CN110468091B - 微生物及其用途 - Google Patents

微生物及其用途 Download PDF

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CN110468091B
CN110468091B CN201810436934.8A CN201810436934A CN110468091B CN 110468091 B CN110468091 B CN 110468091B CN 201810436934 A CN201810436934 A CN 201810436934A CN 110468091 B CN110468091 B CN 110468091B
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叶紫玲
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Shenzhen Aigexin Technology Co ltd
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Abstract

本发明提出了一种微生物。该微生物过表达IDI1、JI‑018‑A、ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1以及ERG20基因。利用根据本发明实施例的微生物发酵生产法尼烯，相对于现有技术，法尼烯的产量显著提高，生产周期短，生产效率高。

Description

微生物及其用途
技术领域
本发明涉及生物工程领域，具体地，本发明涉及微生物及其用途，更具体地，本发明涉及微生物、制备法尼烯的方法以及制备微生物的方法。
背景技术
法尼烯最早从苹果树中分离得到，在植物防御方面起重要作用。由于其是一种重要的化工原料，在新兴航空燃料、橡胶、天然维生素E等领域具有广阔的市场价值，近年来受到广泛关注。
法尼烯在植物中的含量非常低，无法以提取天然产物的方式应用于市场。化学合成法虽然具有原料易得价廉、反应条件温和、反应速率快、产物易与反应体系分离等优点，但由于合成过程中β-法尼烯双键的立体构型难以控制及不同程度的化学试剂残留，其产品的质量、安全性及使用范围都受到了限制。微生物发酵法主要是利用微生物的生物代谢，将葡萄糖等廉价原料转化为法尼烯，这种方法不受季节、地域、气候等因素的影响，原料易获取、生产周期短、工艺操作简单、成本低廉、产物质量可控、产物易纯化、安全性高，并且环境污染较少，因而以微生物生产法尼烯是一种可行的方式。目前合成法尼烯的途径主要有MEP途径和MVA途径，参考图1。在此基础上，可以通过表达法尼烯合酶以生成法尼烯，参考图2。
然而，如何提高微生物生产β-法尼烯的产量，还需进一步研究。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
微生物发酵的最大问题就是如何提升产量，目前的报道都是随机地对微生物进行改造以提升微生物发酵合成法尼烯的产量。然而如何定向改造，才能够快速有效地提高微生物发酵生产法尼烯的产量，目前并没有相关的详细研究。基于上述问题，本申请发明人通过优化法尼烯MVA合成途径中各步骤的酶的含量比，构建了相应的微生物。利用该微生物发酵生产法尼烯，相对于现有技术，法尼烯的产量显著提高，生产周期短，生产效率高。
为此，在本发明的第一方面，本发明提出了一种微生物。根据本发明的实施例，所述微生物过表达IDI1、JI-018-A、ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1以及ERG20基因。利用根据本发明实施例的微生物，相对于现有技术，法尼烯的产量显著提高，生产周期短，生产效率高。
根据本发明的实施例，上述微生物还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20以及J1-018-A基因的表达量的比为2：2：5：2：2：2：2：2：5。发明人发现，所述微生物的上述基因的表达量的比在该比例时，法尼烯的产量进一步提高。
根据本发明的实施例，所述微生物为酵母菌、大肠杆菌或链霉菌。酵母菌发酵方法成熟，大肠杆菌生产周期短，同时发明人克服了导入链霉菌时存在的遗传操作困难、阳性接合子概率低等技术难点，利用本发明实施例的微生物生产法尼烯的产量和效率进一步提高。
在本发明的第二方面，本发明提出了一种制备法尼烯的方法。根据本发明的实施例，所述方法包括：在合适微生物发酵的条件下，将上述微生物进行发酵处理，以便获得法尼烯。利用根据本发明实施例的方法，能够高产量、高效率地获得法尼烯，且法尼烯的纯度高。
根据本发明的实施例，上述方法还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，进一步包括向发酵体系中加入乙酰-CoA。其中，乙酰-CoA作为MVA发酵途径的底物，在发酵过程不必须首先加入或补充。向发酵体系中补充乙酰-CoA，法尼烯的产量进一步提高。
在本发明的第三方面，本发明提出了一种制备上述微生物的方法。根据本发明的实施例，所述方法包括：
将A质粒导入菌株A，所述A质粒携带ERG8、tHMG1以及ERG12基因，以便获得菌株B，其中，所述菌株A为酵母菌株；
将B质粒导入所述菌株B，所述B质粒携带IDI1、ERG10以及MVD1基因，以便获得菌株C；
将C质粒导入所述菌株C，所述C质粒携带Cre酶的基因，以便获得菌株D；
将D质粒导入所述菌株D，所述D质粒携带ERG13以及tHMG1基因，以便获得菌株E；
将E质粒导入所述菌株E，所述E质粒携带J1-018-A以及ERG20基因，以便获得所述微生物，所述微生物为菌株F。
利用根据本发明实施例的方法制备的微生物，扩增速率高，基因表达量的比比较接近上述预定比例，能够高产量、高效率地获得法尼烯，且法尼烯的纯度高。
根据本发明的实施例，上述方法还可进一步包括如下附加技术特征至少之一：
根据本发明的实施例，所述方法进一步包括：将F质粒以及G质粒导入所述菌株F，所述F质粒携带J1-018-A以及tHMG1基因，所述G质粒携带J1-018-A基因，以便获得所述微生物。发明人发现，上述质粒在上述特定结构下，获得的微生物中ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20以及J1-018-A基因的表达量的比无限接近于最优比2：2：5：2：2：2：2：2：5。利用根据本发明实施例的方法制备的微生物，扩增速率高，基因表达量的比无限接近上述预定比例，法尼烯的产量进一步提高。
在本发明的第四方面，本发明提出了一种制备上述任一项所述微生物的方法。根据本发明的实施例，所述方法包括：将H质粒、I质粒、J质粒导入大肠杆菌中，以便获得所述微生物，其中，所述H质粒携带IDI1、ERG10、MVD1、ERG8以及ERG12基因，所述IDI1、ERG10、MVD1、ERG8以及ERG12基因在所述H质粒的拷贝数为1，所述I质粒携带ERG13以及tHMG1基因，所述ERG13基因在所述I质粒的拷贝数为1，所述tHMG1基因在所述I质粒的拷贝数为3，所述J质粒携带ERG20以及J1-018-A基因，所述ERG20基因在所述J质粒的拷贝数为1，所述J1-018-A基因在所述J质粒的拷贝数为3。发明人发现，上述质粒在上述特定结构和拷贝数下，获得的微生物中ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20以及J1-018-A基因的表达量的比无限接近于最优比2：2：5：2：2：2：2：2：5。利用根据本发明实施例的方法制备的微生物，扩增速率高，基因表达量的比无限接近上述预定比例，法尼烯的产量进一步提高。
在本发明的第五方面，本发明提出了一种制备上述任一项所述微生物的方法。根据本发明的实施例，所述方法包括：将K质粒导入菌株A中，以便获得菌株B，其中，菌株A为链霉菌菌株；将L质粒导入菌株B中，以便获得所述微生物；其中，所述K质粒携带IDI1、ERG10、MVD1、ERG8、ERG12、ERG20以及ERG13基因，所述IDI1、ERG10、MVD1、ERG8、ERG12、ERG20以及ERG13基因在所述K质粒的拷贝数为1，所述L质粒携带tHMG1以及J1-018-A基因，所述tHMG1基因在所述L质粒的拷贝数为2，所述J1-018-A基因在所述L质粒的拷贝数为3。发明人发现，上述质粒在上述特定结构和拷贝数下，获得的微生物中ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20以及J1-018-A基因的表达量的比无限接近于最优比2：2：5：2：2：2：2：2：5。利用根据本发明实施例的方法制备的微生物，扩增速率高，基因表达量的比无限接近上述预定比例，法尼烯的产量进一步提高。
附图说明
图1是根据本发明实施例的生产法尼烯途径前体的MEP途径和MVA途径示意图；
图2是根据本发明实施例的微生物中法尼烯合成途径示意图；以及
图3是根据本发明实施例的MVA途径优化后的反应速率对比示意图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
实施例1体外重建MVA途径
前期Zhu等人的研究(Zhu F,et al.,2014.In vitro reconstitution ofmevalonate pathway and targeted engineering of farnesene overproduction inEscherichia coli.Biotechnol Bioeng.111(7):1396-405.)表明MVA途径各蛋白的最佳比例为AtoB:ERG13:tHMG1:ERG12:ERG8:MVD1:IDI1:IspA＝1:10:2:5:5:2:5:2，但这其中的atoB,ispA不是酵母来源的蛋白，因此发明人在此基础上，通过进一步深入研究发现，酵母体内MVA途径的蛋白的最佳比例为ERG10:ERG13:tHMG1:ERG12:ERG8:MVD1:IDI1:ERG20:J1-018-A＝2:2:5:2:2:2:2:2:5，与等摩尔的各蛋白量相比，反应速率有明显提高，参见图3。
实施例2酵母菌中MVA途径优化比例蛋白表达质粒的构建
(1)pZY401
△ChrXII-4：：G418_IDI1_pGAL1pGAL10_ERG10_pGAL7_MVD1_tADH1
质粒pZY401的具体构建过程：以酿酒酵母30000B的基因组为模板，用引物G4011_new_F和G4011_R通过PCR扩增得到片段4011_new(用于整合至ChrXII-4位点的同源左臂，引入NotI酶切位点)；用引物G4012_F和4012_R通过PCR扩增得到片段y4012(终止子tADH1)；用引物4013_F和4013_R通过PCR扩增得到片段y4013(基因MVD1)；用引物4014_F和4014_R通过PCR扩增得到片段y4014(终止子tGAL10以及启动子pGAL7)；用引物4015_F和4015_R通过PCR扩增得到片段y4015(基因ERG10)；用引物4016_F和4016_R通过PCR扩增得到片段y4016(双相启动子pGAL1-pGAL10)；用引物4017_F和4017_R通过PCR扩增得到片段y4017(基因IDI1)；用引物4018_F和4018_R通过PCR扩增得到片段y4018(终止子tCYC1)；载体pUG6为模板，用引物4019_F和G4013_R通过PCR扩增得到片段y4019(筛选标记KanMX)；酿酒酵母30000B的基因组为模板，用引物G4014_F和G4014_new_R通过PCR扩增得到片段4014_new(用于整合至ChrXII-4位点的同源左臂，引入NotI酶切位点)；载体pRS426为模板用引物G4015_new_F和G4015_new_R通过PCR扩增得到片段4015_new(载体骨架)。通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY401，然后在大肠杆菌内扩增，酶切验证以及测序正确后，用NotI线性化质粒pZY401，回收带有目的基因的片段。该敲除盒序列如SEQ ID NO：2所示。
GACTCAATGACTTCGGGATCTAATTCAGCATAAATAATCTCTTCTCCCTCGCCAGCTTCAGCTACAATCTTGCCTCTGGGGTCCACCACTATAGAGTGGCCATATGCATGGTAAGAACTCTGTAGATTCCTTGCGGGAGAACATAACATCACGTACACTTGGTTGTCAACTGCCCTGCTTCTTGCCAGTAAATGCCAATGCAAGGGCCCTGTGACAGTGTTGAATGCACTAGGGTAGATCATGGCAAACGCACCCTTGCGTGCACTTAGCATTGCGAGTTCCGGAAACCTCATGTCATAGCATATACCTACGCCAAACTTACCATATTTCGTGTCAATGGTAGTAGACTTCTCTCCAGGGCTCAAGGTTTCACTTTCATGGAACGATATACCGTTAGGAATGTCTACATCGAAAAGATGGACCTTCCGATGCTTGTCAATCAACTTGCCATCCTCGTTGAAAATTATAGAGGTGTTGTAGATTTTGTCTGTCTTTGGATCCAATTCCGGAATTGTTCCTCCTACCAAGATGATTTTAAACTTATTGGCTAAATTAGACAGAAATTGAACTGACGTCGAAGGCTCTTTCGGATTGATAACTTCTGAATACTTTCTAAACTGATCAGTGGAGTACGGAGAATTGAAGCATTCTGGCAATACGACCAACTTGGTATCCGGCTGTTCCTTCATAGCCCTTTCAATGAACGTTGCAGCCCTTTGAAGATTGGCCATTTTGTCAGGACTCGAGCCTGACAGTTGGACCAACGCAACTTTAATTTTTTGTGAAAGAATCTTCGAAGCACTCATACTGGCGATCTTCACGCCCTCCTGCTATTACAAAAGCTGTGTTTTTACAAGAATCAAATTAAGTTAGCAAGATATCATACAACATTATTGATAATTTCAATATCGTGTTCGTACCTGATGACGTATCTGTGCATTGATAAGGCCCGCATGGTTTCAGAAAGCAGAGCGGAACGATTCCAAATTAGTGGCCTTGTGCTTTGCATGTCAATTGTGTTACCTTCAGCTCGTGGATTTGTTTTATCAATACACAGTCTACAGTCAAGNNNNNNNNNNTATCAAATTTTGCGTTCGAGCGTATAAAATAGCCGCTGTAGCTACTTAAGTTCCTGTTCAGCCGGTAGAGGTGTGGTCAATAAGAGCGACCTCATGCTATACCTGAGAAAGCAACCTGACCTACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTTTTAAAACCTAAGAGTCACTTTAAAATTTGTATACACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCTTATTCCTTTGGTAGACCAGTCTTTGCGTCAATCAAAGATTCGTTTGTTTCTTGTGGGCCTGAATCGACTTGAGTTAAAATCACTCTGGCAACATCCTTTTGCAACTCAAGATCCAATTCACGTGCAGTAAAGTTAGATGATTCAAATTGATGGTTGAAAGCCTCAAGCTGCTCAGTAGTAAATTTCTTGTCCCATCCAGGAACAGAGCCAAACAATTTATAGATAAATGCAAAGAGTTTCGACTCATTTTCAGCTAAGTAGTACAACACAGCATTTGGACCTGCATCAAACGTGTATGCAACGATTGTTTCTCCGTAAAACTGATTAATGGTGTGGCACCAACTGATGATACGCTTGGAAGTGTCATTCATGTAGAATATTGGAGGGAAAGAGTCCAAACATGTGGCATGGAAAGAGTTGGAATCCATCATTGTTTCCTTTGCAAAGGTGGCGAAATCTTTTTCAACAATGGCTTTACGCATGACTTCAAATCTCTTTGGTACGACATGTTCAATTCTTTCTTTAAATAGTTCGGAGGTTGCCACGGTCAATTGCATACCCTGAGTGGAACTCACATCCTTTTTAATATCGCTGACAACTAGGACACAAGCTTTCATCTGAGGCCAGTCAGAGCTGTCTGCGATTTGTACTGCCATGGAATCATGACCATCTTCAGCTTTTCCCATTTCCCAGGCCACGTATCCGCCAAACAACGATCTACAAGCTGAACCAGACCCCTTTCTTGCTATTCTAGATATTTCTGAAGTTGACTGTGGTAATTGGTATAACTTAGCAATTGCAGAGACCAATGCAGCAAAGCCAGCAGCGGAGGAAGCTAAACCAGCTGCTGTAGGAAAGTTATTTTCGGAGACAATGTGGAGTTTCCATTGAGATAATGTGGGCAATGAGGCGTCCTTCGATTCCATTTCCTTTCTTAATTGGCGTAGGTCGCGCAGACAATTTTGAGTTCTTTCATTGTCGATGCTGTGTGGTTCTCCATTTAACCACAAAGTGTCGCGTTCAAACTCAGGTGCAGTAGCCGCAGAGGTCAACGTTCTGAGGTCATCTTGCGATAAAGTCACTGATATGGACGAATTGGTGGGCAGATTCAACTTCGTGTCCCTTTTCCCCCAATACTTAAGGGTTGCGATGTTGACGGGTGCGGTAACGGATGCTGTGTAAACGGTCATTTTTGAGGGAATATTCAACTGTTTTTTTTTATCATGTTGATGCTCTGCATAATAATGCCCATAAATATTTCCGACCTGCTTTTATATCTTTGCTAGCCAAACTAACTGAACATAGCTACACATTATTTTCAGCTTGGCTATTTTGTGAACACTGTATAGCCAGTCCTTCGGATCACGGTCAACAGTTGTCCGAGCGCTTTTTGGACCCTTTCCCTTATTTTTGGGTTAAGGAAAATGACAGAAAATATATCTAATGAGCCTTCGCTCAACAGTGCTCCGAAGTATAGCTTTCCAAAAGGAGAGGCAAAGCAATTTAAGAATGTATGAACAAAATAAAGGGGAAAAATTACCCCCTCTACTTTACCAAACGAATACTACCAATAATATTTACAACTTTTCCTTATGATTTTTTCACTGAAGCGCTTCGCAATAGTTGTGAGCGATATCAAAAGTAACGAAATGAACTTCGCGGCTCGTGCTATATTCTTGTTGCTACCGTCCATATCTTTCCATAGATTTTCAATCTTTGATGTCTCCATGGTGGTACAGAGAACTTGTAAACAATTCGGTCCCTACATGTGAGGAAATTCGCTGTGACACTTTTATCACTGAACTCCAAATTTAAAAAATAGCATAAAATTCGTTATACAGCAAATCTATGTGTTGCAATTAAGAACTAAAAGATATAGAGTGCATATTTTCAAGAAGGATAGTAAGCTGGCAAATCATATCTTTTCAATGACAATAGAGGAAGCACCACCACCACCATTACAAATGGCGGCAACACCGATCTTACCTCCTTCTTGCTGTAAGATGGATAGCAGTGTAACAACCACTCTAGCACCAGAACAACCCAATGGGTGACCTAGAGCAACAGCACCACCATATACATTAACCTTAGATGGGTCTAGCTTCAAAATCTTAGTGTTCACCAAACCGACAACCGAAAAGGCTTCATTGAATTCAAAGTAATCAACAGAATTGATGTCTTCGATGCCAGCATGTTTCAAAGCCTTTGGAACTGCAAGAGATGGAGCCCATGTAAAATCAGCTGGTTGATGAGCGGCCTCACCCCAACCTTTGATAATAGCCAAAGGCTTCAAATTCTTTTCCTTCAAAACTTTTTCGGAAACCAAGATGACGGCTGCAGCACCATCGTTGATTGGAGAAGCGTTAGCGGCAGTAACAGTACCGTTTTCTTTTTGGAAAACAGTCCTTGCAGATCTCAATTTTTCAACGTGTAATCTAGCAGGTTCCTCGTCCTTCGTGACTTGAGTATCAGGCTTACCTCTAAATCCCTTAATGGTAACAGGTACAATTTCATTGTCGAATTTACCTTCCTTTTGAGATTTTTGAGATTTTTGGTAGGATTCGATGGCAAAATTGTCTTGTTGTTCTCTAGTAATATCCCAATCACGGGCACACTTTTCTGCGTGTACACCCATGGCTAGACCATCGTACGCATCGTTCAACCCATCTCTTTCGACACCATCAACAAGAACAGTTTGGCCAAATTTGGCACCCGCACGGGCTGCTGGCATGTAGTATGGTGCGTTAGTCATAGATTCACAACCACCAGCTACGACAACATCAGCATTACCACATTTGATGGATTGAGCACCCAAAATGATTGCCTTCATAGCGGATGCACAGACCTTGTTAACTGTGCTTGCAACGATATGATTACTCAAACCGGCAGCCAAAGCAACTTGTCTGGCCGGAGCTTGGCCCAAATTGGCAGAAAGAACGTTACCAAAAATAATTTCGTCAAAATCCTTGGATGCATCCAATTCTGGAACCTTAGCCAAGGCGCCTTTTAAAGCAACAGCACCCAATTCCACTGCTGTCTTGGAGGATAGAGAACCCTGGAATGAACCAATTGGGGTTCTGGCAGTCGATACAATGTAAACGTTCTGAGACATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGACTGCCGACAACAATAGTATGCCCCATGGTGCAGTATCTAGTTACGCCAAATTAGTGCAAAACCAAACACCTGAAGACATTTTGGAAGAGTTTCCTGAAATTATTCCATTACAACAAAGACCTAATACCCGATCTAGTGAGACGTCAAATGACGAAAGCGGAGAAACATGTTTTTCTGGTCATGATGAGGAGCAAATTAAGTTAATGAATGAAAATTGTATTGTTTTGGATTGGGACGATAATGCTATTGGTGCCGGTACCAAGAAAGTTTGTCATTTAATGGAAAATATTGAAAAGGGTTTACTACATCGTGCATTCTCCGTCTTTATTTTCAATGAACAAGGTGAATTACTTTTACAACAAAGAGCCACTGAAAAAATAACTTTCCCTGATCTTTGGACTAACACATGCTGCTCTCATCCACTATGTATTGATGACGAATTAGGTTTGAAGGGTAAGCTAGACGATAAGATTAAGGGCGCTATTACTGCGGCGGTGAGAAAACTAGATCATGAATTAGGTATTCCAGAAGATGAAACTAAGACAAGGGGTAAGTTTCACTTTTTAAACAGAATCCATTACATGGCACCAAGCAATGAACCATGGGGTGAACATGAAATTGATTACATCCTATTTTATAAGATCAACGCTAAAGAAAACTTGACTGTCAACCCAAACGTCAATGAAGTTAGAGACTTCAAATGGGTTTCACCAAATGATTTGAAAACTATGTTTGCTGACCCAAGTTACAAGTTTACGCCTTGGTTTAAGATTATTTGCGAGAATTACTTATTCAACTGGTGGGAGCAATTAGATGACCTTTCTGAAGTGGAAAATGACAGGCAAATTCATAGAATGCTATAAACAGGCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCACGCTGCAGGTCGACAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTAGAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCTCGAGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTGATATCAGATCCACTAGTGGCAATTCCCCATTAGAGTCAAATAAAAGGATGCCTCACGGAGGTATGTTACCCGCGCTATTTCACATGGCTCATTGAATTAGAGGTGGAATTTGGTGTACCCTCCCCTCCTCATCTGATGAAGTAGTGATCCGACAATTCTTAAAAGTTGTAGACATTACTTTTACCACCAACTAAGTTGTATTTATATTGCTACCCTTATCCTTTTATATCTAACTAGCGCTCATAAGGTTGGGGCAATACTAAAACTGTGTTCTTATTCAACTCATTAAATACGTGGCAGTACGTACCCTATTAGAAACAATAGGAAACAGCAGAGTCGGAAGAAGCCAAATGCCAGATTTGAAGTCCAAAACCTTGTCAAGCCAATCTTTGGGAGCGGCTATTCCTCCAGAAATTGTGTACCAAATACTTACATACCAGTTTAGGGATTTGTTAAGAAATGACCATCCAGGTACAGCAGAAAAGTTTAACGAAAACTTAACTACATTTGTCAAAAGTAATTTAACTGTTAATAAGACATTCTCCCATATTTGTCAAGTTCTCATTTATAGATATTGTAATTTAACTACCGCAAAGAGATTTCACGGTCTTTTACAGACTTTAAAGGGAAATAGATGCCTTTGCAATAAGATTGAAGTAGCCGATTTCCAAGAATTAACGTCTATTGGACTGGGGAGGTCCAGTGAGATGAATAAAATGATTAAAAATCTAACTAACGAAACTCTTCTTGAGTTTCTAATGCTCACAAAAGCAAACTTAAGGGAATTCTTAGCATGTGAAAATATTCAAGACGATTTAGACGATAATATCATAAAGTATATTCTATCTCCAGAAAAAGTCTTGAGTGTAGTGGATTTCTGTGGCTGTTCAGGTACCACTTTCACAGAAAGTTTTATTAAGGCCCTGGATAAATACTACCCTAATAAATCGATTGATCAGTACCGGCTGGAACCTATTCAACAAAATTATCAGATTACCTGCTTAGGCCTAAATGATTGTATTGATCTACCATCACACGTTCTTTGGAAGATCTTAAAAATGTTACCAGAACTACAAAAACTTGATTTATCTCATACCTCCATTGATG(SEQ ID NO：2)。
(2)质粒pZY402
△ChrXI-3：：Hygr_ERG8_pGAL1pGAL10_tHMG1_pGAL7_ERG12
质粒pZY402的具体构建过程：以酿酒酵母30000B的基因组为模板，用引物G4021_new_F和4021_R通过PCR扩增得到片段y4021(用于整合至ChrXI-3位点的同源左臂，引入NotI酶切位点)；用引物4022_F和4022_R通过PCR扩增得到片段y4022(终止子tCYC1)；用引物4023_F和4023_R通过PCR扩增得到片段y4023(基因ERG8)；用引物4024_F和4024_R通过PCR扩增得到片段y4024(双相启动子pGAL1-pGAL10)；用引物4025_F和4025_R通过PCR扩增得到片段y4025(基因tHMG1)；用引物4026_F和4026_R通过PCR扩增得到片段y4026(终止子tGAL10以及启动子pGAL7)；用引物4027_F和4027_R通过PCR扩增得到片段y4027(基因ERG12)；用引物4028_F和4028_R通过PCR扩增得到片段y4028(终止子tADH1)；载体pTM36为模板，用引物4029_F和4029_R通过PCR扩增得到片段y4029(筛选标记HygMX)；酿酒酵母30000B的基因组为模板，用引物40210_F和G4024_new_R通过PCR扩增得到片段y4020(用于整合至ChrXI-3位点的同源左臂，引入NotI酶切位点)；载体pRS426为模板用引物G4025_new_F和G4025_new_R通过PCR扩增得到片段4025_new(载体骨架)。通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY402，然后在大肠杆菌内扩增，酶切验证以及测序正确后，用NotI线性化质粒pZY402，回收带有目的基因的片段，该敲除盒序列如SEQ IDNO：3所示。
GCAGAGAATCCTCGGGTTTGTAAACCATGTTATAGTTAAACCCCGATATTACACCATTAAAATCATTTCTAAATATGCGGTCGTCGATGATCAGCTGCAGGGAAATGCCAAAATATGGACCCCATAGAGGTCTTGGCTTGTTCATATCGTTGGGTCGATTTCTGTTCCCAGTGTTATTTGGTTGAATGGGAACAGTCGCGTCTTGCCCCATCTTGTCTCTGCCGCTTTCGTCGAATCTGACTTTGCAATTGACGTTGCATATCAACGTGCCGGATCCCGGAATAGCGTGATAGTCTACTCCTGTTAGGGCATGTTGTGTTTGTACGACCTGGTTTTGCCACATTTGCAGAAAAACTGTTGCTTGCGCGAAGGGGGTAGCATTAAATATTATTCTGCAGTTGTTTGGATTAAAAAGCTGTACGAATTGGGCCAATTTGTTGGAGTCCGGATCATCCAAATGCGCTAATATCTTCTTCGTAAATGTGGCCGTTATTTGTGCTTGATTTGCGTCATTCATTACCATAGTATTACTATTGGTGTTCATATTTCTGTTATTACTGTTATTAGTTTGGCGTTTTGCCTGACGATCAGCCTGGTGGGGCCAAGTATATACTATAAAAACAGGTTAGTTACTTGCTCTATGCGTTTGCGCATCCTCTTTTTACTTTTTTTTTTTCAGTAAAGCCTAAGCATAAATCGTTTTATACGTACGACACGTTCAACTTTTCTTGGTTAGTAGTGGCAATCTCTGCAATACATACAGGGAGTCATGGTCTATCATCTTGTCCAATCAAAGAAGCATCGGTTCAGATCGAGCAAACTGTAGGGAGAAAGGAAAGTAGAAATGCAGAGTGTGCTATATGTCCAATCTCGGTTTTGTAGTTTGGATGTCATTAGAGATCTACCACCCAACCGGCTGCTTTCATGTGGAACAGAAAAGAAATCGGGGCGCTTCCTCTTCTGTATTCCTTTAATTAACGTTTTTATTCAGCCATCTAACCATCATACCCCCATACGGTAACAAAACCTCTTCTAAGAAAAGAAGTCTCTGCTCCTCCGCCATCTTATTTTTATTCGCTGCGCGCGTTTATTGTCGCATCGCTAGCCAGCAAAAAGTTGGTTGCCTTTTTTTACCTAAAAAAGACACATCTAACTGATTAGTTTTCCGTTTTAGGATATTGACGCCAAGCGTGCGTCTGATTGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCGACAAAGGAAAAGGGGCCTGTTTATTTATCAAGATAAGTTTCCGGATCTTTTTCTTTCCTAACACCCCAGTCAGCCTGAGTTACATCCAGCCATTGAACCTTAGAAAATCTTTTGTCATCAGCGGTTTGAGCCCTAAGATCAACATCTTGCTTAGCAATCACTGCAATGGCGTCATAACCACCAGCACCAGGTATTAAGCAAGTAAGAACTCCTTTTAAGGTCTGGCAATCATCCAATAAGCTAGTTTGTACGGGAGGTTCGATATCGGCACCAGATTCTTTAGTTATTTTTCTAAAGGAACGTCTAATTGTGGCAACTGCATCTCTAACTTCTGTGATCTCAGGATACTTTTGACAGGTACAGTCATTCCTCTCAAGAGACTCAAATATCTGATCGCTGTAATCGTCATGAGTCTCGTGTAAGCGATCTAGTTTAGATAGTCCATCCATAAATCTAGAATTTGCATGATCGAGTTCTGTATATATTTTCAAGCTTTCCGGCATATGCGAATCATACCAATTTTTTACCTTCTGGACCAGTTTTACTGTTTCTGAACCATTCTTAATATCGCCCATCCATAAAGTTAATCCCGAAGGTAAATGGTTACTTTTAATCGTTATATTCCAGTCTTCTTCATTAACCAAATGCGCCAGTTTACTGCCGTAAGTAGCACTTCCAATATCTGGCAAATTAGAGATTAATGCGGGTGGGAATCTTCTATATCTGATAGATCCATATGCTGCCGCCGCTACATCAAACCCGCTTCCAATTTTACCCTGAGCTTGACAATGAGCAACTTGTGATAAATTATGAATAACTTCTCTATATTTGTCTACATTATTTTCCAGGTCCGATACAAAAAAGGAGGCCAAAGCTGTAGTTAAAACTGTGACTAAACCTGCCGAGGAGCCCAGCCCTGTTTTGGGAACTTCTTCAATTCTGTGCGAATGAAAACTCAATCTTCTGTTGCCACGATGTTCGGTAACGCTGTCCTCCTGAGAATGGTAGGCATCATCAGAGAAAATATCAATAACGAACAAGTTTCTATTGCAGTAGTCGTCCATGTTAGGCTTAAAGTAGCTAAATACGTTAGCGATAACTTTTTCAATGAAAGGGTTCTTAGATCCGCCTATCGAAACAGGAATGAAGCCAGTTTTAGGACTTATATGGTACAGCCACTCCCCATCTTTAAATTGTTTACTTTTCACACGCACTTCAAACTTATCAGACTCTTGCAATGAACCGTAAGGATGGGCTACAGCATGCATTCTTGCCGATAATCCGACTACAAATGCTTCATATTTCGGATCTAAAACTAAATATCCACCAGCTAGTAACGCTTTCCCTGGGGCACTGAAGGCTCTCAACTCTGACATTATAGTTTTTTCTCCTTGACGTTAAAGTATAGAGGTATATTAACAATTTTTTGTTGATACTTTTATGACATTTGAATAAGAAGTAATACAAACCGAAAATGTTGAAAGTATTAGTTAAAGTGGTTATGCAGCTTTTGCATTTATATATCTGTTAATAGATCAAAAATCATCGCTTCGCTGATTAATTACCCCAGAAATAAGGCTAAAAAACTAATCGCATTATTATCCTATGGTTGTTAATTTGATTCGTTGATTTGAAGGTTTGTGGGGCCAGGTTACTGCCAATTTTTCCTCTTCATAACCATAAAAGCTAGTATTGTAGAATCTTTATTGTTCGGAGCAGTGCGGCGCGAGGCACATCTGCGTTTCAGGAACGCGACCGGTGAAGACCAGGACGCACGGAGGAGAGTCTTCCGTCGGAGGGCTGTCGCCCGCTCGGCGGCTTCTAATCCGTACTTCAATATAGCAATGAGCAGTTAAGCGTATTACTGAAAGTTCCAAAGAGAAGGTTTTTTTAGGCTAAGATAATGGGGCTCTTTACATTTCCACAACATATAAGTAAGATTAGATATGGATATGTATATGGTGGTATTGCCATGTAATATGATTATTAAACTTCTTTGCGTCCATCCAAAAAAAAAGTAAGAATTTTTGAAAATTCAATATAAATGGTTTTAACCAATAAAACAGTCATTTCTGGATCGAAAGTCAAAAGTTTATCATCTGCGCAATCGAGCTCATCAGGACCTTCATCATCTAGTGAGGAAGATGATTCCCGCGATATTGAAAGCTTGGATAAGAAAATACGTCCTTTAGAAGAATTAGAAGCATTATTAAGTAGTGGAAATACAAAACAATTGAAGAACAAAGAGGTCGCTGCCTTGGTTATTCACGGTAAGTTACCTTTGTACGCTTTGGAGAAAAAATTAGGTGATACTACGAGAGCGGTTGCGGTACGTAGGAAGGCTCTTTCAATTTTGGCAGAAGCTCCTGTATTAGCATCTGATCGTTTACCATATAAAAATTATGACTACGACCGCGTATTTGGCGCTTGTTGTGAAAATGTTATAGGTTACATGCCTTTGCCCGTTGGTGTTATAGGCCCCTTGGTTATCGATGGTACATCTTATCATATACCAATGGCAACTACAGAGGGTTGTTTGGTAGCTTCTGCCATGCGTGGCTGTAAGGCAATCAATGCTGGCGGTGGTGCAACAACTGTTTTAACTAAGGATGGTATGACAAGAGGCCCAGTAGTCCGTTTCCCAACTTTGAAAAGATCTGGTGCCTGTAAGATATGGTTAGACTCAGAAGAGGGACAAAACGCAATTAAAAAAGCTTTTAACTCTACATCAAGATTTGCACGTCTGCAACATATTCAAACTTGTCTAGCAGGAGATTTACTCTTCATGAGATTTAGAACAACTACTGGTGACGCAATGGGTATGAATATGATTTCTAAAGGTGTCGAATACTCATTAAAGCAAATGGTAGAAGAGTATGGCTGGGAAGATATGGAGGTTGTCTCCGTTTCTGGTAACTACTGTACCGACAAAAAACCAGCTGCCATCAACTGGATCGAAGGTCGTGGTAAGAGTGTCGTCGCAGAAGCTACTATTCCTGGTGATGTTGTCAGAAAAGTGTTAAAAAGTGATGTTTCCGCATTGGTTGAGTTGAACATTGCTAAGAATTTGGTTGGATCTGCAATGGCTGGGTCTGTTGGTGGATTTAACGCACATGCAGCTAATTTAGTGACAGCTGTTTTCTTGGCATTAGGACAAGATCCTGCACAAAATGTTGAAAGTTCCAACTGTATAACATTGATGAAAGAAGTGGACGGTGATTTGAGAATTTCCGTATCCATGCCATCCATCGAAGTAGGTACCATCGGTGGTGGTACTGTTCTAGAACCACAAGGTGCCATGTTGGACTTATTAGGTGTAAGAGGCCCGCATGCTACCGCTCCTGGTACCAACGCACGTCAATTAGCAAGAATAGTTGCCTGTGCCGTCTTGGCAGGTGAATTATCCTTATGTGCTGCCCTAGCAGCCGGCCATTTGGTTCAAAGTCATATGACCCACAACAGGAAACCTGCTGAACCAACAAAACCTAACAATTTGGACGCCACTGATATAAATCGTTTGAAAGATGGGTCCGTCACCTGCATTAAATCCTAATTTGCCAGCTTACTATCCTTCTTGAAAATATGCACTCTATATCTTTTAGTTCTTAATTGCAACACATAGATTTGCTGTATAACGAATTTTATGCTATTTTTTAAATTTGGAGTTCAGTGATAAAAGTGTCACAGCGAATTTCCTCACATGTAGGGACCGAATTGTTTACAAGTTCTCTGTACCACCATGGAGACATCAAAGATTGAAAATCTATGGAAAGATATGGACGGTAGCAACAAGAATATAGCACGAGCCGCGAAGTTCATTTCGTTACTTTTGATATCGCTCACAACTATTGCGAAGCGCTTCAGTGAAAAAATCATAAGGAAAAGTTGTAAATATTATTGGTAGTATTCGTTTGGTAAAGTAGAGGGGGTAATTTTTCCCCTTTATTTTGTTCATACATTCTTAAATTGCTTTGCCTCTCCTTTTGGAAAGCTATACTTCGGAGCACTGTTGAGCGAAGGCTCATTAGATATATTTTCTGTCATTTTCCTTAACCCAAAAATAAGGGAAAGGGTCCAAAAAGCGCTCGGACAACTGTTGACCGTGATCCGAAGGACTGGCTATACAGTGTTCACAAAATAGCCAAGCTGAAAATAATGTGTAGCTATGTTCAGTTAGTTTGGCTAGCAAAGATATAAAAGCAGGTCGGAAATATTTATGGGCATTATTATGCAGAGCATCAACATGATAAAAAAAAACAGTTGAATATTCCCTCAAAAATGTCATTACCGTTCTTAACTTCTGCACCGGGAAAGGTTATTATTTTTGGTGAACACTCTGCTGTGTACAACAAGCCTGCCGTCGCTGCTAGTGTGTCTGCGTTGAGAACCTACCTGCTAATAAGCGAGTCATCTGCACCAGATACTATTGAATTGGACTTCCCGGACATTAGCTTTAATCATAAGTGGTCCATCAATGATTTCAATGCCATCACCGAGGATCAAGTAAACTCCCAAAAATTGGCCAAGGCTCAACAAGCCACCGATGGCTTGTCTCAGGAACTCGTTAGTCTTTTGGATCCGTTGTTAGCTCAACTATCCGAATCCTTCCACTACCATGCAGCGTTTTGTTTCCTGTATATGTTTGTTTGCCTATGCCCCCATGCCAAGAATATTAAGTTTTCTTTAAAGTCTACTTTACCCATCGGTGCTGGGTTGGGCTCAAGCGCCTCTATTTCTGTATCACTGGCCTTAGCTATGGCCTACTTGGGGGGGTTAATAGGATCTAATGACTTGGAAAAGCTGTCAGAAAACGATAAGCATATAGTGAATCAATGGGCCTTCATAGGTGAAAAGTGTATTCACGGTACCCCTTCAGGAATAGATAACGCTGTGGCCACTTATGGTAATGCCCTGCTATTTGAAAAAGACTCACATAATGGAACAATAAACACAAACAATTTTAAGTTCTTAGATGATTTCCCAGCCATTCCAATGATCCTAACCTATACTAGAATTCCAAGGTCTACAAAAGATCTTGTTGCTCGCGTTCGTGTGTTGGTCACCGAGAAATTTCCTGAAGTTATGAAGCCAATTCTAGATGCCATGGGTGAATGTGCCCTACAAGGCTTAGAGATCATGACTAAGTTAAGTAAATGTAAAGGCACCGATGACGAGGCTGTAGAAACTAATAATGAACTGTATGAACAACTATTGGAATTGATAAGAATAAATCATGGACTGCTTGTCTCAATCGGTGTTTCTCATCCTGGATTAGAACTTATTAAAAATCTGAGCGATGATTTGAGAATTGGCTCCACAAAACTTACCGGTGCTGGTGGCGGCGGTTGCTCTTTGACTTTGTTACGAAGAGACATTACTCAAGAGCAAATTGACAGTTTCAAAAAGAAATTGCAAGATGATTTTAGTTACGAGACATTTGAAACAGACTTGGGTGGGACTGGCTGCTGTTTGTTAAGCGCAAAAAATTTGAATAAAGATCTTAAAATCAAATCCCTAGTATTCCAATTATTTGAAAATAAAACTACCACAAAGCAACAAATTGACGATCTATTATTGCCAGGAAACACGAATTTACCATGGACTTCATAAGCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGGGCTGCAGGTCGACAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTAGAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTCTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTCAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAGCCGGTCGCGGAGGCAATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCTCGAGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTGATATCAGATCCACTAGTGGTTACGTGGATTGAGCCAGCAATACAGATCATTATTAAACTGTTTTGTACATGATGTTAGTATATAATCGTAAAGCTTTTCTAATATGTATACCTTATACATGGAACTCCACAGAACTTGCAAACATACCAAAAATCCTTTATTCTTGTTCACTCATTTTACATCAAAAAATAATATTTCAGTTATTAAGGAAAATAAAAAAATAGATTAGAGAAGCATTTTGAAGAAATAGTATATTCTTTTATTGAACCTAAGAGCGTGATATTTTTACTCGAAATAAAATACGAAAAATCTATACACTCATCTTTCCGACTACTATTGGCTCCTGCTCAAAAAAAGAGGGAAAAAAAGCTCCAAAATTCTATCTTTTCCTATCGCTCCTGTCCTATCCTTATTACGTTCATTACTATTTTAATACTATCCATTCTTTTATTTTCAGTCTAAAAAAAACATTTCTCATAACGGGAAAAGCAAAAAAATGTCAAGCTTATACATCAAAACACCACTGCATGCATTATCTGCTGGTCCGGATTCTCATGCAAATAGCTCCTATTATGACAATCTTTTATTGCCTTCATTTTCCAATTTATCCAGTAATATCAGCAGGAACAATATCACAACCGATAACAATATAAATTCAGCAAGTCCTAGGAAGTATTCGTTTCATTCGCTCAATGTGTCGCCAATTCTTTCCCCAATATCTTTGGCTAACGAAATACTTGGAAAAAAATCAAATACTGCACCAGCATCGCCACATCATATGGATTACAATCCAATTTCCTCATTAACACCAGGCAATTCACCAGAATTCAACAAGGCAAGCTTATCTCAGATTAGTTTTACAAATCCATTGAACTATGGGTCTGGCTTAGGTTTTTCCTCTAATTCACAACCTCGACTACCATTGTTAGACAGGCTATCGTCCGTCTCTTTATCTAAGAGACCGGAGCGCCCACAACAAAGCCTACCATCACTAAGGCATCTGCAATTATTACCCAGTCCTCTTTTACAAGAAAACGCAGCACGCTTCCCTGATACATCAAAACGTACATCTAATTGGAAAACTGATCTTACCCATTGGTGTAAGGATACAAACTATCAAGACTATGTTAAAATACGCGAAGAAGTGGCTCATTTCAAGCCATTAAGTATACCCAACTTAAC(SEQ ID NO：3)。
(3)质粒pZY410
△ChrXII-2：：G418_pGAL1_ERG13_pGAL10_tHMG1
质粒pZY410的具体构建过程：以酿酒酵母30000B的基因组为模板，用引物4101_F和4101_R通过PCR扩增得到片段4101(用于整合至ChrXII-2位点的同源左臂，引入NotI酶切位点)；以酿酒酵母S288C的基因组为模板，用引物4102_F和4102_R通过PCR扩增得到片段4102(基因tHMG1以及终止子tTHMG1)；以酿酒酵母30000B的基因组为模板，用引物4103_F和4103_R通过PCR扩增得到片段4103(双相启动子pGAL1-pGAL10)；载体pZY403为模板，用引物4104_F和4104_R通过PCR扩增得到片段4104(基因ERG13以及终止子tCYC1)；载体pUG6为模板，用引物4105_F和4105_R通过PCR扩增得到片段4105(筛选标记KanMX)；酿酒酵母30000B的基因组为模板，用引物4106_F和4106_R通过PCR扩增得到片段4106(用于整合至ChrXII-2位点的同源左臂，引入NotI酶切位点)；载体pRS426为模板用引物4107_F和4107_R通过PCR扩增得到片段4107(载体骨架)。通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY410，然后在大肠杆菌内扩增，酶切验证以及测序正确后，用NotI线性化质粒pZY410，回收带有目的基因的片段，该敲除盒序列如SEQ ID NO：4所示。
GACATTGAACTCTTGGCCGACTCTTCTGATGAGGCTTTCGAAAAATCTACTTTCATTAACACAATTGAGTTCATTGTGCACGATATTTGGGTCGAAACGTTGATAAAGAACATAAACTTATTACAAATGTTTGATGCAGATTTGAAATGCTACAATGACAAGTACATAATTTGCAAGCTAAAGGGTCAGTATCCCTCCACAAATATAGTGGACATAATGTGCCGTTTGAAGCATTTTTCCAATTCTATTTTGGAAACTTTCAAATTCGGAATCGAGCTAAAAGAACAAGATCAATGCCACAATAGAAACACTATTATCAACTACGTTCTTTTCAGTAGAGTATTTTCAACGATTGTTTTAGAGATTCAAAAATGCTTTATCTTGATCGTTAAGTTCATGTATTCTGTCAATTTTTTAGAAAAATTTTCCGATGAAGTATTTTTGTCATTTATTGAAATTCTCATAAAAATTGTCTTTGAGCACCAGATTCCGCAGTTATTTTTGGGAATTGATGAAATTATTCAACTCTGGTTAAAGAACAATGAAGGAAAACGGCAACAACTCTTGAGCGCTTGGTGCAACGGAACTGTTCAAGATATGAAGCAAAGCCAACAAAGAGAGTCCTCAAACGCAGAATCTGAATCAATCACTTCAAGTACAGAAGAGGACGAAGAAGGCCTGCAATTCAATAAATGGGATGTGATTGAACCATTTATTGATAATATCAAGGCTTTGAATCAATCAAAATCCCATATGTGACGCAGCGATAAAACCGCGGCATGCAAACATCTACACAATTAGCAAGGGCAATCCATATTTTGTCTTTTCGCGCCCTGGAAAGGCCTAAGTAATGTCGTAAACGCATTCTATCTGTACTTCAACTCTCCTCTGTGCATTGGTTTGTGCAAATCACATTTTACGATACTGCCAGATATATGCAAAAAGAGAAAACCAAGGGACCAGAACAAAGCAAAATTACGATATTCTTCGAATTCCTTCGTGCTTGACTAAGACAAAGGGATGGACGTAGCGATTTTTAGCGGGCCAAGAACTGGTTCCGAAAAAGCACAGGTACACCGAACCCTCAGCTAAGGAGGGACAGCACCGATGCGGAAGGACAAACTTTCTTTTTGCCTATCACAGTATCTTATCGAGCTAACTATTTTCGACACACATGAAAAAGCAGAAATATTAACGAAAAAGAAAAGAAAGACCATGTCATGTACGGGCAATCAGAATCTGTAACAAGCGCCATTTTTTTTTCTGTATCGGGCCCTCCTTACTGCTCTCCTTCCGTGTAACGCGTTATGGCTTCATTGCTCCTACATTTATCGGATGGCGCACATAGCTAAAAAAGGAAGATTTTCACTCATAAGGGAAAGGAACCGCATGACTCAAGAGAAGCATGTGGTTTTTGAGTTTTTTTCGTTGAATTTTCAGGTAAAGCTCAATAGTTATGACAATTACAACAACAGAATTCTTTCTATATATGCACGAACTTGTAATATGGAAGAAATTATGACGTACAAACTATAAAGTAAATATTTTACGTAACACATGGTGCTGTTGTGCTTCTTTTTCAAGAGAATACCAATGACGTATGACTAAGTTTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGATTTATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATATGACTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGATAATTCACCTGCCAAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGGAGCGGTAGCATGCGGGCCTCTTACACCTAATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGATGGTACCTACTTCGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAGTTGGAACTTTCAACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAATTAGCTGCATGTGCGTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAATTCTTAGCAATGTTCAACTCAACCAATGCGGAAACATCACTTTTTAACACTTTTCTGACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACGACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTTCCCAGCCATACTCTTCTACCATTTGCTTTAATGAGTATTCGACACCTTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTCTAAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATCTTGATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAGGCACCAGATCTTTTCAAAGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAAAACAGTTGTTGCACCACCGCCAGCATTGATTGCCTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAACACCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGTCGTAGTCATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGAAAGAGCCTTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGAATAACCAAGGCAGCGACCTCTTTGTTCTTCAATTGTTTTGTATTTCCACTACTTAATAATGCTTCTAATTCTTCTAAAGGACGTATTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATCTTCCTCACTAGATGATGAAGGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTCGATCCAGAAATGACTGTTTTATTGGTTAAAACCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGAAACTCTCAACTAAACTTTGTTGGTGTGGTATTAAAGGAAGACTTAGGCCGCAAAAGCAACAACAATTACACAATACAAACTTGCAAATGACTGAACTAAAAAAACAAAAGACCGCTGAACAAAAAACCAGACCTCAAAATGTCGGTATTAAAGGTATCCAAATTTACATCCCAACTCAATGTGTCAACCAATCTGAGCTAGAGAAATTTGATGGCGTTTCTCAAGGTAAATACACAATTGGTCTGGGCCAAACCAACATGTCTTTTGTCAATGACAGAGAAGATATCTACTCGATGTCCCTAACTGTTTTGTCTAAGTTGATCAAGAGTTACAACATCGACACCAACAAAATTGGTAGATTAGAAGTCGGTACTGAAACTCTGATTGACAAGTCCAAGTCTGTCAAGTCTGTCTTGATGCAATTGTTTGGTGAAAACACTGACGTCGAAGGTATTGACACGCTTAATGCCTGTTACGGTGGTACCAACGCGTTGTTCAACTCTTTGAACTGGATTGAATCTAACGCATGGGATGGTAGAGACGCCATTGTAGTTTGCGGTGATATTGCCATCTACGATAAGGGTGCCGCAAGACCAACCGGTGGTGCCGGTACTGTTGCTATGTGGATCGGTCCTGATGCTCCAATTGTATTTGACTCTGTAAGAGCTTCTTACATGGAACACGCCTACGATTTTTACAAGCCAGATTTCACCAGCGAATATCCTTACGTCGATGGTCATTTTTCATTAACTTGTTACGTCAAGGCTCTTGATCAAGTTTACAAGAGTTATTCCAAGAAGGCTATTTCTAAAGGGTTGGTTAGCGATCCCGCTGGTTCGGATGCTTTGAACGTTTTGAAATATTTCGACTACAACGTTTTCCATGTTCCAACCTGTAAATTGGTCACAAAATCATACGGTAGATTACTATATAACGATTTCAGAGCCAATCCTCAATTGTTCCCAGAAGTTGACGCCGAATTAGCTACTCGCGATTATGACGAATCTTTAACCGATAAGAACATTGAAAAAACTTTTGTTAATGTTGCTAAGCCATTCCACAAAGAGAGAGTTGCCCAATCTTTGATTGTTCCAACAAACACAGGTAACATGTACACCGCATCTGTTTATGCCGCCTTTGCATCTCTATTAAACTATGTTGGATCTGACGACTTACAAGGCAAGCGTGTTGGTTTATTTTCTTACGGTTCCGGTTTAGCTGCATCTCTATATTCTTGCAAAATTGTTGGTGACGTCCAACATATTATCAAGGAATTAGATATTACTAACAAATTAGCCAAGAGAATCACCGAAACTCCAAAGGATTACGAAGCTGCCATCGAATTGAGAGAAAATGCCCATTTGAAGAAGAACTTCAAACCTCAAGGTTCCATTGAGCATTTGCAAAGTGGTGTTTACTACTTGACCAACATCGATGACAAATTTAGAAGATCTTACGATGTTAAAAAATAATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGCACGCTGCAGGTCGACAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTCTAGAGATCTGTTTAGCTTGCCTCGTCCCCGCCGGGTCACCCGGCCAGCGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAACCATGGGTAAGGAAAAGACTCACGTTTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGCAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGTACTGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGCTGTCGATTCGATACTAACGCCGCCATCCAGTGTCGAAAACGAGCTCTCGAGAACCCTTAATATAACTTCGTATAATGTATGCTATACGAAGTTATTAGGTGATATCAGATCCACTAGTGGCTACTATCGGCGACTCTCTCGAAATTTTTCTTAACGCGTCCTTGTACTGCGTCTAACGCTTTTGCCACTTGGATTTCTATTATAGGAAATAGTCTCACTTACTGGGCGACGAATTTTCGCGTTTTGATGAAGCACAGGAAGAATTTCTTTTTTTTTTGGCTTCTTCTGGTTCCGTTTTTTACGCGCACAAATCTAAAAAAAGAAATAATTATAACCTAGTCTCGAAAATTTTCATCGATCCATTCGTTCCTTTTTTTCGATTTTTTCAGATCAAAATTCTTGTTTCTTTCTTTGTCTTAGTTTATATTAAAAGATATTTTGATTTTACTCCTGAACTATTTATTCTTTCTAAGAAGGCCAGAACACTACAGCTGTTTTAACCGACTACGAAGTTCTCCATTCTCGAACACTAGCCTTCATTTACCAAACAGGAACTAGCGTATATCATTAGTCCTTATTCGAAAAGAGATTGGTAGATATTTATTGTAGTTTGTGAGAAGGAGAAAATACTGTCATTGGACTGATAGTTAGAGGACATTAACCTCTCTTACGTTCGCTCAAAAAAATTAAAATAAGCAAGTAGCTGAAATCAAGTCTTTCATACAACGGTCAGACCATGAACACTGATCAACAACCTTATCAGGGCCAAACGGACTATACCCAGGGACCAGGTAACGGGCAAAGTCAGGAACAAGACTATGACCAATATGGCCAGCCTTTGTATCCTTCACAAGCTGATGGTTACTACGATCCAAATGTCGCTGCTGGTACTGAAGCTGATATGTATGGTCAACAACCACCAAACGAGTCTTACGACCAAGACTACACAAACGGTGAATACTATGGTCAACCGCCAAATATGGCTGCTCAAGACGGTGAAAACTTCTCGGATTTTAGCAGTTACGGCCCTCCTGGAACACCTGGATATGATAGCTATGGTGGTCAGTATACCGCTTCTCAAATGAGTTATGGAGAACCAAATTCGTCGGGTACCTCGACTCCAATTTACGGTAATTATGACCCAAATGCTATCGCTATGGCTTTGCCAAATGAACCTTATCCCGCTTGGACTGCTGACTCTCAATCTCCCGTTTCGATCGAGCAAATCGAAGATATCTTTATTGATTTGACCAACAGACTCGGGTTCCAAAGAGACTCCATGAGAAATATGTTTGATCATTTTATGGTTCTCTTGGACTCTAGGTCCTCGAGAATGTCTCCTGATCAAGCTTTACTATCTTTACATGCCGACTACATTGGTGGCGATACTGCTAACTATAAAAAATGGTATTTTGCTGCTCAG(SEQ ID NO：4)。
(4)质粒pZY516
△LEU2：：LEU2(URA3)_TCYC1_J1-018-A_pGAL10pGAL1_ERG20_tERG20
质粒pZY516的具体构建过程：以载体pZY153为模板，用引物5161_F和5161_R通过PCR扩增得到片段5161；以载体pZY513为模板，用引物5162_F和5162_R通过PCR扩增得到片段5162；以载体pZY153为模板，用引物5163_F和5163_R通过PCR扩增得到片段5163；以载体pZY153为模板，用引物5164_F和5164_R通过PCR扩增得到片段5164；通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY516，然后在大肠杆菌内扩增，酶切验证以及测序正确后，用NotI线性化质粒pZY516，回收带有目的基因的片段。该敲除盒序列如SEQ IDNO：5所示。
ATAACGAGAACACACAGGGGCGCTATCGCACAGAATCAAATTCGATGACTGGAAATTTTTTGTTAATTTCAGAGGTCGCCTGACGCATATACCTTTTTCAACTGAAAAATTGGGAGAAAAAGGAAAGGTGAGAGCGCCGGAACCGGCTTTTCATATAGAATAGAGAAGCGTTCATGACTAAATGCTTGCATCACAATACTTGAAGTTGACAATATTATTTAAGGACCTATTGTTTTTTCCAATAGGTGGTTAGCAATCGTCTTACTTTCTAACTTTTCTTACCTTTTACATTTCAGCAATATATATATATATATTTCAAGGATATACCATTCTAATGTCTGCCCCTAAGAAGATCGTCGTTTTGCCAGGTGACCACGTTGGTCAAGAAATCACAGCCGAAGCCATTAAGGTTCTTAAAGCTATTTCTGATGTTCGTTCCAATGTCAAGTTCGATTTCGAAAATCATTTAATTGGTGGTGCTGCTATCGATGCTACAGGTGTTCCACTTCCAGATGAGGCGCTGGAAGCCTCCAAGAAGGCTGATGCCGTTTTGTTAGGTGCTGTGGGTGGTCCTAAATGGGGTACCGGTAGTGTTAGACCTGAACAAGGTTTACTAAAAATCCGTAAAGAACTTCAATTGTACGCCAACTTAAGACCATGTAACTTTGCATCCGACTCTCTTTTAGACTTATCTCCAATCAAGCCACAATTTGCTAAAGGTACTGACTTCGTTGTTGTCAGAGAATTAGTGGGAGGTATTTACTTTGGTAAGAGAAAGGAAGACGATGGTGATGGTGTCGCTTGGGATAGTGAACAATACACCGTTCCAGAAGTGCAAAGAATCACAAGAATGGCCGCTTTCATGGCCCTACAACATGAGCCACCATTGCCTATTTGGTCCTTGGATAAAGCTAATGTTTTGGCCTCTTCAAGATTATGGAGAAAAACTGTGGAGGAAACCATCAAGAACGAATTCCCTACATTGAAGGTTCAACATCAATTGATTGATTCTGCCGCCATGATCCTAGTTAAGAACCCAACCCACCTAAATGGTATTATAATCACCAGCAACATGTTTGGTGATATCATCTCCGATGAAGCCTCCGTTATCCCAGGTTCCTTGGGTTTGTTGCCATCTGCGTCCTTGGCCTCTTTGCCAGACAAGAACACCGCATTTGGTTTGTACGAACCATGCCACGGTTCTGCTCCAGATTTGCCAAAGAATAAGGTCAACCCTATCGCCACTATCTTGTCTGCTGCAATGATGTTGAAATTGTCATTGAACTTGCCTGAAGAAGGTAAGGCCATTGAAGATGCAGTTAAAAAGGTTTTGGATGCAGGTATCAGAACTGGTGATTTAGGTGGTTCCAACAGTACCACCGAAGTCGGTGATGCTGTCGCCGAAGAAGTTAAGAAAATCCTTGCTTAAATTTAACTCCTTAAGTTACTTTAATGATTTAGTTTTTATTATTAATAATTCATGCTCATGACATCTCATATACACGTTTATAAAACTTAAATAGATTGAAAATGTATTAAAGATTCCTCAGGGATTCGATTTTTTTGGAAGTTTTTGTTTTTTTTTCCTTGAGATGCTGTAGTATTTGGGAACAATTATACAATCGAAAGATATATGCTTACATTCGACCGTTTTAGCCGTGATCATTATCCTATAGTAACATAACCTGAAGCATAACTGACACTACTATCATCAATACTTGTCACATGAGAACTCTGTGAATAATTAGGCCACTGAAATTTGATGCCTGAAGGACCGGCATCACGGATTTTCGATAAAGCACTTAGTATCACACTAATTGGCTTTTCGCGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCGACAAAGGAAAAGGGGCCTGTCTAACACTTGCAAGGAAGTGGTCTCTCGCCGTGACGAACGTATCTGCTCAAAGTAGCAGAATGGAAGATATTGCCTGTCAACGCATCAAAGAGCGCAGTAACGAACACGCGCTGGCGATCGTTGATATCAGGGCTGTGCATAAACTTCTCACAAATGCCGTAAAACTCCTTCTCCATGTCAAAGGTAATAGCTCTGGTTAAGTTCTTGGCCAGGTCAGGCGACACGGAAAGGAGCTGCTCTGTGACAGCAACTGCGTTCACGATGGACGCGTCGATAGTCTTGACCTCGTGCACCTCCTTATCATACGAGTAGAGATCGTTGATAAGAATTGAGTGTCTGACGTAAAGATCAGCAAGCTCGTCAAAGGGAGCAAGTTCTTCGCGTGTTTGTTGCACGCCAGAGCCGAAGCGGATAGCTGCAATCATAAATGTCTTGCCAACATCATTGCGACGGTAAAGCATGTAATCAGCCAATGTGGCATAGTGATCCTGCTTAAAGGGTGACTTTACACGACCATCACGGACGAAAGCATGGATAGCATAAAGCAGATCAGGTCCAAGGACGGGATCACATTCCAATACGTCTGACCAGAATTTCTTAAGATGCCTGTTCTTGGGAGCCATGCTAAGAAATTCGTCTACCACTGTGGTTTCGTTTTGAGTAGCTCCTCGCTCAATAACATCGTCGTGGAGGAATAGAAGCAGAACAGACTGGGTCAACAACGCCAATCTGGTAGGAGATGATGTAGGGTAAAGATAAGCACAGCAAGAGATGGCATCCTCAACTAGCTCATCAATCTTCCACTTGCGACGCTCATCCATCAACTCCATGGGTAGTGTGCCACGCTCTGATGCCTTGTCAAGGCTGATTCCGTTGGCCATTTCATATCCAGCTGCCTCGGCGTAAGCCCAGAACTTGGATTGTCTGGTCCAAGGAAGAGAGGTGTTCCATGGGACACCCATATCCTCTGGGTAGATAACAAGAGGGGCATCCTCAGAGACGCTGTCTTCCCGGAACAGAGACTCAAAGTCCAAGGTTGGCACACCTAGGGTGTTTGATCCTACAGGATCAAATGTCAACTTGACTGGTCTAAGAGGAACGTGCTTGTGAGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGGCTTCAGAAAAAGAAATTAGGAGAGAGAGATTCTTGAACGTTTTCCCTAAATTAGTAGAGGAATTGAACGCATCGCTTTTGGCTTACGGTATGCCTAAGGAAGCATGTGACTGGTATGCCCACTCATTGAACTACAACACTCCAGGCGGTAAGCTAAATAGAGGTTTGTCCGTTGTGGACACGTATGCTATTCTCTCCAACAAGACCGTTGAACAATTGGGGCAAGAAGAATACGAAAAGGTTGCCATTCTAGGTTGGTGCATTGAGTTGTTGCAGGCTTACTTCTTGGTCGCCGATGATATGATGGACAAGTCCATTACCAGAAGAGGCCAACCATGTTGGTACAAGGTTCCTGAAGTTGGGGAAATTGCCATCAATGACGCATTCATGTTAGAGGCTGCTATCTACAAGCTTTTGAAATCTCACTTCAGAAACGAAAAATACTACATAGATATCACCGAATTGTTCCATGAGGTCACCTTCCAAACCGAATTGGGCCAATTGATGGACTTAATCACTGCACCTGAAGACAAAGTCGACTTGAGTAAGTTCTCCCTAAAGAAGCACTCCTTCATAGTTACTTTCAAGACTGCTTACTATTCTTTCTACTTGCCTGTCGCATTGGCCATGTACGTTGCCGGTATCACGGATGAAAAGGATTTGAAACAAGCCAGAGATGTCTTGATTCCATTGGGTGAATACTTCCAAATTCAAGATGACTACTTAGACTGCTTCGGTACCCCAGAACAGATCGGTAAGATCGGTACAGATATCCAAGATAACAAATGTTCTTGGGTAATCAACAAGGCATTGGAACTTGCTTCCGCAGAACAAAGAAAGACTTTAGACGAAAATTACGGTAAGAAGGACTCAGTCGCAGAAGCCAAATGCAAAAAGATTTTCAATGACTTGAAAATTGAACAGCTATACCACGAATATGAAGAGTCTATTGCCAAGGATTTGAAGGCCAAAATTTCTCAGGTCGATGAGTCTCGTGGCTTCAAAGCTGATGTCTTAACTGCGTTCTTGAACAAAGTTTACAAGAGAAGCAAATAGAACTAACGCTAATCGATAAAACATTAGATTTCAAACTAGATAAGGACCATGTATAAGAACTATATACTTCCAATATAATATAGTATAAGCTTTAAGATAGTATCTCTCGATCTACCGTTCCACGTGACTAGTCCAAGGATTTTTTTTAAAAAGATTCTCTTTTTTTATGATATTTGTACATAAACTTTATAAATGAAATTCATAATAGAAACGACACGAAATTACAAAATGGAATATGTTCATAGGGTAGACGAAACTATATACGCAATCTACATACATTTATCAAGAAGGAGAAAAAGGAGGATGTAAAGGAATACAGGTAAGCAAATTGATACTAATGGCTCAACGTGATAAGGAAAAAGAATTGCACTTTAACATTAATATTGACAAGGAGGAGGGCACCACACAAAAAGTTAGGTGTAACAGAAAATCATGAAACTATGATTCCTAATTTATATATTGGAGGATTTTCTCTAAAAAAAAAAAAATACAACAAATAAAAAACACTCAATGACCTGACCATTTGATGGAGTTTAAGTCAATACCTTCTTGAACCATTTCCCATAATGGTGAAAGTTCCCTCAAGAATTTTACTCTGTCAGAAACGGCCTTAACGACGTAGTCGACCTCCTCTTCAGTACTAAATCTACCAATACCAAATCTGATGGAAGAATGGGCTAATGCATCATCCTTACCCAGCGCATGTAAAACATAAGAAGGTTCTAGGGAAGCAGATGTACAGGCTGAACCCGAGGATAATGCGATATCCCTTAGTGCCATCAATAAAGATTCTCCTTCCACGTAGGCGAAAGAAACGTTAACACACCCTGGATAACGATGATCTGGAGATCCGTTCAACGTGGTATGTTCAGCGGATAATAGACCTTTGACTAATTTATCGGATAGTCTTTTGATGTGAGCTTGGTCGTTGTC(SEQ ID NO：5)。
(5)质粒pZY517
△URA3：：HIS3_TCYC1_tHMG1_pGAL10_pGAL1_J1-018-A_TPGK1
质粒pZY517的具体构建过程：以载体pZY150为模板，用引物5171_F和5171_R通过PCR扩增得到片段5171；以载体pZY141为模板，用引物5172_F和5172_R通过PCR扩增得到片段5172；以基因J1-018-A为模板，用引物5173_F和5173_R通过PCR扩增得到片段5173；以载体pZY150为模板，用引物5174_F和5174_R通过PCR扩增得到片段5174；以载体pZY150为模板，用引物5175_F和5175_R通过PCR扩增得到片段5175；通过DNA assemble的方法将以上片段在酿酒酵母体内重组构建pZY517，然后在大肠杆菌内扩增，酶切验证以及测序正确后，用NotI线性化质粒pZY517，回收带有目的基因的片段。该敲除盒序列如SEQ ID NO：6所示。
ACGCAGATAATTCCAGGTATTTTGAAGCAGAACTTTTATTTATCATCGAATTGACTATTGCATTATTTCTATTTTGCAAAGAGGAGAAAGAATTAGGAAAGTTCATACTTCAAAAAGTTTTCCAACTTTCTCACACGAAAGGCCTCACGAAAAGGACTGTTCGTAGAATGCTAACATACAAAATTTTGTTAATTTCGTTATGTGCGGATCAGACGGAGTACTTGTCCAAATTAATAAACGATGAGCTGTTAAAAAAGGGGGATATTTTTACCCAAAAATTTTTTGCAACTAATCAAGGTAAGGAATTTTTGAAGAGACTATTTTCATTGACCGAATCAGAGTTTTATAGAGGATTTTTACTAGGAAATGAGAATTTTTGGAAATTTTTAAGAAAAGTTACAGCAATGAAAGAGCAGAGCGAGAGCATTTTTGAATATTTAAATGAATCGATCAAGACAGACAGCAATATTTTGACAAATGAGAACTTCATGTGGGTCCTAGGACTATTAGATGAAATTTCATCAATGGGTGCCGTTGGAAATCACTGGGAAATAGAATACAAGAAATTGACAGAAAGTGGTCATAAAATTGATAAGGAGAATCCATACAAGAAATCGATCGAATTATCATTGAAATCCATTCAACTAACATCACACTTGCTGGAAGATAATAACGATCTGCGTAAAAACGAGATATTCGCTATTATTCAAGCTTTGGCACATCAATGCATCAATCCGTGTAAGCAGATAAGTGAATTTGCAGTGGTAACGCTAGAGCAGACGCTCATCAATAAAATCGAAATTCCAACTAATGAGATGGAATCGGTAGAAGAATTAATTGAGGGCGGATTACTACCGTTGCTAAATTCGAGTGAAACACAGGAAGACCAGAAAATCCTCATTTCATCCATATTAACAATAATTTCAAATGTTTATTTGCATTATTTGAAACTAGGGAAGACAAGCAACGAAACGTTTTTGAAAATTTTGAGTATTTTCAATAAATTTGTAGAGGACTCAGATATTGAAAAAAAGCTACAGCAATTAATACTTGATAAGAAGAGTATTGAGAAGGGCAACGGTTCATCATCTCATGGATCTGCACATGAACAAACACCAGAGTCAAACGACGTTGAAATTGAGGCTACTGCGCCAATTGATGACAATACAGACGATGATAACAAACCGAAGTTATCTGATGTAGAAAAGGATTAAAGATGCTAAGAGATAGTGATGATATTTCATAAATAATGTAATTCTATATATGTTAATTACCTTTTTTGCGAGGCATATTTATGGTGAAGGATAAGTTTTGACCATCAAAGAAGGTTAATGTGGCTGTGGTTTCAGGGTCCATAAAGCTTTTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCCTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAGATCCGTCGAGTTCAAGAGAAAAAAAAAGAAAAAGCAAAAAGAAAAAAGGAAAGCGCGCCTCGTTCAGAATGACACGTATAGAATGATGCATTACCTTGTCATCTTCAGTATCATACTGTTCGTATACATACTTACTGACATTCATAGGTATACATATATACACATGTATATATATCGTATGCTGCAGCTTTAAATAATCGGTGTCACTACATAAGAACACCTTTGGTGGAGGGAACATCGTTGGTACCATTGGGCGAGGTGGCTTCTCTTATGGCAACCGCAAGAGCCTTGAACGCACTCTCACTACGGTGATGATCATTCTTGCCTCGCAGACAATCAACGTGGAGGGTAATTCTGCTAGCCTCTGCAAAGCTTTCAAGAAAATGCGGGATCATCTCGCAAGAGAGATCTCCTACTTTCTCCCTTTGCAAACCAAGTTCGACAACTGCGTACGGCCTGTTCGAAAGATCTACCACCGCTCTGGAAAGTGCCTCATCCAAAGGCGCAAATCCTGATCCAAACCTTTTTACTCCACGCGCCAGTAGGGCCTCTTTAAAAGCTTGACCGAGAGCAATCCCGCAGTCTTCAGTGGTGTGATGGTCGTCTATGTGTAAGTCACCAATGCACTCAACGATTAGCGACCAGCCGGAATGCTTGGCCAGAGCATGTATCATATGGTCCAGAAACCCTATACCTGTGTGGACGTTAATCACTTGCGATTGTGTGGCCTGTTCTGCTACTGCTTCTGCCTCTTTTTCTGGGAAGATCGAGTGCTCTATCGCTAGGGGACCACCCTTTAAAGAGATCGCAATCTGAATCTTGGTTTCATTTGTAATACGCTTTACTAGGGCTTTCTGCTCTGTCATCTTTGCCTTCGTTTATCTTGCCTGCTCATTTTTTAGTATATTCTTCGAAGAAATCACATTACTTTATATAATGTATAATTCATTATGTGATAATGCCAATCGCTAAGAAAAAAAAAGAGTCATCCGCTAGGGGAAAAAAAAAAATGAAAATCATTACCGAGGCATAAAAAAATATAGAGTGTACTAGAGGAGGCCAAGAGTAATAGAAAAAGAAAATTGCGGGAAAGGACTGTGTTATGACTTCCCTGACTAATGCCGTGTTCAAACGATACCTGGCAGTGACTCCTAGCGCTCACCAAGCTCTTAAAACGGGAATTTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACGCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGAGCAAATTAAAGCCTTCGAGCGTCCCAAAACCTTCTCAAGCAAGGTTTTCAGTATAATGTTACATGCGTACACGCGTTTGTACAGAAAAAAAAGAAAAATTTGAAATATAAATAACGTTCTTAATACTAACATAACTATAAAAAAATAAATAGGGACCTAGACTTCAGGTTGTCTAACTCCTTCCTTTTCGGTTAGAGCGGATGTGGGGGGAGGGCGTGAATGTAAGCGTGACATAACTAATTACATGATATCGACAAAGGAAAAGGGGCCTGTTTAGGATTTAATGCAGGTGACGGACCCATCTTTCAAACGATTTATATCAGTGGCGTCCAAATTGTTAGGTTTTGTTGGTTCAGCAGGTTTCCTGTTGTGGGTCATATGACTTTGAACCAAATGGCCGGCTGCTAGGGCAGCACATAAGGATAATTCACCTGCCAAGACGGCACAGGCAACTATTCTTGCTAATTGACGTGCGTTGGTACCAGGAGCGGTAGCATGCGGGCCTCTTACACCTAATAAGTCCAACATGGCACCTTGTGGTTCTAGAACAGTACCACCACCGATGGTACCTACTTCGATGGATGGCATGGATACGGAAATTCTCAAATCACCGTCCACTTCTTTCATCAATGTTATACAGTTGGAACTTTCAACATTTTGTGCAGGATCTTGTCCTAATGCCAAGAAAACAGCTGTCACTAAATTAGCTGCATGTGCGTTAAATCCACCAACAGACCCAGCCATTGCAGATCCAACCAAATTCTTAGCAATGTTCAACTCAACCAATGCGGAAACATCACTTTTTAACACTTTTCTGACAACATCACCAGGAATAGTAGCTTCTGCGACGACACTCTTACCACGACCTTCGATCCAGTTGATGGCAGCTGGTTTTTTGTCGGTACAGTAGTTACCAGAAACGGAGACAACCTCCATATCTTCCCAGCCATACTCTTCTACCATTTGCTTTAATGAGTATTCGACACCTTTAGAAATCATATTCATACCCATTGCGTCACCAGTAGTTGTTCTAAATCTCATGAAGAGTAAATCTCCTGCTAGACAAGTTTGAATATGTTGCAGACGTGCAAATCTTGATGTAGAGTTAAAAGCTTTTTTAATTGCGTTTTGTCCCTCTTCTGAGTCTAACCATATCTTACAGGCACCAGATCTTTTCAAAGTTGGGAAACGGACTACTGGGCCTCTTGTCATACCATCCTTAGTTAAAACAGTTGTTGCACCACCGCCAGCATTGATTGCCTTACAGCCACGCATGGCAGAAGCTACCAAACAACCCTCTGTAGTTGCCATTGGTATATGATAAGATGTACCATCGATAACCAAGGGGCCTATAACACCAACGGGCAAAGGCATGTAACCTATAACATTTTCACAACAAGCGCCAAATACGCGGTCGTAGTCATAATTTTTATATGGTAAACGATCAGATGCTAATACAGGAGCTTCTGCCAAAATTGAAAGAGCCTTCCTACGTACCGCAACCGCTCTCGTAGTATCACCTAATTTTTTCTCCAAAGCGTACAAAGGTAACTTACCGTGAATAACCAAGGCAGCGACCTCTTTGTTCTTCAATTGTTTTGTATTTCCACTACTTAATAATGCTTCTAATTCTTCTAAAGGACGTATTTTCTTATCCAAGCTTTCAATATCGCGGGAATCATCTTCCTCACTAGATGATGAAGGTCCTGATGAGCTCGATTGCGCAGATGATAAACTTTTGACTTTCGATCCAGAAATGACTGTTTTATTGGTTAAAACCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAATGCCTCACAAGCACGTTCCTCTTAGACCAGTCAAGTTGACATTTGATCCTGTAGGATCAAACACCCTAGGTGTGCCAACCTTGGACTTTGAGTCTCTGTTCCGGGAAGACAGCGTCTCTGAGGATGCCCCTCTTGTTATCTACCCAGAGGATATGGGTGTCCCATGGAACACCTCTCTTCCTTGGACCAGACAATCCAAGTTCTGGGCTTACGCCGAGGCAGCTGGATATGAAATGGCCAACGGAATCAGCCTTGACAAGGCATCAGAGCGTGGCACACTACCCATGGAGTTGATGGATGAGCGTCGCAAGTGGAAGATTGATGAGCTAGTTGAGGATGCCATCTCTTGCTGTGCTTATCTTTACCCTACATCATCTCCTACCAGATTGGCGTTGTTGACCCAGTCTGTTCTGCTTCTATTCCTCCACGACGATGTTATTGAGCGAGGAGCTACTCAAAACGAAACCACAGTGGTAGACGAATTTCTTAGCATGGCTCCCAAGAACAGGCATCTTAAGAAATTCTGGTCAGACGTATTGGAATGTGATCCCGTCCTTGGACCTGATCTGCTTTATGCTATCCATGCTTTCGTCCGTGATGGTCGTGTAAAGTCACCCTTTAAGCAGGATCACTATGCCACATTGGCTGATTACATGCTTTACCGTCGCAATGATGTTGGCAAGACATTTATGATTGCAGCTATCCGCTTCGGCTCTGGCGTGCAACAAACACGCGAAGAACTTGCTCCCTTTGACGAGCTTGCTGATCTTTACGTCAGACACTCAATTCTTATCAACGATCTCTACTCGTATGATAAGGAGGTGCACGAGGTCAAGACTATCGACGCGTCCATCGTGAACGCAGTTGCTGTCACAGAGCAGCTCCTTTCCGTGTCGCCTGACCTGGCCAAGAACTTAACCAGAGCTATTACCTTTGACATGGAGAAGGAGTTTTACGGCATTTGTGAGAAGTTTATGCACAGCCCTGATATCAACGATCGCCAGCGCGTGTTCGTTACTGCGCTCTTTGATGCGTTGACAGGCAATATCTTCCATTCTGCTACTTTGAGCAGATACGTTCGTCACGGCGAGAGACCACTTCCTTGCAAGTGTTAGATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAATTACGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCTGCGTTCGTTGTAAATGCATGTATACTAAACTCACAAATTAGAGCTTCAATTTAATTATATCAGTTATTACCCGGGAATCTCGGTCGTAATGATTTCTATAATGACGAAAAAAAAAAAATTGGAAAGAAAAAGCTTCATGGCCTTTATAAAAAGGAACTATCCAATACCTCGCCAGAACCAAGTAACAGTATTTTACGGGGCACAAATCAAGAACAATAAGACAGGACTGTAAAGATGGACGCATTGAACTCCAAAGAACAACAAGAGTTCCAAAAAGTAGTGGAACAAAAGCAAATGAAGGATTTCATGCGTTTGTACTCTAATCTGGTAGAAAGATGTTTCACAGACTGTGTCAATGACTTCACAACATCAAAGCTAACCAATAAGGAACAAACATGCATCATGAAGTGCTCAGAAAAGTTCTTGAAGCATAGCGAACGTGTAGGGCAGCGTTTCCAAGAACAAAACGCTGCCTTGGGACAAGGCTTGGGCCGATAAGGTGTACTGGCGTATATATATCTAATTATGTATCTCTGGTGTAGCCCATTTTTAGCATGTAAATATAAAGAGAAACCATATCTAATCTAACCAAATCCAAACAAAATTCAATAGTTACTATCGCTTTTTTCTTTCTGTATCGCAAATAAGTGAAAATTAAAAAAGAAAGATTAAATTGGAAGTTGGATATGGGCTGGAACAGCAGCAGTAATCGGTATCGGGTTCGCCACTAATGACGTCCTACGATTGCACTCAACAGACCTTGACGCTCACGCCGTAGCGGGCGACAAGTCAAACGGAACAACCGTTGCCGTTCCCATCGGAGTCCGACCTAGGCCGAACTCCGTGAATTTCTGATAACAACGGTCGGTAAAGACTGGTTCCCCAGTATATTTCTTCTCTCAGGAGCAGGGGCCAATGCCAAAAGCGACATTAACCCGGAGGACAAGGCTCCACTGTGTTCCACCGAATTTCCCACCTGATAATATCTGATAACCCGCCCATAGGTGGGGATCCTTCTGTAAACAGGTTTCTTAATCGTAGGAATTACCACTGTTCCACTGCCAATCGCAGCTCCCAGAGTTTCGTTCCCAGCCGCGAGCACCACAGCGTACCATGTGCGCCACGAGGCCTCAAACGTAAAACAATCGAAACGAAAAGAAACAGACTATAGGGGAGTATAGAGACAGCCGGCCAATAAGAAGAGGAAAAAGAAATACTAGCGTTTATCAATGTGGGTCGTTAACATATCCTGTTGACAATGATTACAGGTTAAAAGGTAGCGTAAGTGAATATTAACTATGGATATTCTTATACTTAGATAAGAGACATATAAAACACACGATGATTGATTGATTTTTATGACCAATATATGTAATTCGTAATTCAGATAGTTTTTATACTTTTAATGTGTGCCGAACCATATTACTAGTATATGTAACTACCATGTTATGTTCAATTGGCAGATCTTTAACTCGGCTTTAGTTATCCAAGTTACTTGCAATATTTCCTTCTGCGAGAGTACATTTGCCCTTAAACG(SEQ ID NO：6)。
(6)质粒pZY518
△HIS3：：TRP1_TADH1_J1-018-A_pGAL10pGAL1_tCPS1
质粒pZY518的具体构建过程：以酿酒酵母30000B的基因组为模板，用引物5181_F和5181_R通过PCR扩增得到片段5181；以载体pZY141为模板，用引物5182_F和5182_R通过PCR扩增得到片段5182；以载体pZY513为模板，用引物5183_F和5183_R通过PCR扩增得到片段5183；以载体pZY141为模板，用引物5184_F和5184_R通过PCR扩增得到片段5184；以酿酒酵母30000B的基因组为模板，用引物5185_F和5185_R通过PCR扩增得到片段5185；载体pRS426为模板用引物5186_F和5186_R通过PCR扩增得到片段5186(载体骨架)。通过DNAassemble的方法将以上片段在酿酒酵母体内重组构建pZY518，然后在大肠杆菌内扩增，酶切验证以及测序正确后，用NotI线性化质粒pZY518，回收带有目的基因的片段。该敲除盒序列如SEQ ID NO：7所示。
AAACGTCCAGCCACCCATTTCTTGTGATTTAGTAAAAAACTCTAACGGTTTATCAACGTAAAATATGGGCAGAAGTTCGAGGGCCCCACTGCTTGTCTTGGACACCACAGGCGTCAAAGGAGAGCAGTTTCTTCTCGACATCACAATGAAGTCAACCCCCAGGAAGTAAGCGCTTCTAATAATGGCACCGATATTGTGAGGGTCAGTTATTTCATCCAGATATAACCCGAGAGGAAACTTCTTAGCGTCTGTTTTCGTACCATAAGGCAGTTCATGAGGTATATTTTCGTTATTGAAGCCCAGCTCGTGAATGCTTAATGCTGCTGAACTGGTGTCCATGTCGCCTAGGTACGCAATCTCCACAGGCTGCAAAGGTTTTGTCTCAAGAGCAATGTTATTGTGCACCCCGTAATTGGTCAACAAGTTTAATCTGTGCTTGTCCACCAGCTCTGTCGTAACCTTCAGTTCATCGACTATCTGAAGAAATTTACTAGGAATAGTGCCATGGTACAGCAACCGAGAATGGCAATTTCTACTCGGGTTCAGCAACGCTGCATAAACGCTGTTGGTGCCGTAGACATATTCGAAGATAGGATTATCATTCATAAGTTTCAGAGCAATGTCCTTATTCTGGAACTTGGATTTATGGCTCTTTTGGTTTAATTTCGCCTGATTCTTGATCTCCTTTAGCTTCTCGACGTGGGCCTTTTTCTTGCCATATGGATCCGCTGCACGGTCCTGTTCCCTAGCATGTACGTGAGCGTATTTCCTTTTAAACCACGACGCTTTGTCTTCATTCAACGTTTCCCATTGTTTTTTTCTACTATTGCTTTGCTGTGGGAAAAACTTATCGAAAGATGACGACTTTTTCTTAATTCTCGTTTTAAGAGCTTGGTGAGCGCTAGGAGTCACTGCCAGGTATCGTTTGAACACGGCATTAGTCAGGGAAGTCATAACACAGTCCTTTCCCGCAATTTTCTTTTTCTATTACTCTTGGCCTCCTCTAGTACACTCTATATTTTTTTATGCCTCGGTAATGATTTTCATTTTTTTTTTCCACCTAGCGGATGACTCTTTTTTTTTCTTAGCGATTGGCATTATCACATAATGAATTATACATTATATAAAGTAATGTGATTTCTTCGAAGAATATACTAAATCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATAAACGACATTACTATATATATAATATAGGAAGCATTTAATAGACAGCATCGTAATATATGTGTACTTTGCAGTTATGACGCCAGATGGCAGTAGTGGAAGATATTCTTTATTGAAAAATAGCTTGTCACCTTACGTACAATCTTGATCCGGAGCTTTTCTTTTTTTGCCGATTAAGAATTAATTCGGTCGAAAAAAGAAAAGGAGAGGGCCAAGAGGGAGGGCATTGGTGACTATTGAGCACGTGAGTATACGTGATTAAGCACACAAAGGCAGCTTGGAGTATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGCGGCTTGCAGAGCACAGAGGCCGCAGAATGTGCTCTAGATTCCGATGCTGACTTGCTGGGTATTATATGTGTGCCCAATAGAAAGAGAACAATTGACCCGGTTATTGCAAGGAAAATTTCAAGTCTTGTAAAAGCATATAAAAATAGTTCAGGCACTCCGAAATACTTGGTTGGCGTGTTTCGTAATCAACCTAAGGAGGATGTTTTGGCTCTGGTCAATGATTACGGCATTGATATCGTCCAACTGCATGGAGATGAGTCGTGGCAAGAATACCAAGAGTTCCTCGGTTTGCCAGTTATTAAAAGACTCGTATTTCCAAAAGACTGCAACATACTACTCAGTGCAGCTTCACAGAAACCTCATTCGTTTATTCCCTTGTTTGATTCAGAAGCAGGTGGGACAGGTGAACTTTTGGATTGGAACTCGATTTCTGACTGGGTTGGAAGGCAAGAGAGCCCCGAAAGCTTACATTTTATGTTAGCTGGTGGACTGACGCCAGAAAATGTTGGTGATGCGCTTAGATTAAATGGCGTTATTGGTGTTGATGTAAGCGGAGGTGTGGAGACAAATGGTGTAAAAGACTCTAACAAAATAGCAAATTTCGTCAAAAATGCTAAGAAATAGGTTATTACTGAGTAGTATTTATTTAAGTATTGTTTGTGCACTTGCCTATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATCAGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAGGGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCTGGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAGCGCGCGTAATACGACTCACTATAGGGCGAATTGGGTACCCGGTAGAGGTGTGGTCAATAAGAGCGACCTCATGCTATACCTGAGAAAGCAACCTGACCTACAGGAAAGAGTTACTCAAGAATAAGAATTTTCGTTTTAAAACCTAAGAGTCACTTTAAAATTTGTATACACTTATTTTTTTTATAACTTATTTAATAATAAAAATCATAAATCATAAGAAATTCGCCTAACACTTGCAAGGAAGTGGTCTCTCGCCGTGACGAACGTATCTGCTCAAAGTAGCAGAATGGAAGATATTGCCTGTCAACGCATCAAAGAGCGCAGTAACGAACACGCGCTGGCGATCGTTGATATCAGGGCTGTGCATAAACTTCTCACAAATGCCGTAAAACTCCTTCTCCATGTCAAAGGTAATAGCTCTGGTTAAGTTCTTGGCCAGGTCAGGCGACACGGAAAGGAGCTGCTCTGTGACAGCAACTGCGTTCACGATGGACGCGTCGATAGTCTTGACCTCGTGCACCTCCTTATCATACGAGTAGAGATCGTTGATAAGAATTGAGTGTCTGACGTAAAGATCAGCAAGCTCGTCAAAGGGAGCAAGTTCTTCGCGTGTTTGTTGCACGCCAGAGCCGAAGCGGATAGCTGCAATCATAAATGTCTTGCCAACATCATTGCGACGGTAAAGCATGTAATCAGCCAATGTGGCATAGTGATCCTGCTTAAAGGGTGACTTTACACGACCATCACGGACGAAAGCATGGATAGCATAAAGCAGATCAGGTCCAAGGACGGGATCACATTCCAATACGTCTGACCAGAATTTCTTAAGATGCCTGTTCTTGGGAGCCATGCTAAGAAATTCGTCTACCACTGTGGTTTCGTTTTGAGTAGCTCCTCGCTCAATAACATCGTCGTGGAGGAATAGAAGCAGAACAGACTGGGTCAACAACGCCAATCTGGTAGGAGATGATGTAGGGTAAAGATAAGCACAGCAAGAGATGGCATCCTCAACTAGCTCATCAATCTTCCACTTGCGACGCTCATCCATCAACTCCATGGGTAGTGTGCCACGCTCTGATGCCTTGTCAAGGCTGATTCCGTTGGCCATTTCATATCCAGCTGCCTCGGCGTAAGCCCAGAACTTGGATTGTCTGGTCCAAGGAAGAGAGGTGTTCCATGGGACACCCATATCCTCTGGGTAGATAACAAGAGGGGCATCCTCAGAGACGCTGTCTTCCCGGAACAGAGACTCAAAGTCCAAGGTTGGCACACCTAGGGTGTTTGATCCTACAGGATCAAATGTCAACTTGACTGGTCTAAGAGGAACGTGCTTGTGAGGCATTTATATTGAATTTTCAAAAATTCTTACTTTTTTTTTGGATGGACGCAAAGAAGTTTAATAATCATATTACATGGCAATACCACCATATACATATCCATATCTAATCTTACTTATATGTTGTGGAAATGTAAAGAGCCCCATTATCTTAGCCTAAAAAAACCTTCTCTTTGGAACTTTCAGTAATACGCTTAACTGCTCATTGCTATATTGAAGTACGGATTAGAAGCCGCCGAGCGGGCGACAGCCCTCCGACGGAAGACTCTCCTCCGTGCGTCCTGGTCTTCACCGGTCGCGTTCCTGAAACGCAGATGTGCCTCGCGCCGCACTGCTCCGAACAATAAAGATTCTACAATACTAGCTTTTATGGTTATGAAGAGGAAAAATTGGCAGTAACCTGGCCCCACAAACCTTCAAATCAACGAATCAAATTAACAACCATAGGATAATAATGCGATTAGTTTTTTAGCCTTATTTCTGGGGTAATTAATCAGCGAAGCGATGATTTTTGATCTATTAACAGATATATAAATGCAAAAGCTGCATAACCACTTTAACTAATACTTTCAACATTTTCGGTTTGTATTACTTCTTATTCAAATGTCATAAAAGTATCAACAAAAAATTGTTAATATACCTCTATACTTTAACGTCAAGGAGAAAAAACTATAGCGCAATGATTGAATAGTCAAAGATTTTTTTTTTTTAATTTTTTTTTTTTCATAGAACTTTTTATTTAAATAAATCACGTCTATATATGTATCAGTATAACGTAAAAAAAAAAACACCGTCAGTTAAACAAAACATAAATAAAAAAAAAAAGAAGTGTCAAATCAAGTGTCAAATAAGAGATGGAGGACGGGAAAAAGTTAGTTGTGGTGATAGGTGGCAAGTGGTATTCCGTAAGAACAACAAGAAAAGCATTTCATATTATGGCTGAACTGAGCGAACAAGTGCAAAATTTAAGCATCAACGACAACAACGAGAATGGTTATGTTCCTCCTCACTTAAGAGGAAAACCAAGAAGTGCCAGAAATAACAGTAGCAACTACAATAACAACAACGGCGGCTACAACGGTGGCCGTGGCGGTGGCAGCTTCTTTAGCAACAACCGTCGTGGTGGTTACGGCAACGGTGGTTTCTTCGGTGGAAACAACGGTGGCAGCAGATCTAACGGCCGTTCTGGTGGTAGATGGATCGATGGCAAACATGTCCCAGCTCCAAGAAACGAAAAGGCCGAGATCGCCATATTTGGTGTCCCCGAGGATCCAAATTTCCAATCTTCTGGTATTAACTTCGATAACTACGATGATATTCCAGTGGACGCCTCTGGTAAGGATGTTCCTGAACCAATCACAGAATTTACCTCACCTCCATTGGACGGATTGTTATTGGAAAACATCAAATTGGCCCGTTTCACCAAGCCAACACCTGTGCAAAAATACTCCGTCCCTATCGTTGCCAACGGCAGAGATTTGATGGCCTGTGCGCAGACCGGTTCTGGTAAGACTGGTGGGTTTTTATTCCCAGTGTTGTCCGAATCATTTAAGACTGGACCATCTCCTCAACCAGAGTCTCAAGGCTCCTTTTACCAAAGAAAGGCCTACCCAACTGCTGTCATTATGGCTCCAACTAGAGAGTTGGCCACCCAAATTTTCGATGAAGCCAAGAAATTTACTTATAGATCCTGGGTCAAGGCCTGCGTCGTCTACGGTGGTTCTCCAATTGGTAACCAACTAAGAGAAATTGAACGTGGTTGCGATCTTTTAGTCGCTACTCCAGGTCGTTTGAATGACTTGTTGGAACGTGGTAAAATTTCTTTGGCCAACGTCAAGTATTTGGTTCTAGATGAAGCTGATAGAATGTTGGATATGGGTTTCGAACCTCAAATTAGACATATTGTCGAAGACTGTGATATGACTCCTGTTGGTGAAAGACAAACTCTGATGTTCTCAGCTACTTTTCCCGCTGATATCCAACATTTGGCCCGTGATTTCTTAAGTGACTACATCTTTTTGTCTGTTGGTAGAGTCGGTTCTACTTCAGAAAACATTACTCAAAAAGTCTTATACGTTGAAAATCAAGATAAGAAGTCAGCCTTATTGGATCTATTGTCTGCATCCACTGACGGTTT(SEQ ID NO：7)。
(7)敲除盒pZY521
△GAL80：：URA3
敲除盒pZY521的具体构建过程：以酿酒酵母30000B的基因组为模板，用引物5201_F和5211_R通过PCR扩增得到片段5211(用于整合至GAL80位点的同源左臂)；载体pRS426为模板用引物5212_F和5212_R通过PCR扩增得到片段5212(筛选标记URA3)；以酿酒酵母30000B的基因组为模板，用引物5213_F和5203_R通过PCR扩增得到片段5203(用于整合至GAL80位点的同源右臂)；通过重叠延伸PCR将三个片段连接在一起。该敲除盒序列如SEQ IDNO：8所示。
CAATGGTCTAGGTAGTGGCATTCGAAGCTTCGAAAGCGTGGCCTCGCCTCGAGCGTGAAGTCATATTTTCTCACTCTGCACTGCCGTGCTGTGCCATGTAGCCCACTGTATGCCTCTAGCACCCCAATCGGGAAAATTGTAAACATTAGCAGGTGTCGCCTCGAGGCCCCCGATACATTTTTCAACATCTCCTCGATACTTAAACAGCAAACAAACAAGCAACACCCACGCAAGAAGATTTATTAATACCTCTTGACTCCCGTAGATATTGAAGAGACTAAGTATAGTAACGCATATCCGCACAATACTATGGTTAAGGTCTGGAATATAGTACTACGTCTGGTAGTGTTGCTGTTTTTAGCAGGTAATACCTTGCTGTTAATTTTGATGATTATATCTGGTGCCACGGACCATTACCCCGTGAACAGATTCTATTGGGTGCAGGGCAATACAACAGGCATACCTAATGCTGGGGATGAAACACGTTGGACGTTTTGGGGCGCCTGTCTACAGGATAAAGACGGGTCGGATACCTGCACAAGCAATTTGGCACCTGCATACCCCATTTCCCCAGTAGATAACTTCAACACACACATCAATGTCCCTCACCAGTTTATTTCCAAAAGAGACGCTTTTTACTACCTGACTAGATTTTCATTTTGTTTCTTTTGGATTGCGCTTGCCTTTGTAGGTGTGTCGTTTATCCTTTACGTTTTGACTTGGTGCTCGAAGATGCTTTCAGAGATGGTGCTTATCCTCATGTCTTTTGGGTTTGTCTTCAATACGGCAGCCGTTGTCTTGCAAACGGCCGCCTCTGCCATGGCAAAGAATGCTTTCCATGACGATCATCGTAGTGCCCAATTGGGTGCCTCTATGATGGGTATGGCTTGGGCAAGTGTCTTTTTATGTATCGTGGAATTTATCCTGCTGGTCTTCTGGTCTGTTAGGGCAAGGTTGGCCTCTACTTACTCCATCGACAATTCAAGATACAGAACCTCCTCCAGATGGAATCCCTTCCATAGAGAGAAGGAGCAAGCAACTGACCCAATATTGACTGCCACTGGACCTGAAGACATGCAACAAAGTGCAAGCATAGTGGGGCCTTCTTCCAATGCTAATCCGGTCACTGCCACTGCTGCTACGGAAAACCAACCTAAAGGTATTAACTTCTTCACTATAAGAAAATCACACGAGCGCCCGGACGATGTCTCTGTTTAAATGGCGCAAGTTTTCCGCTTTGTAATATATATTTATACCCCTTTCTTCTCTCCCCTGCAATATAATAGTTTAATTCTAATATTAATAATATCCTATATTTTCTTCATTTACCGGCGCACTCTCGCCCGAACGACCTCAAAATGTCTGCTACATTCATAATAACCAAAAGCTCATAACTTTTTTTTTGAACCTGAATATATATACATCACATGTCACTGCTGGTCCTTGCCGACCAGCGTATACAATCTCGATAGTTGGTTTCCCGTTCTTTCCACTCCCGTCGTACCCAATTCGCCCTATAGTGAGTCGTATTACGCGCGCTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACCCAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGCGCGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATAGGGTAATAACTGATATAATTAAATTGAAGCTCTAATTTGTGAGTTTAGTATACATGCATTTACTTATAATACAGTTTTTTAGTTTTGCTGGCCGCATCTTCTCAAATATGCTTCCCAGCCTGCTTTTCTGTAACGTTCACCCTCTACCTTAGCATCCCTTCCCTTTGCAAATAGTCCTCTTCCAACAATAATAATGTCAGATCCTGTAGAGACCACATCATCCACGGTTCTATACTGTTGACCCAATGCGTCTCCCTTGTCATCTAAACCCACACCGGGTGTCATAATCAACCAATCGTAACCTTCATCTCTTCCACCCATGTCTCTTTGAGCAATAAAGCCGATAACAAAATCTTTGTCGCTCTTCGCAATGTCAACAGTACCCTTAGTATATTCTCCAGTAGATAGGGAGCCCTTGCATGACAATTCTGCTAACATCAAAAGGCCTCTAGGTTCCTTTGTTACTTCTTCTGCCGCCTGCTTCAAACCGCTAACAATACCTGGGCCCACCACACCGTGTGCATTCGTAATGTCTGCCCATTCTGCTATTCTGTATACACCCGCAGAGTACTGCAATTTGACTGTATTACCAATGTCAGCAAATTTTCTGTCTTCGAAGAGTAAAAAATTGTACTTGGCGGATAATGCCTTTAGCGGCTTAACTGTGCCCTCCATGGAAAAATCAGTCAAGATATCCACATGTGTTTTTAGTAAACAAATTTTGGGACCTAATGCTTCAACTAACTCCAGTAATTCCTTGGTGGTACGAACATCCAATGAAGCACACAAGTTTGTTTGCTTTTCGTGCATGATATTAAATAGCTTGGCAGCAACAGGACTAGGATGAGTAGCAGCACGTTCCTTATATGTAGCTTTCGACATGATTTATCTTCGTTTCCTGCAGGTTTTTGTTCTGTGCAGTTGGGTTAAGAATACTGGGCAATTTCATGTTTCTTCAACACTACATATGCGTATATATACCAATCTAAGTCTGTGCTCCTTCCTTCGTTCTTCCTTCTGTTCGGAGATTACCGAATCAAAAAAATTTCAAAGAAACCGAAATCAAAAAAAAGAATAAAAAAAAAATGATGAATTGAATTGAAAAGCTGTGGTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGAAAGCATCTTGCCCTGTGCTTGGCCCCCAGTGCAGCGAACGTTATAAAAACGAATACTGAGTATATATCTATGTAAAACAACCATATCATTTCTTGTTCTGAACTTTGTTTACCTAACTAGTTTTAAATTTCCCTTTTTCGTGCATGCGGGTGTTCTTATTTATTAGCATACTACATTTGAAATATCAAATTTCCTTAGTAGAAAAGTGAGAGAAGGTGCACTGACACAAAAAATAAAATGCTACGTATAACTGTCAAAACTTTGCAGCAGCGGGCATCCTTCCATCATAGCTTCAAACATATTAGCGTTCCTGATCTTCATACCCGTGCTCAAAATGATCAAACAAACTGTTATTGCCAAGAAATAAACGCAAGGCTGCCTTCAAAAACTGATCCATTAGATCCTCATATCAAGCTTCCTCATAGAACGCCCAATTACAATAAGCATGTTTTGCTGTTATCACCGGGTGATAGGTTTGCTCAACCATGGAAGGTAGCATGGAATCATAATTTGGATACTAATACAAATCGGCCATATAATGCCATTAGTAAATTGCGCTCCCATTTAGGTGGTTCTCCAGGAATACTAATAAATGCGGTGCATTTGCAAAATGAATTTATTCCAAGGCCAAAACAACACGATGAATGGCTTTATTTTTTTGTTATTCCTGACATGAAGCTTTATGTAATTAAGGAAACGGACATCGAGGAATTTGCATCTTTTTTAGATGAAGGAGCTATTCAAGCACCAAAGCTATCCTTCCAGGATTATTTAAGCGGTAAGGCCAAGGCTTCCCAACAGGTTCATGAAGTGCATCATAGAAAGCTTACAAGGTTTCAGGGTGAAACTTTTCTAAGAGATTGGAACTTAGTCTGTGGGCATTATAAGAGAGATGCTAAGTGTGGAGAAATGGGACCCGACATAATTGCAGCATTTCAAGATGAAAAGCTTTTTCCTGAGAATAATCTAGCCTTAATTTCTCATATTGGGGGTCATATTTTTGCTGGTAATGTCATTTTCTATAAACTATTCGGCAGAGAAAAGATGCAAAACAAGCTGGATTCATTATGGTTTGGTAAAGTTTACCCACACAACTTGAAGCTGTTATGTGAAAACTTGGAAAACGGTAAAATCATCGACGAAATGTACAGAGGTGGCATATCGATGAATTAAAACGGTTTCCTTTTAAAACAGAAATAAAGATGAGGTGAAATTTTATATTTCTTATATTTAAAGCTATCATATATTCCTTGTACATATATTTATTAAGAAAATACATTAACACGCCTTTATACTTGGTATTATTACTGTAAAATCATTACATGTAATTTGTTCTGACTTCGTTGATTGTTTGAATTATATCCTCCACTTGTACACTCTTTAAATTTTTGGCCAAAAATTCCTGCTCATTCAAGGAAAGTCTCAGGAAATTTTCACATAAATCACCATCAATAACTTTACGTACAGGGGCATAATAGCTTCTGTATTCTTGGTGGTCTC(SEQ ID NO：8)。
实施例3法尼烯产生菌的构建
将质粒pZY402线性化后片段(SEQ ID NO：3)转入酿酒酵母菌株CEN.PK2-1D，根据同源重组来整合到染色体上，其整合位点为ChrXI-3，筛选标记为潮霉素(Hygr)，至此成功构建背景菌株JYF21。
在菌株JYF21转入质粒pZY401线性化后片段(SEQ ID NO：2)，根据同源重组来整合到染色体上，其整合位点为ChrXII-4，筛选标记为G418，至此成功构建菌株JYF22。
向菌株JYF22转入含Cre酶的质粒，使得其丢掉筛选标记，获得菌株JYF23。
在菌株JYF23转入质粒pZY410线性化后片段(SEQ ID NO：4)，根据同源重组来整合到染色体上，其整合位点为ChrXII-2，筛选标记为G418，至此成功构建菌株JYF24。
在菌株JYF24转入质粒pZY516线性化后片段(SEQ ID NO：5)，根据同源重组来整合到染色体上，其整合位点为LEU2，筛选标记为亮氨酸(LEU2)，至此成功构建菌株JYF53。
在菌株JYF53转入质粒pZY517线性化后片段(SEQ ID NO：6)以及质粒pZY518线性化后片段(SEQ ID NO：7)，根据同源重组来整合到染色体上，其整合位点分别为URA3和HIS3，筛选标记分别为组氨酸(HIS3)以及色氨酸(TRP1)，至此成功构建菌株JYF56。
在菌株JYF53转入pZY521敲除盒片段(SEQ ID NO：8)，根据同源重组来整合到染色体上，其整合位点为GAL80，筛选标记为尿嘧啶(URA3)，至此成功构建菌株JYF80。
这些菌株通过摇瓶发酵培养，具体方法如下：将保种管中吸取适量甘油菌到含有5mL种子培养基的PA瓶中，种子培养基配方为：硫酸铵(15g/L)，磷酸二氢钾(8g/L)，七水硫酸镁(6.15g/L)，葡萄糖(20g/L)，维生素溶液(12mL/L)，微量金属溶液(10mL/L)，0.5Msuccinate buffer(pH 5.0)100ml/L。其中，维生素溶液中含有biotin生物素(0.05g/L)、calcium pantothenate泛酸钙(1g/L)、nicotinic acid烟酸(1g/L)、myo-inositol肌醇(5g/L)、thiamine HCl硫胺素HCI(1g/L)、pyridoxal HCl吡啶多酚HCl(1g/L)和p-aminobenzoic acid对氨基苯甲酸(0.2g/L)。微量金属溶液中含有七水硫酸锌(5.75g/L)，四水氯化锰(0.32g/L)、无水硫酸铜(0.32g/L)、六水氯化钴(0.47g/L)、二水钼酸钠(0.48g/L)、二水氯化钙(2.9g/L)、七水硫酸亚铁(2.8g/L)、0.5M EDTA(80mL/L)。30℃摇床摇起一级种子液，过夜培养(一般14-18h)后，再按初始OD为0.1转接到含有50mL发酵培养基的250mL摇瓶中，发酵培养基配方为：硫酸铵(15g/L),磷酸二氢钾(8g/L)，七水硫酸镁(6.15g/L)，葡萄糖(40g/L)维生素溶液(12mL/L)，微量金属溶液(10mL/L)，0.5M succinate buffer(pH5.0)100ml/L。其中维生素溶液中含有biotin生物素(0.05g/L)、calcium pantothenate泛酸钙(1g/L)、nicotinic acid烟酸(1g/L)、myo-inositol肌醇(5g/L)、thiamine HCl硫胺素HCI(1g/L)、pyridoxal HCl吡啶多酚HCl(1g/L)和p-aminobenzoic acid对氨基苯甲酸(0.2g/L)。微量金属溶液中含有七水硫酸锌(5.75g/L)，四水氯化锰(0.32g/L)、无水硫酸铜(0.32g/L)、六水氯化钴(0.47g/L)、二水钼酸钠(0.48g/L)、二水氯化钙(2.9g/L)、七水硫酸亚铁(2.8g/L)、0.5M EDTA(80mL/L)，转接后覆盖20％的有机相(正十二烷或肉豆蔻酸异丙酯)置于30℃摇床开始摇瓶发酵。72h收样，取菌液测OD，有机相用正己烷稀释测产量。
将菌株JYF53、JYF56和JYF80按照上述方法进行发酵，结果显示，在发酵72小时后，JY53的发酵产量为1.3g/L，JYF56和JYF80的产量为2.5g/L.
结论：
1、三株菌均可产生法尼烯，证明发明人构建的利用MVA途径生产法尼烯的重组菌株可行。
2、JYF56和JYF80的产量更高，证明发明人构建的微生物菌株发酵生产法尼烯是相对高效的。
实施例4大肠杆菌中MVA途径优化比例蛋白表达质粒的构建
质粒pJ1-018-18含有来源于MVA途径的IDI1、ERG10、MVD1、ERG8、ERG12，这些基因通过PCR获得，所述基因编码在genebank库中的编号为855986、856079、855779、855260、855248，通过设计引物使这些基因片段依次链接，并在相邻两个基因间形成50bp的重叠序列，利用Gisbson方法(Gibson.D.G.,2011.Enzymatic assembly of overlappingDNAfragments.Methods Enzymol.498.349-61.)将这些基因克隆在pBBRIMCS/p15A(Zhu F,et al.,2014.In vitro reconstitution of mevalonate pathway and targetedengineering of farnesene overproduction in Escherichia coli.BiotechnolBioeng.111(7):1396-405.)上，该阳性克隆测序命名为pJ1-018-18。该质粒控制基因表达的为中等强度的lac启动子，质粒的复制子为p15A复制子。
质粒pJ1-018-19含有来源于MVA途径的ERG13、3个拷贝的tHMG1基因，这些基因通过PCR获得，所述基因编码在genebank库中的编号为854913、除去4-1659bp的854900，通过设计引物使这些基因片段依次链接，并在相邻两个基因间形成50bp的重叠序列，利用Gibson方法将这些基因克隆在pBBR1MCS-2质粒上，该阳性克隆测序命名为pJ1-018-19。该质粒控制基因表达的为中等强度的lac启动子，质粒的复制子为pBBR1MCS复制子。
质粒pJ1-018-20质粒含有基因ERG20(所述基因编码在genebank库中的编号为853272)以及3个拷贝的J1-018-A基因，所述J1-018-A基因序列如SEQ ID NO：1所示，利用Gibson方法将这些基因克隆在pET21a质粒上，该阳性克隆测序命名为pJ1-018-20。该质粒控制基因表达的为强启动子T7，质粒的复制子为pBBR3高拷贝复制子。
ATGCCTCACAAGCACGTTCCTCTTAGACCAGTCAAGTTGACATTTGATCCTGTAGGATCAAACACCCTAGGTGTGCCAACCTTGGACTTTGAGTCTCTGTTCCGGGAAGACAGCGTCTCTGAGGATGCCCCTCTTGTTATCTACCCAGAGGATATGGGTGTCCCATGGAACACCTCTCTTCCTTGGACCAGACAATCCAAGTTCTGGGCTTACGCCGAGGCAGCTGGATATGAAATGGCCAACGGAATCAGCCTTGACAAGGCATCAGAGCGTGGCACACTACCCATGGAGTTGATGGATGAGCGTCGCAAGTGGAAGATTGATGAGCTAGTTGAGGATGCCATCTCTTGCTGTGCTTATCTTTACCCTACATCATCTCCTACCAGATTGGCGTTGTTGACCCAGTCTGTTCTGCTTCTATTCCTCCACGACGATGTTATTGAGCGAGGAGCTACTCAAAACGAAACCACAGTGGTAGACGAATTTCTTAGCATGGCTCCCAAGAACAGGCATCTTAAGAAATTCTGGTCAGACGTATTGGAATGTGATCCCGTCCTTGGACCTGATCTGCTTTATGCTATCCATGCTTTCGTCCGTGATGGTCGTGTAAAGTCACCCTTTAAGCAGGATCACTATGCCACATTGGCTGATTACATGCTTTACCGTCGCAATGATGTTGGCAAGACATTTATGATTGCAGCTATCCGCTTCGGCTCTGGCGTGCAACAAACACGCGAAGAACTTGCTCCCTTTGACGAGCTTGCTGATCTTTACGTCAGACACTCAATTCTTATCAACGATCTCTACTCGTATGATAAGGAGGTGCACGAGGTCAAGACTATCGACGCGTCCATCGTGAACGCAGTTGCTGTCACAGAGCAGCTCCTTTCCGTGTCGCCTGACCTGGCCAAGAACTTAACCAGAGCTATTACCTTTGACATGGAGAAGGAGTTTTACGGCATTTGTGAGAAGTTTATGCACAGCCCTGATATCAACGATCGCCAGCGCGTGTTCGTTACTGCGCTCTTTGATGCGTTGACAGGCAATATCTTCCATTCTGCTACTTTGAGCAGATACGTTCGTCACGGCGAGAGACCACTTCCTTGCAAGTGTTAG(SEQ ID NO：1)。
实施例5法尼烯产生菌的构建
将质粒pJ1-018-18、pJ1-018-19、pJ1-018-20导入大肠杆菌BL21中，构建大肠杆菌J1-018-9，并按照实施例3中的方法进行发酵，结果显示，在诱导后48小时，法尼烯的摇瓶产量达到2.5g/L，说明利用MVA途径能够有效地产生法尼烯。
实施例6链霉菌中MVA途径优化比例蛋白表达质粒的构建
质粒pJ1-018-24含有来源于MVA途径的IDI1、ERG10、MVD1、ERG8、ERG12，ERG20，ERG13这些基因通过PCR获得，通过设计引物使这些基因片段依次链接，并在相邻两个基因间形成50bp的重叠序列，利用Gisbson方法(Gibson.D.G.,2011.Enzymatic assembly ofoverlapping DNAfragments.Methods Enzymol.498.349-61.)将这些基因克隆在pIB139上，该阳性克隆测序命名为pJ1-018-24。该质粒控制基因表达的为低强度的ermE启动子，含有attP位点和Int整合酶，为阿伯拉霉素抗性。
利用PCR-targetting(Gust.B.,2003.PCR-targeted Streptomyces genereplacement identifies a protein domain needed for biosynthesis of thesesquiterpene soil odor geosmin.Proc Natl Acad Sci U S A.100(4):1541–1546.)将质粒pLH1(Qian Liu.,2016.Development of Streptomyces sp.FR-008as an emergingchassis.Synth Syst Biotechnol.1(3):207–214.)的抗性基因替换成硫链丝菌素的抗性基因tsr，将该质粒命名pJ1-018-22。将2个拷贝的tHMG1基因，SPL39启动子(Qian Liu.,2016.Development of Streptomyces sp.FR-008as an emerging chassis.Synth SystBiotechnol.1(3):207–214.)和3个拷贝的J1-018-A基因克隆于pJ1-018-22上，具体方法为通过设计引物使这些基因片段依次链接，并在相邻两个基因间形成50bp的重叠序列，利用Gisbson方法将这些基因克隆在pJ1-018-22上，该阳性克隆测序命名为pJ1-018-25。该质粒控制tHMG1基因表达的为强启动子SPL44，控制J1-018-A基因表达的为强启动子SPL39，含有attP位点和Int整合酶，为硫链丝菌素抗性。
实施例7法尼烯产生菌的构建
将质粒pJ1-018-24通过文献报道的接合转移的方法(Qian Liu.,2016.Development of Streptomyces sp.FR-008as an emerging chassis.Synth SystBiotechnol.1(3):207–214.)转入白色链霉菌J1074中，挑选阳性接合子进行验证，验证成功的接合子命名为J1-018-12，再将质粒pJ1-018-25通过接合转移的方法转入J1-018-12中，得到的阳性接合子命名为J1-018-13。
将J1-018-13菌株进行划菌苔培养，在TSB培养基中进行种子摇瓶培养，培养温度为30度，培养转速为220rpm培养两天后，按照10％的接种量转接新的含有20％IPM覆盖的TSB培养基，继续培养3天后进行检测。结果显示，菌株J1-018-13中能检测到法尼烯的产生，产量为2.45g/L，后续仍持续增加。该产量为目前首次在链霉菌中合成法尼烯的报道产量。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。
SEQUENCE LISTING
<110> 武汉臻智生物科技有限公司
<120> 微生物及其用途
<130> PIDC3182010
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1116
<212> DNA
<213> Artificial
<220>
<223> J1-018-A基因序列
<400> 1
atgcctcaca agcacgttcc tcttagacca gtcaagttga catttgatcc tgtaggatca 60
aacaccctag gtgtgccaac cttggacttt gagtctctgt tccgggaaga cagcgtctct 120
gaggatgccc ctcttgttat ctacccagag gatatgggtg tcccatggaa cacctctctt 180
ccttggacca gacaatccaa gttctgggct tacgccgagg cagctggata tgaaatggcc 240
aacggaatca gccttgacaa ggcatcagag cgtggcacac tacccatgga gttgatggat 300
gagcgtcgca agtggaagat tgatgagcta gttgaggatg ccatctcttg ctgtgcttat 360
ctttacccta catcatctcc taccagattg gcgttgttga cccagtctgt tctgcttcta 420
ttcctccacg acgatgttat tgagcgagga gctactcaaa acgaaaccac agtggtagac 480
gaatttctta gcatggctcc caagaacagg catcttaaga aattctggtc agacgtattg 540
gaatgtgatc ccgtccttgg acctgatctg ctttatgcta tccatgcttt cgtccgtgat 600
ggtcgtgtaa agtcaccctt taagcaggat cactatgcca cattggctga ttacatgctt 660
taccgtcgca atgatgttgg caagacattt atgattgcag ctatccgctt cggctctggc 720
gtgcaacaaa cacgcgaaga acttgctccc tttgacgagc ttgctgatct ttacgtcaga 780
cactcaattc ttatcaacga tctctactcg tatgataagg aggtgcacga ggtcaagact 840
atcgacgcgt ccatcgtgaa cgcagttgct gtcacagagc agctcctttc cgtgtcgcct 900
gacctggcca agaacttaac cagagctatt acctttgaca tggagaagga gttttacggc 960
atttgtgaga agtttatgca cagccctgat atcaacgatc gccagcgcgt gttcgttact 1020
gcgctctttg atgcgttgac aggcaatatc ttccattctg ctactttgag cagatacgtt 1080
cgtcacggcg agagaccact tccttgcaag tgttag 1116
<210> 2
<211> 8928
<212> DNA
<213> Artificial
<220>
<223> 质粒pZY401线性化后片段
<220>
<221> misc_feature
<222> (1070)..(1079)
<223> n is a, c, g, or t
<400> 2
gactcaatga cttcgggatc taattcagca taaataatct cttctccctc gccagcttca 60
gctacaatct tgcctctggg gtccaccact atagagtggc catatgcatg gtaagaactc 120
tgtagattcc ttgcgggaga acataacatc acgtacactt ggttgtcaac tgccctgctt 180
cttgccagta aatgccaatg caagggccct gtgacagtgt tgaatgcact agggtagatc 240
atggcaaacg cacccttgcg tgcacttagc attgcgagtt ccggaaacct catgtcatag 300
catataccta cgccaaactt accatatttc gtgtcaatgg tagtagactt ctctccaggg 360
ctcaaggttt cactttcatg gaacgatata ccgttaggaa tgtctacatc gaaaagatgg 420
accttccgat gcttgtcaat caacttgcca tcctcgttga aaattataga ggtgttgtag 480
attttgtctg tctttggatc caattccgga attgttcctc ctaccaagat gattttaaac 540
ttattggcta aattagacag aaattgaact gacgtcgaag gctctttcgg attgataact 600
tctgaatact ttctaaactg atcagtggag tacggagaat tgaagcattc tggcaatacg 660
accaacttgg tatccggctg ttccttcata gccctttcaa tgaacgttgc agccctttga 720
agattggcca ttttgtcagg actcgagcct gacagttgga ccaacgcaac tttaattttt 780
tgtgaaagaa tcttcgaagc actcatactg gcgatcttca cgccctcctg ctattacaaa 840
agctgtgttt ttacaagaat caaattaagt tagcaagata tcatacaaca ttattgataa 900
tttcaatatc gtgttcgtac ctgatgacgt atctgtgcat tgataaggcc cgcatggttt 960
cagaaagcag agcggaacga ttccaaatta gtggccttgt gctttgcatg tcaattgtgt 1020
taccttcagc tcgtggattt gttttatcaa tacacagtct acagtcaagn nnnnnnnnnt 1080
atcaaatttt gcgttcgagc gtataaaata gccgctgtag ctacttaagt tcctgttcag 1140
ccggtagagg tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc 1200
tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa 1260
aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat aaatcataag 1320
aaattcgctt attcctttgg tagaccagtc tttgcgtcaa tcaaagattc gtttgtttct 1380
tgtgggcctg aatcgacttg agttaaaatc actctggcaa catccttttg caactcaaga 1440
tccaattcac gtgcagtaaa gttagatgat tcaaattgat ggttgaaagc ctcaagctgc 1500
tcagtagtaa atttcttgtc ccatccagga acagagccaa acaatttata gataaatgca 1560
aagagtttcg actcattttc agctaagtag tacaacacag catttggacc tgcatcaaac 1620
gtgtatgcaa cgattgtttc tccgtaaaac tgattaatgg tgtggcacca actgatgata 1680
cgcttggaag tgtcattcat gtagaatatt ggagggaaag agtccaaaca tgtggcatgg 1740
aaagagttgg aatccatcat tgtttccttt gcaaaggtgg cgaaatcttt ttcaacaatg 1800
gctttacgca tgacttcaaa tctctttggt acgacatgtt caattctttc tttaaatagt 1860
tcggaggttg ccacggtcaa ttgcataccc tgagtggaac tcacatcctt tttaatatcg 1920
ctgacaacta ggacacaagc tttcatctga ggccagtcag agctgtctgc gatttgtact 1980
gccatggaat catgaccatc ttcagctttt cccatttccc aggccacgta tccgccaaac 2040
aacgatctac aagctgaacc agaccccttt cttgctattc tagatatttc tgaagttgac 2100
tgtggtaatt ggtataactt agcaattgca gagaccaatg cagcaaagcc agcagcggag 2160
gaagctaaac cagctgctgt aggaaagtta ttttcggaga caatgtggag tttccattga 2220
gataatgtgg gcaatgaggc gtccttcgat tccatttcct ttcttaattg gcgtaggtcg 2280
cgcagacaat tttgagttct ttcattgtcg atgctgtgtg gttctccatt taaccacaaa 2340
gtgtcgcgtt caaactcagg tgcagtagcc gcagaggtca acgttctgag gtcatcttgc 2400
gataaagtca ctgatatgga cgaattggtg ggcagattca acttcgtgtc ccttttcccc 2460
caatacttaa gggttgcgat gttgacgggt gcggtaacgg atgctgtgta aacggtcatt 2520
tttgagggaa tattcaactg ttttttttta tcatgttgat gctctgcata ataatgccca 2580
taaatatttc cgacctgctt ttatatcttt gctagccaaa ctaactgaac atagctacac 2640
attattttca gcttggctat tttgtgaaca ctgtatagcc agtccttcgg atcacggtca 2700
acagttgtcc gagcgctttt tggacccttt cccttatttt tgggttaagg aaaatgacag 2760
aaaatatatc taatgagcct tcgctcaaca gtgctccgaa gtatagcttt ccaaaaggag 2820
aggcaaagca atttaagaat gtatgaacaa aataaagggg aaaaattacc ccctctactt 2880
taccaaacga atactaccaa taatatttac aacttttcct tatgattttt tcactgaagc 2940
gcttcgcaat agttgtgagc gatatcaaaa gtaacgaaat gaacttcgcg gctcgtgcta 3000
tattcttgtt gctaccgtcc atatctttcc atagattttc aatctttgat gtctccatgg 3060
tggtacagag aacttgtaaa caattcggtc cctacatgtg aggaaattcg ctgtgacact 3120
tttatcactg aactccaaat ttaaaaaata gcataaaatt cgttatacag caaatctatg 3180
tgttgcaatt aagaactaaa agatatagag tgcatatttt caagaaggat agtaagctgg 3240
caaatcatat cttttcaatg acaatagagg aagcaccacc accaccatta caaatggcgg 3300
caacaccgat cttacctcct tcttgctgta agatggatag cagtgtaaca accactctag 3360
caccagaaca acccaatggg tgacctagag caacagcacc accatataca ttaaccttag 3420
atgggtctag cttcaaaatc ttagtgttca ccaaaccgac aaccgaaaag gcttcattga 3480
attcaaagta atcaacagaa ttgatgtctt cgatgccagc atgtttcaaa gcctttggaa 3540
ctgcaagaga tggagcccat gtaaaatcag ctggttgatg agcggcctca ccccaacctt 3600
tgataatagc caaaggcttc aaattctttt ccttcaaaac tttttcggaa accaagatga 3660
cggctgcagc accatcgttg attggagaag cgttagcggc agtaacagta ccgttttctt 3720
tttggaaaac agtccttgca gatctcaatt tttcaacgtg taatctagca ggttcctcgt 3780
ccttcgtgac ttgagtatca ggcttacctc taaatccctt aatggtaaca ggtacaattt 3840
cattgtcgaa tttaccttcc ttttgagatt tttgagattt ttggtaggat tcgatggcaa 3900
aattgtcttg ttgttctcta gtaatatccc aatcacgggc acacttttct gcgtgtacac 3960
ccatggctag accatcgtac gcatcgttca acccatctct ttcgacacca tcaacaagaa 4020
cagtttggcc aaatttggca cccgcacggg ctgctggcat gtagtatggt gcgttagtca 4080
tagattcaca accaccagct acgacaacat cagcattacc acatttgatg gattgagcac 4140
ccaaaatgat tgccttcata gcggatgcac agaccttgtt aactgtgctt gcaacgatat 4200
gattactcaa accggcagcc aaagcaactt gtctggccgg agcttggccc aaattggcag 4260
aaagaacgtt accaaaaata atttcgtcaa aatccttgga tgcatccaat tctggaacct 4320
tagccaaggc gccttttaaa gcaacagcac ccaattccac tgctgtcttg gaggatagag 4380
aaccctggaa tgaaccaatt ggggttctgg cagtcgatac aatgtaaacg ttctgagaca 4440
tttatattga attttcaaaa attcttactt tttttttgga tggacgcaaa gaagtttaat 4500
aatcatatta catggcaata ccaccatata catatccata tctaatctta cttatatgtt 4560
gtggaaatgt aaagagcccc attatcttag cctaaaaaaa ccttctcttt ggaactttca 4620
gtaatacgct taactgctca ttgctatatt gaagtacgga ttagaagccg ccgagcgggc 4680
gacagccctc cgacggaaga ctctcctccg tgcgtcctgg tcttcaccgg tcgcgttcct 4740
gaaacgcaga tgtgcctcgc gccgcactgc tccgaacaat aaagattcta caatactagc 4800
ttttatggtt atgaagagga aaaattggca gtaacctggc cccacaaacc ttcaaatcaa 4860
cgaatcaaat taacaaccat aggataataa tgcgattagt tttttagcct tatttctggg 4920
gtaattaatc agcgaagcga tgatttttga tctattaaca gatatataaa tgcaaaagct 4980
gcataaccac tttaactaat actttcaaca ttttcggttt gtattacttc ttattcaaat 5040
gtcataaaag tatcaacaaa aaattgttaa tatacctcta tactttaacg tcaaggagaa 5100
aaaactataa tgactgccga caacaatagt atgccccatg gtgcagtatc tagttacgcc 5160
aaattagtgc aaaaccaaac acctgaagac attttggaag agtttcctga aattattcca 5220
ttacaacaaa gacctaatac ccgatctagt gagacgtcaa atgacgaaag cggagaaaca 5280
tgtttttctg gtcatgatga ggagcaaatt aagttaatga atgaaaattg tattgttttg 5340
gattgggacg ataatgctat tggtgccggt accaagaaag tttgtcattt aatggaaaat 5400
attgaaaagg gtttactaca tcgtgcattc tccgtcttta ttttcaatga acaaggtgaa 5460
ttacttttac aacaaagagc cactgaaaaa ataactttcc ctgatctttg gactaacaca 5520
tgctgctctc atccactatg tattgatgac gaattaggtt tgaagggtaa gctagacgat 5580
aagattaagg gcgctattac tgcggcggtg agaaaactag atcatgaatt aggtattcca 5640
gaagatgaaa ctaagacaag gggtaagttt cactttttaa acagaatcca ttacatggca 5700
ccaagcaatg aaccatgggg tgaacatgaa attgattaca tcctatttta taagatcaac 5760
gctaaagaaa acttgactgt caacccaaac gtcaatgaag ttagagactt caaatgggtt 5820
tcaccaaatg atttgaaaac tatgtttgct gacccaagtt acaagtttac gccttggttt 5880
aagattattt gcgagaatta cttattcaac tggtgggagc aattagatga cctttctgaa 5940
gtggaaaatg acaggcaaat tcatagaatg ctataaacag gccccttttc ctttgtcgat 6000
atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 6060
gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 6120
ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta caaacgcgtg 6180
tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 6240
ttaatttgca cgctgcaggt cgacaaccct taatataact tcgtataatg tatgctatac 6300
gaagttatta ggtctagaga tctgtttagc ttgcctcgtc cccgccgggt cacccggcca 6360
gcgacatgga ggcccagaat accctccttg acagtcttga cgtgcgcagc tcaggggcat 6420
gatgtgactg tcgcccgtac atttagccca tacatcccca tgtataatca tttgcatcca 6480
tacattttga tggccgcacg gcgcgaagca aaaattacgg ctcctcgctg cagacctgcg 6540
agcagggaaa cgctcccctc acagacgcgt tgaattgtcc ccacgccgcg cccctgtaga 6600
gaaatataaa aggttaggat ttgccactga ggttcttctt tcatatactt ccttttaaaa 6660
tcttgctagg atacagttct cacatcacat ccgaacataa acaaccatgg gtaaggaaaa 6720
gactcacgtt tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 6780
atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 6840
cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 6900
tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 6960
tatccgtact cctgatgatg catggttact caccactgcg atccccggca aaacagcatt 7020
ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 7080
cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 7140
tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 7200
tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc 7260
attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 7320
cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 7380
ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 7440
ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 7500
cgatgagttt ttctaatcag tactgacaat aaaaagattc ttgttttcaa gaacttgtca 7560
tttgtatagt ttttttatat tgtagttgtt ctattttaat caaatgttag cgtgatttat 7620
attttttttc gcctcgacat catctgccca gatgcgaagt taagtgcgca gaaagtaata 7680
tcatgcgtca atcgtatgtg aatgctggtc gctatactgc tgtcgattcg atactaacgc 7740
cgccatccag tgtcgaaaac gagctctcga gaacccttaa tataacttcg tataatgtat 7800
gctatacgaa gttattaggt gatatcagat ccactagtgg caattcccca ttagagtcaa 7860
ataaaaggat gcctcacgga ggtatgttac ccgcgctatt tcacatggct cattgaatta 7920
gaggtggaat ttggtgtacc ctcccctcct catctgatga agtagtgatc cgacaattct 7980
taaaagttgt agacattact tttaccacca actaagttgt atttatattg ctacccttat 8040
ccttttatat ctaactagcg ctcataaggt tggggcaata ctaaaactgt gttcttattc 8100
aactcattaa atacgtggca gtacgtaccc tattagaaac aataggaaac agcagagtcg 8160
gaagaagcca aatgccagat ttgaagtcca aaaccttgtc aagccaatct ttgggagcgg 8220
ctattcctcc agaaattgtg taccaaatac ttacatacca gtttagggat ttgttaagaa 8280
atgaccatcc aggtacagca gaaaagttta acgaaaactt aactacattt gtcaaaagta 8340
atttaactgt taataagaca ttctcccata tttgtcaagt tctcatttat agatattgta 8400
atttaactac cgcaaagaga tttcacggtc ttttacagac tttaaaggga aatagatgcc 8460
tttgcaataa gattgaagta gccgatttcc aagaattaac gtctattgga ctggggaggt 8520
ccagtgagat gaataaaatg attaaaaatc taactaacga aactcttctt gagtttctaa 8580
tgctcacaaa agcaaactta agggaattct tagcatgtga aaatattcaa gacgatttag 8640
acgataatat cataaagtat attctatctc cagaaaaagt cttgagtgta gtggatttct 8700
gtggctgttc aggtaccact ttcacagaaa gttttattaa ggccctggat aaatactacc 8760
ctaataaatc gattgatcag taccggctgg aacctattca acaaaattat cagattacct 8820
gcttaggcct aaatgattgt attgatctac catcacacgt tctttggaag atcttaaaaa 8880
tgttaccaga actacaaaaa cttgatttat ctcatacctc cattgatg 8928
<210> 3
<211> 10224
<212> DNA
<213> Artificial
<220>
<223> 质粒pZY402线性化后片段
<400> 3
gcagagaatc ctcgggtttg taaaccatgt tatagttaaa ccccgatatt acaccattaa 60
aatcatttct aaatatgcgg tcgtcgatga tcagctgcag ggaaatgcca aaatatggac 120
cccatagagg tcttggcttg ttcatatcgt tgggtcgatt tctgttccca gtgttatttg 180
gttgaatggg aacagtcgcg tcttgcccca tcttgtctct gccgctttcg tcgaatctga 240
ctttgcaatt gacgttgcat atcaacgtgc cggatcccgg aatagcgtga tagtctactc 300
ctgttagggc atgttgtgtt tgtacgacct ggttttgcca catttgcaga aaaactgttg 360
cttgcgcgaa gggggtagca ttaaatatta ttctgcagtt gtttggatta aaaagctgta 420
cgaattgggc caatttgttg gagtccggat catccaaatg cgctaatatc ttcttcgtaa 480
atgtggccgt tatttgtgct tgatttgcgt cattcattac catagtatta ctattggtgt 540
tcatatttct gttattactg ttattagttt ggcgttttgc ctgacgatca gcctggtggg 600
gccaagtata tactataaaa acaggttagt tacttgctct atgcgtttgc gcatcctctt 660
tttacttttt ttttttcagt aaagcctaag cataaatcgt tttatacgta cgacacgttc 720
aacttttctt ggttagtagt ggcaatctct gcaatacata cagggagtca tggtctatca 780
tcttgtccaa tcaaagaagc atcggttcag atcgagcaaa ctgtagggag aaaggaaagt 840
agaaatgcag agtgtgctat atgtccaatc tcggttttgt agtttggatg tcattagaga 900
tctaccaccc aaccggctgc tttcatgtgg aacagaaaag aaatcggggc gcttcctctt 960
ctgtattcct ttaattaacg tttttattca gccatctaac catcataccc ccatacggta 1020
acaaaacctc ttctaagaaa agaagtctct gctcctccgc catcttattt ttattcgctg 1080
cgcgcgttta ttgtcgcatc gctagccagc aaaaagttgg ttgccttttt ttacctaaaa 1140
aagacacatc taactgatta gttttccgtt ttaggatatt gacgccaagc gtgcgtctga 1200
ttgcaaatta aagccttcga gcgtcccaaa accttctcaa gcaaggtttt cagtataatg 1260
ttacatgcgt acacgcgttt gtacagaaaa aaaagaaaaa tttgaaatat aaataacgtt 1320
cttaatacta acataactat aaaaaaataa atagggacct agacttcagg ttgtctaact 1380
ccttcctttt cggttagagc ggatgtgggg ggagggcgtg aatgtaagcg tgacataact 1440
aattacatga tatcgacaaa ggaaaagggg cctgtttatt tatcaagata agtttccgga 1500
tctttttctt tcctaacacc ccagtcagcc tgagttacat ccagccattg aaccttagaa 1560
aatcttttgt catcagcggt ttgagcccta agatcaacat cttgcttagc aatcactgca 1620
atggcgtcat aaccaccagc accaggtatt aagcaagtaa gaactccttt taaggtctgg 1680
caatcatcca ataagctagt ttgtacggga ggttcgatat cggcaccaga ttctttagtt 1740
atttttctaa aggaacgtct aattgtggca actgcatctc taacttctgt gatctcagga 1800
tacttttgac aggtacagtc attcctctca agagactcaa atatctgatc gctgtaatcg 1860
tcatgagtct cgtgtaagcg atctagttta gatagtccat ccataaatct agaatttgca 1920
tgatcgagtt ctgtatatat tttcaagctt tccggcatat gcgaatcata ccaatttttt 1980
accttctgga ccagttttac tgtttctgaa ccattcttaa tatcgcccat ccataaagtt 2040
aatcccgaag gtaaatggtt acttttaatc gttatattcc agtcttcttc attaaccaaa 2100
tgcgccagtt tactgccgta agtagcactt ccaatatctg gcaaattaga gattaatgcg 2160
ggtgggaatc ttctatatct gatagatcca tatgctgccg ccgctacatc aaacccgctt 2220
ccaattttac cctgagcttg acaatgagca acttgtgata aattatgaat aacttctcta 2280
tatttgtcta cattattttc caggtccgat acaaaaaagg aggccaaagc tgtagttaaa 2340
actgtgacta aacctgccga ggagcccagc cctgttttgg gaacttcttc aattctgtgc 2400
gaatgaaaac tcaatcttct gttgccacga tgttcggtaa cgctgtcctc ctgagaatgg 2460
taggcatcat cagagaaaat atcaataacg aacaagtttc tattgcagta gtcgtccatg 2520
ttaggcttaa agtagctaaa tacgttagcg ataacttttt caatgaaagg gttcttagat 2580
ccgcctatcg aaacaggaat gaagccagtt ttaggactta tatggtacag ccactcccca 2640
tctttaaatt gtttactttt cacacgcact tcaaacttat cagactcttg caatgaaccg 2700
taaggatggg ctacagcatg cattcttgcc gataatccga ctacaaatgc ttcatatttc 2760
ggatctaaaa ctaaatatcc accagctagt aacgctttcc ctggggcact gaaggctctc 2820
aactctgaca ttatagtttt ttctccttga cgttaaagta tagaggtata ttaacaattt 2880
tttgttgata cttttatgac atttgaataa gaagtaatac aaaccgaaaa tgttgaaagt 2940
attagttaaa gtggttatgc agcttttgca tttatatatc tgttaataga tcaaaaatca 3000
tcgcttcgct gattaattac cccagaaata aggctaaaaa actaatcgca ttattatcct 3060
atggttgtta atttgattcg ttgatttgaa ggtttgtggg gccaggttac tgccaatttt 3120
tcctcttcat aaccataaaa gctagtattg tagaatcttt attgttcgga gcagtgcggc 3180
gcgaggcaca tctgcgtttc aggaacgcga ccggtgaaga ccaggacgca cggaggagag 3240
tcttccgtcg gagggctgtc gcccgctcgg cggcttctaa tccgtacttc aatatagcaa 3300
tgagcagtta agcgtattac tgaaagttcc aaagagaagg tttttttagg ctaagataat 3360
ggggctcttt acatttccac aacatataag taagattaga tatggatatg tatatggtgg 3420
tattgccatg taatatgatt attaaacttc tttgcgtcca tccaaaaaaa aagtaagaat 3480
ttttgaaaat tcaatataaa tggttttaac caataaaaca gtcatttctg gatcgaaagt 3540
caaaagttta tcatctgcgc aatcgagctc atcaggacct tcatcatcta gtgaggaaga 3600
tgattcccgc gatattgaaa gcttggataa gaaaatacgt cctttagaag aattagaagc 3660
attattaagt agtggaaata caaaacaatt gaagaacaaa gaggtcgctg ccttggttat 3720
tcacggtaag ttacctttgt acgctttgga gaaaaaatta ggtgatacta cgagagcggt 3780
tgcggtacgt aggaaggctc tttcaatttt ggcagaagct cctgtattag catctgatcg 3840
tttaccatat aaaaattatg actacgaccg cgtatttggc gcttgttgtg aaaatgttat 3900
aggttacatg cctttgcccg ttggtgttat aggccccttg gttatcgatg gtacatctta 3960
tcatatacca atggcaacta cagagggttg tttggtagct tctgccatgc gtggctgtaa 4020
ggcaatcaat gctggcggtg gtgcaacaac tgttttaact aaggatggta tgacaagagg 4080
cccagtagtc cgtttcccaa ctttgaaaag atctggtgcc tgtaagatat ggttagactc 4140
agaagaggga caaaacgcaa ttaaaaaagc ttttaactct acatcaagat ttgcacgtct 4200
gcaacatatt caaacttgtc tagcaggaga tttactcttc atgagattta gaacaactac 4260
tggtgacgca atgggtatga atatgatttc taaaggtgtc gaatactcat taaagcaaat 4320
ggtagaagag tatggctggg aagatatgga ggttgtctcc gtttctggta actactgtac 4380
cgacaaaaaa ccagctgcca tcaactggat cgaaggtcgt ggtaagagtg tcgtcgcaga 4440
agctactatt cctggtgatg ttgtcagaaa agtgttaaaa agtgatgttt ccgcattggt 4500
tgagttgaac attgctaaga atttggttgg atctgcaatg gctgggtctg ttggtggatt 4560
taacgcacat gcagctaatt tagtgacagc tgttttcttg gcattaggac aagatcctgc 4620
acaaaatgtt gaaagttcca actgtataac attgatgaaa gaagtggacg gtgatttgag 4680
aatttccgta tccatgccat ccatcgaagt aggtaccatc ggtggtggta ctgttctaga 4740
accacaaggt gccatgttgg acttattagg tgtaagaggc ccgcatgcta ccgctcctgg 4800
taccaacgca cgtcaattag caagaatagt tgcctgtgcc gtcttggcag gtgaattatc 4860
cttatgtgct gccctagcag ccggccattt ggttcaaagt catatgaccc acaacaggaa 4920
acctgctgaa ccaacaaaac ctaacaattt ggacgccact gatataaatc gtttgaaaga 4980
tgggtccgtc acctgcatta aatcctaatt tgccagctta ctatccttct tgaaaatatg 5040
cactctatat cttttagttc ttaattgcaa cacatagatt tgctgtataa cgaattttat 5100
gctatttttt aaatttggag ttcagtgata aaagtgtcac agcgaatttc ctcacatgta 5160
gggaccgaat tgtttacaag ttctctgtac caccatggag acatcaaaga ttgaaaatct 5220
atggaaagat atggacggta gcaacaagaa tatagcacga gccgcgaagt tcatttcgtt 5280
acttttgata tcgctcacaa ctattgcgaa gcgcttcagt gaaaaaatca taaggaaaag 5340
ttgtaaatat tattggtagt attcgtttgg taaagtagag ggggtaattt ttccccttta 5400
ttttgttcat acattcttaa attgctttgc ctctcctttt ggaaagctat acttcggagc 5460
actgttgagc gaaggctcat tagatatatt ttctgtcatt ttccttaacc caaaaataag 5520
ggaaagggtc caaaaagcgc tcggacaact gttgaccgtg atccgaagga ctggctatac 5580
agtgttcaca aaatagccaa gctgaaaata atgtgtagct atgttcagtt agtttggcta 5640
gcaaagatat aaaagcaggt cggaaatatt tatgggcatt attatgcaga gcatcaacat 5700
gataaaaaaa aacagttgaa tattccctca aaaatgtcat taccgttctt aacttctgca 5760
ccgggaaagg ttattatttt tggtgaacac tctgctgtgt acaacaagcc tgccgtcgct 5820
gctagtgtgt ctgcgttgag aacctacctg ctaataagcg agtcatctgc accagatact 5880
attgaattgg acttcccgga cattagcttt aatcataagt ggtccatcaa tgatttcaat 5940
gccatcaccg aggatcaagt aaactcccaa aaattggcca aggctcaaca agccaccgat 6000
ggcttgtctc aggaactcgt tagtcttttg gatccgttgt tagctcaact atccgaatcc 6060
ttccactacc atgcagcgtt ttgtttcctg tatatgtttg tttgcctatg cccccatgcc 6120
aagaatatta agttttcttt aaagtctact ttacccatcg gtgctgggtt gggctcaagc 6180
gcctctattt ctgtatcact ggccttagct atggcctact tgggggggtt aataggatct 6240
aatgacttgg aaaagctgtc agaaaacgat aagcatatag tgaatcaatg ggccttcata 6300
ggtgaaaagt gtattcacgg taccccttca ggaatagata acgctgtggc cacttatggt 6360
aatgccctgc tatttgaaaa agactcacat aatggaacaa taaacacaaa caattttaag 6420
ttcttagatg atttcccagc cattccaatg atcctaacct atactagaat tccaaggtct 6480
acaaaagatc ttgttgctcg cgttcgtgtg ttggtcaccg agaaatttcc tgaagttatg 6540
aagccaattc tagatgccat gggtgaatgt gccctacaag gcttagagat catgactaag 6600
ttaagtaaat gtaaaggcac cgatgacgag gctgtagaaa ctaataatga actgtatgaa 6660
caactattgg aattgataag aataaatcat ggactgcttg tctcaatcgg tgtttctcat 6720
cctggattag aacttattaa aaatctgagc gatgatttga gaattggctc cacaaaactt 6780
accggtgctg gtggcggcgg ttgctctttg actttgttac gaagagacat tactcaagag 6840
caaattgaca gtttcaaaaa gaaattgcaa gatgatttta gttacgagac atttgaaaca 6900
gacttgggtg ggactggctg ctgtttgtta agcgcaaaaa atttgaataa agatcttaaa 6960
atcaaatccc tagtattcca attatttgaa aataaaacta ccacaaagca acaaattgac 7020
gatctattat tgccaggaaa cacgaattta ccatggactt cataagcgaa tttcttatga 7080
tttatgattt ttattattaa ataagttata aaaaaaataa gtgtatacaa attttaaagt 7140
gactcttagg ttttaaaacg aaaattctta ttcttgagta actctttcct gtaggtcagg 7200
ttgctttctc aggtatagca tgaggtcgct cttattgacc acacctctac cgggctgcag 7260
gtcgacaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctaga 7320
gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga 7380
ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt 7440
acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca 7500
cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc 7560
tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg 7620
atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt 7680
ctcacatcac atccgaacat aaacaaccat gggtaaaaag cctgaactca ccgcgacgtc 7740
tgtcgagaag tttctgatcg aaaagttcga cagcgtctcc gacctgatgc agctctcgga 7800
gggcgaagaa tctcgtgctt tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt 7860
aaatagctgc gccgatggtt tctacaaaga tcgttatgtt tatcggcact ttgcatcggc 7920
cgcgctcccg attccggaag tgcttgacat tggggaattc agcgagagcc tgacctattg 7980
catctcccgc cgtgcacagg gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc 8040
tgttctgcag ccggtcgcgg aggcaatgga tgcgatcgct gcggccgatc ttagccagac 8100
gagcgggttc ggcccattcg gaccgcaagg aatcggtcaa tacactacat ggcgtgattt 8160
catatgcgcg attgctgatc cccatgtgta tcactggcaa actgtgatgg acgacaccgt 8220
cagtgcgtcc gtcgcgcagg ctctcgatga gctgatgctt tgggccgagg actgccccga 8280
agtccggcac ctcgtgcacg cggatttcgg ctccaacaat gtcctgacgg acaatggccg 8340
cataacagcg gtcattgact ggagcgaggc gatgttcggg gattcccaat acgaggtcgc 8400
caacatcttc ttctggaggc cgtggttggc ttgtatggag cagcagacgc gctacttcga 8460
gcggaggcat ccggagcttg caggatcgcc gcggctccgg gcgtatatgc tccgcattgg 8520
tcttgaccaa ctctatcaga gcttggttga cggcaatttc gatgatgcag cttgggcgca 8580
gggtcgatgc gacgcaatcg tccgatccgg agccgggact gtcgggcgta cacaaatcgc 8640
ccgcagaagc gcggccgtct ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa 8700
ccgacgcccc agcactcgtc cgagggcaaa ggaataatca gtactgacaa taaaaagatt 8760
cttgttttca agaacttgtc atttgtatag tttttttata ttgtagttgt tctattttaa 8820
tcaaatgtta gcgtgattta tatttttttt cgcctcgaca tcatctgccc agatgcgaag 8880
ttaagtgcgc agaaagtaat atcatgcgtc aatcgtatgt gaatgctggt cgctatactg 8940
ctgtcgattc gatactaacg ccgccatcca gtgtcgaaaa cgagctctcg agaaccctta 9000
atataacttc gtataatgta tgctatacga agttattagg tgatatcaga tccactagtg 9060
gttacgtgga ttgagccagc aatacagatc attattaaac tgttttgtac atgatgttag 9120
tatataatcg taaagctttt ctaatatgta taccttatac atggaactcc acagaacttg 9180
caaacatacc aaaaatcctt tattcttgtt cactcatttt acatcaaaaa ataatatttc 9240
agttattaag gaaaataaaa aaatagatta gagaagcatt ttgaagaaat agtatattct 9300
tttattgaac ctaagagcgt gatattttta ctcgaaataa aatacgaaaa atctatacac 9360
tcatctttcc gactactatt ggctcctgct caaaaaaaga gggaaaaaaa gctccaaaat 9420
tctatctttt cctatcgctc ctgtcctatc cttattacgt tcattactat tttaatacta 9480
tccattcttt tattttcagt ctaaaaaaaa catttctcat aacgggaaaa gcaaaaaaat 9540
gtcaagctta tacatcaaaa caccactgca tgcattatct gctggtccgg attctcatgc 9600
aaatagctcc tattatgaca atcttttatt gccttcattt tccaatttat ccagtaatat 9660
cagcaggaac aatatcacaa ccgataacaa tataaattca gcaagtccta ggaagtattc 9720
gtttcattcg ctcaatgtgt cgccaattct ttccccaata tctttggcta acgaaatact 9780
tggaaaaaaa tcaaatactg caccagcatc gccacatcat atggattaca atccaatttc 9840
ctcattaaca ccaggcaatt caccagaatt caacaaggca agcttatctc agattagttt 9900
tacaaatcca ttgaactatg ggtctggctt aggtttttcc tctaattcac aacctcgact 9960
accattgtta gacaggctat cgtccgtctc tttatctaag agaccggagc gcccacaaca 10020
aagcctacca tcactaaggc atctgcaatt attacccagt cctcttttac aagaaaacgc 10080
agcacgcttc cctgatacat caaaacgtac atctaattgg aaaactgatc ttacccattg 10140
gtgtaaggat acaaactatc aagactatgt taaaatacgc gaagaagtgg ctcatttcaa 10200
gccattaagt atacccaact taac 10224
<210> 4
<211> 8405
<212> DNA
<213> Artificial
<220>
<223> 质粒pZY410线性化后片段
<400> 4
gacattgaac tcttggccga ctcttctgat gaggctttcg aaaaatctac tttcattaac 60
acaattgagt tcattgtgca cgatatttgg gtcgaaacgt tgataaagaa cataaactta 120
ttacaaatgt ttgatgcaga tttgaaatgc tacaatgaca agtacataat ttgcaagcta 180
aagggtcagt atccctccac aaatatagtg gacataatgt gccgtttgaa gcatttttcc 240
aattctattt tggaaacttt caaattcgga atcgagctaa aagaacaaga tcaatgccac 300
aatagaaaca ctattatcaa ctacgttctt ttcagtagag tattttcaac gattgtttta 360
gagattcaaa aatgctttat cttgatcgtt aagttcatgt attctgtcaa ttttttagaa 420
aaattttccg atgaagtatt tttgtcattt attgaaattc tcataaaaat tgtctttgag 480
caccagattc cgcagttatt tttgggaatt gatgaaatta ttcaactctg gttaaagaac 540
aatgaaggaa aacggcaaca actcttgagc gcttggtgca acggaactgt tcaagatatg 600
aagcaaagcc aacaaagaga gtcctcaaac gcagaatctg aatcaatcac ttcaagtaca 660
gaagaggacg aagaaggcct gcaattcaat aaatgggatg tgattgaacc atttattgat 720
aatatcaagg ctttgaatca atcaaaatcc catatgtgac gcagcgataa aaccgcggca 780
tgcaaacatc tacacaatta gcaagggcaa tccatatttt gtcttttcgc gccctggaaa 840
ggcctaagta atgtcgtaaa cgcattctat ctgtacttca actctcctct gtgcattggt 900
ttgtgcaaat cacattttac gatactgcca gatatatgca aaaagagaaa accaagggac 960
cagaacaaag caaaattacg atattcttcg aattccttcg tgcttgacta agacaaaggg 1020
atggacgtag cgatttttag cgggccaaga actggttccg aaaaagcaca ggtacaccga 1080
accctcagct aaggagggac agcaccgatg cggaaggaca aactttcttt ttgcctatca 1140
cagtatctta tcgagctaac tattttcgac acacatgaaa aagcagaaat attaacgaaa 1200
aagaaaagaa agaccatgtc atgtacgggc aatcagaatc tgtaacaagc gccatttttt 1260
tttctgtatc gggccctcct tactgctctc cttccgtgta acgcgttatg gcttcattgc 1320
tcctacattt atcggatggc gcacatagct aaaaaaggaa gattttcact cataagggaa 1380
aggaaccgca tgactcaaga gaagcatgtg gtttttgagt ttttttcgtt gaattttcag 1440
gtaaagctca atagttatga caattacaac aacagaattc tttctatata tgcacgaact 1500
tgtaatatgg aagaaattat gacgtacaaa ctataaagta aatattttac gtaacacatg 1560
gtgctgttgt gcttcttttt caagagaata ccaatgacgt atgactaagt ttaggattta 1620
atgcaggtga cggacccatc tttcaaacga tttatatcag tggcgtccaa attgttaggt 1680
tttgttggtt cagcaggttt cctgttgtgg gtcatatgac tttgaaccaa atggccggct 1740
gctagggcag cacataagga taattcacct gccaagacgg cacaggcaac tattcttgct 1800
aattgacgtg cgttggtacc aggagcggta gcatgcgggc ctcttacacc taataagtcc 1860
aacatggcac cttgtggttc tagaacagta ccaccaccga tggtacctac ttcgatggat 1920
ggcatggata cggaaattct caaatcaccg tccacttctt tcatcaatgt tatacagttg 1980
gaactttcaa cattttgtgc aggatcttgt cctaatgcca agaaaacagc tgtcactaaa 2040
ttagctgcat gtgcgttaaa tccaccaaca gacccagcca ttgcagatcc aaccaaattc 2100
ttagcaatgt tcaactcaac caatgcggaa acatcacttt ttaacacttt tctgacaaca 2160
tcaccaggaa tagtagcttc tgcgacgaca ctcttaccac gaccttcgat ccagttgatg 2220
gcagctggtt ttttgtcggt acagtagtta ccagaaacgg agacaacctc catatcttcc 2280
cagccatact cttctaccat ttgctttaat gagtattcga cacctttaga aatcatattc 2340
atacccattg cgtcaccagt agttgttcta aatctcatga agagtaaatc tcctgctaga 2400
caagtttgaa tatgttgcag acgtgcaaat cttgatgtag agttaaaagc ttttttaatt 2460
gcgttttgtc cctcttctga gtctaaccat atcttacagg caccagatct tttcaaagtt 2520
gggaaacgga ctactgggcc tcttgtcata ccatccttag ttaaaacagt tgttgcacca 2580
ccgccagcat tgattgcctt acagccacgc atggcagaag ctaccaaaca accctctgta 2640
gttgccattg gtatatgata agatgtacca tcgataacca aggggcctat aacaccaacg 2700
ggcaaaggca tgtaacctat aacattttca caacaagcgc caaatacgcg gtcgtagtca 2760
taatttttat atggtaaacg atcagatgct aatacaggag cttctgccaa aattgaaaga 2820
gccttcctac gtaccgcaac cgctctcgta gtatcaccta attttttctc caaagcgtac 2880
aaaggtaact taccgtgaat aaccaaggca gcgacctctt tgttcttcaa ttgttttgta 2940
tttccactac ttaataatgc ttctaattct tctaaaggac gtattttctt atccaagctt 3000
tcaatatcgc gggaatcatc ttcctcacta gatgatgaag gtcctgatga gctcgattgc 3060
gcagatgata aacttttgac tttcgatcca gaaatgactg ttttattggt taaaaccatt 3120
tatattgaat tttcaaaaat tcttactttt tttttggatg gacgcaaaga agtttaataa 3180
tcatattaca tggcaatacc accatataca tatccatatc taatcttact tatatgttgt 3240
ggaaatgtaa agagccccat tatcttagcc taaaaaaacc ttctctttgg aactttcagt 3300
aatacgctta actgctcatt gctatattga agtacggatt agaagccgcc gagcgggcga 3360
cagccctccg acggaagact ctcctccgtg cgtcctggtc ttcaccggtc gcgttcctga 3420
aacgcagatg tgcctcgcgc cgcactgctc cgaacaataa agattctaca atactagctt 3480
ttatggttat gaagaggaaa aattggcagt aacctggccc cacaaacctt caaatcaacg 3540
aatcaaatta acaaccatag gataataatg cgattagttt tttagcctta tttctggggt 3600
aattaatcag cgaagcgatg atttttgatc tattaacaga tatataaatg caaaagctgc 3660
ataaccactt taactaatac tttcaacatt ttcggtttgt attacttctt attcaaatgt 3720
cataaaagta tcaacaaaaa attgttaata tacctctata ctttaacgtc aaggagaaaa 3780
aactataatg aaactctcaa ctaaactttg ttggtgtggt attaaaggaa gacttaggcc 3840
gcaaaagcaa caacaattac acaatacaaa cttgcaaatg actgaactaa aaaaacaaaa 3900
gaccgctgaa caaaaaacca gacctcaaaa tgtcggtatt aaaggtatcc aaatttacat 3960
cccaactcaa tgtgtcaacc aatctgagct agagaaattt gatggcgttt ctcaaggtaa 4020
atacacaatt ggtctgggcc aaaccaacat gtcttttgtc aatgacagag aagatatcta 4080
ctcgatgtcc ctaactgttt tgtctaagtt gatcaagagt tacaacatcg acaccaacaa 4140
aattggtaga ttagaagtcg gtactgaaac tctgattgac aagtccaagt ctgtcaagtc 4200
tgtcttgatg caattgtttg gtgaaaacac tgacgtcgaa ggtattgaca cgcttaatgc 4260
ctgttacggt ggtaccaacg cgttgttcaa ctctttgaac tggattgaat ctaacgcatg 4320
ggatggtaga gacgccattg tagtttgcgg tgatattgcc atctacgata agggtgccgc 4380
aagaccaacc ggtggtgccg gtactgttgc tatgtggatc ggtcctgatg ctccaattgt 4440
atttgactct gtaagagctt cttacatgga acacgcctac gatttttaca agccagattt 4500
caccagcgaa tatccttacg tcgatggtca tttttcatta acttgttacg tcaaggctct 4560
tgatcaagtt tacaagagtt attccaagaa ggctatttct aaagggttgg ttagcgatcc 4620
cgctggttcg gatgctttga acgttttgaa atatttcgac tacaacgttt tccatgttcc 4680
aacctgtaaa ttggtcacaa aatcatacgg tagattacta tataacgatt tcagagccaa 4740
tcctcaattg ttcccagaag ttgacgccga attagctact cgcgattatg acgaatcttt 4800
aaccgataag aacattgaaa aaacttttgt taatgttgct aagccattcc acaaagagag 4860
agttgcccaa tctttgattg ttccaacaaa cacaggtaac atgtacaccg catctgttta 4920
tgccgccttt gcatctctat taaactatgt tggatctgac gacttacaag gcaagcgtgt 4980
tggtttattt tcttacggtt ccggtttagc tgcatctcta tattcttgca aaattgttgg 5040
tgacgtccaa catattatca aggaattaga tattactaac aaattagcca agagaatcac 5100
cgaaactcca aaggattacg aagctgccat cgaattgaga gaaaatgccc atttgaagaa 5160
gaacttcaaa cctcaaggtt ccattgagca tttgcaaagt ggtgtttact acttgaccaa 5220
catcgatgac aaatttagaa gatcttacga tgttaaaaaa taatcatgta attagttatg 5280
tcacgcttac attcacgccc tccccccaca tccgctctaa ccgaaaagga aggagttaga 5340
caacctgaag tctaggtccc tatttatttt tttatagtta tgttagtatt aagaacgtta 5400
tttatatttc aaatttttct tttttttctg tacagacgcg tgtacgcatg taacattata 5460
ctgaaaacct tgcttgagaa ggttttggga cgctcgaagg ctttaatttg cacgctgcag 5520
gtcgacaacc cttaatataa cttcgtataa tgtatgctat acgaagttat taggtctaga 5580
gatctgttta gcttgcctcg tccccgccgg gtcacccggc cagcgacatg gaggcccaga 5640
ataccctcct tgacagtctt gacgtgcgca gctcaggggc atgatgtgac tgtcgcccgt 5700
acatttagcc catacatccc catgtataat catttgcatc catacatttt gatggccgca 5760
cggcgcgaag caaaaattac ggctcctcgc tgcagacctg cgagcaggga aacgctcccc 5820
tcacagacgc gttgaattgt ccccacgccg cgcccctgta gagaaatata aaaggttagg 5880
atttgccact gaggttcttc tttcatatac ttccttttaa aatcttgcta ggatacagtt 5940
ctcacatcac atccgaacat aaacaaccat gggtaaggaa aagactcacg tttcgaggcc 6000
gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt 6060
cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt 6120
tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa 6180
ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga 6240
tgcatggtta ctcaccactg cgatccccgg caaaacagca ttccaggtat tagaagaata 6300
tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc 6360
gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca 6420
atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg 6480
gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt 6540
cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg 6600
ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg 6660
gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat 6720
tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc 6780
agtactgaca ataaaaagat tcttgttttc aagaacttgt catttgtata gtttttttat 6840
attgtagttg ttctatttta atcaaatgtt agcgtgattt atattttttt tcgcctcgac 6900
atcatctgcc cagatgcgaa gttaagtgcg cagaaagtaa tatcatgcgt caatcgtatg 6960
tgaatgctgg tcgctatact gctgtcgatt cgatactaac gccgccatcc agtgtcgaaa 7020
acgagctctc gagaaccctt aatataactt cgtataatgt atgctatacg aagttattag 7080
gtgatatcag atccactagt ggctactatc ggcgactctc tcgaaatttt tcttaacgcg 7140
tccttgtact gcgtctaacg cttttgccac ttggatttct attataggaa atagtctcac 7200
ttactgggcg acgaattttc gcgttttgat gaagcacagg aagaatttct tttttttttg 7260
gcttcttctg gttccgtttt ttacgcgcac aaatctaaaa aaagaaataa ttataaccta 7320
gtctcgaaaa ttttcatcga tccattcgtt cctttttttc gattttttca gatcaaaatt 7380
cttgtttctt tctttgtctt agtttatatt aaaagatatt ttgattttac tcctgaacta 7440
tttattcttt ctaagaaggc cagaacacta cagctgtttt aaccgactac gaagttctcc 7500
attctcgaac actagccttc atttaccaaa caggaactag cgtatatcat tagtccttat 7560
tcgaaaagag attggtagat atttattgta gtttgtgaga aggagaaaat actgtcattg 7620
gactgatagt tagaggacat taacctctct tacgttcgct caaaaaaatt aaaataagca 7680
agtagctgaa atcaagtctt tcatacaacg gtcagaccat gaacactgat caacaacctt 7740
atcagggcca aacggactat acccagggac caggtaacgg gcaaagtcag gaacaagact 7800
atgaccaata tggccagcct ttgtatcctt cacaagctga tggttactac gatccaaatg 7860
tcgctgctgg tactgaagct gatatgtatg gtcaacaacc accaaacgag tcttacgacc 7920
aagactacac aaacggtgaa tactatggtc aaccgccaaa tatggctgct caagacggtg 7980
aaaacttctc ggattttagc agttacggcc ctcctggaac acctggatat gatagctatg 8040
gtggtcagta taccgcttct caaatgagtt atggagaacc aaattcgtcg ggtacctcga 8100
ctccaattta cggtaattat gacccaaatg ctatcgctat ggctttgcca aatgaacctt 8160
atcccgcttg gactgctgac tctcaatctc ccgtttcgat cgagcaaatc gaagatatct 8220
ttattgattt gaccaacaga ctcgggttcc aaagagactc catgagaaat atgtttgatc 8280
attttatggt tctcttggac tctaggtcct cgagaatgtc tcctgatcaa gctttactat 8340
ctttacatgc cgactacatt ggtggcgata ctgctaacta taaaaaatgg tattttgctg 8400
ctcag 8405
<210> 5
<211> 5878
<212> DNA
<213> Artificial
<220>
<223> 质粒pZY516线性化后片段
<400> 5
ataacgagaa cacacagggg cgctatcgca cagaatcaaa ttcgatgact ggaaattttt 60
tgttaatttc agaggtcgcc tgacgcatat acctttttca actgaaaaat tgggagaaaa 120
aggaaaggtg agagcgccgg aaccggcttt tcatatagaa tagagaagcg ttcatgacta 180
aatgcttgca tcacaatact tgaagttgac aatattattt aaggacctat tgttttttcc 240
aataggtggt tagcaatcgt cttactttct aacttttctt accttttaca tttcagcaat 300
atatatatat atatttcaag gatataccat tctaatgtct gcccctaaga agatcgtcgt 360
tttgccaggt gaccacgttg gtcaagaaat cacagccgaa gccattaagg ttcttaaagc 420
tatttctgat gttcgttcca atgtcaagtt cgatttcgaa aatcatttaa ttggtggtgc 480
tgctatcgat gctacaggtg ttccacttcc agatgaggcg ctggaagcct ccaagaaggc 540
tgatgccgtt ttgttaggtg ctgtgggtgg tcctaaatgg ggtaccggta gtgttagacc 600
tgaacaaggt ttactaaaaa tccgtaaaga acttcaattg tacgccaact taagaccatg 660
taactttgca tccgactctc ttttagactt atctccaatc aagccacaat ttgctaaagg 720
tactgacttc gttgttgtca gagaattagt gggaggtatt tactttggta agagaaagga 780
agacgatggt gatggtgtcg cttgggatag tgaacaatac accgttccag aagtgcaaag 840
aatcacaaga atggccgctt tcatggccct acaacatgag ccaccattgc ctatttggtc 900
cttggataaa gctaatgttt tggcctcttc aagattatgg agaaaaactg tggaggaaac 960
catcaagaac gaattcccta cattgaaggt tcaacatcaa ttgattgatt ctgccgccat 1020
gatcctagtt aagaacccaa cccacctaaa tggtattata atcaccagca acatgtttgg 1080
tgatatcatc tccgatgaag cctccgttat cccaggttcc ttgggtttgt tgccatctgc 1140
gtccttggcc tctttgccag acaagaacac cgcatttggt ttgtacgaac catgccacgg 1200
ttctgctcca gatttgccaa agaataaggt caaccctatc gccactatct tgtctgctgc 1260
aatgatgttg aaattgtcat tgaacttgcc tgaagaaggt aaggccattg aagatgcagt 1320
taaaaaggtt ttggatgcag gtatcagaac tggtgattta ggtggttcca acagtaccac 1380
cgaagtcggt gatgctgtcg ccgaagaagt taagaaaatc cttgcttaaa tttaactcct 1440
taagttactt taatgattta gtttttatta ttaataattc atgctcatga catctcatat 1500
acacgtttat aaaacttaaa tagattgaaa atgtattaaa gattcctcag ggattcgatt 1560
tttttggaag tttttgtttt tttttccttg agatgctgta gtatttggga acaattatac 1620
aatcgaaaga tatatgctta cattcgaccg ttttagccgt gatcattatc ctatagtaac 1680
ataacctgaa gcataactga cactactatc atcaatactt gtcacatgag aactctgtga 1740
ataattaggc cactgaaatt tgatgcctga aggaccggca tcacggattt tcgataaagc 1800
acttagtatc acactaattg gcttttcgcg caaattaaag ccttcgagcg tcccaaaacc 1860
ttctcaagca aggttttcag tataatgtta catgcgtaca cgcgtttgta cagaaaaaaa 1920
agaaaaattt gaaatataaa taacgttctt aatactaaca taactataaa aaaataaata 1980
gggacctaga cttcaggttg tctaactcct tccttttcgg ttagagcgga tgtgggggga 2040
gggcgtgaat gtaagcgtga cataactaat tacatgatat cgacaaagga aaaggggcct 2100
gtctaacact tgcaaggaag tggtctctcg ccgtgacgaa cgtatctgct caaagtagca 2160
gaatggaaga tattgcctgt caacgcatca aagagcgcag taacgaacac gcgctggcga 2220
tcgttgatat cagggctgtg cataaacttc tcacaaatgc cgtaaaactc cttctccatg 2280
tcaaaggtaa tagctctggt taagttcttg gccaggtcag gcgacacgga aaggagctgc 2340
tctgtgacag caactgcgtt cacgatggac gcgtcgatag tcttgacctc gtgcacctcc 2400
ttatcatacg agtagagatc gttgataaga attgagtgtc tgacgtaaag atcagcaagc 2460
tcgtcaaagg gagcaagttc ttcgcgtgtt tgttgcacgc cagagccgaa gcggatagct 2520
gcaatcataa atgtcttgcc aacatcattg cgacggtaaa gcatgtaatc agccaatgtg 2580
gcatagtgat cctgcttaaa gggtgacttt acacgaccat cacggacgaa agcatggata 2640
gcataaagca gatcaggtcc aaggacggga tcacattcca atacgtctga ccagaatttc 2700
ttaagatgcc tgttcttggg agccatgcta agaaattcgt ctaccactgt ggtttcgttt 2760
tgagtagctc ctcgctcaat aacatcgtcg tggaggaata gaagcagaac agactgggtc 2820
aacaacgcca atctggtagg agatgatgta gggtaaagat aagcacagca agagatggca 2880
tcctcaacta gctcatcaat cttccacttg cgacgctcat ccatcaactc catgggtagt 2940
gtgccacgct ctgatgcctt gtcaaggctg attccgttgg ccatttcata tccagctgcc 3000
tcggcgtaag cccagaactt ggattgtctg gtccaaggaa gagaggtgtt ccatgggaca 3060
cccatatcct ctgggtagat aacaagaggg gcatcctcag agacgctgtc ttcccggaac 3120
agagactcaa agtccaaggt tggcacacct agggtgtttg atcctacagg atcaaatgtc 3180
aacttgactg gtctaagagg aacgtgcttg tgaggcattt atattgaatt ttcaaaaatt 3240
cttacttttt ttttggatgg acgcaaagaa gtttaataat catattacat ggcaatacca 3300
ccatatacat atccatatct aatcttactt atatgttgtg gaaatgtaaa gagccccatt 3360
atcttagcct aaaaaaacct tctctttgga actttcagta atacgcttaa ctgctcattg 3420
ctatattgaa gtacggatta gaagccgccg agcgggcgac agccctccga cggaagactc 3480
tcctccgtgc gtcctggtct tcaccggtcg cgttcctgaa acgcagatgt gcctcgcgcc 3540
gcactgctcc gaacaataaa gattctacaa tactagcttt tatggttatg aagaggaaaa 3600
attggcagta acctggcccc acaaaccttc aaatcaacga atcaaattaa caaccatagg 3660
ataataatgc gattagtttt ttagccttat ttctggggta attaatcagc gaagcgatga 3720
tttttgatct attaacagat atataaatgc aaaagctgca taaccacttt aactaatact 3780
ttcaacattt tcggtttgta ttacttctta ttcaaatgtc ataaaagtat caacaaaaaa 3840
ttgttaatat acctctatac tttaacgtca aggagaaaaa actataatgg cttcagaaaa 3900
agaaattagg agagagagat tcttgaacgt tttccctaaa ttagtagagg aattgaacgc 3960
atcgcttttg gcttacggta tgcctaagga agcatgtgac tggtatgccc actcattgaa 4020
ctacaacact ccaggcggta agctaaatag aggtttgtcc gttgtggaca cgtatgctat 4080
tctctccaac aagaccgttg aacaattggg gcaagaagaa tacgaaaagg ttgccattct 4140
aggttggtgc attgagttgt tgcaggctta cttcttggtc gccgatgata tgatggacaa 4200
gtccattacc agaagaggcc aaccatgttg gtacaaggtt cctgaagttg gggaaattgc 4260
catcaatgac gcattcatgt tagaggctgc tatctacaag cttttgaaat ctcacttcag 4320
aaacgaaaaa tactacatag atatcaccga attgttccat gaggtcacct tccaaaccga 4380
attgggccaa ttgatggact taatcactgc acctgaagac aaagtcgact tgagtaagtt 4440
ctccctaaag aagcactcct tcatagttac tttcaagact gcttactatt ctttctactt 4500
gcctgtcgca ttggccatgt acgttgccgg tatcacggat gaaaaggatt tgaaacaagc 4560
cagagatgtc ttgattccat tgggtgaata cttccaaatt caagatgact acttagactg 4620
cttcggtacc ccagaacaga tcggtaagat cggtacagat atccaagata acaaatgttc 4680
ttgggtaatc aacaaggcat tggaacttgc ttccgcagaa caaagaaaga ctttagacga 4740
aaattacggt aagaaggact cagtcgcaga agccaaatgc aaaaagattt tcaatgactt 4800
gaaaattgaa cagctatacc acgaatatga agagtctatt gccaaggatt tgaaggccaa 4860
aatttctcag gtcgatgagt ctcgtggctt caaagctgat gtcttaactg cgttcttgaa 4920
caaagtttac aagagaagca aatagaacta acgctaatcg ataaaacatt agatttcaaa 4980
ctagataagg accatgtata agaactatat acttccaata taatatagta taagctttaa 5040
gatagtatct ctcgatctac cgttccacgt gactagtcca aggatttttt ttaaaaagat 5100
tctctttttt tatgatattt gtacataaac tttataaatg aaattcataa tagaaacgac 5160
acgaaattac aaaatggaat atgttcatag ggtagacgaa actatatacg caatctacat 5220
acatttatca agaaggagaa aaaggaggat gtaaaggaat acaggtaagc aaattgatac 5280
taatggctca acgtgataag gaaaaagaat tgcactttaa cattaatatt gacaaggagg 5340
agggcaccac acaaaaagtt aggtgtaaca gaaaatcatg aaactatgat tcctaattta 5400
tatattggag gattttctct aaaaaaaaaa aaatacaaca aataaaaaac actcaatgac 5460
ctgaccattt gatggagttt aagtcaatac cttcttgaac catttcccat aatggtgaaa 5520
gttccctcaa gaattttact ctgtcagaaa cggccttaac gacgtagtcg acctcctctt 5580
cagtactaaa tctaccaata ccaaatctga tggaagaatg ggctaatgca tcatccttac 5640
ccagcgcatg taaaacataa gaaggttcta gggaagcaga tgtacaggct gaacccgagg 5700
ataatgcgat atcccttagt gccatcaata aagattctcc ttccacgtag gcgaaagaaa 5760
cgttaacaca ccctggataa cgatgatctg gagatccgtt caacgtggta tgttcagcgg 5820
ataatagacc tttgactaat ttatcggata gtcttttgat gtgagcttgg tcgttgtc 5878
<210> 6
<211> 8963
<212> DNA
<213> Artificial
<220>
<223> 质粒pZY517线性化后片段
<400> 6
acgcagataa ttccaggtat tttgaagcag aacttttatt tatcatcgaa ttgactattg 60
cattatttct attttgcaaa gaggagaaag aattaggaaa gttcatactt caaaaagttt 120
tccaactttc tcacacgaaa ggcctcacga aaaggactgt tcgtagaatg ctaacataca 180
aaattttgtt aatttcgtta tgtgcggatc agacggagta cttgtccaaa ttaataaacg 240
atgagctgtt aaaaaagggg gatattttta cccaaaaatt ttttgcaact aatcaaggta 300
aggaattttt gaagagacta ttttcattga ccgaatcaga gttttataga ggatttttac 360
taggaaatga gaatttttgg aaatttttaa gaaaagttac agcaatgaaa gagcagagcg 420
agagcatttt tgaatattta aatgaatcga tcaagacaga cagcaatatt ttgacaaatg 480
agaacttcat gtgggtccta ggactattag atgaaatttc atcaatgggt gccgttggaa 540
atcactggga aatagaatac aagaaattga cagaaagtgg tcataaaatt gataaggaga 600
atccatacaa gaaatcgatc gaattatcat tgaaatccat tcaactaaca tcacacttgc 660
tggaagataa taacgatctg cgtaaaaacg agatattcgc tattattcaa gctttggcac 720
atcaatgcat caatccgtgt aagcagataa gtgaatttgc agtggtaacg ctagagcaga 780
cgctcatcaa taaaatcgaa attccaacta atgagatgga atcggtagaa gaattaattg 840
agggcggatt actaccgttg ctaaattcga gtgaaacaca ggaagaccag aaaatcctca 900
tttcatccat attaacaata atttcaaatg tttatttgca ttatttgaaa ctagggaaga 960
caagcaacga aacgtttttg aaaattttga gtattttcaa taaatttgta gaggactcag 1020
atattgaaaa aaagctacag caattaatac ttgataagaa gagtattgag aagggcaacg 1080
gttcatcatc tcatggatct gcacatgaac aaacaccaga gtcaaacgac gttgaaattg 1140
aggctactgc gccaattgat gacaatacag acgatgataa caaaccgaag ttatctgatg 1200
tagaaaagga ttaaagatgc taagagatag tgatgatatt tcataaataa tgtaattcta 1260
tatatgttaa ttaccttttt tgcgaggcat atttatggtg aaggataagt tttgaccatc 1320
aaagaaggtt aatgtggctg tggtttcagg gtccataaag cttttcaatt catcattttt 1380
tttttattct tttttttgat ttcggtttcc ttgaaatttt tttgattcgg taatctccga 1440
acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac gcatatgtag 1500
tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa caaaaacctg 1560
caggaaacga aggtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc 1620
gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca 1680
gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 1740
caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc 1800
gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 1860
gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 1920
ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 1980
aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 2040
cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 2100
ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 2160
tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg 2220
tttacaattt cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 2280
tagatccgtc gagttcaaga gaaaaaaaaa gaaaaagcaa aaagaaaaaa ggaaagcgcg 2340
cctcgttcag aatgacacgt atagaatgat gcattacctt gtcatcttca gtatcatact 2400
gttcgtatac atacttactg acattcatag gtatacatat atacacatgt atatatatcg 2460
tatgctgcag ctttaaataa tcggtgtcac tacataagaa cacctttggt ggagggaaca 2520
tcgttggtac cattgggcga ggtggcttct cttatggcaa ccgcaagagc cttgaacgca 2580
ctctcactac ggtgatgatc attcttgcct cgcagacaat caacgtggag ggtaattctg 2640
ctagcctctg caaagctttc aagaaaatgc gggatcatct cgcaagagag atctcctact 2700
ttctcccttt gcaaaccaag ttcgacaact gcgtacggcc tgttcgaaag atctaccacc 2760
gctctggaaa gtgcctcatc caaaggcgca aatcctgatc caaacctttt tactccacgc 2820
gccagtaggg cctctttaaa agcttgaccg agagcaatcc cgcagtcttc agtggtgtga 2880
tggtcgtcta tgtgtaagtc accaatgcac tcaacgatta gcgaccagcc ggaatgcttg 2940
gccagagcat gtatcatatg gtccagaaac cctatacctg tgtggacgtt aatcacttgc 3000
gattgtgtgg cctgttctgc tactgcttct gcctcttttt ctgggaagat cgagtgctct 3060
atcgctaggg gaccaccctt taaagagatc gcaatctgaa tcttggtttc atttgtaata 3120
cgctttacta gggctttctg ctctgtcatc tttgccttcg tttatcttgc ctgctcattt 3180
tttagtatat tcttcgaaga aatcacatta ctttatataa tgtataattc attatgtgat 3240
aatgccaatc gctaagaaaa aaaaagagtc atccgctagg ggaaaaaaaa aaatgaaaat 3300
cattaccgag gcataaaaaa atatagagtg tactagagga ggccaagagt aatagaaaaa 3360
gaaaattgcg ggaaaggact gtgttatgac ttccctgact aatgccgtgt tcaaacgata 3420
cctggcagtg actcctagcg ctcaccaagc tcttaaaacg ggaatttatg gtgcactctc 3480
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacgcgct 3540
gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgagcaaa 3600
ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt tttcagtata atgttacatg 3660
cgtacacgcg tttgtacaga aaaaaaagaa aaatttgaaa tataaataac gttcttaata 3720
ctaacataac tataaaaaaa taaataggga cctagacttc aggttgtcta actccttcct 3780
tttcggttag agcggatgtg gggggagggc gtgaatgtaa gcgtgacata actaattaca 3840
tgatatcgac aaaggaaaag gggcctgttt aggatttaat gcaggtgacg gacccatctt 3900
tcaaacgatt tatatcagtg gcgtccaaat tgttaggttt tgttggttca gcaggtttcc 3960
tgttgtgggt catatgactt tgaaccaaat ggccggctgc tagggcagca cataaggata 4020
attcacctgc caagacggca caggcaacta ttcttgctaa ttgacgtgcg ttggtaccag 4080
gagcggtagc atgcgggcct cttacaccta ataagtccaa catggcacct tgtggttcta 4140
gaacagtacc accaccgatg gtacctactt cgatggatgg catggatacg gaaattctca 4200
aatcaccgtc cacttctttc atcaatgtta tacagttgga actttcaaca ttttgtgcag 4260
gatcttgtcc taatgccaag aaaacagctg tcactaaatt agctgcatgt gcgttaaatc 4320
caccaacaga cccagccatt gcagatccaa ccaaattctt agcaatgttc aactcaacca 4380
atgcggaaac atcacttttt aacacttttc tgacaacatc accaggaata gtagcttctg 4440
cgacgacact cttaccacga ccttcgatcc agttgatggc agctggtttt ttgtcggtac 4500
agtagttacc agaaacggag acaacctcca tatcttccca gccatactct tctaccattt 4560
gctttaatga gtattcgaca cctttagaaa tcatattcat acccattgcg tcaccagtag 4620
ttgttctaaa tctcatgaag agtaaatctc ctgctagaca agtttgaata tgttgcagac 4680
gtgcaaatct tgatgtagag ttaaaagctt ttttaattgc gttttgtccc tcttctgagt 4740
ctaaccatat cttacaggca ccagatcttt tcaaagttgg gaaacggact actgggcctc 4800
ttgtcatacc atccttagtt aaaacagttg ttgcaccacc gccagcattg attgccttac 4860
agccacgcat ggcagaagct accaaacaac cctctgtagt tgccattggt atatgataag 4920
atgtaccatc gataaccaag gggcctataa caccaacggg caaaggcatg taacctataa 4980
cattttcaca acaagcgcca aatacgcggt cgtagtcata atttttatat ggtaaacgat 5040
cagatgctaa tacaggagct tctgccaaaa ttgaaagagc cttcctacgt accgcaaccg 5100
ctctcgtagt atcacctaat tttttctcca aagcgtacaa aggtaactta ccgtgaataa 5160
ccaaggcagc gacctctttg ttcttcaatt gttttgtatt tccactactt aataatgctt 5220
ctaattcttc taaaggacgt attttcttat ccaagctttc aatatcgcgg gaatcatctt 5280
cctcactaga tgatgaaggt cctgatgagc tcgattgcgc agatgataaa cttttgactt 5340
tcgatccaga aatgactgtt ttattggtta aaaccattta tattgaattt tcaaaaattc 5400
ttactttttt tttggatgga cgcaaagaag tttaataatc atattacatg gcaataccac 5460
catatacata tccatatcta atcttactta tatgttgtgg aaatgtaaag agccccatta 5520
tcttagccta aaaaaacctt ctctttggaa ctttcagtaa tacgcttaac tgctcattgc 5580
tatattgaag tacggattag aagccgccga gcgggcgaca gccctccgac ggaagactct 5640
cctccgtgcg tcctggtctt caccggtcgc gttcctgaaa cgcagatgtg cctcgcgccg 5700
cactgctccg aacaataaag attctacaat actagctttt atggttatga agaggaaaaa 5760
ttggcagtaa cctggcccca caaaccttca aatcaacgaa tcaaattaac aaccatagga 5820
taataatgcg attagttttt tagccttatt tctggggtaa ttaatcagcg aagcgatgat 5880
ttttgatcta ttaacagata tataaatgca aaagctgcat aaccacttta actaatactt 5940
tcaacatttt cggtttgtat tacttcttat tcaaatgtca taaaagtatc aacaaaaaat 6000
tgttaatata cctctatact ttaacgtcaa ggagaaaaaa ctataatgcc tcacaagcac 6060
gttcctctta gaccagtcaa gttgacattt gatcctgtag gatcaaacac cctaggtgtg 6120
ccaaccttgg actttgagtc tctgttccgg gaagacagcg tctctgagga tgcccctctt 6180
gttatctacc cagaggatat gggtgtccca tggaacacct ctcttccttg gaccagacaa 6240
tccaagttct gggcttacgc cgaggcagct ggatatgaaa tggccaacgg aatcagcctt 6300
gacaaggcat cagagcgtgg cacactaccc atggagttga tggatgagcg tcgcaagtgg 6360
aagattgatg agctagttga ggatgccatc tcttgctgtg cttatcttta ccctacatca 6420
tctcctacca gattggcgtt gttgacccag tctgttctgc ttctattcct ccacgacgat 6480
gttattgagc gaggagctac tcaaaacgaa accacagtgg tagacgaatt tcttagcatg 6540
gctcccaaga acaggcatct taagaaattc tggtcagacg tattggaatg tgatcccgtc 6600
cttggacctg atctgcttta tgctatccat gctttcgtcc gtgatggtcg tgtaaagtca 6660
ccctttaagc aggatcacta tgccacattg gctgattaca tgctttaccg tcgcaatgat 6720
gttggcaaga catttatgat tgcagctatc cgcttcggct ctggcgtgca acaaacacgc 6780
gaagaacttg ctccctttga cgagcttgct gatctttacg tcagacactc aattcttatc 6840
aacgatctct actcgtatga taaggaggtg cacgaggtca agactatcga cgcgtccatc 6900
gtgaacgcag ttgctgtcac agagcagctc ctttccgtgt cgcctgacct ggccaagaac 6960
ttaaccagag ctattacctt tgacatggag aaggagtttt acggcatttg tgagaagttt 7020
atgcacagcc ctgatatcaa cgatcgccag cgcgtgttcg ttactgcgct ctttgatgcg 7080
ttgacaggca atatcttcca ttctgctact ttgagcagat acgttcgtca cggcgagaga 7140
ccacttcctt gcaagtgtta gattgaattg aattgaaatc gatagatcaa tttttttctt 7200
ttctctttcc ccatccttta cgctaaaata atagtttatt ttattttttg aatatttttt 7260
atttatatac gtatatatag actattattt atcttttaat gattattaag atttttatta 7320
aaaaaaatta cgctcctctt ttaatgcctt tatgcagttt tttttcccat tcgatatttc 7380
tatgttcggg ttcagcgtat tttaagttta ataactcgaa aattctgcgt tcgttgtaaa 7440
tgcatgtata ctaaactcac aaattagagc ttcaatttaa ttatatcagt tattacccgg 7500
gaatctcggt cgtaatgatt tctataatga cgaaaaaaaa aaaattggaa agaaaaagct 7560
tcatggcctt tataaaaagg aactatccaa tacctcgcca gaaccaagta acagtatttt 7620
acggggcaca aatcaagaac aataagacag gactgtaaag atggacgcat tgaactccaa 7680
agaacaacaa gagttccaaa aagtagtgga acaaaagcaa atgaaggatt tcatgcgttt 7740
gtactctaat ctggtagaaa gatgtttcac agactgtgtc aatgacttca caacatcaaa 7800
gctaaccaat aaggaacaaa catgcatcat gaagtgctca gaaaagttct tgaagcatag 7860
cgaacgtgta gggcagcgtt tccaagaaca aaacgctgcc ttgggacaag gcttgggccg 7920
ataaggtgta ctggcgtata tatatctaat tatgtatctc tggtgtagcc catttttagc 7980
atgtaaatat aaagagaaac catatctaat ctaaccaaat ccaaacaaaa ttcaatagtt 8040
actatcgctt ttttctttct gtatcgcaaa taagtgaaaa ttaaaaaaga aagattaaat 8100
tggaagttgg atatgggctg gaacagcagc agtaatcggt atcgggttcg ccactaatga 8160
cgtcctacga ttgcactcaa cagaccttga cgctcacgcc gtagcgggcg acaagtcaaa 8220
cggaacaacc gttgccgttc ccatcggagt ccgacctagg ccgaactccg tgaatttctg 8280
ataacaacgg tcggtaaaga ctggttcccc agtatatttc ttctctcagg agcaggggcc 8340
aatgccaaaa gcgacattaa cccggaggac aaggctccac tgtgttccac cgaatttccc 8400
acctgataat atctgataac ccgcccatag gtggggatcc ttctgtaaac aggtttctta 8460
atcgtaggaa ttaccactgt tccactgcca atcgcagctc ccagagtttc gttcccagcc 8520
gcgagcacca cagcgtacca tgtgcgccac gaggcctcaa acgtaaaaca atcgaaacga 8580
aaagaaacag actatagggg agtatagaga cagccggcca ataagaagag gaaaaagaaa 8640
tactagcgtt tatcaatgtg ggtcgttaac atatcctgtt gacaatgatt acaggttaaa 8700
aggtagcgta agtgaatatt aactatggat attcttatac ttagataaga gacatataaa 8760
acacacgatg attgattgat ttttatgacc aatatatgta attcgtaatt cagatagttt 8820
ttatactttt aatgtgtgcc gaaccatatt actagtatat gtaactacca tgttatgttc 8880
aattggcaga tctttaactc ggctttagtt atccaagtta cttgcaatat ttccttctgc 8940
gagagtacat ttgcccttaa acg 8963
<210> 7
<211> 6435
<212> DNA
<213> Artificial
<220>
<223> 质粒pZY518线性化后片段
<400> 7
aaacgtccag ccacccattt cttgtgattt agtaaaaaac tctaacggtt tatcaacgta 60
aaatatgggc agaagttcga gggccccact gcttgtcttg gacaccacag gcgtcaaagg 120
agagcagttt cttctcgaca tcacaatgaa gtcaaccccc aggaagtaag cgcttctaat 180
aatggcaccg atattgtgag ggtcagttat ttcatccaga tataacccga gaggaaactt 240
cttagcgtct gttttcgtac cataaggcag ttcatgaggt atattttcgt tattgaagcc 300
cagctcgtga atgcttaatg ctgctgaact ggtgtccatg tcgcctaggt acgcaatctc 360
cacaggctgc aaaggttttg tctcaagagc aatgttattg tgcaccccgt aattggtcaa 420
caagtttaat ctgtgcttgt ccaccagctc tgtcgtaacc ttcagttcat cgactatctg 480
aagaaattta ctaggaatag tgccatggta cagcaaccga gaatggcaat ttctactcgg 540
gttcagcaac gctgcataaa cgctgttggt gccgtagaca tattcgaaga taggattatc 600
attcataagt ttcagagcaa tgtccttatt ctggaacttg gatttatggc tcttttggtt 660
taatttcgcc tgattcttga tctcctttag cttctcgacg tgggcctttt tcttgccata 720
tggatccgct gcacggtcct gttccctagc atgtacgtga gcgtatttcc ttttaaacca 780
cgacgctttg tcttcattca acgtttccca ttgttttttt ctactattgc tttgctgtgg 840
gaaaaactta tcgaaagatg acgacttttt cttaattctc gttttaagag cttggtgagc 900
gctaggagtc actgccaggt atcgtttgaa cacggcatta gtcagggaag tcataacaca 960
gtcctttccc gcaattttct ttttctatta ctcttggcct cctctagtac actctatatt 1020
tttttatgcc tcggtaatga ttttcatttt ttttttccac ctagcggatg actctttttt 1080
tttcttagcg attggcatta tcacataatg aattatacat tatataaagt aatgtgattt 1140
cttcgaagaa tatactaaat cacagcttgt ctgtaagcgg atgccgggag cagacaagcc 1200
cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca 1260
gagcagattg tactgagagt gcaccataaa cgacattact atatatataa tataggaagc 1320
atttaataga cagcatcgta atatatgtgt actttgcagt tatgacgcca gatggcagta 1380
gtggaagata ttctttattg aaaaatagct tgtcacctta cgtacaatct tgatccggag 1440
cttttctttt tttgccgatt aagaattaat tcggtcgaaa aaagaaaagg agagggccaa 1500
gagggagggc attggtgact attgagcacg tgagtatacg tgattaagca cacaaaggca 1560
gcttggagta tgtctgttat taatttcaca ggtagttctg gtccattggt gaaagtttgc 1620
ggcttgcaga gcacagaggc cgcagaatgt gctctagatt ccgatgctga cttgctgggt 1680
attatatgtg tgcccaatag aaagagaaca attgacccgg ttattgcaag gaaaatttca 1740
agtcttgtaa aagcatataa aaatagttca ggcactccga aatacttggt tggcgtgttt 1800
cgtaatcaac ctaaggagga tgttttggct ctggtcaatg attacggcat tgatatcgtc 1860
caactgcatg gagatgagtc gtggcaagaa taccaagagt tcctcggttt gccagttatt 1920
aaaagactcg tatttccaaa agactgcaac atactactca gtgcagcttc acagaaacct 1980
cattcgttta ttcccttgtt tgattcagaa gcaggtggga caggtgaact tttggattgg 2040
aactcgattt ctgactgggt tggaaggcaa gagagccccg aaagcttaca ttttatgtta 2100
gctggtggac tgacgccaga aaatgttggt gatgcgctta gattaaatgg cgttattggt 2160
gttgatgtaa gcggaggtgt ggagacaaat ggtgtaaaag actctaacaa aatagcaaat 2220
ttcgtcaaaa atgctaagaa ataggttatt actgagtagt atttatttaa gtattgtttg 2280
tgcacttgcc tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca 2340
ggaaattgta aacgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 2400
attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 2460
gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 2520
caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 2580
ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 2640
cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa 2700
agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 2760
cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcg cgccattcgc cattcaggct 2820
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 2880
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 2940
ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac 3000
ccggtagagg tgtggtcaat aagagcgacc tcatgctata cctgagaaag caacctgacc 3060
tacaggaaag agttactcaa gaataagaat tttcgtttta aaacctaaga gtcactttaa 3120
aatttgtata cacttatttt ttttataact tatttaataa taaaaatcat aaatcataag 3180
aaattcgcct aacacttgca aggaagtggt ctctcgccgt gacgaacgta tctgctcaaa 3240
gtagcagaat ggaagatatt gcctgtcaac gcatcaaaga gcgcagtaac gaacacgcgc 3300
tggcgatcgt tgatatcagg gctgtgcata aacttctcac aaatgccgta aaactccttc 3360
tccatgtcaa aggtaatagc tctggttaag ttcttggcca ggtcaggcga cacggaaagg 3420
agctgctctg tgacagcaac tgcgttcacg atggacgcgt cgatagtctt gacctcgtgc 3480
acctccttat catacgagta gagatcgttg ataagaattg agtgtctgac gtaaagatca 3540
gcaagctcgt caaagggagc aagttcttcg cgtgtttgtt gcacgccaga gccgaagcgg 3600
atagctgcaa tcataaatgt cttgccaaca tcattgcgac ggtaaagcat gtaatcagcc 3660
aatgtggcat agtgatcctg cttaaagggt gactttacac gaccatcacg gacgaaagca 3720
tggatagcat aaagcagatc aggtccaagg acgggatcac attccaatac gtctgaccag 3780
aatttcttaa gatgcctgtt cttgggagcc atgctaagaa attcgtctac cactgtggtt 3840
tcgttttgag tagctcctcg ctcaataaca tcgtcgtgga ggaatagaag cagaacagac 3900
tgggtcaaca acgccaatct ggtaggagat gatgtagggt aaagataagc acagcaagag 3960
atggcatcct caactagctc atcaatcttc cacttgcgac gctcatccat caactccatg 4020
ggtagtgtgc cacgctctga tgccttgtca aggctgattc cgttggccat ttcatatcca 4080
gctgcctcgg cgtaagccca gaacttggat tgtctggtcc aaggaagaga ggtgttccat 4140
gggacaccca tatcctctgg gtagataaca agaggggcat cctcagagac gctgtcttcc 4200
cggaacagag actcaaagtc caaggttggc acacctaggg tgtttgatcc tacaggatca 4260
aatgtcaact tgactggtct aagaggaacg tgcttgtgag gcatttatat tgaattttca 4320
aaaattctta cttttttttt ggatggacgc aaagaagttt aataatcata ttacatggca 4380
ataccaccat atacatatcc atatctaatc ttacttatat gttgtggaaa tgtaaagagc 4440
cccattatct tagcctaaaa aaaccttctc tttggaactt tcagtaatac gcttaactgc 4500
tcattgctat attgaagtac ggattagaag ccgccgagcg ggcgacagcc ctccgacgga 4560
agactctcct ccgtgcgtcc tggtcttcac cggtcgcgtt cctgaaacgc agatgtgcct 4620
cgcgccgcac tgctccgaac aataaagatt ctacaatact agcttttatg gttatgaaga 4680
ggaaaaattg gcagtaacct ggccccacaa accttcaaat caacgaatca aattaacaac 4740
cataggataa taatgcgatt agttttttag ccttatttct ggggtaatta atcagcgaag 4800
cgatgatttt tgatctatta acagatatat aaatgcaaaa gctgcataac cactttaact 4860
aatactttca acattttcgg tttgtattac ttcttattca aatgtcataa aagtatcaac 4920
aaaaaattgt taatatacct ctatacttta acgtcaagga gaaaaaacta tagcgcaatg 4980
attgaatagt caaagatttt ttttttttaa tttttttttt ttcatagaac tttttattta 5040
aataaatcac gtctatatat gtatcagtat aacgtaaaaa aaaaaacacc gtcagttaaa 5100
caaaacataa ataaaaaaaa aaagaagtgt caaatcaagt gtcaaataag agatggagga 5160
cgggaaaaag ttagttgtgg tgataggtgg caagtggtat tccgtaagaa caacaagaaa 5220
agcatttcat attatggctg aactgagcga acaagtgcaa aatttaagca tcaacgacaa 5280
caacgagaat ggttatgttc ctcctcactt aagaggaaaa ccaagaagtg ccagaaataa 5340
cagtagcaac tacaataaca acaacggcgg ctacaacggt ggccgtggcg gtggcagctt 5400
ctttagcaac aaccgtcgtg gtggttacgg caacggtggt ttcttcggtg gaaacaacgg 5460
tggcagcaga tctaacggcc gttctggtgg tagatggatc gatggcaaac atgtcccagc 5520
tccaagaaac gaaaaggccg agatcgccat atttggtgtc cccgaggatc caaatttcca 5580
atcttctggt attaacttcg ataactacga tgatattcca gtggacgcct ctggtaagga 5640
tgttcctgaa ccaatcacag aatttacctc acctccattg gacggattgt tattggaaaa 5700
catcaaattg gcccgtttca ccaagccaac acctgtgcaa aaatactccg tccctatcgt 5760
tgccaacggc agagatttga tggcctgtgc gcagaccggt tctggtaaga ctggtgggtt 5820
tttattccca gtgttgtccg aatcatttaa gactggacca tctcctcaac cagagtctca 5880
aggctccttt taccaaagaa aggcctaccc aactgctgtc attatggctc caactagaga 5940
gttggccacc caaattttcg atgaagccaa gaaatttact tatagatcct gggtcaaggc 6000
ctgcgtcgtc tacggtggtt ctccaattgg taaccaacta agagaaattg aacgtggttg 6060
cgatctttta gtcgctactc caggtcgttt gaatgacttg ttggaacgtg gtaaaatttc 6120
tttggccaac gtcaagtatt tggttctaga tgaagctgat agaatgttgg atatgggttt 6180
cgaacctcaa attagacata ttgtcgaaga ctgtgatatg actcctgttg gtgaaagaca 6240
aactctgatg ttctcagcta cttttcccgc tgatatccaa catttggccc gtgatttctt 6300
aagtgactac atctttttgt ctgttggtag agtcggttct acttcagaaa acattactca 6360
aaaagtctta tacgttgaaa atcaagataa gaagtcagcc ttattggatc tattgtctgc 6420
atccactgac ggttt 6435
<210> 8
<211> 4951
<212> DNA
<213> Artificial
<220>
<223> pZY521敲除盒片段
<400> 8
caatggtcta ggtagtggca ttcgaagctt cgaaagcgtg gcctcgcctc gagcgtgaag 60
tcatattttc tcactctgca ctgccgtgct gtgccatgta gcccactgta tgcctctagc 120
accccaatcg ggaaaattgt aaacattagc aggtgtcgcc tcgaggcccc cgatacattt 180
ttcaacatct cctcgatact taaacagcaa acaaacaagc aacacccacg caagaagatt 240
tattaatacc tcttgactcc cgtagatatt gaagagacta agtatagtaa cgcatatccg 300
cacaatacta tggttaaggt ctggaatata gtactacgtc tggtagtgtt gctgttttta 360
gcaggtaata ccttgctgtt aattttgatg attatatctg gtgccacgga ccattacccc 420
gtgaacagat tctattgggt gcagggcaat acaacaggca tacctaatgc tggggatgaa 480
acacgttgga cgttttgggg cgcctgtcta caggataaag acgggtcgga tacctgcaca 540
agcaatttgg cacctgcata ccccatttcc ccagtagata acttcaacac acacatcaat 600
gtccctcacc agtttatttc caaaagagac gctttttact acctgactag attttcattt 660
tgtttctttt ggattgcgct tgcctttgta ggtgtgtcgt ttatccttta cgttttgact 720
tggtgctcga agatgctttc agagatggtg cttatcctca tgtcttttgg gtttgtcttc 780
aatacggcag ccgttgtctt gcaaacggcc gcctctgcca tggcaaagaa tgctttccat 840
gacgatcatc gtagtgccca attgggtgcc tctatgatgg gtatggcttg ggcaagtgtc 900
tttttatgta tcgtggaatt tatcctgctg gtcttctggt ctgttagggc aaggttggcc 960
tctacttact ccatcgacaa ttcaagatac agaacctcct ccagatggaa tcccttccat 1020
agagagaagg agcaagcaac tgacccaata ttgactgcca ctggacctga agacatgcaa 1080
caaagtgcaa gcatagtggg gccttcttcc aatgctaatc cggtcactgc cactgctgct 1140
acggaaaacc aacctaaagg tattaacttc ttcactataa gaaaatcaca cgagcgcccg 1200
gacgatgtct ctgtttaaat ggcgcaagtt ttccgctttg taatatatat ttatacccct 1260
ttcttctctc ccctgcaata taatagttta attctaatat taataatatc ctatattttc 1320
ttcatttacc ggcgcactct cgcccgaacg acctcaaaat gtctgctaca ttcataataa 1380
ccaaaagctc ataacttttt ttttgaacct gaatatatat acatcacatg tcactgctgg 1440
tccttgccga ccagcgtata caatctcgat agttggtttc ccgttctttc cactcccgtc 1500
gtacccaatt cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa 1560
cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct 1620
ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc 1680
agcctgaatg gcgaatggcg cgacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg 1740
gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc tcctttcgct 1800
ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg 1860
ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgattag 1920
ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg 1980
gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caaccctatc 2040
tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg gttaaaaaat 2100
gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttcc 2160
tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata gggtaataac 2220
tgatataatt aaattgaagc tctaatttgt gagtttagta tacatgcatt tacttataat 2280
acagtttttt agttttgctg gccgcatctt ctcaaatatg cttcccagcc tgcttttctg 2340
taacgttcac cctctacctt agcatccctt ccctttgcaa atagtcctct tccaacaata 2400
ataatgtcag atcctgtaga gaccacatca tccacggttc tatactgttg acccaatgcg 2460
tctcccttgt catctaaacc cacaccgggt gtcataatca accaatcgta accttcatct 2520
cttccaccca tgtctctttg agcaataaag ccgataacaa aatctttgtc gctcttcgca 2580
atgtcaacag tacccttagt atattctcca gtagataggg agcccttgca tgacaattct 2640
gctaacatca aaaggcctct aggttccttt gttacttctt ctgccgcctg cttcaaaccg 2700
ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa tgtctgccca ttctgctatt 2760
ctgtatacac ccgcagagta ctgcaatttg actgtattac caatgtcagc aaattttctg 2820
tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct ttagcggctt aactgtgccc 2880
tccatggaaa aatcagtcaa gatatccaca tgtgttttta gtaaacaaat tttgggacct 2940
aatgcttcaa ctaactccag taattccttg gtggtacgaa catccaatga agcacacaag 3000
tttgtttgct tttcgtgcat gatattaaat agcttggcag caacaggact aggatgagta 3060
gcagcacgtt ccttatatgt agctttcgac atgatttatc ttcgtttcct gcaggttttt 3120
gttctgtgca gttgggttaa gaatactggg caatttcatg tttcttcaac actacatatg 3180
cgtatatata ccaatctaag tctgtgctcc ttccttcgtt cttccttctg ttcggagatt 3240
accgaatcaa aaaaatttca aagaaaccga aatcaaaaaa aagaataaaa aaaaaatgat 3300
gaattgaatt gaaaagctgt ggtatggtgc actctcagta caatctgctc tgatgccgca 3360
tagttaagcc agccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg 3420
ctcccggcat ccgcttacag acaagctgtg aaagcatctt gccctgtgct tggcccccag 3480
tgcagcgaac gttataaaaa cgaatactga gtatatatct atgtaaaaca accatatcat 3540
ttcttgttct gaactttgtt tacctaacta gttttaaatt tccctttttc gtgcatgcgg 3600
gtgttcttat ttattagcat actacatttg aaatatcaaa tttccttagt agaaaagtga 3660
gagaaggtgc actgacacaa aaaataaaat gctacgtata actgtcaaaa ctttgcagca 3720
gcgggcatcc ttccatcata gcttcaaaca tattagcgtt cctgatcttc atacccgtgc 3780
tcaaaatgat caaacaaact gttattgcca agaaataaac gcaaggctgc cttcaaaaac 3840
tgatccatta gatcctcata tcaagcttcc tcatagaacg cccaattaca ataagcatgt 3900
tttgctgtta tcaccgggtg ataggtttgc tcaaccatgg aaggtagcat ggaatcataa 3960
tttggatact aatacaaatc ggccatataa tgccattagt aaattgcgct cccatttagg 4020
tggttctcca ggaatactaa taaatgcggt gcatttgcaa aatgaattta ttccaaggcc 4080
aaaacaacac gatgaatggc tttatttttt tgttattcct gacatgaagc tttatgtaat 4140
taaggaaacg gacatcgagg aatttgcatc ttttttagat gaaggagcta ttcaagcacc 4200
aaagctatcc ttccaggatt atttaagcgg taaggccaag gcttcccaac aggttcatga 4260
agtgcatcat agaaagctta caaggtttca gggtgaaact tttctaagag attggaactt 4320
agtctgtggg cattataaga gagatgctaa gtgtggagaa atgggacccg acataattgc 4380
agcatttcaa gatgaaaagc tttttcctga gaataatcta gccttaattt ctcatattgg 4440
gggtcatatt tttgctggta atgtcatttt ctataaacta ttcggcagag aaaagatgca 4500
aaacaagctg gattcattat ggtttggtaa agtttaccca cacaacttga agctgttatg 4560
tgaaaacttg gaaaacggta aaatcatcga cgaaatgtac agaggtggca tatcgatgaa 4620
ttaaaacggt ttccttttaa aacagaaata aagatgaggt gaaattttat atttcttata 4680
tttaaagcta tcatatattc cttgtacata tatttattaa gaaaatacat taacacgcct 4740
ttatacttgg tattattact gtaaaatcat tacatgtaat ttgttctgac ttcgttgatt 4800
gtttgaatta tatcctccac ttgtacactc tttaaatttt tggccaaaaa ttcctgctca 4860
ttcaaggaaa gtctcaggaa attttcacat aaatcaccat caataacttt acgtacaggg 4920
gcataatagc ttctgtattc ttggtggtct c 4951

Claims (7)

1.一种微生物，其特征在于，所述微生物过表达IDI1、J1-018-A、ERG10、ERG13、tHMG1、 ERG12、ERG8、MVD1以及ERG20基因；
所述ERG10、ERG13、tHMG1、ERG12、ERG8、MVD1、IDI1、ERG20以及J1-018-A基因的表达量的比为2：2：5：2：2：2：2：2：5；
所述微生物为酵母、大肠杆菌或链霉菌；
所述J1-018-A基因序列如SEQ ID NO：1所示。
2.一种制备法尼烯的方法，其特征在于，包括：在合适微生物发酵的条件下，将权利要求1所述的微生物进行发酵处理，以便获得法尼烯。
3.根据权利要求2所述的方法，其特征在于，进一步包括向发酵体系中加入乙酰-CoA。
4.一种制备权利要求1所述的微生物的方法，其特征在于，包括：
将A质粒导入菌株A，所述A质粒携带ERG8、tHMG1以及ERG12基因，以便获得菌株B，其中，所述菌株A为酵母菌菌株，
将B质粒导入所述菌株B，所述B质粒携带IDI1、ERG10以及MVD1基因，以便获得菌株C，
将C质粒导入所述菌株C，所述C质粒携带Cre酶的基因，以便获得菌株D，
将D质粒导入所述菌株D，所述D质粒携带ERG13以及tHMG1基因，以便获得菌株E，
将E质粒导入所述菌株E，所述E质粒携带J1-018-A以及ERG20基因，以便获得所述微生物，所述微生物为菌株F。
5.根据权利要求4所述的方法，其特征在于，进一步包括：将F质粒以及G质粒导入所述菌株F，所述F质粒携带J1-018-A以及tHMG1基因，所述G质粒携带J1-018-A基因，以便获得所述微生物。
6.一种制备权利要求1所述的微生物的方法，其特征在于，包括：
将H质粒、I质粒、J质粒导入大肠杆菌中，以便获得所述微生物，
其中，所述H质粒携带IDI1、ERG10、MVD1、ERG8以及ERG12基因，所述IDI1、ERG10、MVD1、 ERG8以及ERG12基因在所述H质粒的拷贝数为1，
所述I质粒携带ERG13以及tHMG1基因，所述ERG13基因在所述I质粒的拷贝数为1，所述tHMG1基因在所述I质粒的拷贝数为3，
所述J质粒携带ERG20以及J1-018-A基因，所述ERG20基因在所述J质粒的拷贝数为1，所述J1-018-A基因在所述J质粒的拷贝数为3。
7.一种制备权利要求1所述的微生物的方法，其特征在于，包括：
将K质粒导入菌株G中，以便获得菌株H，其中，菌株G为链霉菌菌株；
将L质粒导入菌株H中，以便获得所述微生物；
其中，所述K质粒携带IDI1、ERG10、MVD1、ERG8、ERG12、ERG20以及ERG13基因，所述IDI1、 ERG10、MVD1、ERG8、ERG12、ERG20以及ERG13基因在所述K质粒的拷贝数为1，
所述L质粒携带tHMG1以及J1-018-A基因，所述tHMG1基因在所述L质粒的拷贝数为2，所述J1-018-A基因在所述L质粒的拷贝数为3。
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