CN111041038B - 高效生物合成虾青素的集胞藻6803基因工程菌及构建方法及应用 - Google Patents

高效生物合成虾青素的集胞藻6803基因工程菌及构建方法及应用 Download PDF

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CN111041038B
CN111041038B CN201911215898.3A CN201911215898A CN111041038B CN 111041038 B CN111041038 B CN 111041038B CN 201911215898 A CN201911215898 A CN 201911215898A CN 111041038 B CN111041038 B CN 111041038B
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张卫文
刁进进
宋馨宇
陈磊
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Abstract

本发明公开了高效生物合成虾青素的集胞藻6803基因工程菌及构建方法及应用，构建方法为：扩增CrtWD和CrtZA基因；将pTZD与CrtWD基因构建pTZD‑WD；将pBA3031M与CrtZA基因构建pBA3031M‑ZA；并依次转入集胞藻6803中，得到工程菌WD‑ZA；扩增FBS基因，将pJAK与FBS构建pJAK‑FBS转入工程菌WD‑ZA中，得到WD‑ZA‑FBS；扩增基因dxs‑E和基因ispA‑E，构建操纵子片段dxs‑RBS‑ispA，将p0168与dxs‑RBS‑ispA片段构建p0168‑DI转入菌株WD‑ZA‑FBS中，本发明的工程菌最终虾青素的产量达29.6mg/g。

Description

高效生物合成虾青素的集胞藻6803基因工程菌及构建方法及 应用
技术领域
本发明属于工业微生物领域，具体涉及一种生物合成虾青素的基因工程菌的构建方法及其应用。
技术背景
虾青素是一种罕见的二羟基二酮类胡萝卜素，具有比其他类胡萝卜素和维生素E1高得多的抗氧化活性。据报道，虾青素具有多种生物活性，有效清除细胞内的氧自由基、增强细胞再生能力、维持机体平衡和减少衰老细胞的堆积等，已成为国内外保健品市场上一致公认的具有抗氧化活性、抗肿瘤和增强免疫力等多种重要生理和生物学功能的天然保健食品的原料。此外，虾青素也被认为是一种很有前景的候选药物，在治疗心血管疾病、神经系统疾病和糖尿病中存在诸多益处。鉴于虾青素的重要应用价值，其在水产养殖、食品、化妆品和制药行业中的需求正在快速增长。
目前大多数虾青素产品通过化学合成和生物提取两种方式获得。化学合成的虾青素产品包括立体异构体的外消旋混合物，生物活性较低，主要用作动物和水产的养殖饲料。生物提取能够获得活性较高的产品，主要有三种来源，水产品加工工业的废弃物、红发夫酵母和以雨生红球藻为主的微藻。但生物提取涉及程序较复杂，生产成本相应较高，在大规模产业化的经济效益和生产规模上存在局限。近年来，研究者提出利用微生物细胞工厂生物合成虾青素。与传统方法相比，生物合成虾青素具有成本低、操作简单、条件温和、副产物少、绿色环保等优势。已有研究者试图在大肠杆菌和酵母等微生物细胞工厂中，利用合成生物学技术开展虾青素的生物合成的研究，但其产量仍然较低，难以满足工业生产的需求。此外，这些现有的生物合成方法都需要大量生物质作为发酵碳源，异氧培养存在着“与人争粮”的问题。
光合蓝细菌是一类能够利用光能和二氧化碳进行光合作用的自养型细菌，避免了异氧培养过程中有机碳源的消耗，同时又具有生长速度快、培养简单等优势。模式蓝细菌集胞藻6803作为一种绿色、可持续的“自养型细胞工厂”在近年来受到合成生物学领域的青睐，目前已实现了丁醇、3-羟基丙酸等多种生物燃料和高附加值化学品在集胞藻6803中的生物合成。集胞藻6803底盘细胞拥有复杂的膜系统，与大肠杆菌和酵母菌等异养微生物相比，在色素的生物合成中具有显著的优势。因此，利用集胞藻6803作为细胞工厂生产虾青素具有重要意义。但目前，蓝细菌中合成虾青素的研究仅有少量局限于构建虾青素核心途径的报道，且产量很低(培养14天产量仅有4.81mg/g)，无法满足工业化需求。
发明内容
本发明的目的是克服现有技术的不足，提供一种高效生物合成虾青素的集胞藻6803基因工程菌。
本发明的第二个目的是提供一种高效生物合成虾青素的集胞藻6803基因工程菌的构建方法。
本发明的第三个目的是提供一种高效生物合成虾青素的集胞藻6803基因工程菌在生产虾青素中的应用。
本发明的技术方案概述如下：
高效生物合成虾青素的集胞藻6803基因工程菌的构建方法，包括如下步骤：
(1)以质粒pUC57-WD为模板，扩增获得短波毛单胞菌β-类胡萝卜素酮酶基因CrtWD，所述CrtWD基因的核苷酸序列如SEQ ID NO.1所示；以质粒pUC57-ZA为模板，扩增获得产碱杆菌β-胡萝卜素羟化酶基因CrtZA，所述CrtZA基因的核苷酸序列如SEQ ID NO.2所示；
(2)将含有psbA启动子和rbcL终止子的表达载体pTZD，与步骤(1)获得的CrtWD基因片段构建重组表达载体pTZD-WD，所述表达载体pTZD的核苷酸序列如SEQ ID NO.29所示；
(3)将含有cpc560启动子和rbcL终止子的表达载体pBA3031M，与步骤(1)获得的CrtZA基因构建重组表达载体pBA3031M-ZA，所述表达载体pBA3031M的核苷酸序列如SEQ IDNO.30所示；
(4)将所述重组表达载体pTZD-WD与重组表达载体pBA3031M-ZA依次转入集胞藻6803中，涂布于含有氯霉素和壮观霉素的BG11固体平板培养基，挑取阳性转化子进行验证，得到重组集胞藻6803工程菌命名为WD-ZA；
(5)以聚球藻7002基因组为模板，扩增果糖-1,6-二磷酸酶/景天庚酮糖-1,7-二磷酸酶FBS基因，所述FBS基因的核苷酸序列如SEQ ID NO.3所示；
(6)将含有psbA启动子和BBa_B0015终止子的表达载体pJAK，与步骤(5)中获得的FBS基因构建重组表达载体pJAK-FBS，所述表达载体pJAK的核苷酸序列如SEQ ID NO.31所示；
(7)将所述重组表达载体pJAK-FBS转入步骤(4)获得的工程菌WD-ZA中，涂布于含有氯霉素、壮观霉素和卡纳霉素的BG11固体平板培养基，挑取阳性转化子进行验证，得到工程菌株WD-ZA-FBS；
(8)以大肠杆菌DH5α基因组为模板，扩增1-脱氧-D-木酮糖-5-磷酸合成酶基因dxs-E和法呢基焦磷酸合酶基因ispA-E，所述dxs-E基因的核苷酸序列分别如SEQ ID NO.4所示，所述ispA-E基因的核苷酸序列如SEQ ID NO.5所示；利用融合PCR方法，构建操纵子片段dxs-RBS-ispA，所述dxs-RBS-ispA片段的核苷酸序列如SEQ ID NO.26所示；
(9)将含有psbA启动子和rbcL终止子的表达载体p0168，与步骤(8)中获得的dxs-RBS-ispA片段构建重组表达载体p0168-DI，所述表达载体p0168的核苷酸序列如SEQ IDNO.32所示；
(10)将所述重组表达载体p0168-DI转入步骤(7)获得的菌株WD-ZA-FBS中，氯霉素、壮观霉素、卡纳霉素和红霉素的BG11固体平板培养基，挑取阳性转化子进行验证，获得高产菌株WD-ZA-FBS-DI。
上述方法构建的高效生物合成虾青素的集胞藻6803基因工程菌。
上述高效生物合成虾青素的集胞藻6803基因工程菌在生产虾青素中的应用。
本发明的优点：
本发明利用合成生物学方法围绕虾青素生物合成的限速问题系统改造集胞藻6803，得到高效生物合成虾青素的集胞藻6803基因工程菌，命名为WD-ZA-FBS-DI。实验证明，本发明的高效生物合成虾青素的集胞藻6803基因工程菌最终虾青素的产量达到29.6mg/g，为现有技术的6倍多，这对利用光合微生物生产虾青素具有重要的理论及实际意义。
附图说明
图1为本发明通过PCR方法扩增得到的CrtWD和CrtZA基因电泳图及其表达载体验证电泳图。
其中图1A为PCR产物验证电泳图，由左至右3条泳道分别为DNA Maker(M)、CrtWD(lane 1)、CrtZA(lane 2)；
图1B为载体构建验证电泳图，由左至右3条泳道分别为DNA Maker(M)、pTZD-WD载体验证(lane 1)、pBA3031M-ZA载体验证(lane 2)。
图2为本发明通过PCR方法扩增得到的FBS基因电泳图及其表达载体验证电泳图。
其中图2A为PCR产物验证电泳图，由左至右2条泳道分别为DNA Maker(M)、FBS(lane 1)；
图2B为载体构建验证电泳图，由左至右2条泳道分别为DNA Maker(M)、pJAK-FBS载体验证(lane1)结果。
图3为本发明通过PCR方法扩增得到的dxs-E和ispA-E基因电泳图及其表达载体验证电泳图。
其中
图3A为PCR产物验证电泳图，由左至右3条泳道分别为DNA Maker(M)、dxs-E(lane1)、ispA-E(lane 2)；
图3B为融合PCR基因片段电泳图，由左至右2条泳道分别为DNA Maker(M)、dxs-RBS-ispA(lane 1)；
图3C为载体构建验证电泳图，由左至右2条泳道分别为DNA Maker(M)、p0168-DI载体验证(lane 1)结果。
图4为本发明高产虾青素集胞藻6803工程菌产量分析图。图4A为虾青素标品的液相分析图谱；图4B为工程菌株WD-ZA-FBS-DI虾青素产量分析图谱。
具体实施方式
下面的实施例是为了使本领域的技术人员更好地理解本发明但并不用于限制本发明。
本发明各实施例使用集胞藻6803(Synechocystis sp.PCC6803)，购自美国菌株保藏中心ATCC，菌株代码ATCC27184，菌株购买于2012.5.30，https://www.atcc.org/ Products/All/27184.aspx。
聚球藻7002(Synechococcus sp.PCC 7002)，购自美国菌株保藏中心ATCC，菌株代码ATCC27264，菌株购买于2012.5.30，https://www.atcc.org/Products/All/27264.aspx。
大肠杆菌DH5α(Escherichia coli DH5α)感受态细胞购自全式金公司。
表达载体pTZD的构建以pTZ57R/T载体为基础，将复制起始位点替换为p15A，将同源臂slr1704、sll1575、氯霉素抗性基因、启动子(psbA)以及终止子(rbcL)通过酶切连入其中构成，所述pTZ57R/T载体购自美国Thermo Scientific公司；
表达载体pJAK以文献(Using Transcriptomics To Improve Butanol Tolerancein Synechocystis sp.Strain PCC 6803)中报道的pJA2载体为基础，去掉氨苄抗生素序列，并将启动子psbA2替换为psbA构成；
载体p0168以pTZ57R/T载体为基础，将slr0168基因上下游同源臂、红霉素抗性基因、启动子psbA和终止子rbcL连入其中构成；
表达载体pBA3031M为(Re-direction of carbon flux to key precursormalonyl-CoA via artificial small RNAs in photosynthetic Synechocystis sp.PCC6803)文献报道；
质粒pUC57-WD由金唯智生物科技有限公司根据本发明提供的短波毛单胞菌β-类胡萝卜素酮酶基因CrtWD核苷酸序列进行合成；
质粒pUC57-ZA由金唯智生物科技有限公司根据本发明提供的产碱杆菌β-胡萝卜素羟化酶基因CrtZA核苷酸序列合成。
下面结合具体实施例对本发明作进一步说明：
实施例1
菌株WD-ZA的构建：
(1)以序列表中SEQ ID NO.6和SEQ ID NO.7为上下游引物，以质粒pUC57-WD为模板，扩增短波毛单胞菌β-类胡萝卜素酮酶基因CrtWD基因；以序列表中SEQ ID NO.8和SEQID NO.9为上下游引物，以质粒pUC57-ZA为模板，扩增获得产碱杆菌β-胡萝卜素羟化酶基因CrtZA基因；CrtWD基因和CrtZA基因核苷酸序列依次用SEQ ID NO.1和SEQ ID NO.2表示。
(2)以序列表中SEQ ID NO.11和SEQ ID NO.10为上下游引物，以质粒pTZD为模板，扩增获得带有psbA启动子和rbcL终止子的第一骨架片段，第一骨架片段的核苷酸序列如SEQ ID NO.12所示；将第一骨架片段与步骤(1)获得的CrtWD基因片段进行平末端连接，构建重组表达载体pTZD-WD，所述表达载体pTZD的核苷酸序列如SEQ ID NO.29所示；以序列表中SEQ ID NO.13和SEQ ID NO.17为上下游引物进行验证。
(3)以序列表中SEQ ID NO.11与SEQ ID NO.14为上下游引物，以质粒pBA3031M为模板，扩增获得带有cpc560启动子和rbcL终止子的第二骨架片段，第二骨架片段的核苷酸序列如SEQ ID NO.15所示；将第二骨架片段与CrtZA基因片段进行平末端连接，构建表达载体pBA3031M-ZA，以序列表中SEQ ID NO.16和SEQ ID NO.9为上下游引物进行验证。所述表达载体pBA3031M的核苷酸序列如SEQ ID NO.30所示。
(4)无菌条件下，接种集胞藻6803菌液于BG11液体培养基中，放置于30℃光照培养箱中130rpm振荡培养，待菌液OD730nm值长到0.4～0.6时，取1.5mL于3,500rpm离心5min，去上清。在收集的菌体中加入200μL BG 11液体培养基进行重悬并加入表达载体pTZD-WD，放置于30℃培养箱中孵育培养6h后，涂布于含有氯霉素(终浓度为10μg/mL)和壮观霉素(终浓度为10μg/mL)的BG 11固体平板培养基上。光照静置培养10天左右，阳性转化子长出后传代培养获得纯合转化子WD，用同样的方法再将载体pBA3031M-ZA转入菌株WD，获得菌株WD-ZA。
所述液体培养基BG 11：NaNO3 1.5g，K2HPO4·3H2O 0.04g，MgSO4·7H2O 0.075g，EDTA 0.001g，Na2CO3 0.02g，H3BO3 2.86g，MnCl2·4H2O 1.81g，ZnSO4·7H2O 0.222g，NaMoO4·5H2O0.390g，CuSO4·5H2O 0.079g，Co(NO3)2·6H2O0.0494g，CaCl2·2H2O 0.036g，柠檬酸铁铵0.006g，加水至1L。
所述固体培养基BG 11：NaNO3 1.5g，K2HPO4.3H2O 0.04g，MgSO4·7H2O 0.075g，EDTA 0.001g，Na2CO3 0.02g，H3BO 32.86g，MnCl2·4H2O 1.81g，ZnSO4·7H2O 0.222g，NaMoO4·5H2O 0.390g，CuSO4·5H2O 0.079g，Co(NO3)2·6H2O 0.0494g，CaCl2·2H2O0.036g，柠檬酸铁铵0.006g，TES-NaOH(pH8.0)0.008M，Na2S2O3 3g，琼脂15g，加水至1L。
(5)将菌株WD-ZA光照培养5天后，4℃条件下7500rpm离心8min，并用ddH2O洗涤2次。细胞沉淀用0.5mL丙酮重悬浮，放置于细胞破碎仪中进行破碎3min后，10,000rpm离心1min，收集上清。重复提取两遍后，将1.5mL提取液用0.22μm过滤器过滤，然后将上清液进行HPLC定量分析。
菌株WD-ZA虾青素产量6.8mg/g(DCW)。
实施例2
菌株WD-ZA-FBS的构建：
(1)以序列表中SEQ ID NO.17和SEQ ID NO.18分别为上下游引物，以聚球藻7002基因组为模板，扩增获得果糖-1,6-二磷酸酶/景天庚酮糖-1,7-二磷酸酶FBS基因，所述FBS基因核苷酸序列如序列表SEQ ID NO.3所示。
(2)以序列表中SEQ ID NO.28和SEQ ID NO.10分别为上下游引物，以pJAK为模板进行反向扩增，获得带有psbA启动子和BBa_B0015终止子的第三骨架片段，所述第三骨架片段SEQ ID NO.19所示；表达载体pJAK的核苷酸序列如SEQ ID NO.31所示，将第三骨架片段与FBS基因片段进行平末端连接，构建表达载体pJAK-FBS，以序列表中SEQ ID NO.13和SEQID NO.18为上下游引物进行验证。
(3)按照实施例1中步骤(4)的方法，将表达载体pJAK-FBS转入实施例1获得的菌株WD-ZA中，涂布于含有氯霉素(终浓度为10μg/mL)、壮观霉素(终浓度为10μg/mL)和卡那霉素(终浓度为10μg/mL)的BG11固体平板培养基，筛选阳性转化子，获得菌株WD-ZA-FBS。
(4)按照实施例1步骤(5)的方法，对菌株WD-ZA-FBS中虾青素的产量进行检测。
菌株WD-ZA-FBS经培养5天虾青素产量达到11.6mg/g(DCW)。
实施例3
菌株WD-ZA-FBS-DI的构建：
(1)以序列表中SEQ ID NO.20和SEQ ID NO.21分别为上下游引物，以大肠杆菌DH5α基因组作为模板，扩增1-脱氧-D-木酮糖-5-磷酸合成酶基因dxs-E；以序列表中SEQ IDNO.22和SEQ ID NO.23分别为上下游引物，以大肠杆菌DH5α基因组作为模板，扩增法呢基焦磷酸合酶基因ispA-E；所述dxs-E基因的核苷酸序列分别如SEQ ID NO.4所示，以序列表中SEQ ID NO.24和SEQ ID NO.23分别为上下游引物，以ispA-E基因片段作为模板扩增获得含有RBS的ispA-E片段ispA-E-RBS，所述ispA-E基因的核苷酸序列如SEQ ID NO.5所示；所述ispA-E-RBS序列如SEQ ID NO.25所示；以序列表中SEQ ID NO.20和SEQ ID NO.23分别为上下游引物，以ispA-E-RBS片段作为模板，进行融合PCR，获得操纵子片段dxs-RBS-ispA片段，所述dxs-RBS-ispA片段核苷酸序列如序列表中SEQ ID NO.26所示；
(2)以序列表SEQ ID NO.11和SEQ ID NO.10分别为上下游引物，以载体p0168为模板，扩增获得含有psbA启动子和rbcL终止子第四骨架片段，所述第四骨架片段核苷酸序列如SEQ ID NO.27所示；将第四骨架片段与dxs-RBS-ispA片段进行平末端连接，构建表达载体p0168-DI，表达载体p0168的核苷酸序列如SEQ ID NO.32所示。
(3)按照实施例1中步骤(4)的方法，将质粒p0168-DI转入实施例2获得的菌株WD-ZA-FBS中，利用含有氯霉素(终浓度为10μg/mL)、壮观霉素(终浓度为10μg/mL)、卡那霉素(终浓度为10μg/mL)和红霉素(终浓度为10μg/mL)的BG11固体平板培养基筛选阳性转化子，获得高产菌株WD-ZA-FBS-DI。
(4)按照实施例1步骤(5)的提取和检测方法，对步骤(3)中菌株WD-ZA-FBS-DI进行检测分析，经检测发现菌株WD-ZA-FBS-DI经培养5天后，虾青素的产量达到19.7mg/g(DCW)；培养10天后，产量达到29.6mg/g(DCW)。
序列表
<110> 天津大学
<120> 高效生物合成虾青素的集胞藻6803基因工程菌及构建方法及应用
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 735
<212> DNA
<213> 短波毛单胞菌(Brevundimonas sp.)
<400> 1
atgaccgccg ccgtggctga accccggatt gtgccccggc agacttggat tggcttgacc 60
ttggccggca tgattgtggc cggctggggc agtttgcacg tgtacggcgt gtattttcac 120
cgctggggta cctcctcctt agtgattgtg cccgccattg tggccgtgca gacttggttg 180
tccgtgggct tgtttattgt ggcccacgat gccatgcatg gcagtttggc tcccggtcgg 240
ccccggttaa atgccgccgt tggccggtta accttgggct tgtacgccgg ctttcgcttt 300
gatcgcttga aaaccgccca ccacgcccat cacgccgctc ccggcaccgc tgatgatccc 360
gatttttatg cccccgcccc ccgggccttt ttaccttggt ttttgaattt ttttcgcacc 420
tactttggct ggcgcgaaat ggccgtgttg accgccttgg tgttgattgc cttgttcggc 480
ttaggcgccc ggcccgccaa cttgttgacc ttttgggccg cccccgcctt gttgtccgcc 540
ttgcagttgt ttaccttcgg cacttggttg ccccatcggc ataccgatca accctttgcc 600
gatgcccacc atgctcggag ttccggctac ggccccgtgt tgtccttgtt gacttgtttc 660
cactttggcc ggcatcatga acaccattta accccttggc ggccttggtg gcggttatgg 720
cgcggcgaaa gttaa 735
<210> 2
<211> 489
<212> DNA
<213> 产碱杆菌(Alcaligenes sp.)
<400> 2
atgacccagt ttttgatcgt ggtggccact gtgttggtga tggaattgac cgcctactcc 60
gtgcatcggt ggattatgca tggccccttg ggctggggct ggcacaaatc ccaccacgaa 120
gaacatgacc acgccttgga aaaaaatgat ttgtacggcg tggtgtttgc cgtgttggcc 180
accattttgt tcaccgtggg cgcctattgg tggcccgtgt tgtggtggat tgccttgggc 240
atgaccgtgt acggcttgat ttactttatt ttgcatgatg gcttggtgca tcaacggtgg 300
ccctttcggt atattccccg gcggggctat tttcggcggt tgtatcaagc ccaccggttg 360
caccatgccg tggaaggccg ggatcattgt gtgtcctttg gctttattta cgcccccccc 420
gtggataaat tgaagcagga tttgaaacgc tccggcgtgt tgcggcccca agatgaacgg 480
ccctcctaa 489
<210> 3
<211> 1038
<212> DNA
<213> 聚球藻(Synechococcus sp.)
<400> 3
atggaaagca ccctcggttt agaaattatt gaagttgtcg aacaagcggc gatcgcctct 60
gccaaatgga tggggatggg cgaaaaagat accgccgacc aagtggccgt tgaagccatg 120
cgggaacgga tgaatcaaat tcatatgcgc ggtcgcatcg ttatcggtga aggggagcgg 180
gatgatgccc ccatgcttta catcggtgaa gaagtcggta tttgtacccg tgaagatgcc 240
aaagcctatt gcaaccccga tgaactgatc gaaattgaca tcgccgttga cccctgtgaa 300
ggcaccaacc tcgtcgccaa tggtcaaccc ggttccatgg cagtcctcgc catttccgaa 360
aaaggcggtc tgttccatgc ccccgactac tacatgaaaa aattggccgc gcccccagcc 420
gccaaaggaa aagttgacat tcgcaagtca gccaccgaaa acatcaaaat tttgtctgag 480
tgcttaaatc gtagcccaga agaattagtc attatcgtga tggatcgccc ccgtcacaaa 540
gatctgatca aagaaattcg ggccactggc gcccgggttc gtttgattag cgatggggac 600
gtatctgcgg caatttgtgc tgcctttgct gggacgaaca tccatgcgct gatgggaatt 660
ggggccgctc ctgaaggggt gatcagtgcc gccgcgatgc gttgtttggg tggtcacttc 720
caagggcaac tgatttacga tcccgccgat gtgaataccc ccgaaagtgc ggactggaat 780
cgcgaagaaa atatcaaacg cctcaagtcc atgggcgttg aagatccgga caaggtttat 840
gaagcagaag aattagcttc cggtgaaacg gttctgtttg cggcctgtgg gattaccccc 900
ggcaccctca tggaaggggt tcgtctgttc catggtggcg cacgcaccca gtctctggtt 960
atttctagcc aatccatgac ggctcgcttc gtggatacca ttcacatgtg ggacaacccc 1020
caaaatattc aactctag 1038
<210> 4
<211> 1863
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 4
atgagttttg atattgccaa atacccgacc ctggcactgg tcgactccac ccaggagtta 60
cgactgttgc cgaaagagag tttaccgaaa ctctgcgacg aactgcgccg ctatttactc 120
gacagcgtga gccgttccag cgggcacttc gcctccgggc tgggcacggt cgaactgacc 180
gtggcgctgc actatgtcta caacaccccg tttgaccaat tgatttggga tgtggggcat 240
caggcttatc cgcataaaat tttgaccgga cgccgcgaca aaatcggcac catccgtcag 300
aaaggcggtc tgcacccgtt cccgtggcgc ggcgaaagcg aatatgacgt attaagcgtc 360
gggcattcat caacctccat cagtgccgga attggtattg cggttgctgc cgaaaaagaa 420
ggcaaaaatc gccgcaccgt ctgtgtcatt ggcgatggcg cgattaccgc aggcatggcg 480
tttgaagcga tgaatcacgc gggcgatatc cgtcctgata tgctggtgat tctcaacgac 540
aatgaaatgt cgatttccga aaatgtcggc gcgctcaaca accatctggc acagctgctt 600
tccggtaagc tttactcttc actgcgcgaa ggcgggaaaa aagttttctc tggcgtgccg 660
ccaattaaag agctgctcaa acgcaccgaa gaacatatta aaggcatggt agtgcctggc 720
acgttgtttg aagagctggg ctttaactac atcggcccgg tggacggtca cgatgtgctg 780
gggcttatca ccacgctaaa gaacatgcgc gacctgaaag gcccgcagtt cctgcatatc 840
atgaccaaaa aaggtcgtgg ttatgaaccg gcagaaaaag acccgatcac tttccacgcc 900
gtgcctaaat ttgatccctc cagcggttgt ttgccgaaaa gtagcggcgg tttgccgagc 960
tattcaaaaa tctttggcga ctggttgtgc gaaacggcag cgaaagacaa caagctgatg 1020
gcgattactc cggcgatgcg tgaaggttcc ggcatggtcg agttttcacg taaattcccg 1080
gatcgctact tcgacgtggc aattgccgag caacacgcgg tgacctttgc tgcgggtctg 1140
gcgattggtg ggtacaaacc cattgtcgcg atttactcca ctttcctgca acgcgcctat 1200
gatcaggtgc tgcatgacgt ggcgattcaa aagcttccgg tcctgttcgc catcgaccgc 1260
gcgggcattg ttggtgctga cggtcaaacc catcagggtg cttttgatct ctcttacctg 1320
cgctgcatac cggaaatggt cattatgacc ccgagcgatg aaaacgaatg tcgccagatg 1380
ctctataccg gctatcacta taacgatggc ccgtcagcgg tgcgctaccc gcgtggcaac 1440
gcggtcggcg tggaactgac gccgctggaa aaactaccaa ttggcaaagg cattgtgaag 1500
cgtcgtggcg agaaactggc gatccttaac tttggtacgc tgatgccaga agcggcgaaa 1560
gtcgccgaat cgctgaacgc cacgctggtc gatatgcgtt ttgtgaaacc gcttgatgaa 1620
gcgttaattc tggaaatggc cgccagccat gaagcgctgg tcaccgtaga agaaaacgcc 1680
attatgggcg gcgcaggcag cggcgtgaac gaagtgctga tggcccatcg taaaccagta 1740
cccgtgctga acattggcct gccggacttc tttattccgc aaggaactca ggaagaaatg 1800
cgcgccgaac tcggcctcga tgccgctggt atggaagcca aaatcaaggc ctggctggca 1860
taa 1863
<210> 5
<211> 900
<212> DNA
<213> 大肠杆菌(Escherichia coli)
<400> 5
atggactttc cgcagcaact cgaagcctgc gttaagcagg ccaaccaggc gctgagccgt 60
tttatcgccc cactgccctt tcagaacact cccgtggtcg aaaccatgca gtatggcgca 120
ttattaggtg gtaagcgcct gcgacctttc ctggtttatg ccaccggtca tatgtttggc 180
gttagcacaa acacgctgga cgcacccgct gctgccgtag agtgtatcca cgcttactca 240
ttaattcatg atgatttacc ggcgatggat gatgacgatc tgcgccgcgg tttgccgacc 300
tgccatgtga agtttggcga agcaaacgcg attctcgctg gcgacgcttt acaaacgctg 360
gcgttctcga ttctaagcga tgccgatatg ccggaagtgt cggatcgcga cagaatttcg 420
atgatttctg aactggcgag cgccagcggt attgccggaa tgtgcggtgg tcaggcacta 480
gatttagacg cggaaggcaa acacgtacct ctggacgcgc ttgagcgtat tcatcgtcat 540
aaaaccggcg cattgattcg cgccgccgtt cgccttggtg cattaagcgc cggagataaa 600
gggcgtcgtg ctctgccagt actcgacaag tacgcagaga gcatcggcct tgccttccag 660
gttcaagatg acatcctgga tgtggtagga gatactgcaa cgttgggaaa acgccagggt 720
gccgaccagc aacttggtaa aagtacctac cctgcacttc tgggtcttga gcaagcccgg 780
aagaaagccc gggatctgat cgacgatgcc cgtcagtcgc tgaaacaact ggctgaacag 840
tcactcgata cctcggcact ggaagcgcta gcggactaca tcatccagcg taataaataa 900
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
atgaccgccg ccgtggctga ac 22
<210> 7
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ttaactttcg ccgcgccata acc 23
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
atgacccagt ttttgatcgt gg 22
<210> 9
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ttaggagggc cgttcatctt gg 22
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
actgttcctc cttctttatt tttg 24
<210> 11
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
accggtgttt ggattgtcgg agttg 25
<210> 12
<211> 3064
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
accggtgttt ggattgtcgg agttgtactc gtccgttaag gatgaacagt tcttcggggt 60
tgagtctgct aactaattag ccattaacag cggcttaact aacagttagt cattggcaat 120
tgtcaaaaaa ttgttaatca gccaaaaccc actgcttact gatgttcaac ttcgacagct 180
tacgccccgc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca 240
tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg 300
ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc 360
acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc 420
tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa 480
tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt 540
tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca 600
ccgtctttca ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga 660
ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata 720
tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt 780
tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt 840
ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt 900
atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa 960
aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag 1020
tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta 1080
ctgatttagt gtatgatggt gtttttgagg tgctccagtg gcttctgttt ctatcagcta 1140
tagtggatag cgccaaacca gataacctcg tatggattgt cacttttgaa atataaagtg 1200
acttttttgt caatgaaatt taaaatttct taaaattaat tgggtaaaca gtaacatttg 1260
tgacggtcta ctccggttaa atcccctaac gtagcttcta attcattcac taggaataaa 1320
tactcatgag tgttttcccc gcagaaacct gtcccgtgtg tggcgtcacc attgaaaatg 1380
gctccaaggt ggttttttcc tctgggccgg cgggaacgag ggcccggctc tgggccaggg 1440
tctgcaattt tgcccgtaac accagttgca tcaatcaaga tgaagcggcg atcggtaatg 1500
tttccagccg ggattattac gactagtatt tttatgtcta gactgttttt gctcgactaa 1560
attaagtcaa attttgccca gttttcccat gtttatgtct ggctttggcc aaaggggagc 1620
tgggctctcg cttgaatatt aatcttgctt attgttgggg ctgtggtaaa tccgactagg 1680
catgggcttc agttatccac tagggcagaa ggatttaatg gaagcggtgt gagtgtggga 1740
atctagttga gatcgttttg gtctgcgcgt aatctcttgc tctgaaaacg aaaaaaccgc 1800
cttgcagggc ggtttttcga aggttctctg agctaccaac tctttgaacc gaggtaactg 1860
gcttggagga gcgcagtcac caaaacttgt cctttcagtt tagccttaac cggcgcatga 1920
cttcaagact aactcctcta aatcaattac cagtggctgc tgccagtggt gcttttgcat 1980
gtctttccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcggactgaa 2040
cggggggttc gtgcatacag tccagcttgg agcgaactgc ctacccggaa ctgagtgtca 2100
ggcgtggaat gagacaaacg cggccataac agcggaatga caccggtaaa ccgaaaggca 2160
ggaacaggag agcgcacgag ggagccgcca ggggaaacgc ctggtatctt tatagtcctg 2220
tcgggtttcg ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga 2280
gcctatggaa aagggaagct ttacaatgtg gaagtgccgg cccaaacgat gattcctgtg 2340
gcggttgacc aaggacaaat cacattgaga aggggagaaa ccagggcggc cagtctccgc 2400
accactacgg ccctccgtca atccgatggt gttacgatta ttcctgctgg cagtgaaatt 2460
attggtcagt ttcgcccgat cgccaatggt aatgcgatgc aatttgtggc ccaacaattg 2520
gtgctcaata atggcgaata tttaccgatt aatgcccgtt cccgtgagct tgtcggtttt 2580
caaacggtca ataagggggc gagtgcttcc gatgtgatta tggggacttt ggctggggcc 2640
ggcacggcaa caatcatttc cggtaccacc ggcgatcgcc gtattacacc tctggaagtg 2700
cttgggggag ccgcggcggg ggccttggcc ggttggggtc tgcccaccgc agggattgtt 2760
ggcggtggct cggaagaatt gttaaccgtt tccagccgag atttgaccct aatgttgcaa 2820
tctcccctcc gcatcggtaa tggccaagga caaacggtgc ccactaataa caatagtaac 2880
tggcgtaatg ctagttggta ggataaagta ctattcagat agaacgagaa atgagcttgt 2940
tctatccgcc cggggctgag gggaattcga tctcaatgaa tattggttga cacgggcgta 3000
taagacatgt tatactgttg aataacaagt ttaccgttcc caaaaataaa gaaggaggaa 3060
cagt 3064
<210> 13
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
aaagtactat tcagatagaa cg 22
<210> 14
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
tgaattaatc tcctacttga ctt 23
<210> 15
<211> 5944
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
accggtgttt ggattgtcgg agttgtactc gtccgttaag gatgaacagt tcttcggggt 60
tgagtctgct aactaattag ccattaacag cggcttaact aacagttagt cattggcaat 120
tgtcaaaaaa ttgttaatca gccaaaaccc actgcttact gatgttcaac ttcgacagca 180
aaaccccgtt aatttttctt acccatgggg aagtttgctg gttcaatagg ccgagttgta 240
gtcggcagtc tatcttgaga acaattgttt ttacatgaca gatagtgggc taagaataac 300
tttgctcaaa ccatttggta aaactgctca atggacgagc cgattttcac cccggcaatt 360
ttgttgaacc tcccacttct ccggtggtca ttctcaagga gttggtggct aagttgtacc 420
gggaacaaaa taaagtgcag gatctgttgg gggccatggg ctacgccctg cggagtttac 480
ataatctgaa tcagtttctg gaattgaccc ctctgatggc caccagggtc accgatgccg 540
atggcagtgc tttagttcta atgagagagg gggaaatatc tatctttgaa caaatccatg 600
gccacaaaaa tagtcttaag ggcaccatta aaggcgcttt gcagaaggcc cgccaggtta 660
acctcacatt ggattcttcc actgtcctga gttattttga ccgtcaattg cgccaagaat 720
taccggcgat cgcctgttat aacaccccca ttttgagtga attcactggc cgtcgtttta 780
caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 840
cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 900
cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 960
atttttgtta aatcagctca ttttttaacc aataggccga aatcggcaaa atcccttata 1020
aatcaaaaga atagaccgag atagggttga gtgttgttcc agtttggaac aagagtccac 1080
tattaaagaa cgtggactcc aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc 1140
cactacgtga accatcaccc taatcaagtt ttttggggtc gaggtgccgt aaagcactaa 1200
atcggaaccc taaagggagc ccccgattta gagcttgacg gggaaagccg gcgaacgtgg 1260
cgagaaagga agggaagaaa gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg 1320
tcacgctgcg cgtaaccacc acacccgccg cgcttaatgc gccgctacag ggcgcgtcag 1380
gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt 1440
caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa 1500
ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt 1560
gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt 1620
tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt 1680
ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg 1740
tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga 1800
atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa 1860
gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga 1920
caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa 1980
ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca 2040
ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta 2100
ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac 2160
ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc 2220
gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag 2280
ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga 2340
taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca tatatacttt 2400
agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata 2460
atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag 2520
aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa 2580
caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 2640
ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt ctagtgtagc 2700
cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 2760
tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 2820
gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 2880
ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 2940
gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 3000
caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 3060
ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 3120
tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg 3180
ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 3240
agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 3300
aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat 3360
gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg 3420
tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt 3480
tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg 3540
ccaagctcta atacgactca ctatagggaa agcttgcttt gcaaaaggaa tttgcccagc 3600
aactaggtgg taactttgtt tggttggccg atgaatggtt tctcattgcc cgtcaacctt 3660
tgccaccgga aagccattac gaagactatc cccaaattgg caacggggta gggtctatcc 3720
gtcaattcat taaggagttt caacaacaag ctgcggaatt tctccccccg gcgatcgccg 3780
aagccaagac gttgacctgg gtagtgggca atgctgtaga acaagcgttt gagctactgg 3840
tggaacagtt aaatcaggta aagggtttaa cagttaattt agcgccctta aacagtgact 3900
attggggtca ggaaattacg gtgacgggac tattgacggg gcaggattta atcgctaaat 3960
tggcaggcag agatttaggg gatggtattc tattgcctgc tctgatgttg aaacatgatg 4020
atactcgctt cttggatgac ctccgggtgg ccgatgtggc tcaaaagttg ggcacaacca 4080
tttaccctgt ggctgatgtg gccagtttgt tggaacattg tgttcagccc atggctgtct 4140
cccgccattg ctaatataaa cgcagaaagg cccacccgaa ggtgagccag tgtgactcta 4200
gtagagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc 4260
ctttcgtttt atttgatgcc tggttatttg ccgactacct tggtgatctc gcctttcacg 4320
tagtggacaa attcttccaa ctgatctgcg cgcgaggcca agcgatcttc ttcttgtcca 4380
agataagcct gtctagcttc aagtatgacg ggctgatact gggccggcag gcgctccatt 4440
gcccagtcgg cagcgacatc cttcggcgcg attttgccgg ttactgcgct gtaccaaatg 4500
cgggacaacg taagcactac atttcgctca tcgccagccc agtcgggcgg cgagttccat 4560
agcgttaagg tttcatttag cgcctcaaat agatcctgtt caggaaccgg atcaaagagt 4620
tcctccgccg ctggacctac caaggcaacg ctatgttctc ttgcttttgt cagcaagata 4680
gccagatcaa tgtcgatcgt ggctggctcg aagatacctg caagaatgtc attgcgctgc 4740
cattctccaa attgcagttc gcgcttagct ggataacgcc acggaatgat gtcgtcgtgc 4800
acaacaatgg tgacttctac agcgcggaga atctcgctct ctccagggga agccgaagtt 4860
tccaaaaggt cgttgatcaa agctcgccgc gttgtttcat caagccttac ggtcaccgta 4920
accagcaaat caatatcact gtgtggcttc aggccgccat ccactgcgga gccgtacaaa 4980
tgtacggcca gcaacgtcgg ttcgagatgg cgctcgatga cgccaactac ctctgatagt 5040
tgagtcgata cttcggcgat caccgcttcc ctcatgatgt ttaactttgt tttagggcga 5100
ctgccctgct gcgtaacatc gttgctgctc cataacatca aacatcgacc cacggcgtaa 5160
cgcgcttgct gcttggatgc ccgaggcata gactgtaccc caaaaaaaca gtcataacaa 5220
gccatgaaaa ccgccactgc gccgttacca ccgctgcgtt cggtcaaggt tctggaccag 5280
ttgcgtgagc gcatacgcta cttgcattac agcttacgaa ccgaacaggc ttatgtccac 5340
tgggttcgtg ccttcatccg tttccacggt gtgcgggatc cgatacctgt agagaagagt 5400
ccctgaatat caaaatggtg ggataaaaag ctcaaaaagg aaagtaggct gtggttccct 5460
aggcaacagt cttccctacc ccactggaaa ctaaaaaaac gagaaaagtt cgcaccgaac 5520
atcaattgca taattttagc cctaaaacat aagctgaacg aaactggttg tcttcccttc 5580
ccaatccagg acaatctgag aatcccctgc aacattactt aacaaaaaag caggaataaa 5640
attaacaaga tgtaacagac ataagtccca tcaccgttgt ataaagttaa ctgtgggatt 5700
gcaaaagcat tcaagcctag gcgctgagct gtttgagcat cccggtggcc cttgtcgctg 5760
cctccgtgtt tctccctgga tttatttagg taatatctct cataaatccc cgggtagtta 5820
acgaaagtta atggagatca gtaacaataa ctctagggtc attactttgg actccctcag 5880
tttatccggg ggaattgtgt ttaagaaaat cccaactcat aaagtcaagt aggagattaa 5940
ttca 5944
<210> 16
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acctgtagag aagagtccct g 21
<210> 17
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
atggaaagca ccctcggttt ag 22
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ctagagttga atattttggg gg 22
<210> 19
<211> 7020
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
tactagtagc ggccgctgca gccaggcatc aaataaaacg aaaggctcag tcgaaagact 60
gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct ctactagagt cacactggct 120
caccttcggg tgggcctttc tgcgtttata atgcagtccg gcaaaaaagg gcaaggtgtc 180
accaccctgc cctttttctt taaaaccgaa aagattactt cgcgttatgc aggcttcctc 240
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 300
ggcggtaatc aattgagttc ttttaccctc agccgaaatg cctgccgttg ctagacattg 360
ccagccagtg cccgtcactc ccgtactaac tgtcacgaac ccctgcaata actgtcacgc 420
ccccctgcaa taactgtcac gaacccctgc aataactgtc acgcccccaa acctgcaaac 480
ccagcagggg cgggggctgg cggggtgttg gaaaaatcca tccatgatta tctaagaata 540
atccactagg cgcggttatc agcgcccttg tggggcgctg ctgcccttgc ccaatatgcc 600
cggccagagg ccggatagct ggtctattcg ctgcgctagg ctacacaccg ccccaccgct 660
gcgcggcagg gggaaaggcg ggcaaagccc gctaaacccc acaccaaacc ccgcagaaat 720
acgctggagc gcttttagcc gctttagcgg cctttccccc tacccgaagg gtgggggcgc 780
gtgtgcagcc ccgcagggcc tgtctcggtc gatcattcag cccggctcat ccttctggcg 840
tggcggcaga ccgaacaagg cgcggtcgtg gtcgcgttca aggtacgcat ccattgccgc 900
catgagccga tcctccggcc actcgctgct gttcaccttg gccaaaatca tggcccccac 960
cagcaccttg cgccttgttt cgttcttgcg ctcttgctgc tgttcccttg cccgcacccg 1020
ctgaatttcg gcattgattc gcgctcgttg ttcttcgagc ttggccagcc gatccgccgc 1080
cttgttgctc cccttaacca tcttgacacc ccattgttaa tgtgctgtct cgtaggctat 1140
catggaggca cagcggcggc aatcccgacc ctactttgta ggggagggcg cacttaccgg 1200
tttctcttcg agaaactggc ctaacggcca cccttcgggc ggtgcgctct ccgagggcca 1260
ttgcatggag ccgaaaagca aaagcaacag cgaggcagca tggcgattta tcaccttacg 1320
gcgaaaaccg gcagcaggtc gggcggccaa tcggccaggg ccaaggccga ctacatccag 1380
cgcgaaggca agtatgcccg cgacatggat gaagtcttgc acgccgaatc cgggcacatg 1440
ccggagttcg tcgagcggcc cgccgactac tgggatgctg ccgacctgta tgaacgcgcc 1500
aatgggcggc tgttcaagga ggtcgaattt gccctgccgg tcgagctgac cctcgaccag 1560
cagaaggcgc tggcgtccga gttcgcccag cacctgaccg gtgccgagcg cctgccgtat 1620
acgctggcca tccatgccgg tggcggcgag aacccgcact gccacctgat gatctccgag 1680
cggatcaatg acggcatcga gcggcccgcc gctcagtggt tcaagcggta caacggcaag 1740
accccggaga agggcggggc acagaagacc gaagcgctca agcccaaggc atggcttgag 1800
cagacccgcg aggcatgggc cgaccatgcc aaccgggcat tagagcgggc tggccacgac 1860
gcccgcattg accacagaac acttgaggcg cagggcatcg agcgcctgcc cggtgttcac 1920
ctggggccga acgtggtgga gatggaaggc cggggcatcc gcaccgaccg ggcagacgtg 1980
gccctgaaca tcgacaccgc caacgcccag atcatcgact tacaggaata ccgggaggca 2040
atagaccatg aacgcaatcg acagagtgaa gaaatccaga ggcatcaacg agttagcgga 2100
gcagatcgaa ccgctggccc agagcatggc gacactggcc gacgaagccc ggcaggtcat 2160
gagccagacc cagcaggcca gcgaggcgca ggcggcggag tggctgaaag cccagcgcca 2220
gacaggggcg gcatgggtgg agctggccaa agagttgcgg gaggtagccg ccgaggtgag 2280
cagcgccgcg cagagcgccc ggagcgcgtc gcgggggtgg cactggaagc tatggctaac 2340
cgtgatgctg gcttccatga tgcctacggt ggtgctgctg atcgcatcgt tgctcttgct 2400
cgacctgacg ccactgacaa ccgaggacgg ctcgatctgg ctgcgcttgg tggcccgatg 2460
aagaacgaca ggactttgca ggccataggc cgacagctca aggccatggg ctgtgagcgc 2520
ttcgatatcg gcgtcaggga cgccaccacc ggccagatga tgaaccggga atggtcagcc 2580
gccgaagtgc tccagaacac gccatggctc aagcggatga atgcccaggg caatgacgtg 2640
tatatcaggc ccgccgagca ggagcggcat ggtctggtgc tggtggacga cctcagcgag 2700
tttgacctgg atgacatgaa agccgagggc cgggagcctg ccctggtagt ggaaaccagc 2760
ccgaagaact atcaggcatg ggtcaaggtg gccgacgccg caggcggtga acttcggggg 2820
cagattgccc ggacgctggc cagcgagtac gacgccgacc cggccagcgc cgacagccgc 2880
cactatggcc gcttggcggg cttcaccaac cgcaaggaca agcacaccac ccgcgccggt 2940
tatcagccgt gggtgctgct gcgtgaatcc aagggcaaga ccgccaccgc tggcccggcg 3000
ctggtgcagc aggctggcca gcagatcgag caggcccagc ggcagcagga gaaggcccgc 3060
aggctggcca gcctcgaact gcccgagcgg cagcttagcc gccaccggcg cacggcgctg 3120
gacgagtacc gcagcgagat ggccgggctg gtcaagcgct tcggtgatga cctcagcaag 3180
tgcgacttta tcgccgcgca gaagctggcc agccggggcc gcagtgccga ggaaatcggc 3240
aaggccatgg ccgaggccag cccagcgctg gcagagcgca agcccggcca cgaagcggat 3300
tacatcgagc gcaccgtcag caaggtcatg ggtctgccca gcgtccagct tgcgcgggcc 3360
gagctggcac gggcaccggc accccgccag cgaggcatgg acaggggcgg gccagatttc 3420
agcatgtagt gcttgcgttg gtactcacgc ctgttatact atgagtactc acgcacagaa 3480
gggggtttta tggaatacga aaaaagcgct tcagggtcgg tctacctgat caaaagtgac 3540
aagggctatt ggttgcccgg tggctttggt tatacgtcaa acaaggccga ggctggccgc 3600
ttttcagtcg ctgatatggc cagccttaac cttgacggct gcaccttgtc cttgttccgc 3660
gaagacaagc ctttcggccc cggcaagttt ctcggtgact gatatgaaag accaaaagga 3720
caagcagacc ggcgacctgc tggccagccc tgacgctgta cgccaagcgc gatatgccga 3780
gcgcatgaag gccaaaggga tgcgtcagcg caagttctgg ctgaccgacg acgaatacga 3840
ggcgctgcgc gagtgcctgg aagaactcag agcggcgcag ggcgggggta gtgaccccgc 3900
cagcgcctaa ccaccaactg cctgcaaagg aggcaatcaa tggctaccca taagcctatc 3960
aatattctgg aggcgttcgc agcagcgccg ccaccgctgg actacgtttt gcccaacatg 4020
gtggccggta cggtcggggc gctggtgtcg cccggtggtg ccggtaaatc catgctggcc 4080
ctgcaactgg ccgcacagat tgcaggcggg ccggatctgc tggaggtggg cgaactgccc 4140
accggcccgg tgatctacct gcccgccgaa gacccgccca ccgccattca tcaccgcctg 4200
cacgcccttg gggcgcacct cagcgccgag gaacggcaag ccgtggctga cggcctgctg 4260
atccagccgc tgatcggcag cctgcccaac atcatggccc cggagtggtt cgacggcctc 4320
aagcgcgccg ccgagggccg ccgcctgatg gtgctggaca cgctgcgccg gttccacatc 4380
gaggaagaaa acgccagcgg ccccatggcc caggtcatcg gtcgcatgga ggccatcgcc 4440
gccgataccg ggtgctctat cgtgttcctg caccatgcca gcaagggcgc ggccatgatg 4500
ggcgcaggcg accagcagca ggccagccgg ggcagctcgg tactggtcga taacatccgc 4560
tggcagtcct acctgtcgag catgaccagc gccgaggccg aggaatgggg tgtggacgac 4620
gaccagcgcc ggttcttcgt ccgcttcggt gtgagcaagg ccaactatgg cgcaccgttc 4680
gctgatcggt ggttcaggcg gcatgacggc ggggtgctca agcccgccgt gctggagagg 4740
cagcgcaaga gcaagggggt gccccgtggt gaagcctaag aacaagcaca gcctcagcca 4800
cgtccggcac gacccggcgc actgtctggc ccccggcctg ttccgtgccc tcaagcgggg 4860
cgagcgcaag cgcagcaagc tggacgtgac gtatgactac ggcgacggca agcggatcga 4920
gttcagcggc ccggagccgc tgggcgctga tgatctgcgc atcctgcaag ggctggtggc 4980
catggctggg cctaatggcc tagtgcttgg cccggaaccc aagaccgaag gcggacggca 5040
gctccggctg ttcctggaac ccaagtggga ggccgtcacc gctgatgcca tggtggtcaa 5100
aggtagctat cgggcgctgg caaaggaaat cggggcagag gtcgatagtg gtggggcgct 5160
caagcacata caggactgca tcgagcgcct ttggaaggta tccatcatcg cccagaatgg 5220
ccgcaagcgg caggggtttc ggctgctgtc ggagtacgcc agcgacgagg cggacgggcg 5280
cctgtacgtg gccctgaacc ccttgatcgc gcaggccgtc atgggtggcg gccagcatgt 5340
gcgcatcagc atggacgagg tgcgggcgct ggacagcgaa accgcccgcc tgctgcacca 5400
gcggctgtgt ggctggatcg accccggcaa aaccggcaag gcttccatag ataccttgtg 5460
cggctatgtc tggccgtcag aggccagtgg ttcgaccatg cgcaagcgcc gccagcgggt 5520
gcgcgaggcg ttgccggagc tggtcgcgct gggctggacg gtaaccgagt tcgcggcggg 5580
caagtacgac atcacccggc ccaaggcggc aggctgaccc cccccactct attgtaaaca 5640
agacattttt atcttttata ttcaatggct tattttcctg ctaattggta ataccatggt 5700
accgctctgc cagtgttaca accaattaac caattctgat tagaaaaact catcgagcat 5760
caaatgaaac tgcaatttat tcatatcagg attatcaata ccatattttt gaaaaagccg 5820
tttctgtaat gaaggagaaa actcaccgag gcagttccat aggatggcaa gatcctggta 5880
tcggtctgcg attccgactc gtccaacatc aatacaacct attaatttcc cctcgtcaaa 5940
aataaggtta tcaagtgaga aatcaccatg agtgacgact gaatccggtg agaatggcaa 6000
aagcttatgc atttctttcc agacttgttc aacaggccag ccattacgct cgtcatcaaa 6060
atcactcgca tcaaccaaac cgttattcat tcgtgattgc gcctgagcga gacgaaatac 6120
gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa tgcaaccggc gcaggaacac 6180
tgccagcgca tcaacaatat tttcacctga atcaggatat tcttctaata cctggaatgc 6240
tgttttcccg gggatcgcag tggtgagtaa ccatgcatca tcaggagtac ggataaaatg 6300
cttgatggtc ggaagaggca taaattccgt cagccagttt agtctgacca tctcatctgt 6360
aacatcattg gcaacgctac ctttgccatg tttcagaaac aactctggcg catcgggctt 6420
cccatacaat cgatagattg tcgcacctga ttgcccgaca ttatcgcgag cccatttata 6480
cccatataaa tcagcatcca tgttggaatt taatcgcggc ctggagcaag acgtttcccg 6540
ttgaatatgg ctcataacac cccttgtatt actgtttatg taagcagaca gttttattgt 6600
tcatgatgat atatttttat cttgtgcaat gtaacatcag agattttgag acacaacgtg 6660
gctttgttga ataaatcgaa cttttgctga gttgaaggat cagctcgagc acatttcccc 6720
gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc tataaaaata 6780
ggcgtatcac gaggcagaat ttcagataaa aaaaatcctt agctttcgct aaggatgatt 6840
tctggaattg ggcgcgccaa aaagtactat tcagatagaa cgagaaatga gcttgttcta 6900
tccgcccggg gctgagggga attcgatctc aatgaatatt ggttgacacg ggcgtataag 6960
acatgttata ctgttgaata acaagtttac cgttcccaaa aataaagaag gaggaacagt 7020
<210> 20
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
atgagttttg atattgccaa atacc 25
<210> 21
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
ttatgccagc caggccttga ttttgg 26
<210> 22
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
atggactttc cgcagcaact cg 22
<210> 23
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ttatttatta cgctggatga tgtag 25
<210> 24
<211> 52
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aaggcctggc tggcataaag gaggtaatat atggactttc cgcagcaact cg 52
<210> 25
<211> 930
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
aaggcctggc tggcataaag gaggtaatat atggactttc cgcagcaact cgaagcctgc 60
gttaagcagg ccaaccaggc gctgagccgt tttatcgccc cactgccctt tcagaacact 120
cccgtggtcg aaaccatgca gtatggcgca ttattaggtg gtaagcgcct gcgacctttc 180
ctggtttatg ccaccggtca tatgtttggc gttagcacaa acacgctgga cgcacccgct 240
gctgccgtag agtgtatcca cgcttactca ttaattcatg atgatttacc ggcgatggat 300
gatgacgatc tgcgccgcgg tttgccgacc tgccatgtga agtttggcga agcaaacgcg 360
attctcgctg gcgacgcttt acaaacgctg gcgttctcga ttctaagcga tgccgatatg 420
ccggaagtgt cggatcgcga cagaatttcg atgatttctg aactggcgag cgccagcggt 480
attgccggaa tgtgcggtgg tcaggcacta gatttagacg cggaaggcaa acacgtacct 540
ctggacgcgc ttgagcgtat tcatcgtcat aaaaccggcg cattgattcg cgccgccgtt 600
cgccttggtg cattaagcgc cggagataaa gggcgtcgtg ctctgccagt actcgacaag 660
tacgcagaga gcatcggcct tgccttccag gttcaagatg acatcctgga tgtggtagga 720
gatactgcaa cgttgggaaa acgccagggt gccgaccagc aacttggtaa aagtacctac 780
cctgcacttc tgggtcttga gcaagcccgg aagaaagccc gggatctgat cgacgatgcc 840
cgtcagtcgc tgaaacaact ggctgaacag tcactcgata cctcggcact ggaagcgcta 900
gcggactaca tcatccagcg taataaataa 930
<210> 26
<211> 2775
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
atgagttttg atattgccaa atacccgacc ctggcactgg tcgactccac ccaggagtta 60
cgactgttgc cgaaagagag tttaccgaaa ctctgcgacg aactgcgccg ctatttactc 120
gacagcgtga gccgttccag cgggcacttc gcctccgggc tgggcacggt cgaactgacc 180
gtggcgctgc actatgtcta caacaccccg tttgaccaat tgatttggga tgtggggcat 240
caggcttatc cgcataaaat tttgaccgga cgccgcgaca aaatcggcac catccgtcag 300
aaaggcggtc tgcacccgtt cccgtggcgc ggcgaaagcg aatatgacgt attaagcgtc 360
gggcattcat caacctccat cagtgccgga attggtattg cggttgctgc cgaaaaagaa 420
ggcaaaaatc gccgcaccgt ctgtgtcatt ggcgatggcg cgattaccgc aggcatggcg 480
tttgaagcga tgaatcacgc gggcgatatc cgtcctgata tgctggtgat tctcaacgac 540
aatgaaatgt cgatttccga aaatgtcggc gcgctcaaca accatctggc acagctgctt 600
tccggtaagc tttactcttc actgcgcgaa ggcgggaaaa aagttttctc tggcgtgccg 660
ccaattaaag agctgctcaa acgcaccgaa gaacatatta aaggcatggt agtgcctggc 720
acgttgtttg aagagctggg ctttaactac atcggcccgg tggacggtca cgatgtgctg 780
gggcttatca ccacgctaaa gaacatgcgc gacctgaaag gcccgcagtt cctgcatatc 840
atgaccaaaa aaggtcgtgg ttatgaaccg gcagaaaaag acccgatcac tttccacgcc 900
gtgcctaaat ttgatccctc cagcggttgt ttgccgaaaa gtagcggcgg tttgccgagc 960
tattcaaaaa tctttggcga ctggttgtgc gaaacggcag cgaaagacaa caagctgatg 1020
gcgattactc cggcgatgcg tgaaggttcc ggcatggtcg agttttcacg taaattcccg 1080
gatcgctact tcgacgtggc aattgccgag caacacgcgg tgacctttgc tgcgggtctg 1140
gcgattggtg ggtacaaacc cattgtcgcg atttactcca ctttcctgca acgcgcctat 1200
gatcaggtgc tgcatgacgt ggcgattcaa aagcttccgg tcctgttcgc catcgaccgc 1260
gcgggcattg ttggtgctga cggtcaaacc catcagggtg cttttgatct ctcttacctg 1320
cgctgcatac cggaaatggt cattatgacc ccgagcgatg aaaacgaatg tcgccagatg 1380
ctctataccg gctatcacta taacgatggc ccgtcagcgg tgcgctaccc gcgtggcaac 1440
gcggtcggcg tggaactgac gccgctggaa aaactaccaa ttggcaaagg cattgtgaag 1500
cgtcgtggcg agaaactggc gatccttaac tttggtacgc tgatgccaga agcggcgaaa 1560
gtcgccgaat cgctgaacgc cacgctggtc gatatgcgtt ttgtgaaacc gcttgatgaa 1620
gcgttaattc tggaaatggc cgccagccat gaagcgctgg tcaccgtaga agaaaacgcc 1680
attatgggcg gcgcaggcag cggcgtgaac gaagtgctga tggcccatcg taaaccagta 1740
cccgtgctga acattggcct gccggacttc tttattccgc aaggaactca ggaagaaatg 1800
cgcgccgaac tcggcctcga tgccgctggt atggaagcca aaatcaaggc ctggctggca 1860
taaaggaggt aatatatgga ctttccgcag caactcgaag cctgcgttaa gcaggccaac 1920
caggcgctga gccgttttat cgccccactg ccctttcaga acactcccgt ggtcgaaacc 1980
atgcagtatg gcgcattatt aggtggtaag cgcctgcgac ctttcctggt ttatgccacc 2040
ggtcatatgt ttggcgttag cacaaacacg ctggacgcac ccgctgctgc cgtagagtgt 2100
atccacgctt actcattaat tcatgatgat ttaccggcga tggatgatga cgatctgcgc 2160
cgcggtttgc cgacctgcca tgtgaagttt ggcgaagcaa acgcgattct cgctggcgac 2220
gctttacaaa cgctggcgtt ctcgattcta agcgatgccg atatgccgga agtgtcggat 2280
cgcgacagaa tttcgatgat ttctgaactg gcgagcgcca gcggtattgc cggaatgtgc 2340
ggtggtcagg cactagattt agacgcggaa ggcaaacacg tacctctgga cgcgcttgag 2400
cgtattcatc gtcataaaac cggcgcattg attcgcgccg ccgttcgcct tggtgcatta 2460
agcgccggag ataaagggcg tcgtgctctg ccagtactcg acaagtacgc agagagcatc 2520
ggccttgcct tccaggttca agatgacatc ctggatgtgg taggagatac tgcaacgttg 2580
ggaaaacgcc agggtgccga ccagcaactt ggtaaaagta cctaccctgc acttctgggt 2640
cttgagcaag cccggaagaa agcccgggat ctgatcgacg atgcccgtca gtcgctgaaa 2700
caactggctg aacagtcact cgatacctcg gcactggaag cgctagcgga ctacatcatc 2760
cagcgtaata aataa 2775
<210> 27
<211> 3273
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
accggtgttt ggattgtcgg agttgtactc gtccgttaag gatgaacagt tcttcggggt 60
tgagtctgct aactaattag ccattaacag cggcttaact aacagttagt cattggcaat 120
tgtcaaaaaa ttgttaatca gccaaaaccc actgcttact gatgttcaac ttcgacagca 180
acagtggcgg ggatttatat ggagaccaag cccaatttcg tttgcgaatt tacaccagcg 240
ccggtttttc ccccgatggc attgccagtt tactacccac agaatttgaa cggtattttc 300
aactccaagc ggaagatatt acgggacgga cagttatcct aacccaaact ggtgttgatt 360
atgaaattcc cggctttggt ctggtgcagg tgttggggct ggcggatttg gccggggttc 420
aggacagcta tgacctgact tacatcgaag atcatgacaa ctattacgac attatcctca 480
aaggggacga agccgcagtt cgccaaatta agagggttgc tttgccctcc gaaggggatt 540
attcggcggt ttataatccc ggtggccccg gcaatgatcc agagaatggt cccccagggc 600
cctttactgt gtccagtagt ccccaggtaa ttaaggtaac ggataccatc ggccagccca 660
ccaaagtctc ctatgtggaa gtggatggcc ccgtattgcg taatcccttc agtggtactc 720
ccattgggca agaggtgggt ttagcggtta aagatgaatt cactggccgt cgttttacaa 780
cgtcgtgact gggaaaactt tccataggct ccgcccccct gacaagcatc acgaaatctg 840
acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg cgtttcccct 900
ggcggctccc tcgtgcgctc tcctgttcct gcctttcggt ttaccggtgt cattccgctg 960
ttatggccgc gtttgtctca ttccacgcct gacactcagt tccgggtagg cagttcgctc 1020
caagctggac tgtatgcacg aaccccccgt tcagtccgac cgctgcgcct tatccggtaa 1080
ctatcgtctt gagtccaacc cggaaagaca tgcaaaagca ccactggcag cagccactgg 1140
taattgattt agaggagtta gtcttgaagt catgcgccgg ttaaggctaa actgaaagga 1200
caagttttgg tgactgcgct cctccaagcc agttacctcg gttcaaagag ttggtagctc 1260
agagaacctt cgaaaaaccg ccctgcaagg cggttttttc gttttcagag caagagatta 1320
cgcgcagacc aaaacgatct caaagcttat tcccggtatg gatggcaccg atgcggaatc 1380
ccaacagatt gcctttgaca acaatgtggc ctggaataac ctgggggatt tgtccaccac 1440
cacccaacgg gcctacactt cggctattag cacagacaca gtgcagagtg tttatggcgt 1500
taatctggaa aaaaacgata acattcccat tgtttttgcg tggcccattt ttcccaccac 1560
ccttaatccc acagattttc aggtaatgct taacacgggg gaaattgtca ccccggtgat 1620
cgcctctttg attcccaaca gtgaatacaa cgaacggcaa acggtagtaa ttacgggcaa 1680
ttttggtaat cgtttaaccc caggcacgga gggagcgatt tatcccgttt ccgtaggcac 1740
agtgttggac agtactcctt tggaaatggt gggacccaac ggcccggtca gtgcggtggg 1800
tattaccatt gatagtctca acccctacgt ggccggcaat ggtcccaaaa ttgtcgccgc 1860
taagttagac cgcttcagtg acctggggga aggggctccc ctctggttag ccaccaatca 1920
aaattataaa cgcagaaagg cccacccgaa ggtgagccag tgtgactcta gtagagagcg 1980
ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt 2040
atttgatgcc tggcgtgcta taattatact aattttataa ggaggaaaaa atatgggcat 2100
ttttagtatt tttgtaatca gcacagttca ttatcaacca aacaaaaaat aagtggttat 2160
aatgaatcgt taataagcaa aattcatata accaaattaa agagggttat aatgaacgag 2220
aaaaatataa aacacagtca aaactttatt acttcaaaac ataatataga taaaataatg 2280
acaaatataa gattaaatga acatgataat atctttgaaa tcggctcagg aaaaggccat 2340
tttacccttg aattagtaaa gaggtgtaat ttcgtaactg ccattgaaat agaccataaa 2400
ttatgcaaaa ctacagaaaa taaacttgtt gatcacgata atttccaagt tttaaacaag 2460
gatatattgc agtttaaatt tcctaaaaac caatcctata aaatatatgg taatatacct 2520
tataacataa gtacggatat aatacgcaaa attgtttttg atagtatagc taatgagatt 2580
tatttaatcg tggaatacgg gtttgctaaa agattattaa atacaaaacg ctcattggca 2640
ttacttttaa tggcagaagt tgatatttct atattaagta tggttccaag agaatatttt 2700
catcctaaac ctaaagtgaa tagctcactt atcagattaa gtagaaaaaa atcaagaata 2760
tcacacaaag ataaacaaaa gtataattat ttcgttatga aatgggttaa caaagaatac 2820
aagaaaatat ttacaaaaaa tcaatttaac aattccttaa aacatgcagg aattgacgat 2880
ttaaacaata ttagctttga acaattctta tctcttttca atagctataa attatttaat 2940
aagtaagggc ccgtcgactg cagaggctgt cagaccaagt ttactcatat atactttaga 3000
ttgatttacg tctcattttc gccaaaagtt ggcccagggc ttcccggtat caacagggac 3060
accaggattt atttattctg cgaagtgatc ttccgtcaca ggtaggatcc gataaagtac 3120
tattcagata gaacgagaaa tgagcttgtt ctatccgccc ggggctgagg ggaattcgat 3180
ctcaatgaat attggttgac acgggcgtat aagacatgtt atactgttga ataacaagtt 3240
taccgttccc aaaaataaag aaggaggaac agt 3273
<210> 28
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
tactagtagc ggccgctgca gcc 23
<210> 29
<211> 3086
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
ctagattccc acactcacac cgcttccatt aaatccttct gccctagtgg ataactgaag 60
cccatgccta gtcggattta ccacagcccc aacaataagc aagattaata ttcaagcgag 120
agcccagctc ccctttggcc aaagccagac ataaacatgg gaaaactggg caaaatttga 180
cttaatttag tcgagcaaaa acagtctaga cataaaaata ctagtcgtaa taatcccggc 240
tggaaacatt accgatcgcc gcttcatctt gattgatgca actggtgtta cgggcaaaat 300
tgcagaccct ggcccagagc cgggccctcg ttcccgccgg cccagaggaa aaaaccacct 360
tggagccatt ttcaatggtg acgccacaca cgggacaggt ttctgcgggg aaaacactca 420
tgagtattta ttcctagtga atgaattaga agctacgtta ggggatttaa ccggagtaga 480
ccgtcacaaa tgttactgtt tacccaatta attttaagaa attttaaatt tcattgacaa 540
aaaagtcact ttatatttca aaagtgacaa tccatacgag gttatctggt ttggcgctat 600
ccactatagc tgatagaaac agaagccact ggagcacctc aaaaacacca tcatacacta 660
aatcagtaag ttggcagcat cacccgacgc actttgcgcc gaataaatac ctgtgacgga 720
agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga agccctgggc 780
caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact ttcaccataa 840
tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag gagctaagga 900
agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg 960
taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca 1020
gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc 1080
ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa 1140
agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca 1200
aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca 1260
catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt 1320
tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt 1380
aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac 1440
gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg 1500
cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg 1560
ggcgtaagct gtcgaagttg aacatcagta agcagtgggt tttggctgat taacaatttt 1620
ttgacaattg ccaatgacta actgttagtt aagccgctgt taatggctaa ttagttagca 1680
gactcaaccc cgaagaactg ttcatcctta acggacgagt acaactccga caatccaaac 1740
accggtctcg agcggaatcg gatccgatac tgttcctcct tctttatttt tgggaacggt 1800
aaacttgtta ttcaacagta taacatgtct tatacgcccg tgtcaaccaa tattcattga 1860
gatcgaattc ccctcagccc cgggcggata gaacaagctc atttctcgtt ctatctgaat 1920
agtactttat cctaccaact agcattacgc cagttactat tgttattagt gggcaccgtt 1980
tgtccttggc cattaccgat gcggagggga gattgcaaca ttagggtcaa atctcggctg 2040
gaaacggtta acaattcttc cgagccaccg ccaacaatcc ctgcggtggg cagaccccaa 2100
ccggccaagg cccccgccgc ggctccccca agcacttcca gaggtgtaat acggcgatcg 2160
ccggtggtac cggaaatgat tgttgccgtg ccggccccag ccaaagtccc cataatcaca 2220
tcggaagcac tcgccccctt attgaccgtt tgaaaaccga caagctcacg ggaacgggca 2280
ttaatcggta aatattcgcc attattgagc accaattgtt gggccacaaa ttgcatcgca 2340
ttaccattgg cgatcgggcg aaactgacca ataatttcac tgccagcagg aataatcgta 2400
acaccatcgg attgacggag ggccgtagtg gtgcggagac tggccgccct ggtttctccc 2460
cttctcaatg tgatttgtcc ttggtcaacc gccacaggaa tcatcgtttg ggccggcact 2520
tccacattgt aaagcttccc ttttccatag gctccgcccc cctgacaagc atcacgaaat 2580
ctgacgctca aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc 2640
cctggcggct ccctcgtgcg ctctcctgtt cctgcctttc ggtttaccgg tgtcattccg 2700
ctgttatggc cgcgtttgtc tcattccacg cctgacactc agttccgggt aggcagttcg 2760
ctccaagctg gactgtatgc acgaaccccc cgttcagtcc gaccgctgcg ccttatccgg 2820
taactatcgt cttgagtcca acccggaaag acatgcaaaa gcaccactgg cagcagccac 2880
tggtaattga tttagaggag ttagtcttga agtcatgcgc cggttaaggc taaactgaaa 2940
ggacaagttt tggtgactgc gctcctccaa gccagttacc tcggttcaaa gagttggtag 3000
ctcagagaac cttcgaaaaa ccgccctgca aggcggtttt ttcgttttca gagcaagaga 3060
ttacgcgcag accaaaacga tctcaa 3086
<210> 30
<211> 6002
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
aagcttgctt tgcaaaagga atttgcccag caactaggtg gtaactttgt ttggttggcc 60
gatgaatggt ttctcattgc ccgtcaacct ttgccaccgg aaagccatta cgaagactat 120
ccccaaattg gcaacggggt agggtctatc cgtcaattca ttaaggagtt tcaacaacaa 180
gctgcggaat ttctcccccc ggcgatcgcc gaagccaaga cgttgacctg ggtagtgggc 240
aatgctgtag aacaagcgtt tgagctactg gtggaacagt taaatcaggt aaagggttta 300
acagttaatt tagcgccctt aaacagtgac tattggggtc aggaaattac ggtgacggga 360
ctattgacgg ggcaggattt aatcgctaaa ttggcaggca gagatttagg ggatggtatt 420
ctattgcctg ctctgatgtt gaaacatgat gatactcgct tcttggatga cctccgggtg 480
gccgatgtgg ctcaaaagtt gggcacaacc atttaccctg tggctgatgt ggccagtttg 540
ttggaacatt gtgttcagcc catggctgtc tcccgccatt gctaatataa acgcagaaag 600
gcccacccga aggtgagcca gtgtgactct agtagagagc gttcaccgac aaacaacaga 660
taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc ctggttattt 720
gccgactacc ttggtgatct cgcctttcac gtagtggaca aattcttcca actgatctgc 780
gcgcgaggcc aagcgatctt cttcttgtcc aagataagcc tgtctagctt caagtatgac 840
gggctgatac tgggccggca ggcgctccat tgcccagtcg gcagcgacat ccttcggcgc 900
gattttgccg gttactgcgc tgtaccaaat gcgggacaac gtaagcacta catttcgctc 960
atcgccagcc cagtcgggcg gcgagttcca tagcgttaag gtttcattta gcgcctcaaa 1020
tagatcctgt tcaggaaccg gatcaaagag ttcctccgcc gctggaccta ccaaggcaac 1080
gctatgttct cttgcttttg tcagcaagat agccagatca atgtcgatcg tggctggctc 1140
gaagatacct gcaagaatgt cattgcgctg ccattctcca aattgcagtt cgcgcttagc 1200
tggataacgc cacggaatga tgtcgtcgtg cacaacaatg gtgacttcta cagcgcggag 1260
aatctcgctc tctccagggg aagccgaagt ttccaaaagg tcgttgatca aagctcgccg 1320
cgttgtttca tcaagcctta cggtcaccgt aaccagcaaa tcaatatcac tgtgtggctt 1380
caggccgcca tccactgcgg agccgtacaa atgtacggcc agcaacgtcg gttcgagatg 1440
gcgctcgatg acgccaacta cctctgatag ttgagtcgat acttcggcga tcaccgcttc 1500
cctcatgatg tttaactttg ttttagggcg actgccctgc tgcgtaacat cgttgctgct 1560
ccataacatc aaacatcgac ccacggcgta acgcgcttgc tgcttggatg cccgaggcat 1620
agactgtacc ccaaaaaaac agtcataaca agccatgaaa accgccactg cgccgttacc 1680
accgctgcgt tcggtcaagg ttctggacca gttgcgtgag cgcatacgct acttgcatta 1740
cagcttacga accgaacagg cttatgtcca ctgggttcgt gccttcatcc gtttccacgg 1800
tgtgcgggat ccgatacctg tagagaagag tccctgaata tcaaaatggt gggataaaaa 1860
gctcaaaaag gaaagtaggc tgtggttccc taggcaacag tcttccctac cccactggaa 1920
actaaaaaaa cgagaaaagt tcgcaccgaa catcaattgc ataattttag ccctaaaaca 1980
taagctgaac gaaactggtt gtcttccctt cccaatccag gacaatctga gaatcccctg 2040
caacattact taacaaaaaa gcaggaataa aattaacaag atgtaacaga cataagtccc 2100
atcaccgttg tataaagtta actgtgggat tgcaaaagca ttcaagccta ggcgctgagc 2160
tgtttgagca tcccggtggc ccttgtcgct gcctccgtgt ttctccctgg atttatttag 2220
gtaatatctc tcataaatcc ccgggtagtt aacgaaagtt aatggagatc agtaacaata 2280
actctagggt cattactttg gactccctca gtttatccgg gggaattgtg tttaagaaaa 2340
tcccaactca taaagtcaag taggagatta attcactcga gaagatcttg atatccatat 2400
gaggcctaat ctagatgcat tcgcgagggt accaccggtg tttggattgt cggagttgta 2460
ctcgtccgtt aaggatgaac agttcttcgg ggttgagtct gctaactaat tagccattaa 2520
cagcggctta actaacagtt agtcattggc aattgtcaaa aaattgttaa tcagccaaaa 2580
cccactgctt actgatgttc aacttcgaca gcaaaacccc gttaattttt cttacccatg 2640
gggaagtttg ctggttcaat aggccgagtt gtagtcggca gtctatcttg agaacaattg 2700
tttttacatg acagatagtg ggctaagaat aactttgctc aaaccatttg gtaaaactgc 2760
tcaatggacg agccgatttt caccccggca attttgttga acctcccact tctccggtgg 2820
tcattctcaa ggagttggtg gctaagttgt accgggaaca aaataaagtg caggatctgt 2880
tgggggccat gggctacgcc ctgcggagtt tacataatct gaatcagttt ctggaattga 2940
cccctctgat ggccaccagg gtcaccgatg ccgatggcag tgctttagtt ctaatgagag 3000
agggggaaat atctatcttt gaacaaatcc atggccacaa aaatagtctt aagggcacca 3060
ttaaaggcgc tttgcagaag gcccgccagg ttaacctcac attggattct tccactgtcc 3120
tgagttattt tgaccgtcaa ttgcgccaag aattaccggc gatcgcctgt tataacaccc 3180
ccattttgag tgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 3240
gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 3300
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggaaattg 3360
taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc tcatttttta 3420
accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc gagatagggt 3480
tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac tccaacgtca 3540
aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca ccctaatcaa 3600
gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg agcccccgat 3660
ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag 3720
gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc accacacccg 3780
ccgcgcttaa tgcgccgcta cagggcgcgt caggtggcac ttttcgggga aatgtgcgcg 3840
gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 3900
aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 3960
gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 4020
cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 4080
tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 4140
tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 4200
agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 4260
cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 4320
tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 4380
ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 4440
tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 4500
cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 4560
actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 4620
ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 4680
tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 4740
ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 4800
aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 4860
ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 4920
agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 4980
ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 5040
tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 5100
cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 5160
ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 5220
gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 5280
ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 5340
aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 5400
cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 5460
ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 5520
gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 5580
ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 5640
ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 5700
gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 5760
cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 5820
ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 5880
aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 5940
ttcacacagg aaacagctat gaccatgatt acgccaagct ctaatacgac tcactatagg 6000
ga 6002
<210> 31
<211> 7047
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
gcgattccga ctcgtccaac atcaatacaa cctattaatt tcccctcgtc aaaaataagg 60
ttatcaagtg agaaatcacc atgagtgacg actgaatccg gtgagaatgg caaaagctta 120
tgcatttctt tccagacttg ttcaacaggc cagccattac gctcgtcatc aaaatcactc 180
gcatcaacca aaccgttatt cattcgtgat tgcgcctgag cgagacgaaa tacgcgatcg 240
ctgttaaaag gacaattaca aacaggaatc gaatgcaacc ggcgcaggaa cactgccagc 300
gcatcaacaa tattttcacc tgaatcagga tattcttcta atacctggaa tgctgttttc 360
ccggggatcg cagtggtgag taaccatgca tcatcaggag tacggataaa atgcttgatg 420
gtcggaagag gcataaattc cgtcagccag tttagtctga ccatctcatc tgtaacatca 480
ttggcaacgc tacctttgcc atgtttcaga aacaactctg gcgcatcggg cttcccatac 540
aatcgataga ttgtcgcacc tgattgcccg acattatcgc gagcccattt atacccatat 600
aaatcagcat ccatgttgga atttaatcgc ggcctggagc aagacgtttc ccgttgaata 660
tggctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat 720
gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt 780
tgaataaatc gaacttttgc tgagttgaag gatcagctcg agcacatttc cccgaaaagt 840
gccacctgac gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat 900
cacgaggcag aatttcagat aaaaaaaatc cttagctttc gctaaggatg atttctggaa 960
ttgggcgcgc caaaaagtac tattcagata gaacgagaaa tgagcttgtt ctatccgccc 1020
ggggctgagg ggaattcgat ctcaatgaat attggttgac acgggcgtat aagacatgtt 1080
atactgttga ataacaagtt taccgttccc aaaaataaag aaggaggaac agttctaggc 1140
tctagaccgg atccacgcgt tactagtagc ggccgctgca gccaggcatc aaataaaacg 1200
aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct 1260
ctactagagt cacactggct caccttcggg tgggcctttc tgcgtttata atgcagtccg 1320
gcaaaaaagg gcaaggtgtc accaccctgc cctttttctt taaaaccgaa aagattactt 1380
cgcgttatgc aggcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 1440
gcggtatcag ctcactcaaa ggcggtaatc aattgagttc ttttaccctc agccgaaatg 1500
cctgccgttg ctagacattg ccagccagtg cccgtcactc ccgtactaac tgtcacgaac 1560
ccctgcaata actgtcacgc ccccctgcaa taactgtcac gaacccctgc aataactgtc 1620
acgcccccaa acctgcaaac ccagcagggg cgggggctgg cggggtgttg gaaaaatcca 1680
tccatgatta tctaagaata atccactagg cgcggttatc agcgcccttg tggggcgctg 1740
ctgcccttgc ccaatatgcc cggccagagg ccggatagct ggtctattcg ctgcgctagg 1800
ctacacaccg ccccaccgct gcgcggcagg gggaaaggcg ggcaaagccc gctaaacccc 1860
acaccaaacc ccgcagaaat acgctggagc gcttttagcc gctttagcgg cctttccccc 1920
tacccgaagg gtgggggcgc gtgtgcagcc ccgcagggcc tgtctcggtc gatcattcag 1980
cccggctcat ccttctggcg tggcggcaga ccgaacaagg cgcggtcgtg gtcgcgttca 2040
aggtacgcat ccattgccgc catgagccga tcctccggcc actcgctgct gttcaccttg 2100
gccaaaatca tggcccccac cagcaccttg cgccttgttt cgttcttgcg ctcttgctgc 2160
tgttcccttg cccgcacccg ctgaatttcg gcattgattc gcgctcgttg ttcttcgagc 2220
ttggccagcc gatccgccgc cttgttgctc cccttaacca tcttgacacc ccattgttaa 2280
tgtgctgtct cgtaggctat catggaggca cagcggcggc aatcccgacc ctactttgta 2340
ggggagggcg cacttaccgg tttctcttcg agaaactggc ctaacggcca cccttcgggc 2400
ggtgcgctct ccgagggcca ttgcatggag ccgaaaagca aaagcaacag cgaggcagca 2460
tggcgattta tcaccttacg gcgaaaaccg gcagcaggtc gggcggccaa tcggccaggg 2520
ccaaggccga ctacatccag cgcgaaggca agtatgcccg cgacatggat gaagtcttgc 2580
acgccgaatc cgggcacatg ccggagttcg tcgagcggcc cgccgactac tgggatgctg 2640
ccgacctgta tgaacgcgcc aatgggcggc tgttcaagga ggtcgaattt gccctgccgg 2700
tcgagctgac cctcgaccag cagaaggcgc tggcgtccga gttcgcccag cacctgaccg 2760
gtgccgagcg cctgccgtat acgctggcca tccatgccgg tggcggcgag aacccgcact 2820
gccacctgat gatctccgag cggatcaatg acggcatcga gcggcccgcc gctcagtggt 2880
tcaagcggta caacggcaag accccggaga agggcggggc acagaagacc gaagcgctca 2940
agcccaaggc atggcttgag cagacccgcg aggcatgggc cgaccatgcc aaccgggcat 3000
tagagcgggc tggccacgac gcccgcattg accacagaac acttgaggcg cagggcatcg 3060
agcgcctgcc cggtgttcac ctggggccga acgtggtgga gatggaaggc cggggcatcc 3120
gcaccgaccg ggcagacgtg gccctgaaca tcgacaccgc caacgcccag atcatcgact 3180
tacaggaata ccgggaggca atagaccatg aacgcaatcg acagagtgaa gaaatccaga 3240
ggcatcaacg agttagcgga gcagatcgaa ccgctggccc agagcatggc gacactggcc 3300
gacgaagccc ggcaggtcat gagccagacc cagcaggcca gcgaggcgca ggcggcggag 3360
tggctgaaag cccagcgcca gacaggggcg gcatgggtgg agctggccaa agagttgcgg 3420
gaggtagccg ccgaggtgag cagcgccgcg cagagcgccc ggagcgcgtc gcgggggtgg 3480
cactggaagc tatggctaac cgtgatgctg gcttccatga tgcctacggt ggtgctgctg 3540
atcgcatcgt tgctcttgct cgacctgacg ccactgacaa ccgaggacgg ctcgatctgg 3600
ctgcgcttgg tggcccgatg aagaacgaca ggactttgca ggccataggc cgacagctca 3660
aggccatggg ctgtgagcgc ttcgatatcg gcgtcaggga cgccaccacc ggccagatga 3720
tgaaccggga atggtcagcc gccgaagtgc tccagaacac gccatggctc aagcggatga 3780
atgcccaggg caatgacgtg tatatcaggc ccgccgagca ggagcggcat ggtctggtgc 3840
tggtggacga cctcagcgag tttgacctgg atgacatgaa agccgagggc cgggagcctg 3900
ccctggtagt ggaaaccagc ccgaagaact atcaggcatg ggtcaaggtg gccgacgccg 3960
caggcggtga acttcggggg cagattgccc ggacgctggc cagcgagtac gacgccgacc 4020
cggccagcgc cgacagccgc cactatggcc gcttggcggg cttcaccaac cgcaaggaca 4080
agcacaccac ccgcgccggt tatcagccgt gggtgctgct gcgtgaatcc aagggcaaga 4140
ccgccaccgc tggcccggcg ctggtgcagc aggctggcca gcagatcgag caggcccagc 4200
ggcagcagga gaaggcccgc aggctggcca gcctcgaact gcccgagcgg cagcttagcc 4260
gccaccggcg cacggcgctg gacgagtacc gcagcgagat ggccgggctg gtcaagcgct 4320
tcggtgatga cctcagcaag tgcgacttta tcgccgcgca gaagctggcc agccggggcc 4380
gcagtgccga ggaaatcggc aaggccatgg ccgaggccag cccagcgctg gcagagcgca 4440
agcccggcca cgaagcggat tacatcgagc gcaccgtcag caaggtcatg ggtctgccca 4500
gcgtccagct tgcgcgggcc gagctggcac gggcaccggc accccgccag cgaggcatgg 4560
acaggggcgg gccagatttc agcatgtagt gcttgcgttg gtactcacgc ctgttatact 4620
atgagtactc acgcacagaa gggggtttta tggaatacga aaaaagcgct tcagggtcgg 4680
tctacctgat caaaagtgac aagggctatt ggttgcccgg tggctttggt tatacgtcaa 4740
acaaggccga ggctggccgc ttttcagtcg ctgatatggc cagccttaac cttgacggct 4800
gcaccttgtc cttgttccgc gaagacaagc ctttcggccc cggcaagttt ctcggtgact 4860
gatatgaaag accaaaagga caagcagacc ggcgacctgc tggccagccc tgacgctgta 4920
cgccaagcgc gatatgccga gcgcatgaag gccaaaggga tgcgtcagcg caagttctgg 4980
ctgaccgacg acgaatacga ggcgctgcgc gagtgcctgg aagaactcag agcggcgcag 5040
ggcgggggta gtgaccccgc cagcgcctaa ccaccaactg cctgcaaagg aggcaatcaa 5100
tggctaccca taagcctatc aatattctgg aggcgttcgc agcagcgccg ccaccgctgg 5160
actacgtttt gcccaacatg gtggccggta cggtcggggc gctggtgtcg cccggtggtg 5220
ccggtaaatc catgctggcc ctgcaactgg ccgcacagat tgcaggcggg ccggatctgc 5280
tggaggtggg cgaactgccc accggcccgg tgatctacct gcccgccgaa gacccgccca 5340
ccgccattca tcaccgcctg cacgcccttg gggcgcacct cagcgccgag gaacggcaag 5400
ccgtggctga cggcctgctg atccagccgc tgatcggcag cctgcccaac atcatggccc 5460
cggagtggtt cgacggcctc aagcgcgccg ccgagggccg ccgcctgatg gtgctggaca 5520
cgctgcgccg gttccacatc gaggaagaaa acgccagcgg ccccatggcc caggtcatcg 5580
gtcgcatgga ggccatcgcc gccgataccg ggtgctctat cgtgttcctg caccatgcca 5640
gcaagggcgc ggccatgatg ggcgcaggcg accagcagca ggccagccgg ggcagctcgg 5700
tactggtcga taacatccgc tggcagtcct acctgtcgag catgaccagc gccgaggccg 5760
aggaatgggg tgtggacgac gaccagcgcc ggttcttcgt ccgcttcggt gtgagcaagg 5820
ccaactatgg cgcaccgttc gctgatcggt ggttcaggcg gcatgacggc ggggtgctca 5880
agcccgccgt gctggagagg cagcgcaaga gcaagggggt gccccgtggt gaagcctaag 5940
aacaagcaca gcctcagcca cgtccggcac gacccggcgc actgtctggc ccccggcctg 6000
ttccgtgccc tcaagcgggg cgagcgcaag cgcagcaagc tggacgtgac gtatgactac 6060
ggcgacggca agcggatcga gttcagcggc ccggagccgc tgggcgctga tgatctgcgc 6120
atcctgcaag ggctggtggc catggctggg cctaatggcc tagtgcttgg cccggaaccc 6180
aagaccgaag gcggacggca gctccggctg ttcctggaac ccaagtggga ggccgtcacc 6240
gctgatgcca tggtggtcaa aggtagctat cgggcgctgg caaaggaaat cggggcagag 6300
gtcgatagtg gtggggcgct caagcacata caggactgca tcgagcgcct ttggaaggta 6360
tccatcatcg cccagaatgg ccgcaagcgg caggggtttc ggctgctgtc ggagtacgcc 6420
agcgacgagg cggacgggcg cctgtacgtg gccctgaacc ccttgatcgc gcaggccgtc 6480
atgggtggcg gccagcatgt gcgcatcagc atggacgagg tgcgggcgct ggacagcgaa 6540
accgcccgcc tgctgcacca gcggctgtgt ggctggatcg accccggcaa aaccggcaag 6600
gcttccatag ataccttgtg cggctatgtc tggccgtcag aggccagtgg ttcgaccatg 6660
cgcaagcgcc gccagcgggt gcgcgaggcg ttgccggagc tggtcgcgct gggctggacg 6720
gtaaccgagt tcgcggcggg caagtacgac atcacccggc ccaaggcggc aggctgaccc 6780
cccccactct attgtaaaca agacattttt atcttttata ttcaatggct tattttcctg 6840
ctaattggta ataccatggt accgctctgc cagtgttaca accaattaac caattctgat 6900
tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 6960
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 7020
aggatggcaa gatcctggta tcggtct 7047
<210> 32
<211> 3331
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
tttccatagg ctccgccccc ctgacaagca tcacgaaatc tgacgctcaa atcagtggtg 60
gcgaaacccg acaggactat aaagatacca ggcgtttccc ctggcggctc cctcgtgcgc 120
tctcctgttc ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct 180
cattccacgc ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca 240
cgaacccccc gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa 300
cccggaaaga catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt 360
tagtcttgaa gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg 420
ctcctccaag ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac 480
cgccctgcaa ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat 540
ctcaaagctt attcccggta tggatggcac cgatgcggaa tcccaacaga ttgcctttga 600
caacaatgtg gcctggaata acctggggga tttgtccacc accacccaac gggcctacac 660
ttcggctatt agcacagaca cagtgcagag tgtttatggc gttaatctgg aaaaaaacga 720
taacattccc attgtttttg cgtggcccat ttttcccacc acccttaatc ccacagattt 780
tcaggtaatg cttaacacgg gggaaattgt caccccggtg atcgcctctt tgattcccaa 840
cagtgaatac aacgaacggc aaacggtagt aattacgggc aattttggta atcgtttaac 900
cccaggcacg gagggagcga tttatcccgt ttccgtaggc acagtgttgg acagtactcc 960
tttggaaatg gtgggaccca acggcccggt cagtgcggtg ggtattacca ttgatagtct 1020
caacccctac gtggccggca atggtcccaa aattgtcgcc gctaagttag accgcttcag 1080
tgacctgggg gaaggggctc ccctctggtt agccaccaat caaaattata aacgcagaaa 1140
ggcccacccg aaggtgagcc agtgtgactc tagtagagag cgttcaccga caaacaacag 1200
ataaaacgaa aggcccagtc tttcgactga gcctttcgtt ttatttgatg cctggcgtgc 1260
tataattata ctaattttat aaggaggaaa aaatatgggc atttttagta tttttgtaat 1320
cagcacagtt cattatcaac caaacaaaaa ataagtggtt ataatgaatc gttaataagc 1380
aaaattcata taaccaaatt aaagagggtt ataatgaacg agaaaaatat aaaacacagt 1440
caaaacttta ttacttcaaa acataatata gataaaataa tgacaaatat aagattaaat 1500
gaacatgata atatctttga aatcggctca ggaaaaggcc attttaccct tgaattagta 1560
aagaggtgta atttcgtaac tgccattgaa atagaccata aattatgcaa aactacagaa 1620
aataaacttg ttgatcacga taatttccaa gttttaaaca aggatatatt gcagtttaaa 1680
tttcctaaaa accaatccta taaaatatat ggtaatatac cttataacat aagtacggat 1740
ataatacgca aaattgtttt tgatagtata gctaatgaga tttatttaat cgtggaatac 1800
gggtttgcta aaagattatt aaatacaaaa cgctcattgg cattactttt aatggcagaa 1860
gttgatattt ctatattaag tatggttcca agagaatatt ttcatcctaa acctaaagtg 1920
aatagctcac ttatcagatt aagtagaaaa aaatcaagaa tatcacacaa agataaacaa 1980
aagtataatt atttcgttat gaaatgggtt aacaaagaat acaagaaaat atttacaaaa 2040
aatcaattta acaattcctt aaaacatgca ggaattgacg atttaaacaa tattagcttt 2100
gaacaattct tatctctttt caatagctat aaattattta ataagtaagg gcccgtcgac 2160
tgcagaggct gtcagaccaa gtttactcat atatacttta gattgattta cgtctcattt 2220
tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc 2280
tgcgaagtga tcttccgtca caggtaggat ccgataaagt actattcaga tagaacgaga 2340
aatgagcttg ttctatccgc ccggggctga ggggaattcg atctcaatga atattggttg 2400
acacgggcgt ataagacatg ttatactgtt gaataacaag tttaccgttc ccaaaaataa 2460
agaaggagga acagtctcga gaagatcttg atatccatat gaggcctaat ctagatgcat 2520
tcgcgagggt accaccggtg tttggattgt cggagttgta ctcgtccgtt aaggatgaac 2580
agttcttcgg ggttgagtct gctaactaat tagccattaa cagcggctta actaacagtt 2640
agtcattggc aattgtcaaa aaattgttaa tcagccaaaa cccactgctt actgatgttc 2700
aacttcgaca gcaacagtgg cggggattta tatggagacc aagcccaatt tcgtttgcga 2760
atttacacca gcgccggttt ttcccccgat ggcattgcca gtttactacc cacagaattt 2820
gaacggtatt ttcaactcca agcggaagat attacgggac ggacagttat cctaacccaa 2880
actggtgttg attatgaaat tcccggcttt ggtctggtgc aggtgttggg gctggcggat 2940
ttggccgggg ttcaggacag ctatgacctg acttacatcg aagatcatga caactattac 3000
gacattatcc tcaaagggga cgaagccgca gttcgccaaa ttaagagggt tgctttgccc 3060
tccgaagggg attattcggc ggtttataat cccggtggcc ccggcaatga tccagagaat 3120
ggtcccccag ggccctttac tgtgtccagt agtccccagg taattaaggt aacggatacc 3180
atcggccagc ccaccaaagt ctcctatgtg gaagtggatg gccccgtatt gcgtaatccc 3240
ttcagtggta ctcccattgg gcaagaggtg ggtttagcgg ttaaagatga attcactggc 3300
cgtcgtttta caacgtcgtg actgggaaaa c 3331

Claims (3)

1.高效生物合成虾青素的集胞藻6803基因工程菌的构建方法，其特征是包括如下步骤：
(1)以质粒pUC57-WD为模板，扩增获得短波毛单胞菌β-类胡萝卜素酮酶基因CrtWD，所述CrtWD基因的核苷酸序列如SEQ ID NO.1所示；以质粒pUC57-ZA为模板，扩增获得产碱杆菌β-胡萝卜素羟化酶基因CrtZA，所述CrtZA基因的核苷酸序列如SEQ ID NO.2所示；
(2)将含有psbA启动子和rbcL终止子的表达载体pTZD，与步骤(1)获得的CrtWD基因片段构建重组表达载体pTZD-WD，所述表达载体pTZD的核苷酸序列如SEQ ID NO.29所示；
(3)将含有cpc560启动子和rbcL终止子的表达载体pBA3031M，与步骤(1)获得的CrtZA基因构建重组表达载体pBA3031M-ZA，所述表达载体pBA3031M的核苷酸序列如SEQ IDNO.30所示；
(4)将所述重组表达载体pTZD-WD与重组表达载体pBA3031M-ZA依次转入集胞藻6803中，涂布于含有氯霉素和壮观霉素的BG11固体平板培养基，挑取阳性转化子进行验证，得到重组集胞藻6803工程菌命名为WD-ZA；
(5)以聚球藻7002基因组为模板，扩增果糖-1,6-二磷酸酶/景天庚酮糖-1,7-二磷酸酶FBS基因，所述FBS基因的核苷酸序列如SEQ ID NO.3所示；
(6)将含有psbA启动子和BBa_B0015终止子的表达载体pJAK，与步骤(5)中获得的FBS基因构建重组表达载体pJAK-FBS，所述表达载体pJAK的核苷酸序列如SEQ ID NO.31所示；
(7)将所述重组表达载体pJAK-FBS转入步骤(4)获得的工程菌WD-ZA中，涂布于含有氯霉素、壮观霉素和卡纳霉素的BG11固体平板培养基，挑取阳性转化子进行验证，得到工程菌株WD-ZA-FBS；
(8)以大肠杆菌DH5α基因组为模板，扩增1-脱氧-D-木酮糖-5-磷酸合成酶基因dxs-E和法呢基焦磷酸合酶基因ispA-E，所述dxs-E基因的核苷酸序列分别如SEQ ID NO.4所示，所述ispA-E基因的核苷酸序列如SEQ ID NO.5所示；利用融合PCR方法，构建操纵子片段dxs-RBS-ispA，所述dxs-RBS-ispA片段的核苷酸序列如SEQ ID NO.26所示；
(9)将含有psbA启动子和rbcL终止子的表达载体p0168，与步骤(8)中获得的dxs-RBS-ispA片段构建重组表达载体p0168-DI，所述表达载体p0168的核苷酸序列如SEQ ID NO.32所示；
(10)将所述重组表达载体p0168-DI转入步骤(7)获得的菌株WD-ZA-FBS中，氯霉素、壮观霉素、卡纳霉素和红霉素的BG11固体平板培养基，挑取阳性转化子进行验证，获得高产菌株WD-ZA-FBS-DI。
2.权利要求1的方法构建的高效生物合成虾青素的集胞藻6803基因工程菌。
3.权利要求2的高效生物合成虾青素的集胞藻6803基因工程菌在生产虾青素中的应用。
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