys Thr Gly Leu Gly Ser Ser Ala Gly
145 150 155 160
Leu Val Thr Val Leu Thr Thr Ala Leu Ala Ser Phe Phe Val Ser Asp
165 170 175
Leu Glu Asn Asn Val Asp Lys Tyr Arg Glu Val Ile His Asn Leu Ala
180 185 190
Gln Val Ala His Cys Gln Ala Gln Gly Lys Ile Gly Ser Gly Phe Asp
195 200 205
Val Ala Ala Ala Ala Tyr Gly Ser Ile Arg Tyr Arg Arg Phe Pro Pro
210 215 220
Ala Leu Ile Ser Asn Leu Pro Asp Ile Gly Ser Ala Thr Tyr Gly Ser
225 230 235 240
Lys Leu Ala His Leu Val Asp Glu Glu Asp Trp Asn Ile Thr Ile Lys
245 250 255
Ser Asn His Leu Pro Ser Gly Leu Thr Leu Trp Met Gly Asp Ile Lys
260 265 270
Asn Gly Ser Glu Thr Val Lys Leu Val Gln Lys Val Lys Asn Trp Tyr
275 280 285
Asp Ser His Met Pro Glu Ser Leu Lys Ile Tyr Thr Glu Leu Asp His
290 295 300
Ala Asn Ser Arg Phe Met Asp Gly Leu Ser Lys Leu Asp Arg Leu His
305 310 315 320
Glu Thr His Asp Asp Tyr Ser Asp Gln Ile Phe Glu Ser Leu Glu Arg
325 330 335
Asn Asp Cys Thr Cys Gln Lys Tyr Pro Glu Ile Thr Glu Val Arg Asp
340 345 350
Ala Val Ala Thr Ile Arg Arg Ser Phe Arg Lys Ile Thr Lys Glu Ser
355 360 365
Gly Ala Asp Ile Glu Pro Pro Val Gln Thr Ser Leu Leu Asp Asp Cys
370 375 380
Gln Thr Leu Lys Gly Val Leu Thr Cys Leu Ile Pro Gly Ala Gly Gly
385 390 395 400
Tyr Asp Ala Ile Ala Val Ile Thr Lys Gln Asp Val Asp Leu Arg Ala
405 410 415
Gln Thr Ala Asn Asp Lys Arg Phe Ser Lys Val Gln Trp Leu Asp Val
420 425 430
Thr Gln Ala Asp Trp Gly Val Arg Lys Glu Lys Asp Pro Glu Thr Tyr
435 440 445
Leu Asp Lys
450
<210> 43
<211> 396
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> PMD编码的氨基酸序列
<400> 43
Met Thr Val Tyr Thr Ala Ser Val Thr Ala Pro Val Asn Ile Ala Thr
1 5 10 15
Leu Lys Tyr Trp Gly Lys Arg Asp Thr Lys Leu Asn Leu Pro Thr Asn
20 25 30
Ser Ser Ile Ser Val Thr Leu Ser Gln Asp Asp Leu Arg Thr Leu Thr
35 40 45
Ser Ala Ala Thr Ala Pro Glu Phe Glu Arg Asp Thr Leu Trp Leu Asn
50 55 60
Gly Glu Pro His Ser Ile Asp Asn Glu Arg Thr Gln Asn Cys Leu Arg
65 70 75 80
Asp Leu Arg Gln Leu Arg Lys Glu Met Glu Ser Lys Asp Ala Ser Leu
85 90 95
Pro Thr Leu Ser Gln Trp Lys Leu His Ile Val Ser Glu Asn Asn Phe
100 105 110
Pro Thr Ala Ala Gly Leu Ala Ser Ser Ala Ala Gly Phe Ala Ala Leu
115 120 125
Val Ser Ala Ile Ala Lys Leu Tyr Gln Leu Pro Gln Ser Thr Ser Glu
130 135 140
Ile Ser Arg Ile Ala Arg Lys Gly Ser Gly Ser Ala Cys Arg Ser Leu
145 150 155 160
Phe Gly Gly Tyr Val Ala Trp Glu Met Gly Lys Ala Glu Asp Gly His
165 170 175
Asp Ser Met Ala Val Gln Ile Ala Asp Ser Ser Asp Trp Pro Gln Met
180 185 190
Lys Ala Cys Val Leu Val Val Ser Asp Ile Lys Lys Asp Val Ser Ser
195 200 205
Thr Gln Gly Met Gln Leu Thr Val Ala Thr Ser Glu Leu Phe Lys Glu
210 215 220
Arg Ile Glu His Val Val Pro Lys Arg Phe Glu Val Met Arg Lys Ala
225 230 235 240
Ile Val Glu Lys Asp Phe Ala Thr Phe Ala Lys Glu Thr Met Met Asp
245 250 255
Ser Asn Ser Phe His Ala Thr Cys Leu Asp Ser Phe Pro Pro Ile Phe
260 265 270
Tyr Met Asn Asp Thr Ser Lys Arg Ile Ile Ser Trp Cys His Thr Ile
275 280 285
Asn Gln Phe Tyr Gly Glu Thr Ile Val Ala Tyr Thr Phe Asp Ala Gly
290 295 300
Pro Asn Ala Val Leu Tyr Tyr Leu Ala Glu Asn Glu Ser Lys Leu Phe
305 310 315 320
Ala Phe Ile Tyr Lys Leu Phe Gly Ser Val Pro Gly Trp Asp Lys Lys
325 330 335
Phe Thr Thr Glu Gln Leu Glu Ala Phe Asn His Gln Phe Glu Ser Ser
340 345 350
Asn Phe Thr Ala Arg Glu Leu Asp Leu Glu Leu Gln Lys Asp Val Ala
355 360 365
Arg Val Ile Leu Thr Gln Val Gly Ser Gly Pro Gln Glu Thr Asn Glu
370 375 380
Ser Leu Ile Asp Ala Lys Thr Gly Leu Pro Lys Glu
385 390 395
<210> 44
<211> 182
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> Idi编码的氨基酸序列
<400> 44
Met Gln Thr Glu His Val Ile Leu Leu Asn Ala Gln Gly Val Pro Thr
1 5 10 15
Gly Thr Leu Glu Lys Tyr Ala Ala His Thr Ala Asp Thr Arg Leu His
20 25 30
Leu Ala Phe Ser Ser Trp Leu Phe Asn Ala Lys Gly Gln Leu Leu Val
35 40 45
Thr Arg Arg Ala Leu Ser Lys Lys Ala Trp Pro Gly Val Trp Thr Asn
50 55 60
Ser Val Cys Gly His Pro Gln Leu Gly Glu Ser Asn Glu Asp Ala Val
65 70 75 80
Ile Arg Arg Cys Arg Tyr Glu Leu Gly Val Glu Ile Thr Pro Pro Glu
85 90 95
Ser Ile Tyr Pro Asp Phe Arg Tyr Arg Ala Thr Asp Pro Ser Gly Ile
100 105 110
Val Glu Asn Glu Val Cys Pro Val Phe Ala Ala Arg Thr Thr Ser Ala
115 120 125
Leu Gln Ile Asn Asp Asp Glu Val Met Asp Tyr Gln Trp Cys Asp Leu
130 135 140
Ala Asp Val Leu His Gly Ile Asp Ala Thr Pro Trp Ala Phe Ser Pro
145 150 155 160
Trp Met Val Met Gln Ala Thr Asn Arg Glu Ala Arg Lys Arg Leu Ser
165 170 175
Ala Phe Thr Gln Leu Lys
180
<210> 45
<211> 297
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> GPPS编码的氨基酸序列
<400> 45
Met Glu Phe Asp Phe Asn Lys Tyr Met Asp Ser Lys Ala Met Thr Val
1 5 10 15
Asn Glu Ala Leu Asn Lys Ala Ile Pro Leu Arg Tyr Pro Gln Lys Ile
20 25 30
Tyr Glu Ser Met Arg Tyr Ser Leu Leu Ala Gly Gly Lys Arg Val Arg
35 40 45
Pro Val Leu Cys Ile Ala Ala Cys Glu Leu Val Gly Gly Thr Glu Glu
50 55 60
Leu Ala Ile Pro Thr Ala Cys Ala Ile Glu Met Ile His Thr Met Ser
65 70 75 80
Leu Met His Asp Asp Leu Pro Cys Ile Asp Asn Asp Asp Leu Arg Arg
85 90 95
Gly Lys Pro Thr Asn His Lys Ile Phe Gly Glu Asp Thr Ala Val Thr
100 105 110
Ala Gly Asn Ala Leu His Ser Tyr Ala Phe Glu His Ile Ala Val Ser
115 120 125
Thr Ser Lys Thr Val Gly Ala Asp Arg Ile Leu Arg Met Val Ser Glu
130 135 140
Leu Gly Arg Ala Thr Gly Ser Glu Gly Val Met Gly Gly Gln Met Val
145 150 155 160
Asp Ile Ala Ser Glu Gly Asp Pro Ser Ile Asp Leu Gln Thr Leu Glu
165 170 175
Trp Ile His Ile His Lys Thr Ala Met Leu Leu Glu Cys Ser Val Val
180 185 190
Cys Gly Ala Ile Ile Gly Gly Ala Ser Glu Ile Val Ile Glu Arg Ala
195 200 205
Arg Arg Tyr Ala Arg Cys Val Gly Leu Leu Phe Gln Val Val Asp Asp
210 215 220
Ile Leu Asp Val Thr Lys Ser Ser Asp Glu Leu Gly Lys Thr Ala Gly
225 230 235 240
Lys Asp Leu Ile Ser Asp Lys Ala Thr Tyr Pro Lys Leu Met Gly Leu
245 250 255
Glu Lys Ala Lys Glu Phe Ser Asp Glu Leu Leu Asn Arg Ala Lys Gly
260 265 270
Glu Leu Ser Cys Phe Asp Pro Val Lys Ala Ala Pro Leu Leu Gly Leu
275 280 285
Ala Asp Tyr Val Ala Phe Arg Gln Asn
290 295
<210> 46
<211> 567
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> GES编码的氨基酸序列
<400> 46
Met Ser Cys Ala Arg Ile Thr Val Thr Leu Pro Tyr Arg Ser Ala Lys
1 5 10 15
Thr Ser Ile Gln Arg Gly Ile Thr His Tyr Pro Ala Leu Ile Arg Pro
20 25 30
Arg Phe Ser Ala Cys Thr Pro Leu Ala Ser Ala Met Pro Leu Ser Ser
35 40 45
Thr Pro Leu Ile Asn Gly Asp Asn Ser Gln Arg Lys Asn Thr Arg Gln
50 55 60
His Met Glu Glu Ser Ser Ser Lys Arg Arg Glu Tyr Leu Leu Glu Glu
65 70 75 80
Thr Thr Arg Lys Leu Gln Arg Asn Asp Thr Glu Ser Val Glu Lys Leu
85 90 95
Lys Leu Ile Asp Asn Ile Gln Gln Leu Gly Ile Gly Tyr Tyr Phe Glu
100 105 110
Asp Ala Ile Asn Ala Val Leu Arg Ser Pro Phe Ser Thr Gly Glu Glu
115 120 125
Asp Leu Phe Thr Ala Ala Leu Arg Phe Arg Leu Leu Arg His Asn Gly
130 135 140
Ile Glu Ile Ser Pro Glu Ile Phe Leu Lys Phe Lys Asp Glu Arg Gly
145 150 155 160
Lys Phe Asp Glu Ser Asp Thr Leu Gly Leu Leu Ser Leu Tyr Glu Ala
165 170 175
Ser Asn Leu Gly Val Ala Gly Glu Glu Ile Leu Glu Glu Ala Met Glu
180 185 190
Phe Ala Glu Ala Arg Leu Arg Arg Ser Leu Ser Glu Pro Ala Ala Pro
195 200 205
Leu His Gly Glu Val Ala Gln Ala Leu Asp Val Pro Arg His Leu Arg
210 215 220
Met Ala Arg Leu Glu Ala Arg Arg Phe Ile Glu Gln Tyr Gly Lys Gln
225 230 235 240
Ser Asp His Asp Gly Asp Leu Leu Glu Leu Ala Ile Leu Asp Tyr Asn
245 250 255
Gln Val Gln Ala Gln His Gln Ser Glu Leu Thr Glu Ile Ile Arg Trp
260 265 270
Trp Lys Glu Leu Gly Leu Val Asp Lys Leu Ser Phe Gly Arg Asp Arg
275 280 285
Pro Leu Glu Cys Phe Leu Trp Thr Val Gly Leu Leu Pro Glu Pro Lys
290 295 300
Tyr Ser Ser Val Arg Ile Glu Leu Ala Lys Ala Ile Ser Ile Leu Leu
305 310 315 320
Val Ile Asp Asp Ile Phe Asp Thr Tyr Gly Glu Met Asp Asp Leu Ile
325 330 335
Leu Phe Thr Asp Ala Ile Arg Arg Trp Asp Leu Glu Ala Met Glu Gly
340 345 350
Leu Pro Glu Tyr Met Lys Ile Cys Tyr Met Ala Leu Tyr Asn Thr Thr
355 360 365
Asn Glu Val Cys Tyr Lys Val Leu Arg Asp Thr Gly Arg Ile Val Leu
370 375 380
Leu Asn Leu Lys Ser Thr Trp Ile Asp Met Ile Glu Gly Phe Met Glu
385 390 395 400
Glu Ala Lys Trp Phe Asn Gly Gly Ser Ala Pro Lys Leu Glu Glu Tyr
405 410 415
Ile Glu Asn Gly Val Ser Thr Ala Gly Ala Tyr Met Ala Phe Ala His
420 425 430
Ile Phe Phe Leu Ile Gly Glu Gly Val Thr His Gln Asn Ser Gln Leu
435 440 445
Phe Thr Gln Lys Pro Tyr Pro Lys Val Phe Ser Ala Ala Gly Arg Ile
450 455 460
Leu Arg Leu Trp Asp Asp Leu Gly Thr Ala Lys Glu Glu Gln Glu Arg
465 470 475 480
Gly Asp Leu Ala Ser Cys Val Gln Leu Phe Met Lys Glu Lys Ser Leu
485 490 495
Thr Glu Glu Glu Ala Arg Ser Arg Ile Leu Glu Glu Ile Lys Gly Leu
500 505 510
Trp Arg Asp Leu Asn Gly Glu Leu Val Tyr Asn Lys Asn Leu Pro Leu
515 520 525
Ser Ile Ile Lys Val Ala Leu Asn Met Ala Arg Ala Ser Gln Val Val
530 535 540
Tyr Lys His Asp Gln Asp Thr Tyr Phe Ser Ser Val Asp Asn Tyr Val
545 550 555 560
Asp Ala Leu Phe Phe Thr Gln
565
<210> 47
<211> 373
<212> PRT
<213> 人工序列(Artificial Sequence)
<220>
<223> geoA编码的氨基酸序列
<400> 47
Met Asn Asp Thr Gln Asp Phe Ile Ser Ala Gln Ala Ala Val Leu Arg
1 5 10 15
Gln Val Gly Gly Pro Leu Ala Val Glu Pro Val Arg Ile Ser Met Pro
20 25 30
Lys Gly Asp Glu Val Leu Ile Arg Ile Ala Gly Val Gly Val Cys His
35 40 45
Thr Asp Leu Val Cys Arg Asp Gly Phe Pro Val Pro Leu Pro Ile Val
50 55 60
Leu Gly His Glu Gly Ser Gly Thr Val Glu Ala Val Gly Glu Gln Val
65 70 75 80
Arg Thr Leu Lys Pro Gly Asp Arg Val Val Leu Ser Phe Asn Ser Cys
85 90 95
Gly His Cys Gly Asn Cys His Asp Gly His Pro Ser Asn Cys Leu Gln
100 105 110
Met Leu Pro Leu Asn Phe Gly Gly Ala Gln Arg Val Asp Gly Gly Gln
115 120 125
Val Leu Asp Gly Ala Gly His Pro Val Gln Ser Met Phe Phe Gly Gln
130 135 140
Ser Ser Phe Gly Thr His Ala Val Ala Arg Glu Ile Asn Ala Val Lys
145 150 155 160
Val Gly Asp Asp Leu Pro Leu Glu Leu Leu Gly Pro Leu Gly Cys Gly
165 170 175
Ile Gln Thr Gly Ala Gly Ala Ala Ile Asn Ser Leu Gly Ile Gly Pro
180 185 190
Gly Gln Ser Leu Ala Ile Phe Gly Gly Gly Gly Val Gly Leu Ser Ala
195 200 205
Leu Leu Gly Ala Arg Ala Val Gly Ala Asp Arg Val Val Val Ile Glu
210 215 220
Pro Asn Ala Ala Arg Arg Ala Leu Ala Leu Glu Leu Gly Ala Ser His
225 230 235 240
Ala Leu Asp Pro His Ala Glu Gly Asp Leu Val Ala Ala Ile Lys Ala
245 250 255
Ala Thr Gly Gly Gly Ala Thr His Ser Leu Asp Thr Thr Gly Leu Pro
260 265 270
Pro Val Ile Gly Ser Ala Ile Ala Cys Thr Leu Pro Gly Gly Thr Val
275 280 285
Gly Met Val Gly Leu Pro Ala Pro Asp Ala Pro Val Pro Ala Thr Leu
290 295 300
Leu Asp Leu Leu Ser Lys Ser Val Thr Leu Arg Pro Ile Thr Glu Gly
305 310 315 320
Asp Ala Asp Pro Gln Arg Phe Ile Pro Arg Met Leu Asp Phe His Arg
325 330 335
Ala Gly Lys Phe Pro Phe Asp Arg Leu Ile Thr Arg Tyr Arg Phe Asp
340 345 350
Gln Ile Asn Glu Ala Leu His Ala Thr Glu Lys Gly Glu Ala Ile Lys
355 360 365
Pro Val Leu Val Phe
370

Claims (9)

1.一种构建用于生产柠檬醛的重组菌的方法，其包括以下步骤：
1)构建用于生成IPP(异戊二烯焦磷酸)和DMAPP(二甲基烯丙基焦磷酸)的表达载体，用以表达乙酰乙酰CoA硫解酶(优选SEQ ID NO:38)、3-羟基-3-甲基戊二酰-CoA合酶(优选SEQID NO:39)、3-羟基-3-甲基戊二酰-CoA还原酶(优选SEQ ID NO:40)、MVA激酶(优选SEQ IDNO:41)、磷酸MVA激酶(优选SEQ ID NO:42)、MVA焦磷酸脱羧酶(优选SEQ ID NO:43)以及IPP异构酶(优选SEQ ID NO:44)；
2)构建包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体，用以表达香叶酯二磷酸合成酶(优选SEQ ID NO:45)、香叶醇合成酶(优选SEQ ID NO:46)、香叶醇氧化酶(优选SEQ ID NO:47)以及IPP异构酶(优选SEQ ID NO:44)；
3)将步骤1)和步骤2)中构建的表达载体共转入出发菌株中，得到重组菌。
2.根据权利要求1所述的方法，其中，所述出发菌株为大肠杆菌BW25113。
3.根据权利要求1所述的方法，其中，所述用于生成IPP和DMAPP的表达载体包括合成IPP和DMAPP的各个酶的编码序列：编码乙酰乙酰辅酶A硫解酶(acetoacetyl-CoAthiolase)的基因、编码3-羟基-3-甲基戊二酰-CoA合酶(3-Hydroxy-3-methylglutaryl-CoA synthase)的基因、编码3-羟基-3-甲基戊二酰-CoA还原酶(3-Hydroxy-3-methylglutaryl-CoA reductase)的基因、编码MVA激酶(MVA kinase)的基因、编码磷酸MVA激酶(phospho MVA kinase)的基因、编码MVA焦磷酸脱羧酶(MVA diphosphatedecarboxylase)的基因和编码IPP异构酶的基因。
4.根据权利要求3所述的方法，其中，所述用于生成IPP和DMAPP的表达载体包括以下基因：atoB(优选SEQ ID NO:1)、HMGS(优选SEQ ID NO:2)、HMGR(优选SEQ ID NO:3)、MK(优选SEQ ID NO:4)、PMK(优选SEQ ID NO:5)、PMD(优选SEQ ID NO:6)和idi(优选SEQ ID NO:7)，
其中，atoB、HMGS、HMGR共用一套调控元件，受诱导型启动子(优选lac UV5启动子(SEQID NO:31))调控；MK、PMK、PMD和idi共用一套调控元件，受诱导型启动子(优选pTac启动子(SEQ ID NO:32))调控。
5.根据权利要求1所述的方法，其中，所述包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体包含表达GPPS-GES-geoA-idi的基因盒序列，所述基因盒包括六个部分：组成型启动子(优选sp2启动子(SEQ ID NO:36))或诱导型启动子，绝缘子RiboJ(SEQ ID NO:37)，带有核糖体结合序列的GPPS编码序列(优选SEQ ID NO:9)，带有rbs1的GES序列(优选SEQ ID NO:10)，带有rbs2的geoA序列(优选SEQ ID NO:11)，以及带有rbs3的idi序列(优选SEQ ID NO:12)。
6.根据权利要求5所述的方法，其中，所述包含GPPS编码基因、GES编码基因、geoA编码基因和idi编码基因的表达载体为pTALE-GPPS-GES-geoA-idi质粒(SEQ ID NO:13)。
7.一种表达载体，其包含序列SEQ ID NO:13。
8.重组菌，其由如权利要求1至6中任一项所述的方法构建。
9.一种生产柠檬醛的方法，所述方法包括使用如权利要求8所述的重组菌发酵生产柠檬醛。
CN202010157998.1A 2020-03-09 2020-03-09 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用 Active CN113355340B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202010157998.1A CN113355340B (zh) 2020-03-09 2020-03-09 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202010157998.1A CN113355340B (zh) 2020-03-09 2020-03-09 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用

Publications (2)

Publication Number Publication Date
CN113355340A true CN113355340A (zh) 2021-09-07
CN113355340B CN113355340B (zh) 2023-05-09

Family

ID=77524332

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202010157998.1A Active CN113355340B (zh) 2020-03-09 2020-03-09 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用

Country Status (1)

Country Link
CN (1) CN113355340B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113652440A (zh) * 2021-08-05 2021-11-16 昆明理工大学 3-酮脂酰辅酶a硫解酶基因rkacaa1-2及其应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2492667A (en) * 1947-04-12 1949-12-27 Miles Lab Production of citric acid by fermentation
JP2009515541A (ja) * 2005-11-17 2009-04-16 ビーエーエスエフ ソシエタス・ヨーロピア シトロネラールの生産法
CN110669713A (zh) * 2019-10-18 2020-01-10 中国科学院青岛生物能源与过程研究所 一种合成d-柠檬烯的基因工程菌及其构建方法与应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US2492667A (en) * 1947-04-12 1949-12-27 Miles Lab Production of citric acid by fermentation
JP2009515541A (ja) * 2005-11-17 2009-04-16 ビーエーエスエフ ソシエタス・ヨーロピア シトロネラールの生産法
CN110669713A (zh) * 2019-10-18 2020-01-10 中国科学院青岛生物能源与过程研究所 一种合成d-柠檬烯的基因工程菌及其构建方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JIA ZHOU ET AL.: "Engineering Escherichia coli for selective geraniol production withminimized endogenous dehydrogenation", 《JOURNAL OF BIOTECHNOLOGY》 *
应向贤 等: "化学-酶法不对称氢化柠檬醛合成(R)-香茅醛", 发酵科技通讯 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113652440A (zh) * 2021-08-05 2021-11-16 昆明理工大学 3-酮脂酰辅酶a硫解酶基因rkacaa1-2及其应用
CN113652440B (zh) * 2021-08-05 2023-04-21 昆明理工大学 3-酮脂酰辅酶a硫解酶基因rkacaa1-2及其应用

Also Published As

Publication number Publication date
CN113355340B (zh) 2023-05-09

Similar Documents

Publication Publication Date Title
CN106190937B9 (zh) 一种构建重组大肠杆菌生物合成2’-岩藻乳糖的方法
CN108753636B (zh) 一种生产酪醇及羟基酪醇的酵母及构建方法
EP1366176B2 (fr) Genes synthetiques et plasmides bacteriens depourvus de cpg
TWI250210B (en) An isolated DNA sequence coding for an enzyme involved in the mevalonate pathway or the pathway from isopentenyl pyrophosphate to farnesyl pyrophosphate
JP5074185B2 (ja) イソプレノイドの生成
TW201111512A (en) Improved isoprene production using the DXP and MVA pathway
CN107435049B (zh) 一种生产红景天苷的重组大肠杆菌及构建方法及应用
HUE033564T2 (hu) Eljárás (+)-zizaen elõállítására
CN109988722B (zh) 一种重组酿酒酵母菌株及其应用和生产酪醇和/或红景天苷的方法
CN114381416B (zh) 一种高产5-氨基乙酰丙酸的重组大肠杆菌菌株及其应用
CN113355340B (zh) 一种构建生产柠檬醛的重组菌的方法及由其构建的重组菌及其应用
CN109266596A (zh) 高效利用脂肪酸合成甘氨酸的大肠杆菌重组菌及其构建方法与应用
CN112501095B (zh) 一种合成3-岩藻乳糖的重组大肠杆菌构建方法及其应用
CN113355300B (zh) 芳香族异戊烯基转移酶突变体、用于其表达的重组菌的构建方法及由其构建的重组菌
KR102043356B1 (ko) 마크로포미나 파세올리나로부터의 리그닌 분해 효소 및 이의 용도
CN107287172B (zh) 一种利用大肠杆菌发酵生产胸苷磷酸化酶的方法
CN113832167B (zh) 一个基因及其在提高苯乙醇和色醇产量中的用途
CN113249240B (zh) 一种高产羟基酪醇的酿酒酵母及其构建方法
CN104894150B (zh) 天山雪莲细胞苯丙氨酸解氨酶基因SiPAL及其编码产物和应用
US11352652B2 (en) Method for producing 4-aminocinnamic acid, and vector and host cell used in same
CN108795832B (zh) 一种内源l-门冬酰胺酶ii基因敲除的宿主菌、其制备方法及其应用
CN115873881A (zh) 一种产1,3-丁二醇的基因工程菌及其应用
KR101863239B1 (ko) 아세트산을 유일 탄소원으로 이용할 수 있는 미생물
KR20090060124A (ko) Ｔｈｅｒｍｏｃｏｃｃｕｓ ｓｐｐ.로부터 분리된 신규한 수소화효소, 이를 암호화하는 유전자 및 그 유전자를 갖는 미생물을 이용하여 수소를 생산하는 방법
CN107287221B (zh) 人工合成的编码胸苷磷酸化酶蛋白的基因及其应用

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