CN113614229A - 遗传修饰的梭菌属细菌、其制备和用途 - Google Patents

遗传修饰的梭菌属细菌、其制备和用途 Download PDF

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CN113614229A
CN113614229A CN201980088931.2A CN201980088931A CN113614229A CN 113614229 A CN113614229 A CN 113614229A CN 201980088931 A CN201980088931 A CN 201980088931A CN 113614229 A CN113614229 A CN 113614229A
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雷米·霍克
格拉迪斯·沙尔捷
弗朗索瓦·瓦塞尔斯
尼古拉斯·洛普斯费雷拉
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Abstract

本发明涉及梭菌属(Clostridium)细菌、通常为梭菌属的产溶剂细菌、特别是在野生状态下具有编码酰胺醇–O–乙酰转移酶的基因的细菌的遗传修饰。因此，本发明涉及允许此类遗传修饰、特别是编码或控制酰胺醇–O–乙酰转移酶转录的序列的消除或修饰的方法、工具和试剂盒，涉及所获得的遗传修饰的细菌，并涉及其用途，特别是优选地在工业规模上用于生产溶剂的用途。

Description

遗传修饰的梭菌属细菌、其制备和用途
本发明涉及梭菌属(Clostridium)细菌、通常为产溶剂梭菌属细菌、特别是在野生型中具有编码酰胺醇–O–乙酰转移酶的基因的细菌的遗传修饰。因此，本发明涉及允许此类遗传修饰、特别是编码酰胺醇–O–乙酰转移酶的序列或控制编码酰胺醇–O–乙酰转移酶的序列转录的序列的移除或修饰的方法、工具和试剂盒，涉及所获得的遗传修饰的细菌，并涉及其用途，特别是优选地在工业规模上用于生产溶剂的用途。
背景技术
梭菌属含有属于厚壁菌门的革兰氏阳性、严格厌氧、产孢子的细菌。出于几个原因，梭菌对于科学界来说是一个重要组群。首先是许多严重疾病(例如破伤风、肉毒梭菌中毒)由这个家族的致病成员的感染造成(Gonzales等，2014)。其次是在生物技术中使用所谓的产酸或产溶剂菌株的可能性(John&Wood，1986和Moon等，2016)。这些非致病性梭菌天然具有在被称为ABE的发酵过程中转化各种不同糖类以产生感兴趣的化学物质，尤其是丙酮、丁醇和乙醇的能力(John&Wood，1986)。同样地，在某些特定菌种中，将丙酮以不同比例还原成异丙醇的IBE发酵是可能的(Chen等，1986，George等，1983)，这是由于在这些菌株的基因组中存在编码仲醇脱氢酶的基因(s-ADH；Ismael等，1993；Hiu等，1987)。
所述产溶剂梭菌菌种显示出显著的表型相似性，使得在现代测序技术出现之前难以将它们分类(Rogers等，2006)。由于可以对这些细菌的完整基因组进行测序，现在可以将该细菌属分为4个主要种：丙酮丁醇梭菌(C.acetobutylicum)，糖乙酸多丁醇梭菌(C.saccharoperbutylacetonicum)，糖丁醇梭菌(C.saccharobutylicum)和拜氏梭菌(C.beijerinckii)。一份最近的出版物在对30个菌株的完整基因组进行比较分析后，将这些产溶剂梭菌分成4个主要进化枝(图1)。
具体来说，这些研究组将菌种丙酮丁醇梭菌和拜氏梭菌分开，并将丙酮丁醇梭菌ATCC 824(也被命名为DSM 792或LMG 5710)和拜氏梭菌NCIMB 8052作为用于研究ABE型发酵的模式菌株。
天然能够进行IBE发酵的梭菌菌株很少，并且大部分属于拜氏梭菌种(Zhang等，2018，表1)。这些菌株通常选自丁醇梭菌(C.butylicum)LMD 27.6、金黄丁酸梭菌(C.aurantibutylicum)NCIB 10659、拜氏梭菌LMD 27.6、拜氏梭菌VPI2968、拜氏梭菌NRRLB–593、拜氏梭菌ATCC 6014、拜氏梭菌McClung 3081、异丙醇梭菌(C.isopropylicum)IAM19239、拜氏梭菌DSM 6423、梭菌属菌种A1424、拜氏梭菌optinoii和拜氏梭菌BGS1。
然而，到目前为止尚无已被遗传修饰过的能够天然产生异丙醇、特别是能够天然进行IBE发酵的梭菌属细菌菌株，特别是被遗传修饰以使其对属于酰胺醇类型的抗生素例如氯霉素或甲砜霉素敏感、优选地能够优化异丙醇生产的菌株。
发明内容
本发明人在本发明的背景中首次描述了一种遗传修饰的拜氏梭菌细菌以及允许对梭菌属细菌进行遗传修饰的工具，所述细菌通常是天然(即在野生型中)能够产生异丙醇、特别是天然能够进行IBE发酵的产溶剂梭菌属细菌，特别是在野生型中包含为所述细菌提供对一种或多种抗生素的抗性的基因、特别是编码酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶或甲砜霉素–O–乙酰转移酶的基因的细菌。
根据本发明的优选的遗传修饰的细菌是不表达提供对一种或多种抗生素的抗性的酶的细菌，特别是不表达酰胺醇–O–乙酰转移酶的细菌，例如缺少或不能表达catB基因的细菌。
根据本发明的优选的遗传修饰的细菌是在本说明书中被鉴定为拜氏梭菌DSM6423ΔcatB的细菌，其于2018年12月6日在比利时微生物协调保藏中心(Belgian Co–ordinated Collections of Micro–organisms)(“BCCM”，K.L.Ledeganckstraat 35,B–9000Gent–Belgium)登记在保藏号LMG P–31151下(也被标识为拜氏梭菌IFP962ΔcatB)。本说明书还涉及其任何衍生细菌、克隆、突变体或遗传修饰形式。
本发明人描述的一个特定主题内容是一种核酸，其识别(至少部分结合)并优选地靶向、即识别并允许切割感兴趣的细菌的基因组中下述序列的至少一条链：i)编码允许所述感兴趣的细菌在含有抗生素、通常为属于酰胺醇类别的抗生素、优选地选自氯霉素、甲砜霉素、叠氮氯霉素和氟苯尼考的培养基中生长的酶，通常为酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶或甲砜霉素–O–乙酰转移酶的序列；ii)控制编码所述酶的序列的转录的序列；或iii)在编码所述酶的序列两侧的序列。
本发明人还描述了这种核酸的用途，其用于转化和/或遗传修饰梭菌属细菌，优选地天然能够产生异丙醇的梭菌属细菌，特别是能够进行IBE发酵的梭菌属细菌。
具体来说，本发明人描述了识别拜氏梭菌DSM 6423的基因组中序列SEQ ID NO：18的catB基因或与其具有至少70％同一性的序列的核酸的用途，其用于转化和/或遗传修饰拜氏梭菌DSM 6423细菌。
所述能够在野生型中产生异丙醇的细菌可以是例如选自拜氏梭菌细菌、二醇梭菌(C.diolis)细菌、微紫色梭菌(C.puniceum)细菌、丁酸梭菌(C.butyricum)细菌、糖乙酸多丁醇梭菌细菌、肉毒梭菌(C.botulinum)细菌、德雷克氏梭菌(C.drakei)细菌、粪味梭菌(C.scatologenes)细菌、产气荚膜梭菌(C.perfringens)细菌和突尼斯梭菌(C.tunisiense)细菌的细菌，优选为选自拜氏梭菌细菌、二醇梭菌细菌、微紫色梭菌细菌和糖乙酸多丁醇梭菌细菌的细菌。天然能够产生异丙醇的特别优选的细菌是拜氏梭菌细菌。
根据一个特定方面，所述识别并优选地靶向i)编码酰胺醇–O–乙酰转移酶的序列、ii)控制这个序列的转录的序列或iii)在这个序列两侧的序列的核酸，被用于转化选自DSM6423、LMG 7814、LMG 7815、NRRL B–593、NCCB 27006的拜氏梭菌的进化分枝和与菌株DSM6423具有至少97％同一性的进化分枝。
本发明人还描述了一种转化并优选地遗传修饰梭菌属细菌的方法。这种方法包括通过在所述细菌中引入核酸来转化这种细菌的步骤，所述核酸识别并优选地靶向i)编码感兴趣的酶、优选为酰胺醇–O–乙酰转移酶的序列、ii)控制编码所述酶的序列的转录的序列或iii)在编码所述酶的序列两侧的序列。这种方法通常使用遗传修饰工具来进行，例如使用选自CRISPR工具、基于II类内含子的工具和等位基因交换工具的遗传修饰工具。还描述了使用这种方法转化和遗传修饰的细菌，其实例是拜氏梭菌DSM 6423ΔcatB细菌。
本发明人描述的另一方面涉及根据本发明所述的遗传修饰的细菌、优选为登记在保藏号LMG P–31151下的拜氏梭菌DSM 6423ΔcatB细菌或其遗传修饰形式的用途，其用于优选地在工业规模上生产溶剂、优选为异丙醇，或溶剂的混合物。
最后，本说明书涉及试剂盒，特别是包含下述组分的试剂盒：本文中描述的核酸和遗传修饰工具，所述遗传修饰工具特别选自CRISPR工具、基于II类内含子的工具和等位基因交换工具的遗传工具的元件、作为向导RNA(gRNA)的核酸、作为修复模板的核酸、至少一个引物对和允许由所述工具编码的蛋白质的表达的诱导物。
具体实施方式
尽管在工业上已使用超过一个世纪，但对梭菌属细菌的了解仍受限于对它们进行遗传修饰时遇到的困难。
近年来已设计了不同的遗传工具来优化这个属的菌株，最新的一代是基于成簇规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白(Cas)技术的使用。这种方法基于使用一种被称为核酸酶的酶(在CRISPR/Cas遗传工具的情况下通常是Cas-型核酸酶，例如来自于酿脓链球菌(Streptococcus pyogenes)的Cas9蛋白)，它在RNA分子指导下在DNA分子(感兴趣的靶序列)中进行双链切割。向导RNA(gRNA)的序列决定所述核酸酶的切割位点，赋予其极高的特异性(图17)。
由于必需DNA分子中的双链切割对生物体来说是致命的，因此生物体的存活将取决于它修复所述切割的能力(参见Cui&Bikard,2016)。在梭菌属细菌中，双链断裂的修复依赖于同源重组机制，需要被切割序列的完整拷贝。通过为细菌提供允许这种修复并同时修饰原始序列的DNA片段，有可能迫使所述微生物将所需的变化整合到其基因组中。通过修饰靶序列或PAM位点，已进行的修饰必须不再允许Cas9-gRNA核糖核蛋白复合体靶向基因组DNA(图18)。
已描述了不同方法以尝试制造在梭菌属细菌中有功能的这种遗传工具。事实上，已知这些微生物由于转化和同源重组频率低而难以进行遗传修饰。几种方法是基于使用在拜氏梭菌和扬氏梭菌(C.ljungdahlii)中组成性表达(Wang等，2015；Huang等，2016)或在拜氏梭菌、糖乙酸多丁醇梭菌和自养产乙醇梭菌(C.authoethanogenum)中在诱导型启动子控制之下表达(Wang等，2016；Nagaraju等，2016；Wang等，2017)的Cas9。其他作者描述了使用所述核酸酶的改良版本Cas9n，其在基因组内进行单链而不是双链切割(Xu等，2015；Li等，2016)。这种选择是由于观察到在测试的实验条件下，Cas9对于在梭菌属细菌中使用来说毒性过大。大多数上述工具依靠使用单一质粒。最后，当在微生物的基因组内鉴定到内源CRISPR/Cas系统时，也可以使用它们，正如在巴氏梭菌(C.pasteurianum)中那样(Pyne等，2016)。
除非使用(如上述最后一种情况)待修饰菌株的内源机制，否则基于CRISPR技术的工具的主要缺点是显著限制了可以插入到细菌基因组中的感兴趣的核酸的尺寸(以及因此编码序列或基因的数目)(根据Xu等，2015，最大约1.8kb)。
基于使用显著解决了这一问题的两种不同的核酸、通常为两种质粒(参见WO2017064439，Wasels等，2017和图3)，本发明人已开发并描述了一种用于修饰细菌的更加强有力的遗传工具，其适合于细菌、通常为属于厚壁菌门的细菌、特别是梭菌属细菌。在特定实施方式中，这种工具的第一核酸允许cas9的表达，并且特异性针对待进行的修饰的第二核酸含有一个或多个gRNA表达盒以及允许被Cas9靶向的细菌DNA的一部分被感兴趣的序列代替的修复模板。通过将cas9和/或gRNA表达盒置于诱导型启动子的控制之下，所述系统的毒性受到限制。本发明人最近改进了这种工具，使其能够非常显著地提高转化效率，并因此以有用的数目和数量(特别是在选择用于工业规模生产的鲁棒菌株的情况下)获得感兴趣的遗传修饰的细菌(参见FR 18/54835)。在这种改进的工具中，至少一个核酸包含置于诱导型启动子控制之下的编码抗CRISPR蛋白(“acr”)的序列。这种抗CRISPR蛋白抑制DNA核酸内切酶/向导RNA复合体的活性。所述蛋白的表达受到调控，以允许其仅在所述细菌的转化阶段中表达。
在本说明书的上下文中，属于厚壁菌门的细菌被理解为意味着属于梭菌纲、柔膜菌纲、杆菌纲或Togobacteria，优选地属于梭菌纲或杆菌纲的细菌。
属于厚壁菌门的具体细菌包括例如梭菌属细菌、芽孢杆菌属细菌或乳杆菌属细菌。
“芽孢杆菌属细菌”具体来说意味着解淀粉芽孢杆菌(B.amyloliquefaciens)、苏云金芽孢杆菌(B.thurigiensis)、凝结芽孢杆菌(B.coagulans)、蜡样芽孢杆菌(B.cereus)、炭疽芽孢杆菌(B.anthracis)或枯草芽孢杆菌(B.subtilis)。
“梭菌属细菌”具体来说意味着工业上感兴趣的梭菌属菌种，通常为产溶剂或产乙酸梭菌属细菌。表述“梭菌属细菌”涵盖了野生型细菌以及从其衍生的菌株，它们进行了目的在于改进其性能的遗传修饰(例如过表达ctfA、ctfB和adc基因)，但尚未暴露于CRISPR系统。
“工业上感兴趣的梭菌属菌种”意味着能够从糖或单糖，通常从包含5个碳原子的糖例如木糖、阿拉伯糖或果糖，从包含6个碳原子的糖例如葡萄糖或甘露糖，从多糖例如纤维素或半纤维素，和/或从可以被梭菌属细菌同化和使用的任何其他碳源(例如CO、CO2和甲醇)，通过发酵产生溶剂和酸例如丁酸或乙酸的菌种。感兴趣的产溶剂细菌的实例是产生丙酮、丁醇、乙醇和/或异丙醇的梭菌属细菌，例如在文献中被标识为“ABE菌株”[执行允许产生丙酮、丁醇和乙醇的发酵的菌株]和“IBE菌株”[执行允许产生异丙醇(通过丙酮的还原)、丁醇和乙醇的发酵的菌株]的菌株。产溶剂梭菌属细菌可以例如选自丙酮丁醇梭菌、解纤维梭菌(C.cellulolyticum)、植物发酵梭菌(C.phytofermentans)、拜氏梭菌、糖丁醇梭菌、糖乙酸多丁醇梭菌、产芽胞梭菌(C.sporogenes)、丁酸梭菌、金黄丁酸梭菌和酪丁酸梭菌(C.tyrobutyricum)，最优选地选自丙酮丁醇梭菌、拜氏梭菌、丁酸梭菌、酪丁酸梭菌和解纤维梭菌，甚至更优选地选自丙酮丁醇梭菌和拜氏梭菌。
天然产生异丙醇，通常在基因组中具有编码将丙酮还原成异丙醇的伯醇/仲醇脱氢酶的adh基因的梭菌属细菌，在遗传和功能两方面与能够在天然状态下进行ABE发酵的细菌区分开。
有利的是，在本发明的情形中，本发明人成功地对一种天然产异丙醇的梭菌属细菌拜氏梭菌DSM 6423细菌以及参比菌株丙酮丁醇梭菌DSM 792进行了遗传修饰。
因此，本发明人第一次描述了一种已被遗传修饰的天然(即在野生型中)能够产生异丙醇、特别是天然能够进行IBE发酵的产溶剂梭菌属细菌，以及使所述细菌能够被获得的工具，特别是遗传工具。这些工具的优点在于显著促进能够在野生型中产生异丙醇、特别是进行IBE发酵的细菌，特别是那些带有编码负责抗生素抗性的酶的基因的细菌的转化和遗传修饰。
实验部分中描述的一部分工作在能够进行IBE发酵的菌株即拜氏梭菌菌株DSM6423中进行，所述菌株的基因组和转录组分析最近已被本发明人描述(Mátéde Gerando等，2018)。
具体来说，在该菌株的基因组组装中，本发明人发现除了染色体之外，还存在可移动遗传元件(登记号PRJEB11626–https://www.ebi.ac.uk/ena/data/view/PRJEB11626)：两个天然质粒(pNF1和pNF2)和一个线性噬菌体(Φ6423)。
在本发明的特定实施方式中，本发明人成功地从拜氏梭菌菌株DSM 6423中缺失了它的天然质粒pNF2。
在另一个特定实施方式中，他们成功地缺失了拜氏梭菌菌株DSM 6423的染色体中最初存在的upp基因。因此，这些实验证实了所述工具以及更广义来说由本发明人在本文中描述的技术对能够在野生型中产生异丙醇、特别是进行IBE发酵的细菌进行遗传修饰的可能用途。
在特别有利的实施方式中，本发明人特别成功地使作为编码负责酰胺醇类抗生素抗性的酶的基因的天然携带者(在野生型中携带)的细菌对这些抗生素敏感。
在本发明的情形中，感兴趣的酰胺醇类抗生素的实例是氯霉素、甲砜霉素、叠氮氯霉素和氟苯尼考(Schwarz S.等，2004)，特别是氯霉素和甲砜霉素。
因此，本发明的第一方面涉及一种可用于对感兴趣的细菌进行遗传转化和/或修饰的遗传工具，所述感兴趣的细菌通常是本文中描述的属于厚壁菌门的细菌，例如梭菌属、芽孢杆菌属或乳杆菌属的细菌，优选为天然(即在野生型中)能够产生异丙醇、特别是天然能够进行IBE发酵的产溶剂梭菌属细菌，优选为天然对一种或多种抗生素具有抗性的细菌，例如拜氏梭菌细菌。优选的细菌在野生型中具有细菌染色体和至少一个不同于染色体DNA的DNA分子两者。
根据一个特定方面，这种遗传工具由识别(至少部分结合)并优选地靶向、即识别并允许切割感兴趣的细菌的基因组中下述序列的至少一条链的核酸(在本文中也被称为“感兴趣的核酸”)构成：i)允许所述感兴趣的细菌在含有其赋予抗性的抗生素的培养基中生长的酶的编码序列，ii)控制允许所述感兴趣的细菌在含有其赋予抗性的抗生素的培养基中生长的酶的编码序列的转录的序列，或iii)在允许所述感兴趣的细菌在含有其赋予抗性的抗生素的培养基中生长的酶的编码序列两侧的序列。这种感兴趣的核酸在本发明的情形中通常用于从所述细菌的基因组缺失所述被识别的序列或修饰其表达，例如调节/调控其表达，特别是抑制它，优选地修饰它以使所述细菌不能表达来自于所述序列的蛋白，特别是功能性蛋白。所述被识别序列在本文中也被称为“靶序列”或“被靶向序列”。
在特定实施方式中，所述感兴趣的核酸包含至少一个与所述靶序列互补的区域，其与所述细菌基因组内被靶向的DNA区域/部分/序列具有100％同一性或至少80％同一性，优选地85％、90％、95％、96％、97％、98％或99％同一性，并且能够杂交到与所述区域/部分/序列互补的序列的全部或一部分，通常杂交到包含至少1个核苷酸，优选地至少1、2、3、4、5、10、14、15、20、25、30、35或40个核苷酸，通常为1、10或20至1000个核苷酸之间，例如1、10或20至900、800、700、600、500、400、300或200个核苷酸之间，1、10或20至100个核苷酸之间，1、10或20至50个核苷酸之间或1、10或20至40个核苷酸之间，例如10至40个核苷酸之间、10至30个核苷酸之间、10至20个核苷酸之间、20至30个核苷酸之间、15至40个核苷酸之间、15至30个核苷酸之间或15至20个核苷酸之间的序列，优选杂交到包含14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列。与所述感兴趣的核酸内存在的靶序列互补的区域可以对应于在本文中描述的CRISPR工具中使用的向导RNA(gRNA)的“SDS”区域。
在另一个特定实施方式中，所述感兴趣的核酸包含至少两个各自与靶序列互补的区域，其与所述细菌基因组内的所述被靶向的DNA区域/部分/序列具有100％同一性或至少80％同一性，优选地至少85％、90％、95％、96％、97％、98％或99％同一性。这些区域能够杂交到与所述区域/部分/序列互补的序列的全部或一部分，通常杂交到如上所述包含至少1个核苷酸，优选地至少100个核苷酸，通常为100至1000个核苷酸之间的序列。所述与感兴趣的核酸内存在的靶序列互补的区域可以识别、优选地靶向本文中描述的遗传修饰工具例如
Figure BDA0003160802810000101
遗传工具、
Figure BDA0003160802810000102
遗传工具或
Figure BDA0003160802810000103
型等位基因交换工具中所述被靶向序列的5′和3′侧翼区。
通常，所述靶序列是在感兴趣的梭菌属细菌的基因组中编码酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶或甲砜霉素–O–乙酰转移酶的序列、控制这种序列的转录的序列或在这种序列两侧的序列，所述细菌能够在含有属于酰胺醇类的一种或多种抗生素例如氯霉素和/或甲砜霉素的培养基中生长。
在特定实施方式中，所述被识别的序列是对应于来自于拜氏梭菌DSM 6423的编码氯霉素–O–乙酰转移酶的catB基因(CIBE_3859)的序列SEQ ID NO：18，或与所述氯霉素–O–乙酰转移酶具有至少70％、75％、80％、85％、90％或95％同一性的氨基酸序列，或包含序列SEQ ID NO：18的全部或至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列。换句话说，所述被识别的序列可以是包含序列SEQ ID NO：18的至少1个核苷酸，优选地至少1、2、3、4、5、10、15、20、25、30、35或40个核苷酸，通常为1至40个核苷酸之间的序列，优选为包含序列SEQ ID NO：18的14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰转移酶具有至少70％同一性的氨基酸序列的实例对应于在NCBI数据库中下述条目下标识的序列：WP_077843937.1，SEQ ID NO：44(WP_063843219.1)，SEQ ID NO：45(WP_078116092.1)，SEQ ID NO：46(WP_077840383.1)，SEQ ID NO：47(WP_077307770.1)，SEQ ID NO：48(WP_103699368.1)，SEQ ID NO：49(WP_087701812.1)，SEQ ID NO：50(WP_017210112.1)，SEQ ID NO：51(WP_077831818.1)，SEQ IDNO：52(WP_012059398.1)，SEQ ID NO：53(WP_077363893.1)，SEQ ID NO：54(WP_015393553.1)，SEQ ID NO：55(WP_023973814.1)，SEQ ID NO：56(WP_026887895.1)，SEQ IDNO：57(AWK51568.1)，SEQ ID NO：58(WP_003359882.1)，SEQ ID NO：59(WP_091687918.1)，SEQ ID NO：60(WP_055668544.1)，SEQ ID NO：61(KGK90159.1)，SEQ ID NO：62(WP_032079033.1)，SEQ ID NO：63(WP_029163167.1)，SEQ ID NO：64(WP_017414356.1)，SEQ IDNO：65(WP_073285202.1)，SEQ ID NO：66(WP_063843220.1)，以及SEQ ID NO：67(WP_021281995.1)。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰转移酶具有至少75％同一性的氨基酸序列的实例对应于序列WP_077843937.1、WP_063843219.1、WP_078116092.1、WP_077840383.1、WP_077307770.1、WP_103699368.1、WP_087701812.1、WP_017210112.1、WP_077831818.1、WP_012059398.1、WP_077363893.1、WP_015393553.1、WP_023973814.1、WP_026887895.1AWK51568.1、WP_003359882.1、WP_091687918.1、WP_055668544.1和KGK90159.1。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰转移酶具有至少90％同一性的氨基酸序列的实例是序列WP_077843937.1、WP_063843219.1、WP_078116092.1、WP_077840383.1、WP_077307770.1、WP_103699368.1、WP_087701812.1、WP_017210112.1、WP_077831818.1、WP_012059398.1、WP_077363893.1、WP_015393553.1、WP_023973814.1、WP_026887895.1和AWK51568.1。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰转移酶具有至少95％同一性的氨基酸序列的实例对应于序列WP_077843937.1、WP_063843219.1、WP_078116092.1、WP_077840383.1、WP_077307770.1、WP_103699368.1、WP_087701812.1、WP_017210112.1、WP_077831818.1、WP_012059398.1、WP_077363893.1、WP_015393553.1、WP_023973814.1和WP_026887895.1。
与序列SEQ ID NO：18编码的氯霉素–O–乙酰转移酶具有至少99％同一性的优选氨基酸序列是序列WP_077843937.1、SEQ ID NO：44(WP_063843219.1)和SEQ ID NO：45(WP_078116092.1)。
与SEQ ID NO：18同一的具体序列是在NCBI数据库中在条目WP_077843937.1下标识的序列。
在特定实施方式中，所述靶序列是对应于来自于产气荚膜梭菌的编码氯霉素–O–乙酰转移酶的catQ基因的序列SEQ ID NO：68，其氨基酸序列对应于SEQ ID NO：66(WP_063843220.1)，或与所述氯霉素–O–乙酰转移酶具有至少70％、75％、80％、85％、90％或95％同一性的序列，或包含序列SEQ ID NO：68的全部或至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％的序列。
换句话说，所述被识别的序列可以是包含序列SEQ ID NO：68的至少1个核苷酸，优选地至少1、2、3、4、5、10、15、20、25、30、35或40个核苷酸，通常为1至40个核苷酸之间的序列，优选为包含序列SEQ ID NO：68的14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个核苷酸的序列。
在又一个特定实施方式中，所述被识别的序列选自本领域技术人员已知的天然存在于梭菌属细菌中或被人工引入到此类细菌中的核酸序列catB(SEQ ID NO：18)、catQ(SEQID NO：68)、catD(SEQ ID NO：69，Schwarz S.等，2004)或catP(SEQ ID NO：70，Schwarz S.等，2004)。
正如上文指明的，根据另一个实施方式，所述靶序列也可以是控制如上所述的编码序列(编码允许所述感兴趣的细菌在含有其赋予抗性的抗生素的培养基中生长的酶)的转录的序列，通常为启动子序列例如catB基因的启动子序列(SEQ ID NO：73)或catQ基因的启动子序列(SEQ ID NO：74)。
然后，所述用作遗传工具的感兴趣的核酸识别并因此通常能够结合到控制如上所述的编码序列的转录的序列。
根据另一个实施方式，所述靶序列可以是在如上所述的编码序列(编码允许所述感兴趣的细菌在含有其赋予抗性的抗生素的培养基中生长的酶)两侧的序列，例如在序列SEQ ID NO：18的catB基因或与其具有至少70％同一性的序列两侧的序列。这种侧翼序列通常包含1、10或20至1000个核苷酸，例如1、10或20至900、800、700、600、500、400、300或200个核苷酸之间，1、10或20至100个核苷酸之间，1、10或20至50个核苷酸之间或1、10或20至40个核苷酸之间，例如10至40个核苷酸之间、10至30个核苷酸之间、10至20个核苷酸之间、20至30个核苷酸之间、15至40个核苷酸之间、15至30个核苷酸之间或15至20个核苷酸之间。
根据一种特定情况，所述靶序列对应于在此类编码序列两侧的一对序列，每个侧翼序列通常包含至少20个核苷酸，通常为100至1000个核苷酸之间，优选为200至800个核苷酸之间。
在本发明的意义上，“核酸”意味着任何天然、合成、半合成或重组的DNA或RNA分子，其任选被化学修饰(即包含非天然碱基、含有例如修饰的连键、修饰的碱基和/或修饰的糖的修饰的核苷酸)，或者被优化以使得从所述编码序列合成的转录本的密码子是打算在其中使用的梭菌属细菌中最常发现的密码子。在梭菌属的情况下，所述优化的密码子通常是富含腺嘌呤(“A”)和胸腺嘧啶(“T”)碱基的密码子。
在本文中描述的肽序列中，氨基酸用对应于下述命名法的单字母编码表示：C：半胱氨酸；D：天冬氨酸；E：谷氨酸；F：苯丙氨酸；G：甘氨酸；H：组氨酸；I：异亮氨酸；K：赖氨酸；L：亮氨酸；M：甲硫氨酸；N：天冬酰胺；P：脯氨酸；Q：谷氨酰胺；R：精氨酸；S：丝氨酸；T：苏氨酸；V：缬氨酸；W：色氨酸和Y：酪氨酸。
在本发明的情形中，所述用作遗传工具以转化和/或遗传修饰感兴趣的细菌的感兴趣的核酸是在梭菌属细菌、特别是天然能够产生异丙醇、特别是天然能够进行IBE发酵的产溶剂梭菌属细菌的基因组中i)识别感兴趣的酶的编码序列、ii)控制所述感兴趣的酶的编码序列的转录或iii)在所述感兴趣的酶的编码序列两侧的DNA片段，所述感兴趣的酶优选为酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶或甲砜霉素–O–乙酰转移酶。
所述能够天然产生异丙醇的细菌可以是例如选自拜氏梭菌、二醇梭菌细菌、微紫色梭菌细菌、丁酸梭菌细菌、糖乙酸多丁醇梭菌细菌、肉毒梭菌细菌、德雷克氏梭菌细菌、粪味梭菌细菌、产气荚膜梭菌细菌和突尼斯梭菌细菌的细菌，优选为选自拜氏梭菌细菌、二醇梭菌(细菌、微紫色梭菌细菌和糖乙酸多丁醇梭菌细菌的细菌。在野生型中能够产生异丙醇的特别优选的细菌是拜氏梭菌细菌。
根据一种特定情况，所述梭菌属细菌是拜氏梭菌细菌，其进化分枝选自DSM 6423、LMG 7814、LMG 7815、NCCB 27006和与菌株DSM 6423具有至少90％、95％、96％、97％、98％或99％同一性的进化分枝。
正如上文指明的，根据本发明所述的感兴趣的核酸能够缺失在所述细菌的基因组中识别的序列(“靶序列”)或修饰它的表达，例如调节它，特别是抑制它，优选地修饰它以使所述细菌不能从所述序列表达蛋白，优选为酰胺醇–O–乙酰转移酶，特别是有功能的蛋白。
这种感兴趣的核酸通常采取表达盒(或“构建物”)的形式，例如包含操作性连接(在本领域技术人员所理解的意义上)到一个或多个感兴趣的(编码)序列的转录启动子的核酸，例如包含其表达产物有助于在所述细菌内实现感兴趣的功能的几个感兴趣的编码序列的操纵子，或另外还包含活化序列和/或转录终止子的核酸；或采取包含一个或多个如上所定义的表达盒的环状或线状单链或双链载体的形式，例如质粒、噬菌体、粘粒、人工或合成染色体。优选地，所述载体是质粒。
所述感兴趣的核酸、优选为表达盒或载体，可以通过专业技术人员公知的常规技术来构建，并且可以包含一个或多个启动子、细菌复制原点(ORI序列)、终止序列、选择基因例如抗生素抗性基因和允许所述表达盒或载体靶向插入的序列(例如“侧翼区”)。此外，这些表达盒和载体可以通过专业技术人员公知的技术整合到基因组中。
感兴趣的ORI序列可以选自pIP404、pAMβ1、pCB102、repH(丙酮丁醇梭菌中的复制原点)、ColE1或rep(大肠埃希氏杆菌中的复制原点)或允许所述载体、通常为质粒在梭菌细胞中维持的任何其他复制原点。
感兴趣的终止序列可以选自adc、thl、bcs操纵子或专业技术人员公知的允许在梭菌中转录终止的任何其他终止子。
感兴趣的选择基因(抗性基因)可以选自ermB、catP、bla、tetA、tetM和/或提供针对氨苄青霉素、红霉素、氯霉素、甲砜霉素、四环素或专业技术人员公知的可用于选择梭菌属细菌的任何其他抗生素的抗性的任何其他基因。
在所述被识别的酶编码序列是为细菌提供氯霉素和/或甲砜霉素抗性的序列的特定实施方式中，所述选择基因不是氯霉素和/或甲砜霉素抗性基因，并且优选地不是catB、catQ、catD或catP基因中的任一者。
在特定实施方式中，所述感兴趣的核酸包含靶向编码感兴趣的酶、特别是酰胺醇–O–乙酰转移酶、控制所述酶的转录或在所述酶的编码序列两侧的序列(“靶序列”、“被靶向序列”或“被识别的序列”)的一个或多个向导RNA(gRNA)，和/或修饰模板(在本文中也被称为“编辑模板”)，例如能够消除或修饰所述靶序列的全部或一部分优选地以便抑制或压制所述靶序列的表达的模板，通常为包含与位于如上所述的靶序列的上游和下游的序列同源(对应)的序列的模板，通常序列(与所述位于靶序列上游和下游的序列同源)各自包含10或20个碱基对至1000、1500或2000个碱基对之间，例如100、200、300、400或500个碱基对至1000、1200、1300、1400或1500个碱基对之间，优选地100至1500或100至1000个碱基对之间，甚至更优选地500至1000个碱基对或200至800个碱基对之间。
特别感兴趣的核酸采取包含一个或多个表达盒的载体的形式，每个表达盒编码至少一个向导RNA(gRNA)。
根据本发明所述的特定遗传工具包含几个(至少两个)如上所述的感兴趣的核酸，所述感兴趣的核酸彼此不同。
在特定实施方式中，所述用作遗传工具以转化和/或遗传修饰感兴趣的细菌、通常为梭菌属细菌的感兴趣的核酸，是识别为所述细菌提供对一种或多种抗生素的抗性的酶的编码序列、控制所述酶的编码序列的转录的序列或在所述编码序列两侧的序列，并且能够缺失该细菌的基因组中的所述序列或使其无功能的核酸，特别是在被Dam–和Dcm–型甲基转移酶(从表现出dam–dcm–基因型的大肠埃希氏杆菌细菌制备)识别的基序的水平上不表现出甲基化的核酸。
当所述待转化和/或遗传修饰的感兴趣的细菌是拜氏梭菌细菌，特别是属于进化分枝DSM 6423、LMG 7814、LMG 7815、NRRL B–593和NCCB 27006之一的拜氏梭菌细菌时，所述用作遗传工具例如质粒的感兴趣的核酸是在被Dam–和Dcm–型甲基转移酶识别的基序的水平上不表现出甲基化的核酸，通常为其中GATC基序的腺苷(“A”)和/或CCWGG基序(W可以对应于腺苷(“A”)或胸苷(“T”))的第二个胞苷“C”被去甲基化的核酸。
被Dam–和Dcm–型甲基转移酶识别的基序不表现出甲基化的核酸通常可以从具有dam–dcm–基因型的大肠埃希氏杆菌细菌(例如大肠埃希氏杆菌INV 110，Invitrogen)制备。这种核酸可能含有例如由EcoKI型甲基转移酶进行的其他甲基化，后一种酶靶向AAC(N6)GTGC和GCAC(N6)GTT基序(N可以对应于任何碱基)的腺嘌呤(“A”)。
在优选实施方式中，所述被靶向的序列对应于编码酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶的基因例如catB基因、控制该基因的转录的序列或在该基因两侧的序列。
在本发明的情形中用作遗传工具的特别感兴趣的核酸是例如载体，优选为质粒，例如在本说明书的实验部分中描述的序列SEQ ID NO：21的质粒pCas9ind–ΔcatB或序列SEQ ID NO：38的质粒pCas9ind–gRNA_catB，特别是所述序列的在被Dam–和Dcm–型甲基转移酶识别的基序处不表现出甲基化的版本。
本说明书还涉及本文中描述的感兴趣的核酸的用途，其用于转化和/或遗传修饰感兴趣的细菌，特别是能够在野生型中产生异丙醇、特别是能够在野生型中进行IBE发酵的产溶剂梭菌属细菌。
能够在野生型中产生异丙醇、特别是能够在野生型中进行IBE发酵的细菌可以是例如选自拜氏梭菌、二醇梭菌细菌、微紫色梭菌细菌、丁酸梭菌细菌、糖乙酸多丁醇梭菌细菌、肉毒梭菌细菌、德雷克氏梭菌细菌、粪味梭菌细菌、产气荚膜梭菌细菌和突尼斯梭菌细菌的细菌，优选为选自拜氏梭菌细菌、二醇梭菌细菌、微紫色梭菌细菌和糖乙酸多丁醇梭菌细菌的细菌。
(天然)能够在野生型中产生异丙醇、特别是能够在野生型中进行IBE发酵的特别优选的细菌是拜氏梭菌细菌。
所述感兴趣的产乙酸细菌是从CO2和H2产生酸和/或溶剂的细菌。产乙酸梭菌属细菌可以例如选自醋酸梭菌(C.aceticum)、嗜热醋酸梭菌(C.thermoaceticum)、扬氏梭菌、自养产乙醇梭菌(C.autoethanogenum)、艰难梭菌(C.difficile)、粪味梭菌和食一氧化碳梭菌(C.carboxydivorans)。
在特定实施方式中，所述有关的梭菌属细菌是“ABE菌株”，优选为丙酮丁醇梭菌菌株DSM 792(也被称为ATCC菌株824或LMG 5710)或拜氏梭菌菌株NCIMB 8052。
在另一个特定实施方式中，所述有关的梭菌属细菌是“IBE菌株”，通常为本说明书中鉴定的拜氏梭菌细菌之一，例如其进化分枝选自DSM 6423、LMG 7814、LMG 7815、NRRL B–593、NCCB 27006的拜氏梭菌或金黄丁酸梭菌DSZM 793细菌(Georges等，1983)，和与菌株DSM 6423显示出至少90％、95％、96％、97％、98％或99％的同一性的此类拜氏梭菌或金黄丁酸梭菌细菌的进化分枝。特别优选的拜氏梭菌细菌或拜氏梭菌细菌的进化分枝缺少pNF2质粒。
一方面是LMG 7814、LMG 7815、NRRL B–593和NCCB 27006进化分枝的相应基因组，另一方面是DSZM 793的基因组，显示出与DSM 6423进化分枝的基因组至少97％的序列同一性百分数。
本发明人已进行了发酵试验，确认了进化分枝DSM 6423、LMG 7815和NCCB 27006的拜氏梭菌细菌能够在野生型中产生异丙醇(参见表1)。
[表1]
Figure BDA0003160802810000191
Figure BDA0003160802810000201
使用天然产异丙醇菌株拜氏梭菌DSM 6423、LMG 7815和NCCB 27006的葡萄糖发酵试验的概述
在本发明的特别优选实施方式中，所述拜氏梭菌细菌是DSM 6423进化分枝细菌。
在本发明的又一个实施方式中，所述拜氏梭菌细菌是拜氏梭菌IFP963ΔcatBΔpNF2菌株(于2019年2月20日在BCCM–LMG保藏中心登记在保藏号LMG P–31277下，并且在本文中也被称为拜氏梭菌DSM 6423ΔcatBΔpNF2)。
根据一个特定实施方式，所述待转化并优选地遗传修饰的细菌是已暴露于使用根据本发明的核酸或遗传工具的第一转化步骤和第一遗传修饰步骤，使得可以缺失在野生型中天然存在于所述细菌中的至少一个染色体外DNA分子(通常为至少一个质粒)的细菌。
本发明人描述的另一方面涉及一种使用根据本发明的遗传工具，通常使用如上所述的根据本发明的感兴趣的核酸来转化并优选地还遗传修饰梭菌属细菌的方法。所述方法包括通过将本文中描述的感兴趣的核酸引入到所述细菌中来转化所述细菌的步骤。所述方法还可以包括获得、回收、选择或分离所述转化的细菌，即具有所需重组/修饰/优化的细菌的步骤。
在特定实施方式中，所述用于转化并优选地遗传修饰梭菌属细菌的方法涉及遗传修饰工具，例如选自CRISPR、基于使用II类内含子的工具(例如
Figure BDA0003160802810000202
工具或
Figure BDA0003160802810000203
工具)和等位基因交换工具(例如
Figure BDA0003160802810000204
工具)的遗传修饰工具，并包括通过将如上所述根据本发明的感兴趣的核酸引入到所述细菌中来转化所述细菌的步骤。
通常，本发明有利地在所述被选择用于转化并优选地遗传修饰梭菌属细菌的遗传修饰工具旨在用于在野生型中带有负责对一种或多种抗生素的抗性的酶的编码基因的细菌例如拜氏梭菌的情况下实施，并且所述遗传工具的实施包括在允许该细菌在野生型中有抗性的抗生素的抗性标记物表达的核酸的帮助下转化所述细菌的步骤，和/或在所述抗生素(所述细菌在野生型中对其有抗性)的帮助下选择所述转化和/或遗传修饰的细菌的步骤。
可以例如使用选自CRISPR工具、基于使用II类内含子的工具和等位基因交换工具的遗传修饰工具通过本发明有利地实现的修饰，主要在于缺失为所述细菌提供针对一种或多种抗生素的抗性的酶的编码序列，或主要在于使该序列无功能。可以通过本发明有利地实现的另一种修饰主要在于对细菌进行遗传修饰以便提高它的性能，例如它的生产感兴趣的溶剂或溶剂混合物的性能，所述细菌先前已通过本发明进行修饰，使其对它在野生型中有抗性的抗生素敏感。
在优选实施方式中，根据本发明所述的方法是基于成簇规则间隔短回文重复序列(CRISPR)技术/遗传工具、特别是CRISPR/Cas(CRISPR相关蛋白)遗传工具的使用(实施)。
这种方法是基于使用被称为核酸酶的酶(在CRISPR/Cas遗传工具的情况下通常是Cas–型核酸酶，例如来自于酿脓链球菌的CRISPR相关蛋白9(Cas9蛋白))，其在RNA分子指导下在DNA分子(感兴趣的靶序列)中制造双链切割。所述向导RNA(gRNA)的序列决定所述核酸酶的切割位点，为其提供非常高的特异性。由于对微生物的存活来说必需DNA分子内的双链切割事实上对生物体是致死的，因此所述生物体的存活取决于它修复所述切割的能力(参见例如Cui&Bikard，2016)。在梭菌属细菌中，双链断裂的修复依靠同源重组机制，需要被切割序列的完整拷贝。通过为所述细菌提供在修饰原始序列的同时允许发生修复的DNA片段，可以迫使所述微生物将所需变化整合在它的基因组中。
本发明可以在梭菌属细菌中使用常规的CRISPR/Cas遗传工具来实施，使用如Wang等(2015)所描述的包含核酸酶、gRNA和修复模板的单一质粒。所述CRISPR/Cas系统含有两个不同的必需元件，即i)核酸内切酶，在这种情况下是CRISPR相关核酸酶Cas，以及ii)向导RNA。所述向导RNA采取嵌合RNA的形式，其由细菌CRISPR RNA(crRNA)和tracrRNA(反式激活CRISPR RNA)的组合构成。所述gRNA将充当Cas蛋白的向导的对应于“间隔物序列”的crRNA的靶向特异性与crRNA的构象性质组合在单一转录本中。当所述gRNA和Cas蛋白在细胞中同时表达时，由于提供的修复模板，所述靶基因组序列可以被永久性地修饰。专业技术人员可以使用公知的技术，根据待靶向的染色体区域或可移动遗传元件容易地确定所述gRNA的序列和结构(参见例如DiCarlo等，2013的论文)。
所述遗传工具的元件(核酸或gRNA)在所述细菌中的引入通过专业技术人员已知的任何直接或间接方法来进行，例如通过转化、接合、微注射、转染、电穿孔等，优选地通过电穿孔(Mermelstein等，1993)。
本发明人最近已开发并描述了一种基于使用两种质粒的用于修饰细菌的遗传工具，其适合于梭菌属细菌并且可以在本发明的情形中使用(参见WO2017/064439，Wasels等，2017，和与本说明书相关的图15)。
在特定实施方式中，这种工具的“第一”质粒允许Cas核酸酶的表达，并且特异性针对待进行的修饰的“第二”质粒含有一个或多个gRNA表达盒(通常靶向细菌DNA的不同区域)，以及允许通过同源重组机制将细菌DNA中被Cas靶向的部分用感兴趣的序列代替的修复模板。所述cas基因和/或gRNA表达盒被置于本领域技术人员已知的组成型或诱导型、优选为诱导型，并且优选地不同但可以通过相同的诱导剂诱导的表达启动子(例如在申请WO2017/064439中描述的并通过参考并入本说明书)的控制之下。
所述gRNA可以是天然RNA、合成的RNA或通过重组技术产生的RNA。这些gRNA可以通过专业技术人员已知的任何方法来制备，例如化学合成、体内转录或扩增技术。当使用多个gRNA时，每个gRNA的表达可以通过不同的启动子控制。优选地，用于所有gRNA的启动子是相同的。在特定实施方式中，相同的启动子可用于表达几个、例如仅仅一些，或者换句话说全部或某些打算表达的gRNA。
在适合于在本发明的情形中使用的另一个特定实施方式中，ii)所述“第一”和“第二”核酸中的至少一者还编码一个或多个向导RNA(gRNA)，或者所述遗传工具还包含一个或多个向导RNA，每个向导RNA包含结合Cas酶的RNA结构和与所述细菌DNA的被靶向部分互补的序列，和iii)所述“第一”和“第二”核酸中的至少一者还包含在诱导型启动子控制之下的编码抗CRISPR蛋白的序列，或者所述遗传工具还包含在诱导型启动子控制之下的编码抗CRISPR蛋白的“第三”核酸，所述启动子优选地不同于控制Cas和/或RNA表达的启动子，并且可以通过另一种诱导剂诱导。
在优选实施方式中，所述抗CRISPR蛋白优选地在将所述遗传工具的核酸序列引入到感兴趣的细菌菌株的阶段中能够抑制、优选地中和所述核酸酶的作用。
一种涉及CRISPR技术，可以在本发明的情形中实施以转化并通常通过同源重组遗传修饰梭菌属细菌的特定方法包括下述步骤：
a)在诱导抗CRISPR蛋白的表达的试剂存在下，将本发明人描述的CRISPR遗传工具引入到所述细菌中，以及
b)将在步骤a)结束时获得的转化的细菌在不含用于诱导抗CRISPR蛋白的表达的试剂的培养基上(或在不涉及所述试剂的条件下)培养，通常允许Cas/gRNA核糖核蛋白复合体的表达。
在特定实施方式中，所述方法在步骤b)期间或之后还包括诱导控制Cas和/或向导RNA的表达的诱导型启动子(在此类启动子存在于所述遗传工具中的情况下)的步骤，以便在所述遗传工具被引入到所述细菌中之后允许所述细菌的感兴趣的遗传修饰。所述诱导使用允许解除与所选诱导型启动子相关的表达抑制的物质来进行。
在另一个特定实施方式中，所述方法还包括除去所述含有修复模板的核酸(然后所述细菌细胞被认为“清除”了所述核酸)和/或除去在步骤a)中使用所述遗传工具引入的向导RNA或编码向导RNA的序列的另外的步骤c)。
在又一个特定实施方式中，所述方法在步骤b)或步骤c)后，包括在诱导抗CRISPR蛋白的表达的试剂存在下，引入第n例如第三、第四、第五等的核酸的一个或多个另外的步骤，所述核酸含有不同于已引入的修复模板的修复模板和允许所述不同的修复模板中包含的感兴趣的序列整合到细菌基因组的被靶向区域中的一个或多个向导RNA表达盒，每个另外的步骤之后跟有将由此转化的细菌在不含所述用于诱导抗CRISPR蛋白的表达的试剂的培养基上进行培养，通常允许Cas/gRNA核糖核蛋白复合体的表达的步骤。
在根据本发明的方法的特定实施方式中，所述细菌使用例如上文描述的CRISPR工具或方法来转化，所述CRISPR工具或方法使用(例如编码)负责切割感兴趣的靶序列的至少一条链的酶，其中在特定实施方式中所述酶是核酸酶，优选为Cas–型核酸酶，优选地选自Cas9酶和MAD7酶。优选地，所述感兴趣的靶序列是为所述细菌提供对一种或多种抗生素、优选为属于酰胺醇类的一种或多种抗生素的抗性的酶、通常为酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶的编码序列例如catB基因，控制所述编码序列的转录的序列，或在所述编码序列两侧的序列。
适合用于本发明的Cas9蛋白的实例包括但不限于来自于酿脓链球菌(参见申请WO2017/064439的SEQ ID NO：1和NCBI登记号WP_010922251.1)、嗜热链球菌(Streptococcus thermophilus)、变形链球菌(Streptococcus mutans)、空肠弯曲杆菌(Campylobacter jejuni)、多杀巴氏杆菌(Pasteurella multocida)、新凶手弗朗西斯菌(Francisella novicida)、脑膜炎奈瑟氏菌(Neisseria meningitidis)、乳糖奈瑟氏菌(Neisseria lactamica)和嗜肺军团菌(Legionella pneumophila)的Cas9蛋白(参见Fonfara等，2013；Makarova等，2015)。
也被称为“Cas12”或“Cpf1”的MAD7核酸酶(其氨基酸序列对应于SEQ ID NO：72)，也可以通过与本领域技术人员已知的能够与此类核酸酶结合的gRNA的组合，有利地用于本发明的情形(参见Garcia–Doval等，2017和Stella S.等，2017)。
根据一个特定方面，所述编码MAD7核酸酶的序列是被优化以容易地在梭菌属菌株中表达的序列，优选为序列SEQ ID NO：71。
在使用时，所述CRISPR蛋白通常是“抗Cas”蛋白，即能够抑制或阻止/中和Cas的作用的蛋白，和/或能够抑制或阻止/中和CRISPR/Cas系统、例如当所述核酸酶是Cas9核酸酶时II型CRISPR/Cas系统的作用的蛋白。
有利情况下，所述抗CRISPR蛋白是例如选自AcrIIA1、AcrIIA2、AcrIIA3、AcrIIA4、AcrIIA5、AcrIIC1、AcrIIC2和AcrIIC3的“抗Cas9”蛋白(Pawluk等，2018)。优选地，所述“抗Cas9”蛋白是AcrIIA2或AcrIIA4。这种蛋白通常能够例如通过与所述Cas9酶结合，非常显著地限制、理想情况下阻止Cas9的作用。
可以有利地使用的另一种抗CRISPR蛋白是“抗MAD7”蛋白，例如AcrVA1蛋白(Marino等，2018)。
与所述被靶向DNA部分(“被识别序列”)相似，所述编辑/修复模板自身可以包含对应于天然和/或合成的编码和/或非编码序列的一个或多个核酸序列或核酸序列部分。所述模板也可以包含一个或多个“外来”序列，即在属于梭菌属的细菌的基因组或所述属的特定菌种的基因组中天然不存在的序列。所述模板也可以包括序列的组合。
在本发明中使用的遗传工具允许所述修复模板指导感兴趣的核酸并入到细菌基因组中，所述感兴趣的核酸通常是包含至少1个碱基对(bp)，优选地至少1、2、3、4、5、10、15、20、50、100、1,000、10,000、100 000或1 000 000bp，通常在1bp至20kb之间例如2、3、4、5、6、7、8、9、10、11、12或13kb，或在1bp至10kb之间，优选地在10bp至10kb之间或1kb至10kb之间，例如1bp至5kb之间、2kb至5kb之间或2.5或3kb至5kb之间的DNA序列或序列的部分。
在特定实施方式中，所述感兴趣的DNA序列的表达允许所述梭菌属细菌发酵(通常同时地)几种不同的糖，例如至少两种不同的糖，通常为在包含5个碳原子的糖(例如葡萄糖或甘露糖)和/或包含6个碳原子的糖(例如木糖、阿拉伯糖或果糖)中的至少两种不同的糖，优选为至少三种不同的糖，例如选自葡萄糖、木糖和甘露糖；葡萄糖、阿拉伯糖和甘露糖；以及葡萄糖、木糖和阿拉伯糖。
在另一个特定实施方式中，所述感兴趣的DNA序列编码至少一种感兴趣的产物，优选为促进梭菌属细菌的溶剂生产的产物，通常为至少一种感兴趣的蛋白，例如酶、膜蛋白例如转运蛋白、用于其他蛋白的成熟蛋白(伴侣蛋白)、转录因子或其组合。
在另一个实施方式中，根据本发明所述的方法是基于II类内含子的使用，并实施例如
Figure BDA0003160802810000271
遗传技术/工具或
Figure BDA0003160802810000272
遗传工具。
所述
Figure BDA0003160802810000273
技术依赖于使用可重编程的II类内含子(基于乳酸乳球菌(Lactococcus lactis)的Ll.ltrB内含子)，其能够在所需基因座处快速整合细菌基因组(Chen等，2005，Wang等，2013)，目的通常是失活被靶向的基因。识别被编辑区域以及通过反向剪接插入到基因组中的机制一方面是基于所述内含子与所述区域之间的同源性，另一方面是基于蛋白质(ltrA)的活性。
Figure BDA0003160802810000274
技术是基于类似的方法，并补充了在内含子序列中添加选择标记物(Heap等，2007)。这种标记物允许选择所述内含子在基因组中的整合，并因此便于获得所需突变体。这种遗传系统也利用I类内含子。事实上，所述选择标记物(被称为反转录转座活化的标记物或RAM)被这种遗传元件中断，这阻止了选择标记物从所述质粒表达(所述系统的更精确的描述：Zhong等)。这种遗传元件的剪接发生在整合到基因组中之前，产生具有活化形式的抗性基因的染色体。所述系统的优化版本包括在该基因上游和下游的FLP/FRT位点，允许使用FRT重组酶移除所述抗性基因(Heap等，2010)。
在另一个实施方式中，根据本发明所述的方法是基于使用等位基因交换工具，并实施例如
Figure BDA0003160802810000275
遗传技术/工具。
Figure BDA0003160802810000276
技术是基于使用营养缺陷突变体(对于尿嘧啶营养缺陷来说，在丙酮丁醇梭菌ATCC 824中通过缺失pyrE基因，这也引起对5-氟乳清酸(A–5–FO)的抗性；Heap等，2012)。所述系统使用专业技术人员公知的等位基因交换机制。在用假自杀(极低拷贝)载体转化后，后者通过第一次等位基因交换事件在细菌染色体中的整合通过最初存在于所述质粒上的抗性基因来选择。所述整合步骤可以以两种不同方式进行，即在pyrE基因座内或在另一个基因座内：
在pyrE基因座处整合的情况下，所述pyrE基因也被放置在所述质粒上，但不表达(没有有功能的启动子)。第二次重组恢复了有功能的pyrE基因，并因此可以通过营养缺陷(不含尿嘧啶的最低培养基)来选择。由于无功能的pyrE基因也具有可选择特点(对A–5–FO的敏感性)，因此可以在同一模型上发生其他整合，使pyrE的状态在有功能与无功能之间依次轮换。
在另一个基因座处整合的情况下，靶向允许反选择标记物在重组后表达的基因组区域(通常在另一个基因、优选为高表达基因之后的操纵子中)。然后通过营养缺陷(不含尿嘧啶的最低培养基)选择该第二次重组。
在所描述的基于使用II类内含子并实施
Figure BDA0003160802810000281
遗传技术/工具或
Figure BDA0003160802810000282
遗传工具或基于使用等位基因交换工具并实施例如
Figure BDA0003160802810000283
遗传技术/工具的实施方式中，所述被靶向的序列优选为感兴趣的酶、优选为上文所解释的酰胺醇–O–乙酰转移酶的编码序列两侧的序列。
本发明的另一个主题内容涉及利用本发明人在本文中描述的方法获得的转化和/或遗传修饰的细菌，通常为属于本发明人描述的物种或对应于本发明人描述的进化分枝之一的梭菌属细菌，及其任何衍生细菌、克隆、突变体或遗传修饰形式。
由此转化和/或遗传修饰的本发明典型的细菌是不再表达为一种或多种抗生素提供抗性的酶的细菌，特别是不再表达酰胺醇–O–乙酰转移酶的细菌，例如在野生型中表达catB基因而由于本发明在被转化和/或遗传修饰后缺少所述catB基因或不能表达所述catB基因的细菌。所述利用本发明而被转化和/或遗传修饰的细菌被赋予对酰胺醇、例如对本文中描述的酰胺醇、特别是对氯霉素或甲砜霉素的敏感性。
根据本发明的优选的遗传修饰的细菌的具体实例是在本说明书中被鉴定为拜氏梭菌DSM 6423ΔcatB的细菌，其于2018年12月6日在比利时微生物协调保藏中心(BelgianCo–ordinated Collections of Micro–organisms)(“BCCM”，K.L.Ledeganckstraat 35,B–9000Gent–Belgium)登记在保藏号LMG P–31151下。本说明书还涉及保留了对酰胺醇例如甲砜霉素和/或氯霉素的敏感性的所述细菌的任何衍生细菌、克隆、突变体或遗传修饰形式。
根据特定实施方式，所述根据本发明的不表达为一种或多种抗生素提供抗性的酶、特别是酰胺醇–O–乙酰转移酶例如氯霉素–O–乙酰转移酶的转化和/或遗传修饰的细菌例如拜氏梭菌DSM 6423ΔcatB细菌，仍然能够被转化并优选地被遗传修饰。这可以使用在本说明书中，例如在实验部分中描述的核酸例如质粒来进行。可以有利地使用的核酸的实例是序列SEQ ID NO：23的质粒pCas9acr(描述在本说明书的实验部分中)。
事实上，本发明的一个特定方面涉及根据本发明所述的遗传修饰的细菌，优选为在编号LMG P–31151下保藏的拜氏梭菌DSM 6423ΔcatB细菌或其遗传修饰的形式的用途，其例如使用本文中描述的遗传工具或方法之一，通过自主引入到其基因组中的感兴趣的核酸的表达，用于优选地在工业规模上生产一种或多种溶剂，优选至少异丙醇。
本发明还涉及一种试剂盒，其包含：(i)根据本发明所述的感兴趣的核酸，通常为DNA片段，其识别在梭菌属细菌、特别是本文中所描述的能够进行IBE发酵的细菌中感兴趣的酶的编码序列或控制所述编码序列的转录的序列；和(ii)至少一种工具，优选为几种工具，其选自用于转化并通常遗传修饰梭菌属细菌以便产生所述细菌的改进变体的遗传修饰工具的元件、作为gRNA的核酸、作为修复模板的核酸、至少一个引物对例如在本发明的情形中描述的引物对和允许所述工具编码的蛋白质例如Cas9或MAD7型核酸酶的表达的诱导物。
所述用于转化并通常遗传修饰梭菌属细菌的遗传修饰工具可以选自例如上文所解释的CRISPR工具、基于II类内含子的工具和等位基因交换工具。
所述试剂盒还可以包含一种或多种诱导物，其为在所述遗传工具内任选使用的所选诱导型启动子定制，以控制所使用的核酸酶和/或一种或多种向导RNA的表达。
根据本发明所述的特定试剂盒允许表达包含标签的核酸酶。
根据本发明所述的试剂盒还可以包含一种或多种消耗品，例如培养基、至少一种感受态梭菌属细菌(即被调制以用于转化)、至少一种gRNA、核酸酶、一种或多种选择分子或说明性资料。
本说明书还涉及根据本发明所述的试剂盒或该试剂盒的一种或多种元件的用途，其用于实施本文中描述的梭菌属细菌的转化和理想情况下遗传修饰的方法，和/或用于使用梭菌属细菌、优选为天然产生异丙醇的梭菌属细菌，优选地在工业规模上生产溶剂或生物燃料或其混合物。
可以生产的溶剂通常是丙酮、丁醇、乙醇、异丙醇或其混合物，通常为乙醇/异丙醇、丁醇/异丙醇或乙醇/丁醇混合物，优选为异丙醇/丁醇混合物。
根据本发明转化的细菌的使用通常允许在工业规模上每年生产至少100吨丙酮、至少100吨乙醇、至少1000吨异丙醇、至少1800吨丁醇或至少40 000吨它们的混合物。
下面的实施例和附图旨在更充分地说明本发明，但不限制其范围。
附图说明
[图1]图1示出了来自于Poehlein等，2017的30种产溶剂梭菌属菌株的分类。注意进化分枝拜氏梭菌NRRL B–593在文献中也被称为拜氏梭菌DSM 6423。
[图2]图2示出了pCas9ind–ΔcatB质粒图谱。
[图3]图3示出了pCas9acr质粒图谱。
[图4]图4示出了pEC750S–uppHR质粒图谱。
[图5]图5示出了pEX–A2–gRNA–upp质粒图谱。
[图6]图6示出了pEC750S–Δupp质粒图谱。
[图7]图7示出了pEC750C–Δupp质粒图谱。
[图8]图8示出了pGRNA–pNF2图谱。
[图9]图9示出了在源自于拜氏梭菌菌株DSM 6423的细菌转化的克隆中catB基因的PCR扩增。
如果所述菌株仍具有catB基因，扩增产物约为1.5kb，或者如果该基因被缺失，则扩增产物约为900bp。
[图10]图10示出了拜氏梭菌菌株DSM6423 WT和ΔcatB在2YTG培养基和2YTG甲砜霉素选择性培养基上的生长。
[图11]图11示出了在拜氏梭菌菌株DSM 6423的含有pCas9acr和具有或不具有修复模板的靶向upp的gRNA表达质粒的转化体中CRISPR/Cas9acr系统的诱导。图例说明：Em，红霉素；Tm，甲砜霉素；aTc，无水四环素；ND，未稀释。
[图12A]图12示出了通过CRISPR/Cas9系统进行的拜氏梭菌DSM 6423的upp基因座的修饰。图12A代表了upp基因座的遗传架构：基因，gRNA靶位点和与基因组DNA上的相应同源区相关的修复模板。也标出了用于PCR验证的引物杂交位点(RH010和RH011)。
[图12B]图12示出了通过CRISPR/Cas9系统进行的拜氏梭菌DSM 6423的upp基因座的修饰。图12B示出了使用引物RH010和RH011对upp基因座进行的扩增。在野生型基因的情况下预期扩增产物为1680bp，与此相比对于修饰的upp基因来说扩增产物为1090bp。M，100bp–3kb尺寸的标志物(Lonza)；WT，野生型菌株。
[图13]图13示出了验证拜氏梭菌菌株6423ΔcatB中质粒pCas9ind.的存在的PCR扩增。
[图14]图14示出了在CRISPR–Cas9系统在含有aTc的培养基上诱导之前(阳性对照1和2)和诱导之后，验证天然质粒pNF2的存在或不存在的PCR扩增(≈900bp)。
[图15]图15示出了被改造以适应于梭菌属细菌的基于使用两种质粒的用于细菌修饰的遗传工具(参见WO2017/064439，Wasels等，2017)。
[图16]图16示出了pCas9ind–gRNA_catB质粒图谱。
[图17]图17示出了作为遗传工具用于基因组编辑的CRISPR/Cas9系统，其使用Cas9核酸酶在基因组DNA中产生一个或多个gRNA指导的双链切割。
gRNA，向导RNA；PAM，前间区序列邻近基序。图从Jinek等，2012修改。
[图18]图18示出了由Cas9诱导的双链断裂的同源重组修复。PAM，前间区序列邻近基序。
[图19]图19示出了CRISPR/Cas9在梭菌属中的使用。
ermB，红霉素抗性基因；catP(SEQ ID NO：70)，甲砜霉素/氯霉素抗性基因；tetR，该基因的表达产物阻遏从Pcm–tetO2/1、Pcm–2tetO1和Pcm–tetO2/1无水四环素“aTc”诱导型启动子的转录(Dong等，2012)；miniPthl，组成型启动子(Dong等，2012)。
[图20]图20示出了pCas9acr质粒图谱(SEQ ID NO：23)。
ermB，红霉素抗性基因；rep，大肠埃希氏杆菌中的复制原点；repH，丙酮丁醇梭菌中的复制原点；Tthl，硫解酶终止子；miniPthl，组成型启动子(Dong等，2012)；Pcm–tetO2/1，被tetR的产物阻遏并且可以被无水四环素“aTc”诱导的启动子(Dong等，2012)；Pbgal，被lacR的产物阻遏并且可以被乳糖诱导的启动子(Hartman等，2011)；acrIIA4，编码抗CRISPR蛋白AcrII14的基因；bgaR，该基因的表达产物阻遏从Pbgal的转录。
[图21]图21示出了含有pCas9ind(SEQ ID NO：22)或pCas9acr(SEQ ID NO：23)的丙酮丁醇梭菌DSM 792的相对转化率。频率被表示为在转化中使用的每μg DNA获得的转化体的数目相对于pEC750C(SEQ ID NO：106)的转化频率，并代表至少两个独立实验的平均值。
[图22]图22示出了在含有pCas9acr和具有(SEQ ID NO：79和SEQ ID NO：80)或不具有(SEQ ID NO：105)修复模板的靶向bdhB的gRNA表达质粒的DSM 792菌株转化体中CRISPR/Cas9系统的诱导。Em，红霉素；Tm，甲砜霉素；aTc，无水四环素；ND，未稀释。
[图23A]图23示出了通过CRISPR/Cas9系统进行的丙酮丁醇梭菌DSM792的bdh基因座的修饰。图23A示出了bdh基因座的遗传架构。修复模板与基因组DNA之间的同源性用浅灰色平行四边形突出。也示出了引物V1和V2的杂交位点。
[图23B]图23示出了通过CRISPR/Cas9系统进行的丙酮丁醇梭菌DSM792的bdh基因座的修饰。图23B示出了使用引物V1和V2进行的bdh基因座的扩增。M，2–log尺寸标志物(NEB)；P，pGRNA–ΔbdhAΔbdhB质粒；WT，野生型菌株。
[图24]图24示出了20μg pCas9ind质粒在拜氏梭菌菌株DSM6423中的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条表示三份生物学平行样的平均值的标准误差。
[图25]图25示出了NF3质粒图谱。
[图26]图26示出了pEC751S质粒图谱。
[图27]图27示出了pNF3S质粒图谱。
[图28]图28示出了pNF3E质粒图谱。
[图29]图29示出了pNF3C质粒图谱。
[图30]图30示出了质粒pCas9ind在拜氏梭菌DSM 6423的三种菌株中的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条是两份生物学平行样的平均值的标准偏差。
[图31]图31示出了质粒pEC750C在源自于拜氏梭菌DSM 6423的两种菌株中的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条是两份生物学平行样的平均值的标准偏差。
[图32]图32示出了质粒pEC750C、pNF3C、pFW01和pNF3E在拜氏梭菌菌株IFP963ΔcatBΔpNF2中的转化效率(以每μg转化的DNA观察到的菌落数为单位)。误差条是三份生物学平行样的平均值的标准偏差。
[图33]图33示出了质粒pFW01、pNF3E和pNF3S在拜氏梭菌菌株NCIMB 8052中的转化效率(以每μg转化的DNA观察到的菌落数为单位)。
实施例
实施例1
材料和方法
培养条件
丙酮丁醇梭菌DSM 792生长在2YTG培养基(胰蛋白胨16g.l–1，酵母提取物10g.l–1，葡萄糖5g.l–1，NaCl 4g.l–1)中。大肠埃希氏杆菌NEB10B生长在LB培养基(胰蛋白胨10g.l–1，酵母提取物5g.l–1，NaCl 5g.l–1)中。固体培养基通过向液体培养基添加15g.l–1琼脂来制造。在需要时使用红霉素(在2YTG或LB培养基中浓度分别为40或500mg.l–1)、氯霉素(在固体或液体LB培养基中分别为25或12.5mg.l–1)和甲砜霉素(在2YTG培养基中15mg.l–1)。
核酸的操作
所有酶和试剂盒按照制造商的推荐使用。
质粒的构建
图20中示出的pCas9acr质粒(SEQ ID NO：23)通过将由Eurofins Genomics合成的含有在启动子Pbgal控制之下的bgaR和acrIIA4的片段(SEQ ID NO：81)克隆在pCas9ind载体的SacI位点处来构建(Wasels等，2017)。
pGRNAind质粒(SEQ ID NO：82)通过将由Eurofins Genomics合成的在Pcm–2tetO1启动子控制之下的gRNA的表达盒(SEQ ID NO：83)(Dong等，2012)克隆在pEC750C载体(SEQID NO：106)的SacI位点处来构建(Wasels等，2017)。
pGRNA–xylB(SEQ ID NO：102)、pGRNA–xylR(SEQ ID NO：103)、pGRNA–glcG(SEQ IDNO：104)和pGRNA–bdhB(SEQ ID NO：105)质粒通过将相应的引物对5′–TCATGATTTCTCCATATTAGCTAG–3′和5′–AAACCTAGCTAATATGGAGAAATC–3′、5′–TCATGTTACACTTGGAACAGGCGT–3′和5′–AAACACGCCTGTTCCAAGTGTAAC–3′、5′–TCATTTCCGGCAGTAGGATCCCCA–3′和5′–AAACTGGGGATCCTACTGCCGGAA–3′、5′–TCATGCTTATTACGACATAACACA–3′和5′–AAACTGTGTTATGTCGTAATAAGC–3′克隆在BsaI消化的pGRNAind质粒(SEQ ID NO：82)中来构建。
pGRNA–ΔbdhB质粒(SEQ ID NO：79)通过将一方面使用引物5′–ATGCATGGATCCAAACGAACCCAAAAAGAAAGTTTC–3′和5′–GGTTGATTTCAAATCTGTGTAAACCTACCG–3′，另一方面使用引物5′–ACACAGATTTGAAATCAACCACTTTAACCC–3′和5′–ATGCATGTCGACTCTTAAGAACATGTATAAAGTATGG–3′获得的PCR产物通过重叠PCR组装而得到的DNA片段克隆到用BamHI和SacI消化的pGRNA–bdhB载体中来构建。
pGRNA–ΔbdhAΔbdhB质粒(SEQ ID NO：80)通过将一方面使用引物5′–ATGCATGGATCCAAACGAACCCAAAAAGAAAGTTTC–3′和5′–GCTAAGTTTTAAATCTGTGTAAACCTACCG–3′，另一方面使用引物5′–ACACAGATTTAAAACTTAGCATACTTCTTACC–3′和5′–ATGCATGTCGACCTTCTAATCTCCTCTACTATTTTAG–3′获得的PCR产物通过重叠PCR组装而得到的DNA片段克隆到用BamHI和SacI消化的pGRNA–bdhB载体中来构建。
转化
丙酮丁醇梭菌DSM 792按照由Mermelstein等，1993描述的方案来转化。用含有gRNA表达盒的质粒转化的已含有Cas9表达质粒(pCas9ind或pCas9acr)的丙酮丁醇梭菌DSM792转化体的选择在含有红霉素(40mg.l–1)、甲砜霉素(15mg.l–1)和乳糖(40nM)的固体2YTG培养基上进行。
cas9表达的诱导
cas9表达的诱导通过将得到的转化体在含有红霉素(40mg.l–1)、甲砜霉素(15mg.l–1)和cas9和gRNA表达诱导剂aTc(1mg.l–1)的固体2YTG培养基上生长来进行。
bdh基因座的扩增
丙酮丁醇梭菌DSM 792在bdhA和bdhB基因座处的基因组编辑，通过使用
Figure BDA0003160802810000361
高保真DNA聚合酶(NEB)和引物V1(5′–ACACATTGAAGGGAGCTTTT–3′)和V2(5′–GGCAACAACATCAGGCCTTT–3′)的PCR来控制。
结果
转化效率
为了评估acrIIA4基因的插入对cas9表达质粒的转化效率的影响，将不同的gRNA表达质粒转化到含有pCas9ind(SEQ ID NO：22)或pCas9acr(SEQ ID NO：23)的DSM 792菌株中，并在增补有乳糖的培养基上选择转化体。得到的转化效率呈现在图21中。
ΔbdhB和ΔbdhAΔbdhB突变体的产生
将含有靶向bdhB的gRNA表达盒的靶向质粒(pGRNA–bdhB–SEQ ID NO：105)以及含有允许单独缺失bdhB基因(pGRNA–ΔbdhB–SEQ ID NO：79)或bdhA和bdhB两个基因(pGRNA–ΔbdhAΔbdhB–SEQ ID NO：80)的修复模板的两种衍生质粒转化到含有pCas9ind(SEQ IDNO：22)或pCas9acr(SEQ ID NO：23)的DSM 792菌株中。得到的转化效率呈现在表2中：
[表2]
Figure BDA0003160802810000371
靶向bdhB的质粒对含有pCas9ind或pCas9acr的菌株DSM 792的转化频率。频率被表示为在转化中使用的每μg DNA得到的转化体的数目，并代表至少两个独立实验的平均值。
所述得到的转化体通过在增补有无水四环素aTc的培养基上传代，经历CRISPR/Cas9系统表达的诱导阶段(图22)。
所需变化通过对来自于两个aTc抗性菌落的基因组DNA进行PCR得以确认(图23)。
结论
在Wasels等(2017)中描述的基于CRISPR/Cas9的遗传工具使用两个质粒：
–第一质粒pCas9ind含有在aTc诱导型启动子控制之下的cas9，和
–源自于pEC750C的第二质粒含有gRNA表达盒(在第二个aTc诱导型启动子的控制之下)和编辑模板，以修复由所述系统诱导的双链断裂。
然而，本发明人观察到某些gRNA仍显得毒性过高，尽管它们的表达以及Cas9的表达已使用aTc诱导型启动子进行控制，因此限制了通过所述遗传工具进行细菌转化和因此染色体修饰的效率。
为了改进这种遗传工具，通过插入在乳糖诱导型启动子控制之下的抗CRISPR基因acrIIA4，对cas9表达质粒进行修饰。由此显著提高了不同gRNA表达质粒的转化效率，允许获得所有测试的质粒的转化体。
也可以使用不能被引入到含有pCas9ind的DSM 792菌株中的质粒，对丙酮丁醇梭菌DSM 792基因组内的bdhB基因座进行编辑。观察到的修饰效率与以前观察到的(Wasels等，2017)相同，其中100％的测试的菌落被修饰。
总而言之，cas9表达质粒的修饰允许更好地控制Cas9–gRNA核糖核蛋白复合体，有利地便于获得其中Cas9的作用可以被触发的转化体，以便获得感兴趣的突变体。
实施例2
材料和方法
培养条件
拜氏梭菌DSM 6423生长在2YTG培养基(胰蛋白胨16g.L–1，酵母提取物10g.L–1，葡萄糖5g.L–1，NaCl 4g.L–1)中。大肠埃希氏杆菌NEB 10–β和INV110生长在LB培养基(胰蛋白胨10g.L–1，酵母提取物5g.L–1，NaCl 5g.L–1)中。固体培养基通过向液体培养基添加15g.l–1琼脂来制造。在需要时使用红霉素(在2YTG或LB培养基中浓度分别为20或500mg.L–1)、氯霉素(在固体或液体LB培养基中分别为25或12.5mg.L–1)和甲砜霉素(在2YTG培养基中15mg.L–1)或壮观霉素(在LB或2YTG培养基中浓度分别为100或650mg.L–1)。
核酸和质粒载体
所有酶和试剂盒按照制造商的推荐使用。
菌落PCR试验遵照下述方案：
将拜氏梭菌DSM 6423的分离的菌落重悬浮在100μL 10mM Tris pH 7.5，5mM EDTA中。将该溶液在不搅拌的情况下在98℃加热10min。然后将0.5μL该细菌裂解液作为PCR模板，用于使用Phire(Thermo Scientific)、Phusion(Thermo Scientific)、Q5(NEB)或KAPA2G Robust(Sigma–Aldrich)聚合酶的10μL反应中。
下面详述了在所有构建物中使用的引物的名单(名称/DNA序列)：
ΔcatB_fwd：TGTTATGGATTATAAGCGGCTCGAGGACGTCAAACCATGTTAATCATTGC
ΔcatB_rev：AATCTATCACTGATAGGGACTCGAGCAATTTCACCAAAGAATTCGCTAGC
ΔcatB_gRNA_rev：AATCTATCACTGATAGGGACTCGAGGGGCAAAAGTGTAAAGACAAGCTTC
RH076：CATATAATAAAAGGAAACCTCTTGATCG
RH077：ATTGCCAGCCTAACACTTGG
RH001：ATCTCCATGGACGCGTGACGTCGACATAAGGTACCAGGAATTAGAGCAGC
RH002：TCTATCTCCAGCTCTAGACCATTATTATTCCTCCAAGTTTGCT
RH003：ATAATGGTCTAGAGCTGGAGATAGATTATTTGGTACTAAG
RH004：TATGACCATGATTACGAATTCGAGCTCGAAGCGCTTATTATTGCATTAGC
pEX–fwd：CAGATTGTACTGAGAGTGCACC
pEX–rev：GTGAGCGGATAACAATTTCACAC
pEC750C–fwd：CAATATTCCACAATATTATATTATAAGCTAGC
M13–rev：CAGGAAACAGCTATGAC
RH010：CGGATATTGCATTACCAGTAGC
RH011：TTATCAATCTCTTACACATGGAGC
RH025：TAGTATGCCGCCATTATTACGACA
RH134：GTCGACGTGGAATTGTGAGC
pNF2_fwd：GGGCGCACTTATACACCACC
pNF2_rev：TGCTACGCACCCCCTAAAGG
RH021：ACTTGGGTCGACCACGATAAAACAAGGTTTTAAGG
RH022：TACCAGGGATCCGTATTAATGTAACTATGATATCAATTCTTG
aad9–fwd2：ATGCATGGTCCCAATGAATAGGTTTACACTTACTTTAG TTTTATGG
aad9–rev：ATGCGAGTTAACAACTTCTAAAATCTGATTACCAATTAGRH031：ATGCATGGATCCCAATGAATAGGTTTACACTTACTTTAGTTTTATGG
RH032：ATGCGAGAGCTCAACTTCTAAAATCTGATTACCAATTAG
RH138：ATGCATGGATCCGTCTGACAGTTACCAGGTCC
RH139：ATGCGAGAGCTCCAATTGTTCAAAAAAATAATGGCGGAG
RH140：ATGCATGGATCCCGGCAGTTTTTCTTTTTCGG
RH141：ATGCGAGAGCTCGGTTAAATACTAGTTTTTAGTTACAGAC
制备了下述9种质粒载体：
–1号质粒：pEX–A258–ΔcatB(SEQ ID NO：17)
它含有被克隆到质粒pEX–A258中的合成的DNA片段ΔcatB。该ΔcatB片段包含i)在无水四环素诱导型启动子控制之下的靶向拜氏梭菌DSM6423的catB基因(编码氯霉素–O–乙酰转移酶的氯霉素抗性基因–SEQ ID NO：18)的向导RNA表达盒(表达盒：SEQ ID NO：19)，和ii)包含位于catB基因的上游和下游的400个同源bp的编辑模板(SEQ ID NO：20)。
–2号质粒：pCas9ind–ΔcatB(参见图2和SEQ ID NO：21)
它含有通过PCR扩增(引物ΔcatB_fwd和ΔcatB_rev)并在用XhoI限制性酶消化各个DNA后克隆到pCas9ind(描述在专利申请WO2017/064439中–SEQ ID NO：22)中的ΔcatB片段。
–3号质粒：pCas9acr(参见图3和SEQ ID NO：23)
–4号质粒：pEC750S–uppHR(参见图4和SEQ ID NO：24)
它含有用于缺失upp基因并由在upp基因上游和下游的两个同源DNA片段(尺寸分别为：500(SEQ ID NO：26)个和377(SEQ ID NO：27)个碱基对)构成的修复模板(SEQ ID NO：25)。所述组装体使用Gibson克隆系统(New England Biolabs，Gibson组装主混合物2X)来获得。为此目的，使用相应的引物RH001/RH002和RH003/RH004，从菌株DSM 6423的基因组DNA(参见Matéde Gerando等，2018和登记号PRJEB11626(https://www.ebi.ac.uk/ena/data/view/PRJEB11626))通过PCR来扩增所述上游和下游部分。然后将这两个片段组装到之前通过限制性酶(SalI和SacI限制性酶)线性化的pEC750S中。
–5号质粒：pEX–A2–gRNA–upp(参见图5和SEQ ID NO：28)
这个质粒包含gRNA–upp DNA片段，其对应于插入到名为pEX–A2的复制质粒中的在组成型启动子(序列SEQ ID NO：30的非编码RNA)控制之下的靶向upp基因的向导RNA(靶向upp的前间区序列(SEQ ID NO：31))的表达盒(SEQ ID NO：29)。
–6号质粒：pEC750S–Δupp(参见图6和SEQ ID NO：32)
它具有质粒pEC750S–uppHR(SEQ ID NO：24)作为基础，并另外含有包含在组成型启动子控制之下的靶向upp基因的向导RNA表达盒的DNA片段。
将这个片段插入到pEX–A2中，被命名为pEX–A2–gRNA–upp。然后将所述插入片段用引物pEX–fwd和pEX–rev通过PCR进行扩增，并用限制性酶XhoI和NcoI消化。最后，通过连接到先前用相同的限制性酶消化的pEC750S–uppHR中将该片段进行克隆，以获得pEC750S–Δupp。
–7号质粒：pEC750C–Δupp(参见图7和SEQ ID NO：33)
然后用引物pEC750C–fwd和M13–rev扩增含有所述向导RNA以及修复模板的表达盒。将所述扩增子用限制性酶XhoI和SacI消化，然后通过酶法连接到pEC750C中进行克隆，以获得pEC750C–Δupp。
–8号质粒：pGRNA–pNF2(参见图8和SEQ ID NO：34)
该质粒具有pEC750C作为其基础，并含有靶向pNF2质粒的向导RNA表达盒(SEQ IDNO：118)。
–9号质粒：pCas9ind–gRNA_catB(参见图16和SEQ ID NO：38)
它含有通过PCR扩增(引物ΔcatB_fwd和ΔcatB_gRN A_rev)的靶向catB基因座的向导RNA的编码序列，并将所述编码序列在用限制性酶XhoI消化各个DNA并连接后克隆到pCas9ind(描述在专利申请WO2017/064439中)中。
–10号质粒：pNF3(参见图25和SEQ ID NO：119)
它含有使用引物RH021和RH022扩增的pNF2的一部分，具体来说包括复制原点和编码质粒复制蛋白(CIBE_p20001)的基因。然后将该PCR产物在SalI和BamHI限制性位点处克隆到质粒pUC19(SEQ ID NO：117)中。
–11号质粒：pEC751S(参见图26和SEQ ID NO：121)
它含有pEC750C(SEQ ID NO：106)的除了catP氯霉素抗性基因(SEQ ID NO：70)之外的所有元件。后者被提供壮观霉素抗性的粪肠球菌(Enterococcus faecalis)aad9基因(SEQ ID NO：130)代替。这个元件使用引物aad9–fwd2和aad9–rev从质粒pMTL007S–E1(SEQID NO：120)扩增，并克隆到pEC750C的AvaII和HpaI位点中代替catP基因(SEQ ID NO：70)。
–12号质粒：pNF3S(参见图27和SEQ ID NO：123)
它含有pNF3的所有元件，并在BamHI与SacI位点之间插入aad9基因(使用引物RH031和RH032从pEC751S扩增)。
–13号质粒：pNF3E(参见图28和SEQ ID NO：124)
它含有pNF3的所有元件，并插入在miniPthl启动子控制之下的艰难梭菌ermB基因(SEQ ID NO：131)。该元件使用引物RH138和RH139从pFW01扩增，并克隆在pNF3E的BamHI与SacI位点之间。
–14号质粒：pNF3C(参见图29和SEQ ID NO：125)
它含有pNF3的所有元件并插入有产气荚膜梭菌catP基因(SEQ ID NO：70)。该元件使用引物RH140和RH141从pEC750C扩增并克隆在pNF3E的BamHI与SacI位点之间。
结果1
拜氏梭菌菌株DSM 6423的转化
将质粒引入到大肠埃希氏杆菌dam–dcm–菌株(INV110，Invitrogen)中并复制。这允许除去pCas9ind–ΔcatB质粒上的Dam–和Dcm–型甲基化，然后按照Mermelstein等(1993)描述的方案将其通过转化引入到菌株DSM 6423中，并对所述方案做出了下述修改：将菌株在OD600为0.8时用更大量的质粒(20μg)并使用下述电穿孔参数进行转化：100Ω，25μF，1400V。铺展在含有红霉素(20μg/mL)的皮氏培养皿上，由此得到含有pCas9ind–ΔcatB质粒的拜氏梭菌DSM 6423转化体。
cas9表达的诱导和获得拜氏梭菌菌株DSM 6423ΔcatB
然后将几个红霉素抗性菌落转移到100μL培养基(2YTG)中，并在培养基中连续稀释到104的稀释倍数。对于每个菌落，将8μL每种稀释液置于含有红霉素和无水四环素(200ng/mL)的皮氏培养皿上，以诱导Cas9核酸酶基因的表达。
在提取基因组DNA后，在该平板上生长的克隆中catB基因的缺失通过PCR，使用引物RH076和RH077来验证(参见图9)。
拜氏梭菌菌株DSM 6423ΔcatB对甲砜霉素的敏感性的验证
为了确保catB基因的缺失提供对甲砜霉素的新的敏感性，在琼脂培养基上进行了比较分析。拜氏梭菌DSM 6423和拜氏梭菌DSM 6423ΔcatB的预培养物生长在2YTG培养基上，然后将100μL这些预培养物在增补有浓度为15mg/L的甲砜霉素或不含甲砜霉素的2YTG琼脂培养基上铺板。图10显示只有初始的拜氏梭菌DSM 6423菌株能够在增补甲砜霉素的培养基上生长。
在拜氏梭菌菌株DSM 6423ΔcatB中通过CRISPR–Cas9工具缺失upp基因
将拜氏梭菌菌株DSM 6423ΔcatB的一个克隆预先用在dam–和dcm–型甲基转移酶识别的基序处不表现出甲基化的pCas9acr载体(从具有dam–dcm–基因型的大肠埃希氏杆菌细菌制备)转化。维持在拜氏梭菌菌株DSM 6423中的质粒pCas9acr的存在，通过使用引物RH025和RH134的菌落PCR来验证。
然后将红霉素抗性克隆用先前去甲基化的pEC750C–Δupp转化。将得到的菌落在含有红霉素(20μg/mL)、甲砜霉素(15μg/mL)和乳糖(40mM)的培养基上选择。
然后将这些克隆中的几个重悬浮在100μL培养基(2YTG)中，并在培养基中连续稀释(至104的稀释倍数)。将5μL每种稀释液置于含有红霉素、甲砜霉素和无水四环素(200ng/mL)的皮氏培养皿上(参见图11)。
对于每个克隆来说，使用被设计用于扩增upp基因座的引物，通过菌落PCR测试两个aTc抗性菌落(参见图12)。
在拜氏梭菌菌株DSM 6423ΔcatB中通过CRISPR–Cas9工具缺失天然质粒pNF2
将拜氏梭菌菌株DSM 6423ΔcatB的一个克隆预先用在Dam–和Dcm–型甲基转移酶识别的基序处不表现出甲基化的pCas9ind载体(从具有dam–dcm–基因型的大肠埃希氏杆菌细菌制备)转化。拜氏梭菌菌株DSM 6423中质粒pCas9ind的存在，使用引物pCas9ind_fwd(SEQID NO：42)和pCas9ind_rev(SEQ ID NO：43)通过PCR来验证(参见图13)。
然后将红霉素抗性克隆用于转化从具有dam–dcm–基因型的大肠埃希氏杆菌细菌制备的pGRNA–pNF2。
将在含有红霉素(20μg/mL)和甲砜霉素(15μg/mL)的培养基上获得的几个克隆重悬浮在培养基中并连续稀释至104的稀释倍数。将8μL每种稀释液置于含有红霉素、甲砜霉素和无水四环素(200ng/mL)的皮氏培养皿上，以诱导CRISPR/Cas9表达。
天然质粒pNF2的不存在使用引物pNF2_fwd(SEQ ID NO：39)和pNF2_rev(SEQ IDNO：40)，通过PCR来验证(参见图14)。
结论
在本工作的过程中，本发明人成功地在拜氏梭菌菌株DSM 6423中引入并维持了不同的质粒。他们成功地在使用单个质粒的基础上使用CRISPR–Cas9工具缺失了catB基因。得到的重组菌株对甲砜霉素的敏感性通过琼脂试验得以确认。
这种缺失允许他们使用在专利申请FR1854835中描述的需要两个质粒的CRISPR–Cas9工具。进行了演示本申请的重要性的两个实例：upp基因的缺失和对拜氏梭菌菌株DSM6423来说非必需的天然质粒的移除。
结果2
拜氏梭菌菌株的转化
在大肠埃希氏杆菌菌株NEB 10–β中制备的质粒也被用于转化拜氏梭菌菌株NCIMB8052。相反，对于拜氏梭菌DSM 6423来说，将质粒预先引入到大肠埃希氏杆菌dam–dcm–菌株(INV110，Invitrogen)中并复制。这允许在将感兴趣的质粒通过转化引入到菌株DSM 6423中之前移除所述质粒上的Dam–和Dcm–型甲基化。
对于每种菌株来说转化类似地进行，即按照Mermelstein等，1992描述的方案并进行了下述修改：将菌株在OD600为0.6–0.8时用更大量的质粒(5–20μg)转化，并且电穿孔参数为100Ω、25μF、1400V。在2YTG中再生3h后，将细菌在含有所需抗生素(红霉素：20–40μg/mL；甲砜霉素：15μg/mL；壮观霉素：650μg/mL)的皮氏培养皿(2YTG琼脂)上铺板。
拜氏梭菌DSM 6423菌株的转化效率的比较
转化在下述拜氏梭菌菌株中以两份生物平行样进行：DSM 6423野生型，DSM 6423ΔcatB和DSM 6423ΔcatBΔpNF2(图30)。为此使用了pCas9ind载体，所述载体由于不允许良好的转化效率而尤其难以用于修饰细菌。它还含有提供红霉素抗性的基因，所有三种菌株都对红霉素敏感。
结果表明转化效率提高约15-20倍，这可归因于天然质粒pNF2的丧失。
也测试了提供甲砜霉素抗性的质粒pEC750C的转化效率，但仅仅在DSM 6423ΔcatB(IFP962ΔcatB)和DSM 6423ΔcatBΔpNF2(IFP963ΔcatBΔpNF2)菌株中，因为野生型菌株对这种抗生素有抗性(图31)。对于这个质粒来说，转化效率的提高甚至更加显著(提高约2000倍)。
pNF3质粒与其他质粒的转化效率的比较
为了确定含有天然质粒pNF2的复制原点的质粒的转化效率，将质粒pNF3E和pNF3C引入到拜氏梭菌菌株DSM 6423ΔcatBΔpNF2中。含有红霉素或氯霉素抗性基因的载体的使用允许在抗性基因的本性的基础上比较载体的转化效率。也转化了质粒pFW01和pEC750C。这两种质粒含有针对不同抗生素(分别为红霉素和甲砜霉素)的抗性基因，并且常用于转化拜氏梭菌和丙酮丁醇梭菌。
正如在图32中所示，基于pNF3的载体显示出出色的转化效率，并且特别地可用于拜氏梭菌DSM 6423ΔcatBΔpNF2。具体来说，pNF3E(其含有红霉素抗性基因)与包含相同抗性基因的pFW01相比显示出明显更高的转化效率。该同一种质粒不能被引入到野生型拜氏梭菌DSM 6423菌株中(在两份生物平行样中使用5μg转化质粒获得0个菌落)，证实了存在天然质粒pNF2的影响。
pNF3质粒在其他菌株/菌种中的可转化性的验证
为了说明在其他产溶剂梭菌属菌株中使用这种新质粒的可能性，本发明人在ABE菌株拜氏梭菌NCIMB 8052中进行了质粒pFW01、pNF3E和pNF3S的转化效率的比较性分析(图33)。由于NCIMB 8052菌株天然对甲砜霉素具有抗性，因此使用提供壮观霉素抗性的pNF3S代替pNF3C。
结果证实了NCIMB 8052菌株可以用基于pNF3的质粒转化，证明了这些载体适用于广泛意义上的拜氏梭菌菌种。
也在来自于丙酮丁醇梭菌的参比菌株DSM 792中测试了所述基于pNF3的合成载体套装的适用性。转化测定法显示了用pNF3C质粒转化这种菌株的可能性(转化效率为每μg转化的DNA观察到3个菌落，相比于pEC750C质粒的120个菌落/μg)。
pNF3质粒与申请FR18/73492中描述的遗传工具的相容性的验证
专利申请FR18/73492描述了ΔcatB菌株以及需要使用红霉素抗性基因和甲砜霉素抗性基因的双质粒CRISPR/Cas9系统的用途。为了证实所述新的pNF3质粒套装的价值，将pNF3C载体转化到已含有pCas9acr质粒的ΔcatB菌株中。所述在两份平行样中进行的转化显示出转化效率为0.625±0.125个菌落/μg DNA(平均值±标准误差)，证实了在ΔcatB菌株中基于pNF3C的载体可以与pCas9acr相组合使用。
与这些结果相平行，所述pNF2质粒的包括其复制原点(SEQ ID NO：118)的部分可以成功地重新用于产生新的穿梭载体套装(SEQ ID NO：119、123、124和125)，它们可以被任意修饰，以特别是允许它们在大肠埃希氏杆菌菌株中的复制以及它们在拜氏梭菌DSM 6423中的重新引入。这些新的载体表现出有利的转化效率，以在例如拜氏梭菌DSM 6423及其衍生株中进行基因编辑，特别是使用包含两种不同核酸的CRISPR/Cas9工具进行基因编辑。
这些新的载体也已成功地在另一株拜氏梭菌(NCIMB 8052)和梭菌属菌种(特别是丙酮丁醇梭菌)中试验，证实了它们在厚壁菌门的其他生物体中的适用性。也在芽孢杆菌属中进行了试验。
结论
这些结果证实，天然质粒pNF2的缺失显著提高了含有它的细菌的转化效率(对于pFW01来说提高约15倍，对于pEC750C来说提高约2000倍)。在已知难以转化的梭菌属细菌的情况下，特别是对于天然受制于低转化效率(低于5个菌落/μg质粒)的菌株拜氏梭菌DSM6423来说，这个结果是特别令人感兴趣的。
参考文献
–Banerjee,A.,Leang,C.,Ueki,T.,Nevin,K.P.,&Lovley,D.R.(2014)，用于扬氏梭菌的代谢工程的乳糖诱导型系统(Lactose–inducible system for metabolicengineering of Clostridium ljungdahlii)，Applied and environmentalmicrobiology,80(8),2410–2416.
–Chen J.–S.,Hiu S.F.(1986)，通过拜氏梭菌(同义词，丁醇梭菌)生产丙酮–丁醇–异丙醇(Acetone–butanol–isopropanol production by Clostridium beijerinckii(synonym,Clostridium butylicum))，Biotechnol.Lett.8:371–376.
–Cui,L.,&Bikard,D.(2016)，大肠埃希氏杆菌染色体中Cas9切割的后果(Consequences of Cas9 cleavage in the chromosome of Escherichia coli)，Nucleicacids research,44(9),4243–4251.
–Currie,D.H.,Herring,C.D.,Guss,A.M.,Olson,D.G.,Hogsett,D.A.,&Lynd,L.R.(2013)，来自于热纤维梭菌的工程化全长CipA在解糖嗜热厌氧杆菌中的功能性异源表达(Functional heterologous expression of an engineered full length CipA fromClostridium thermocellum in Thermoanaerobacterium saccharolyticum)，Biotechnology for biofuels,6(1),32.
–DiCarlo,J.E.,Norville,J.E.,Mali,P.,Rios,X.,Aach,J.,&Church,G.M.(2013)，酿酒酵母中使用CRISPR–Cas系统的基因组工程(Genome engineering inSaccharomyces cerevisiae using CRISPR–Cas systems)，Nucleic acids research,41(7),4336–4343.
–Dong,H.,Tao,W.,Zhang,Y.,&Li,Y.(2012)，用于产溶剂丙酮丁醇梭菌的无水四环素诱导型基因表达系统的开发：一种用于菌株工程的有用工具(Development of ananhydrotetracycline–inducible gene expression system for solvent–producingClostridium acetobutylicum:Auseful tool for strain engineering)，Metabolicengineering,14(1),59–67.
–Dong,D.,Guo,M.,Wang,S.,Zhu,Y.,Wang,S.,Xiong,Z.,...&Huang,Z.(2017)，CRISPR–SpyCas9被抗CRISPR蛋白抑制的结构基础(Structural basis of CRISPR–SpyCas9inhibition by an anti–CRISPR protein)，Nature,546(7658),436.
–Dupuy,B.,Mani,N.,Katayama,S.,&Sonenshein,A.L.(2005)，UV诱导型产气荚膜梭菌细菌素基因被新的σ因子的转录激活(Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novelσfactor)，Molecular microbiology,55(4),1196–1206.
–Egholm,M.,Buchardt,O.,Nielsen,P.E.,&Berg,R.H.(1992)，肽核酸(PNA)，具有非手性肽骨架的寡核苷酸类似物(Peptide nucleic acids(PNA).Oligonucleotideanalogs with an achiral peptide backbone)，Journal of the American ChemicalSociety,114(5),1895–1897.
–Fonfara,I.,Le Rhun,A.,Chylinski,K.,Makarova,K.S.,Lecrivain,A.L.,Bzdrenga,J.,...&Charpentier,E.(2013)，Cas9的系统发育决定了双重RNA和Cas9在直系同源II型CRISPR-Cas系统之间的功能互换性(Phylogeny of Cas9 determinesfunctional exchangeability of dual–RNA and Cas9 among orthologous type IICRISPR–Cas systems)，Nucleic acids research,42(4),2577–2590.
–Garcia–Doval C,Jinek M.，2类CRISPR相关核酸酶的分子体系结构和机制(Molecular architectures and mechanisms of Class 2CRISPR–associatednucleases)，Curr Opin Struct Biol.2017Dec；47:157–166.doi:10.1016/j.sbi.2017.10.015Ajouter au projet Citavi par DOI.Epub 2017年11月3日.综述。
–George H.A.,Johnson J.L.,Moore W.E.C.,Holdeman,L.V.,Chen J.S.(1983)，通过拜氏梭菌(同义词，丁醇梭菌)和金黄丁酸梭菌生产丙酮、异丙醇和丁醇(Acetone,Isopropanol,and Butanol Production by Clostridium beijerinckii(syn.Clostridium butylicum)and Clostridium aurantibutyricum)，Appl.Env.Microbiol.45:1160–1163.
–Gonzales y Tucker RD,Frazee B，来自于第一线的观点：注射吸毒者中梭菌感染的急诊医学展望(View from the front lines:an emergency medicine perspectiveon clostridial infections in injection drug users)，Anaerobe.2014年12月；30:108–15.
–Hartman,A.H.,Liu,H.,&Melville,S.B.(2011)，用于产气荚膜梭菌中的受控基因表达的乳糖诱导型启动子系统的构建和表征(Construction and characterization ofa lactose–inducible promoter system for controlled gene expression inClostridium perfringens)，Applied and environmental microbiology,77(2),471–478.
–Heap,J.T.,Ehsaan,M.,Cooksley,C.M.,Ng,Y.K.,Cartman,S.T.,Winzer,K.,&Minton,N.P.(2012)，来自于不含反选择标记物的质粒的DNA在细菌染色体中的整合(Integration of DNA into bacterial chromosomes from plasmids without acounter–selection marker)，Nucleic acids research,40(8),e59–e59.
–Heap,J.T.,Kuehne,S.A.,Ehsaan,M.,Cartman,S.T.,Cooksley,C.M.,Scott,J.C.,&Minton,N.P.(2010)，ClosTron：梭菌属中精炼且简化的诱变(The ClosTron:mutagenesis in Clostridium refined and streamlined)，Journal ofmicrobiological methods,80(1),49–55.
–Heap,J.T.,Pennington,O.J.,Cartman,S.T.,Carter,G.P.,&Minton,N.P.(2007)，ClosTron：用于梭菌属的通用基因敲除系统(The ClosTron:a universal geneknock–out system for the genus Clostridium)，Journal of microbiologicalmethods,70(3),452–464.
–Heap,J.T.,Pennington,O.J.,Cartman,S.T.,&Minton,N.P.(2009)，用于梭菌穿梭质粒的模块化系统(A modular system for Clostridium shuttle plasmids)，Journalof microbiological methods,78(1),79–85.
–Hidalgo–Cantabrana,C.,O’Flaherty,S.,&Barrangou,R.(2017)，下一代乳酸细菌的基于CRISPR的工程化(CRISPR–based engineering of next–generation lacticacid bacteria)，Current opinion in microbiology,37,79–87.
–Hiu S.F.,Zhu C.–X.,Yan R.–T.,Chen J.–S.(1987)，丁醇–乙醇脱氢酶和丁醇–乙醇–异丙醇脱氢酶：两株拜氏梭菌(丁醇梭菌)菌株中的不同醇脱氢酶(Butanol–ethanoldehydrogenase and butanol–ethanol–isopropanol dehydrogenase:different alcoholdehydrogenases in two strains of Clostridium beijerinckii(Clostridiumbutylicum))，Appl.Env.Microbiol.53:697–703.
–Huang,H.,Chai,C.,Li,N.,Rowe,P.,Minton,N.P.,Yang,S.,&Gu,Y.(2016)，一种自养气体发酵细菌扬氏梭菌中的基于CRISPR/Cas9的高效基因组编辑(CRISPR/Cas9–basedefficient genome editing in Clostridium ljungdahlii,an autotrophic gas–fermenting bacterium)，ACS synthetic biology,5(12),1355–1361.
–Huggins,A.S.,Bannam,T.L.和Rood,J.I.(1992)，来自于丁酸梭菌的catB基因的比较性序列分析(Comparative sequence analysis of the catB gene fromClostridium butyricum)，Antimicrob.Agents Chemother.36,2548–2551.
–Ismaiel A.A.,Zhu C.X.,Colby G.D.,Chen,J.S.(1993)，来自于两株拜氏梭菌菌株的伯醇-仲醇脱氢酶的纯化和表征(Purification and characterization of aprimary–secondary alcohol dehydrogenase from two strains of Clostridiumbeijerinckii)，J.Bacteriol.175:5097–5105.
–Jinek,M.,Chylinski,K.,Fonfara,I.,Hauer,M.,Doudna,J.A.,&Charpentier,E.(2012)，适应性细菌免疫中的可编程双重RNA指导的DNA核酸内切酶(A programmabledual–RNA–guided DNA endonuclease in adaptive bacterial immunity)，Science,337(6096),816–821.
–Jones D.T.,Woods D.R.(1986)，再论丙酮–丁醇发酵(Acetone–butanolfermentation revisited)，Microbiological Reviews50:484–524.
–Kolek J.,Sedlar K.,Provaznik I.,Patakova P.(2016)，Dam和Dcm甲基化阻止基因转移到巴氏梭菌NRRL B–598中：用于电转化、接合和超声穿孔的方法的开发(Dam andDcm methylations prevent gene transfer into Clostridium pasteurianum NRRL B–598:development of methods for electrotransformation,conjugation,andsonoporation)，Biotechnol Biofuels.9:14.
–Li,Q.,Chen,J.,Minton,N.P.,Zhang,Y.,Wen,Z.,Liu,J.,...&Gu,Y.(2016)，丙酮丁醇梭菌和拜氏梭菌中基于CRISPR的基因组编辑和表达控制系统(CRISPR-basedgenome editing and expression control systems in Clostridium acetobutylicumand Clostridium beijerinckii)，Biotechnology journal,11(7),961–972.
–Makarova,K.S.,Haft,D.H.,Barrangou,R.,Brouns,S.J.,Charpentier,E.,Horvath,P.,...&Van Der Oost,J.(2011)，CRISPR–Cas系统的演变和分类(Evolution andclassification of the CRISPR–Cas systems)，Nature Reviews Microbiology,9(6),467.
–Makarova,K.S.,Wolf,Y.I.,Alkhnbashi,O.S.,Costa,F.,Shah,S.A.,Saunders,S.J.,...&Horvath,P.(2015)，更新的CRISPR–Cas系统的进化分类(An updatedevolutionary classification of CRISPR–Cas systems)，Nature ReviewsMicrobiology,13(11),722.
–Marino,N.D.,Zhang,J.Y.,Borges,A.L.,Sousa,A.A.,Leon,L.M.,Rauch,B.J.,...&Bondy–Denomy,J.(2018)，广泛的I型和V型CRISPR–Cas抑制剂的发现(Discoveryof widespread type I and type V CRISPR–Cas inhibitors)，Science,362(6411),240–242.
–Mátéde Gérando,H.,Wasels,F.,Bisson,A.,Clément,B.,Bidard,F.,JourdierE.,Lopez–Contreras A.,Lopes Ferreira N.(2018)，天然异丙醇产生菌拜氏梭菌DSM6423的基因组和转录组(Genome and transcriptome of the natural isopropanolproducer Clostridium beijerinckii DSM 6423)，BMC genomics.19:242.
–Mearls,E.B.,Olson,D.G.,Herring,C.D.,&Lynd,L.R.(2015)，用于热纤维梭菌的可调控的基于质粒的基因表达系统的开发(Development of a regulatable plasmid–based gene expression system for Clostridium thermocellum)，Appliedmicrobiology and biotechnology,99(18),7589–7599.
–Mermelstein L.D.,Welker N.E.,Bennett G.N.,Papoutsakis E.T.(1993)，克隆的同源发酵基因在丙酮丁醇梭菌ATCC 824中的表达(Expression of clonedhomologous fermentative genes in Clostridium acetobutylicum ATCC 824)，10:190–195.
–Mermelstein L.D.,Welker N.E.,Bennett G.N.,Papoutsakis E.T.(1993)，克隆的同源发酵基因在丙酮丁醇梭菌ATCC 824中的表达(Expression of clonedhomologous fermentative genes in Clostridium acetobutylicum ATCC 824)，10:190–195.
–Moon HG,Jang YS,Cho C,Lee J,Binkley R,Lee SY，梭菌丁醇发酵的一百年(One hundred years of clostridial butanol fermentation)，FEMS MicrobiolLett.2016年2月；363(3).
–Nagaraju,S.,Davies,N.K.,Walker,D.J.F.,
Figure BDA0003160802810000551
M.,&Simpson,S.D.(2016)，自养产乙醇梭菌使用CRISPR/Cas9的基因组编辑(Genome editing of Clostridiumautoethanogenum using CRISPR/Cas9)，Biotechnology for biofuels,9(1),219.
–Nariya,H.,Miyata,S.,Kuwahara,T.,&Okabe,A.(2011)，用于产气荚膜梭菌的木糖诱导型基因表达系统的开发和表征(Development and characterization of axylose–inducible gene expression system for Clostridium perfringens)，Appliedand environmental microbiology,77(23),8439–8441.
–Newcomb,M.,Millen,J.,Chen,C.Y.,&Wu,J.D.(2011)，热纤维梭菌中celC基因簇的共转录(Co–transcription of the celC gene cluster in Clostridiumthermocellum)，Applied microbiology and biotechnology,90(2),625–634.
–Pawluk,A.,Davidson,A.R.,&Maxwell,K.L.(2018)，抗CRISPR：发现、机制和功能(Anti–CRISPR:Discovery,mechanism and function)，Nature Reviews Microbiology,16(1),12
–Poehlein A.,Solano J.D.M.,Flitsch S.K.,Krabben P.,Winzer K.,ReidS.J.,Jones D.T.,Green E.,Minton N.P.,Daniel R.,Dürre P.(2017)，通过比较性基因组分析再论微生物溶剂形成(Microbial solvent formation revisited by comparativegenome analysis)，Biotechnol Biofuels.10:58.
–Pyne,M.E.,Bruder,M.R.,Moo–Young,M.,Chung,D.A.,&Chou,C.P.(2016)，利用异源和内源CRISPR-Cas机器在梭菌中进行高效无标记基因组编辑(Harnessingheterologous and endogenous CRISPR–Cas machineries for efficient markerlessgenome editing in Clostridium)，Scientific reports,6.
–Rauch,B.J.,Silvis,M.R.,Hultquist,J.F.,Waters,C.S.,McGregor,M.J.,Krogan,N.J.,&Bondy–Denomy,J.(2017)，使用噬菌体蛋白抑制CRISPR–Cas9(Inhibitionof CRISPR–Cas9 with bacteriophage proteins)，Cell,168(1–2),150–158.
–Rajewska M.,Wegrzyn K,Konieczny I.,FEMS Microbiol Rev.2012Mar；36(2)，富含AT的区域和重复序列——细菌复制子的复制原点的必需元件(AT–rich region andrepeated sequences–the essential elements of replication origins of bacterialreplicons):408–34.
–Ransom,E.M.,Ellermeier,C.D.,&Weiss,D.S.(2015)，使用mCherry红色荧光蛋白研究艰难梭菌中的蛋白质定位和基因表达(Use of mCherry red fluorescent proteinfor studies of protein localization and gene expression in Clostridiumdifficile)，Applied and environmental microbiology,81(5),1652–1660.
–Rogers P.,Chen J.–S.,Zidwick M.(2006)，《原核生物》(The prokaryotes)，第3版，第1卷，Dworkin M主编(Springer,美国纽约，2006)，第3版，第1卷,第672–755页.
–Schwarz S,Kehrenberg C,Doublet B,Cloeckaert A.，细菌对氯霉素和氟苯尼考的抗性的分子基础(Molecular basis of bacterial resistance to chloramphénicoland florfenicol)，FEMS Microbiol Rev.2004年11月；28(5):519–42.
–Stella S,Alcón P,Montoya G，2类CRISPR–Cas RNA指导的核酸内切酶：基因组编辑的瑞士军刀(Class 2CRISPR–Cas RNA–guided endonucleases:Swiss Army knivesof genome editing)，Nat Struct Mol Biol.2017年11月；24(11):882–892.doi:10.1038/nsmb.3486
–Wang,S.,Dong,S.,Wang,P.,Tao,Y.,&Wang,Y.(2017)，使用CRISPR–Cas9系统在糖乙酸多丁醇梭菌N1–4中进行基因组编辑(Genome Editing in Clostridiumsaccharoperbutylacetonicum N1–4with the CRISPR–Cas9 System)，Applied andEnvironmental Microbiology,83(10),e00233–17.
–Wang Y,Li X,Milne CB等，使用可移动II类内含子的基因敲除系统(Targetron)的开发和拜氏梭菌中酸生产途径的遗传破坏(Development of a gene knockout systemusing mobile group II introns(Targetron)and genetic disruption of acidproduction pathways in Clostridium beijerinckii)，Appl Environ Microbiol.2013；79(19):5853–63.
–Wang,Y.等，使用CRISPR/Cas9系统在拜氏梭菌中进行无标记染色体基因缺失(Markerless chromosomal gene deletion in Clostridium beijerinckii usingCRISPR/Cas9 system)，J.Biotechnol.2015.200:1–5.
–Wang,Y.,Zhang,Z.T.,Seo,S.O.,Lynn,P.,Lu,T.,Jin,Y.S.,&Blaschek,H.P.(2016)，使用CRISPR–Cas9的细菌基因组编辑：作为实例的拜氏梭菌中的缺失、整合、单核苷酸修饰和所需的“清洁”突变体选择(Bacterial genome editing with CRISPR–Cas9:deletion,Integration,single nucleotide modification,and desirable“clean”mutant selection in Clostridium beijerinckii as an example)，ACS syntheticbiology,5(7),721–732.
–Wasels,F.,Jean–Marie,J.,Collas,F.,López–Contreras,A.M.,&Ferreira,N.L.(2017)，用于丙酮丁醇梭菌的双质粒诱导型CRISPR/Cas9基因组编辑工具(A two–plasmid inducible CRISPR/Cas9 genome editing tool for Clostridiumacetobutylicum)，Journal of microbiological methods.140:5–11.
–Xu,T.,Li,Y.,Shi,Z.,Hemme,C.L.,Li,Y.,Zhu,Y.,...&Zhou,J.(2015)，通过CRISPR–Cas9切口酶在解纤维梭菌中进行高效基因组编辑(Efficient genome editing inClostridium cellulolyticum via CRISPR–Cas9 nickase)，Applied and environmentalmicrobiology,81(13),4423–4431.
–Yadav,R.,Kumar,V.,Baweja,M.,&Shukla,P.(2018)，用于先进益生菌的基因编辑和遗传工程方法：综述(Gene editing and genetic engineering approaches foradvanced probiotics:A Review)，Critical reviews in food science and nutrition,58(10),1735–1746.
–Yue Chen,Bruce A.McClane,Derek J.Fisher,Julian I.Rood,PhalguniGupta，使用可移动II类内含子构建A型产气荚膜梭菌的α毒素基因敲除突变体(Construction of an Alpha Toxin Gene Knockout Mutant of Clostridiumperfringens Type A by Use of a Mobile Group II Intron)，Appl.Environ.Microbiol.Nov 2005,71(11)7542–7547；DOI:10.1128/AEM.71.11.7542–7547.2005.
–Zhang,J.,Liu,Y.J.,Cui,G.Z.,&Cui,Q.(2015)，一种为解纤维梭菌开发的新的阿拉伯糖诱导型遗传操作系统(A novel arabinose–inducible genetic operationsystem developed for Clostridium cellulolyticum)，Biotechnology for biofuels,8(1),36.
–Zhang C.,Tinggang L.Jianzhong H.(2018)，产丁醇–异丙醇的拜氏梭菌菌株BGS1的表征和基因组分析(Characterization and genome analysis of a butanol–isopropanol–producing Clostridium beijerinckii strain BGS1)，BiotechnolBiofuels(2018)11:280.
–Zhong,J.,Karberg,M.,&Lambowitz,A.M.(2003)，使用含有反转录转座激活的可选择标记的II类内含子(targetron)载体进行定点和随机细菌基因破坏(Targeted andrandom bacterial gene disruption using a group II intron(targetron)vectorcontaining a retrotransposition-activated selectable marker)，Nucleic acidsresearch,31(6),1656–1664.
序列表
<110> IFP新能源公司（IFP Energies nouvelles）
<120> 遗传修饰的梭菌属细菌、其制备和用途
<130> B2903PC00
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ataatggtct agagctggag atagattatt tggtactaag 40
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tatgaccatg attacgaatt cgagctcgaa gcgcttatta ttgcattagc 50
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cagattgtac tgagagtgca cc 22
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gtgagcggat aacaatttca cac 23
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<212> DNA
<213> 人工序列
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<223> 引物pEC750C-fwd
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caatattcca caatattata ttataagcta gc 32
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<212> DNA
<213> 人工序列
<220>
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<400> 12
caggaaacag ctatgac 17
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cggatattgc attaccagta gc 22
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ttatcaatct cttacacatg gagc 24
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tagtatgccg ccattattac gaca 24
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<220>
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<400> 16
gtcgacgtgg aattgtgagc 20
<210> 17
<211> 3658
<212> DNA
<213> 人工序列
<220>
<223> pEX-A258-ΔcatB
<400> 17
ctcgagctgc agcaaaaaaa gcaccgactc ggtgccactt tttcaagttg ataacggact 60
agccttattt taacttgcta tttctagctc taaaactgtg gtctctcttt tcgttgatgg 120
tggaatgata agggtttgca ccttaatttc tcctattgag aaaatcgtct cttctcagac 180
gtcaaaccat gttaatcatt gcttttatca aaaataggat ccactctatc attgatagag 240
tttgaaactc tatcattgat agagtataat atctttgttc atgtacatca tgctatctgt 300
gagttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa 360
gtggcaccga gtcggtgctt tttttgaagc ttgtctttac acttttgccc attaattttt 420
gagttcctta tttttaggga gcttttatta tttttatcat gaaaatttca taaaatactc 480
ataaactaag gatgtcttca taatcagatt agtactccat tttcaatcca tttaatctgg 540
gaatatgata ttttaattac gtattattta agatatatta acgtgtaata taataccccg 600
caaatattaa ttatcacata catatccccc ctttattggg gcattttttg tacccattat 660
tttagtattg tgcagtactt aaataaaaaa atgccgcaaa ttcattttta ttgaataatg 720
cggtatttct tctattcttt atttttatta ctctataaat aatgtaatca agacatgact 780
atctaaatat atgatatctt aattcataat tcgggcctcc taaaaatttt cgtaattcta 840
ttttagaagg cttttttccg tgacctagcc atttcaatct cctttttaca atgatattta 900
cgctttagtt tattatagca cattctgtaa taccgaacta ttcaattttc agagaccatt 960
ttttattgat tcataactta agaatactac gaattactct aatattttac tttttcttat 1020
ctcttgttat tttaacatcg gaattactac taatattaat ttttattttt ccatccgcat 1080
ttgctccaac atttttttaa ctatactttc cttttgttaa taaattatgt tattgttgaa 1140
caatataaga aaagtgcgta acatttttta ttaaaaataa ttaggtattt ctatctgtgg 1200
ggtaccctcg aggtggcagc tctagagcta gcgaattctt tggtgaaatt gttatccgct 1260
cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg 1320
agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct 1380
gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg 1440
gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc 1500
ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg 1560
aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct 1620
ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca 1680
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct 1740
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc 1800
gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt 1860
tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc 1920
cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc 1980
cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg 2040
gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc 2100
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag 2160
cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga 2220
tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat 2280
tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag 2340
ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat 2400
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc 2460
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat 2520
accgcgcgaa ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag 2580
ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg 2640
ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc 2700
tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca 2760
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg 2820
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc 2880
actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta 2940
ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc 3000
aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg 3060
ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc 3120
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc 3180
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat 3240
actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag 3300
cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc 3360
ccgaaaagtg ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa 3420
taggcgtatc acgaggccct ttcgtctcgc gcgtttcggt gatgacggtg aaaacctctg 3480
acacatgcag ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca 3540
agcccgtcag ggcgcgtcag cgggtgttgg cgggtgtcgg ggctggctta actatgcggc 3600
atcagagcag attgtactga gagtttggca attggtcgac ctcgagggcg cgcccgta 3658
<210> 18
<211> 660
<212> DNA
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 18
atgaatttta atttgataga tattaatcat tggagtagaa agccatactt tgaacattat 60
ttaaacaatg tgaaatgtac ttatagtatg actgccaata tagaaataac tgatttattg 120
tatgaaatta aacttaaaaa tattaaattt tatcctaccc ttatttatat gattgcaact 180
gtggttaata agcataaaga attccgtatt tgttttgatc atgaaggtag tttaggatat 240
tgggatagca tgaatccaag ctatactatt tttcataaag aaaacgaaac attttcaagt 300
atttggacgg aatataacaa aagtttttta cgtttttata gtgattatct tgacgatata 360
aaaaactatg gaaatatcat gaagtttact ccgaaatcaa atgaacctga caatacattt 420
tctgtatcaa gcattccttg ggtgagtttt acaggattta acttgaatgt gtataatgaa 480
ggaacatatt taattcctat ttttactgca ggaaagtatt tcaaacaaga aaataaaata 540
tttattccta tatcaataca agtacatcat gctatctgtg acggttatca tgctagtaga 600
tttattaatg aaatgcaaga attagcattt agttttcaag aatggttaga aaataaataa 660
<210> 19
<211> 160
<212> DNA
<213> 人工序列
<220>
<223> gRNA表达盒
<400> 19
actctatcat tgatagagtt tgaaactcta tcattgatag agtataatat ctttgttcat 60
gtacatcatg ctatctgtga gttttagagc tagaaatagc aagttaaaat aaggctagtc 120
cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt 160
<210> 20
<211> 808
<212> DNA
<213> 人工序列
<220>
<223> 编辑模板
<400> 20
gtctttacac ttttgcccat taatttttga gttccttatt tttagggagc ttttattatt 60
tttatcatga aaatttcata aaatactcat aaactaagga tgtcttcata atcagattag 120
tactccattt tcaatccatt taatctggga atatgatatt ttaattacgt attatttaag 180
atatattaac gtgtaatata ataccccgca aatattaatt atcacataca tatcccccct 240
ttattggggc attttttgta cccattattt tagtattgtg cagtacttaa ataaaaaaat 300
gccgcaaatt catttttatt gaataatgcg gtatttcttc tattctttat ttttattact 360
ctataaataa tgtaatcaag acatgactat ctaaatatat gatatcttaa ttcataattc 420
gggcctccta aaaattttcg taattctatt ttagaaggct tttttccgtg acctagccat 480
ttcaatctcc tttttacaat gatatttacg ctttagttta ttatagcaca ttctgtaata 540
ccgaactatt caattttcag agaccatttt ttattgattc ataacttaag aatactacga 600
attactctaa tattttactt tttcttatct cttgttattt taacatcgga attactacta 660
atattaattt ttatttttcc atccgcattt gctccaacat ttttttaact atactttcct 720
tttgttaata aattatgtta ttgttgaaca atataagaaa agtgcgtaac attttttatt 780
aaaaataatt aggtatttct atctgtgg 808
<210> 21
<211> 9954
<212> DNA
<213> 人工序列
<220>
<223> pCas9ind-ΔcatB
<400> 21
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtatattgat aaaaataata 4200
atagtgggta taattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260
gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa 4320
cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380
ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440
tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta 4500
gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag 4560
aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac 4620
tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg 4680
aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc 4740
cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800
tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860
cttaaaagca gcataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920
tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120
gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180
ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240
acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300
aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360
agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600
agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca 6720
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa 6960
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta 7080
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta 7140
tgagtcccta ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat 7200
tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 7260
agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 7320
aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 7380
taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 7440
aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 7500
caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 7560
taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 7620
tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 7680
ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 7740
tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 7800
atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 7860
agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 7920
agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 7980
agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 8040
atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 8220
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 8280
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700
taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760
tatggattat aagcggctcg aggacgtcaa accatgttaa tcattgcttt tatcaaaaat 8820
aggatccact ctatcattga tagagtttga aactctatca ttgatagagt ataatatctt 8880
tgttcatgta catcatgcta tctgtgagtt ttagagctag aaatagcaag ttaaaataag 8940
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttgtc 9000
tttacacttt tgcccattaa tttttgagtt ccttattttt agggagcttt tattattttt 9060
atcatgaaaa tttcataaaa tactcataaa ctaaggatgt cttcataatc agattagtac 9120
tccattttca atccatttaa tctgggaata tgatatttta attacgtatt atttaagata 9180
tattaacgtg taatataata ccccgcaaat attaattatc acatacatat ccccccttta 9240
ttggggcatt ttttgtaccc attattttag tattgtgcag tacttaaata aaaaaatgcc 9300
gcaaattcat ttttattgaa taatgcggta tttcttctat tctttatttt tattactcta 9360
taaataatgt aatcaagaca tgactatcta aatatatgat atcttaattc ataattcggg 9420
cctcctaaaa attttcgtaa ttctatttta gaaggctttt ttccgtgacc tagccatttc 9480
aatctccttt ttacaatgat atttacgctt tagtttatta tagcacattc tgtaataccg 9540
aactattcaa ttttcagaga ccatttttta ttgattcata acttaagaat actacgaatt 9600
actctaatat tttacttttt cttatctctt gttattttaa catcggaatt actactaata 9660
ttaattttta tttttccatc cgcatttgct ccaacatttt tttaactata ctttcctttt 9720
gttaataaat tatgttattg ttgaacaata taagaaaagt gcgtaacatt ttttattaaa 9780
aataattagg tatttctatc tgtggggtac cctcgaggtg gcagctctag agctagcgaa 9840
ttctttggtg aaattgctcg agtccctatc agtgatagat tgaaactcta tcattgatag 9900
agtataatat ctttgttcat tagagcgata aacttgaatt tgagagggaa cttc 9954
<210> 22
<211> 8874
<212> DNA
<213> 人工序列
<220>
<223> pCas9ind
<400> 22
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtatattgat aaaaataata 4200
atagtgggta taattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260
gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa 4320
cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380
ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440
tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta 4500
gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag 4560
aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac 4620
tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg 4680
aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc 4740
cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800
tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860
cttaaaagca gcataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920
tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120
gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180
ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240
acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300
aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360
agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600
agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca 6720
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa 6960
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta 7080
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta 7140
tgagtcccta ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat 7200
tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 7260
agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 7320
aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 7380
taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 7440
aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 7500
caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 7560
taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 7620
tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 7680
ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 7740
tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 7800
atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 7860
agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 7920
agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 7980
agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 8040
atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 8220
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 8280
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700
taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760
tatggattat aagcggctcg agtccctatc agtgatagat tgaaactcta tcattgatag 8820
agtataatat ctttgttcat tagagcgata aacttgaatt tgagagggaa cttc 8874
<210> 23
<211> 10534
<212> DNA
<213> 人工序列
<220>
<223> pCas9acr
<400> 23
cgaattcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc 60
cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct 120
aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc 180
agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt 240
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 300
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 360
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 420
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 480
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 540
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 600
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 660
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 720
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 780
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 840
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 900
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 960
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 1020
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 1080
tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat 1140
caatctaaag tatatatgag taaacttggt ctgacagtta ccaggtccac tgccgggcct 1200
cttgcgggat caaaagaaaa acgaaatgat acaccaatca gtgcaaaaaa agatataatg 1260
ggagataaga cggttcgtgt tcgtgctgac ttgcaccata tcataaaaat cgaaacagca 1320
aagaatggcg gaaacgtaaa agaagttatg gaaataagac ttagaagcaa acttaagagt 1380
gtgttgatag tgcagtatct taaaattttg tataatagga attgaagtta aattagatgc 1440
taaaaatttg taattaagaa ggagtgatta catgaacaaa aatataaaat attctcaaaa 1500
ctttttaacg agtgaaaaag tactcaacca aataataaaa caattgaatt taaaagaaac 1560
cgataccgtt tacgaaattg gaacaggtaa agggcattta acgacgaaac tggctaaaat 1620
aagtaaacag gtaacgtcta ttgaattaga cagtcatcta ttcaacttat cgtcagaaaa 1680
attaaaactg aatactcgtg tcactttaat tcaccaagat attctacagt ttcaattccc 1740
taacaaacag aggtataaaa ttgttgggag tattccttac catttaagca cacaaattat 1800
taaaaaagtg gtttttgaaa gccatgcgtc tgacatctat ctgattgttg aagaaggatt 1860
ctacaagcgt accttggata ttcaccgaac actagggttg ctcttgcaca ctcaagtctc 1920
gattcagcaa ttgcttaagc tgccagcgga atgctttcat cctaaaccaa aagtaaacag 1980
tgtcttaata aaacttaccc gccataccac agatgttcca gataaatatt ggaagctata 2040
tacgtacttt gtttcaaaat gggtcaatcg agaatatcgt caactgttta ctaaaaatca 2100
gtttcatcaa gcaatgaaac acgccaaagt aaacaattta agtaccgtta cttatgagca 2160
agtattgtct atttttaata gttatctatt atttaacggg aggaaataat tctatgagtc 2220
cctaggcagg cctccgccat tatttttttg aacaattgac aattcatttc ttatttttta 2280
ttaagtgata gtcaaaaggc ataacagtgc tgaatagaaa gaaatttaca gaaaagaaaa 2340
ttatagaatt tagtatgatt aattatactc atttatgaat gtttaattga atacaaaaaa 2400
aaatacttgt tatgtattca attacgggtt aaaatataga caagttgaaa aatttaataa 2460
aaaaataagt cctcagctct tatatattaa gctaccaact tagtatataa gccaaaactt 2520
aaatgtgcta ccaacacatc aagccgttag agaactctat ctatagcaat atttcaaatg 2580
taccgacata caagagaaac attaactata tatattcaat ttatgagatt atcttaacag 2640
atataaatgt aaattgcaat aagtaagatt tagaagttta tagcctttgt gtattggaag 2700
cagtacgcaa aggctttttt atttgataaa aattagaagt atatttattt tttcataatt 2760
aatttatgaa aatgaaaggg ggtgagcaaa gtgacagagg aaagcagtat cttatcaaat 2820
aacaaggtat tagcaatatc attattgact ttagcagtaa acattatgac ttttatagtg 2880
cttgtagcta agtagtacga aagggggagc tttaaaaagc tccttggaat acatagaatt 2940
cataaattaa tttatgaaaa gaagggcgta tatgaaaact tgtaaaaatt gcaaagagtt 3000
tattaaagat actgaaatat gcaaaataca ttcgttgatg attcatgata aaacagtagc 3060
aacctattgc agtaaataca atgagtcaag atgtttacat aaagggaaag tccaatgtat 3120
taattgttca aagatgaacc gatatggatg gtgtgccata aaaatgagat gttttacaga 3180
ggaagaacag aaaaaagaac gtacatgcat taaatattat gcaaggagct ttaaaaaagc 3240
tcatgtaaag aagagtaaaa agaaaaaata atttatttat taatttaata ttgagagtgc 3300
cgacacagta tgcactaaaa aatatatctg tggtgtagtg agccgataca aaaggatagt 3360
cactcgcatt ttcataatac atcttatgtt atgattatgt gtcggtggga cttcacgacg 3420
aaaacccaca ataaaaaaag agttcggggt agggttaagc atagttgagg caactaaaca 3480
atcaagctag gatatgcagt agcagaccgt aaggtcgttg tttaggtgtg ttgtaataca 3540
tacgctatta agatgtaaaa atacggatac caatgaaggg aaaagtataa tttttggatg 3600
tagtttgttt gttcatctat gggcaaacta cgtccaaagc cgtttccaaa tctgctaaaa 3660
agtatatcct ttctaaaatc aaagtcaagt atgaaatcat aaataaagtt taattttgaa 3720
gttattatga tattatgttt ttctattaaa ataaattaag tatatagaat agtttaataa 3780
tagtatatac ttaatgtgat aagtgtctga cagtgtcaca gaaaggatga ttgttatgga 3840
ttataagcgg ctcgagtccc tatcagtgat agattgaaac tctatcattg atagagtata 3900
atatctttgt tcattagagc gataaacttg aatttgagag ggaacttcca tggataaaaa 3960
gtacagtatt ggtctagaca taggaactaa ctctgttggg tgggctgtta taacagatga 4020
atataaagtt ccatcaaaaa aatttaaagt attaggaaac actgatagac attcaataaa 4080
aaaaaacttg ataggtgctt tattattcga ttcaggagag actgctgaag ctacacgttt 4140
aaaaagaaca gctagacgta gatatacaag aagaaaaaat aggatatgtt atcttcaaga 4200
aatttttagt aatgaaatgg caaaagttga tgattcattc tttcacagac tagaagaaag 4260
tttcttagtt gaagaagata agaagcatga aagacaccct atttttggta atatcgtaga 4320
tgaagtagca tatcatgaga agtatccaac tatctatcat ttaagaaaga aattagttga 4380
ttctacagat aaagctgatc tgagattaat atatttagct ttagctcata tgattaaatt 4440
tagaggacat tttttaatag aaggtgattt aaacccagac aacagcgatg tagataaatt 4500
atttatccaa ttagttcaaa cttataatca attattcgaa gagaatccaa ttaatgcaag 4560
tggtgtagac gctaaggcta tattatcagc tagattatca aaatctagaa gattagaaaa 4620
tctaatagct caacttcctg gagaaaagaa aaatggactt tttgggaacc taatagctct 4680
ctcactcgga ctaacaccaa attttaaaag caattttgat cttgctgaag acgcaaagtt 4740
acaactatca aaggatacat acgatgatga tttagataat ttgttagctc aaataggtga 4800
tcaatatgct gatttgtttc ttgcagcaaa aaacttaagt gatgcaattt tactatcaga 4860
tatacttaga gtaaatacag aaataacaaa ggctccttta tcagcaagta tgattaaacg 4920
atatgatgag catcatcaag atttaacatt attaaaggca cttgtaagac aacaattacc 4980
agaaaaatat aaagaaattt tctttgatca atctaaaaat ggatatgctg gatatataga 5040
cggtggagca agtcaagaag agttttataa atttataaag cctattttag aaaaaatgga 5100
tggaactgaa gaattacttg ttaaacttaa cagagaagat ttacttagaa aacaaagaac 5160
ttttgataat ggttcaattc ctcaccaaat tcatttagga gaattacatg ctatactaag 5220
aagacaagaa gatttttatc catttcttaa agataataga gaaaaaattg aaaaaatttt 5280
aacttttaga ataccatatt atgtaggacc acttgcaagg ggaaattcaa gatttgcatg 5340
gatgactaga aaatcagaag aaactataac cccgtggaat tttgaagaag tagtagataa 5400
aggagctagt gctcaatcat ttatagaaag aatgacaaat tttgataaga atcttcctaa 5460
cgaaaaggtt ttgccaaagc atagccttct ttatgagtat tttacagttt ataatgagct 5520
tactaaagta aaatacgtta cagaaggaat gagaaaacca gcatttttgt ctggtgaaca 5580
aaagaaagca atagtagacc tattatttaa aacaaatagg aaggttaccg taaagcaact 5640
taaagaagat tacttcaaaa aaattgaatg ctttgatagt gttgaaatat caggagttga 5700
agatagattt aatgcttcac ttggtacata tcacgatctc ttaaaaatta taaaagataa 5760
ggatttttta gataatgaag aaaatgaaga tattcttgaa gatatagtat taacattgac 5820
actttttgaa gatagagaaa tgatagaaga aagattaaaa acatatgcac atctttttga 5880
tgataaggtt atgaagcaac ttaaaagaag aagatataca ggttggggac gtttgtcaag 5940
aaagctaatt aatggtatta gagataaaca atcaggaaag actattctcg attttcttaa 6000
atcagatgga tttgctaata gaaactttat gcaattaatt catgatgatt ctcttacttt 6060
caaagaggat attcaaaagg ctcaagtttc tggacaaggc gatagcttac acgaacacat 6120
tgctaacctt gcagggagcc ccgctatcaa aaaaggaatt ttacaaacag ttaaagttgt 6180
agatgaactt gttaaagtta tgggaagaca caaacctgag aatatagtta tagaaatggc 6240
cagagaaaat caaacaacac aaaaaggaca aaaaaattct agagagagaa tgaagagaat 6300
tgaagaagga ataaaagagc taggatcaca aatattaaaa gaacatccag ttgaaaatac 6360
tcaattgcaa aatgaaaagt tatatttgta ttacttacaa aatggaagag atatgtatgt 6420
tgatcaagaa ctcgatatta atagattaag tgactatgat gttgatcata ttgttcctca 6480
atcattttta aaagatgatt caatcgataa caaagtatta actagatcag ataaaaatag 6540
aggaaagtca gataatgtac catctgaaga agttgttaaa aaaatgaaga actattggag 6600
acaactttta aatgcaaagc taattacaca aagaaaattt gacaatttaa caaaagcaga 6660
aagaggagga ttaagcgaat tagacaaagc tggatttata aaaagacaac ttgttgagac 6720
aagacaaata actaagcatg ttgctcaaat acttgattca agaatgaata caaaatatga 6780
tgaaaatgat aaattaatca gagaagtaaa agtaataaca ttaaagtcaa aattagtatc 6840
agatttcaga aaggattttc aattttacaa agttcgtgaa ataaataact atcatcatgc 6900
tcatgatgca tacttaaatg ctgttgtagg aactgctctt attaagaaat atcctaaact 6960
agaaagcgaa tttgtttatg gagattataa agtttatgat gtgcgcaaaa tgatcgcgaa 7020
atccgaacaa gaaatcggta aggctacagc aaaatatttc ttttatagta atataatgaa 7080
tttttttaag acagaaataa ctttggctaa tggtgaaatc agaaaaagac cacttatcga 7140
aacaaatgga gagacaggag aaatagtatg ggataaagga agagattttg ctactgttag 7200
aaaagtacta agtatgccac aagtaaatat cgtaaagaaa actgaagttc aaactggagg 7260
tttctctaag gaatcaattt tacctaagag aaattcagat aagttaattg caaggaaaaa 7320
agattgggac ccaaaaaaat acggtggttt tgatagtcca acagttgcct atagtgttct 7380
tgtagtagcg aaagttgaga aaggtaagtc aaaaaagttg aaaagcgtaa aagaacttct 7440
tggtatcaca attatggaaa gatcttcatt tgaaaaaaat ccaattgact ttttagaagc 7500
taagggttat aaagaagtta aaaaggattt aatcataaaa ctaccaaagt atagtctatt 7560
tgaactcgaa aacggaagaa aacgaatgct cgctagcgca ggagaacttc aaaaaggaaa 7620
tgaacttgcg ctgccatcaa agtatgtaaa tttcttatat ttagcttctc attatgagaa 7680
attaaaagga tcaccagagg ataatgaaca aaagcaacta tttgtagaac aacacaaaca 7740
ttatttagat gaaataatag aacaaatatc tgaattttct aaaagagtta tacttgccga 7800
cgcaaatcta gataaggtgc tttcagcgta taataaacac agagataaac caataagaga 7860
acaagcagaa aacattatcc atctttttac attaactaat cttggtgcac cagctgcatt 7920
taagtacttt gatacaacaa tagatagaaa aagatacaca tctactaaag aagtattaga 7980
cgcaacttta atacatcaat ctattacagg gctttatgaa acaagaattg atttaagtca 8040
actaggcgga gattaagtcg acaaagtatt gttaaaaata actctgtaga attataaatt 8100
agttctacag agttattttt tgacccgggt atattgataa aaataataat agtgggtata 8160
attaagttgt taggaggtta gttagaatga tgtcaagatt agataaaagt aaagtgatta 8220
acagcgcatt agagctgctt aatgaggtcg gaatcgaagg tttaacaacc cgtaaactcg 8280
cccagaagct aggtgtagag cagcctacat tgtattggca tgtaaaaaat aagcgggctt 8340
tgctcgacgc cttagccatt gagatgttag ataggcacca tactcacttt tgccctttag 8400
aaggggaaag ctggcaagat tttttacgta ataacgctaa aagttttaga tgtgctttac 8460
taagtcatcg cgatggagca aaagtacatt taggtacacg gcctacagaa aaacagtatg 8520
aaactctcga aaatcaatta gcctttttat gccaacaagg tttttcacta gagaatgcat 8580
tatatgcact cagcgctgtg gggcatttta ctttaggttg cgtattggaa gatcaagagc 8640
atcaagtcgc taaagaagaa agggaaacac ctactactga tagtatgccg ccattattac 8700
gacaagctat cgaattattt gatcaccaag gtgcagagcc agccttctta ttcggccttg 8760
aattgatcat atgcggatta gaaaaacaac ttaaatgtga aagtgggtct taaaagcagc 8820
ataacctttt tccgtgatgg taacttcacg gtaaccaaga tgtcgagttg agctcttagt 8880
tcaactcact ttttaaggtg attgtttgca tgtcattata aaattcttct tcatcctcgt 8940
attcttgatt ccaaccgttt ttaaatgcag atatgaattt ttcaactatt gattcatttt 9000
cactttcaga aattacatac tcgtttccat cattattaac tctaataatt agctgtgtta 9060
tactattgct atccgtacca ctcaatttca ctgtgtaatc tttgtttttt atttctctaa 9120
ttaagtcatt aatattcatt tcagccctcc tgtgaaattg ttatccgctc acaattccac 9180
gtcgactacc gcggattcta gattctgcag tatcttcatg gtattcattt tttaatatca 9240
ttttaccctc ccaatacatt taaaataatt atgtattcat gaaacatgat tgtatattta 9300
agaaacataa ttccatataa atcatttttc aaaatagttt ttacccataa ttaaatgtta 9360
atatgtaaat taatctttta gaatagttaa aaagttctaa aatatgttat aatgtttctt 9420
ataatcttat aaattttaat aactaatata taaagatatt tctttaaaat attcttatat 9480
ttagaagaat ttattttaaa ataaaaagct tttatgttga taaactgctt tgcaaagctc 9540
tcatgtaaat gtttaatata agactactat aaaattggct aattttatag gttaggaggt 9600
agaaatgcaa atattgtgga aaaagtatgt taaagaaaac tttgaaatga atgtagatga 9660
atgtggtata gaacaaggta taccaggatt aggatataac tatgaagtat tgaaaaatgc 9720
tgttattcat tacgtaacta agggatatgg aacttttaaa tttaatggta aggtatataa 9780
cttaaaacaa ggtgatattt ttatactact aaaaggtatg caagttgagt atgtggcttc 9840
tattgatgat ccttgggaat actactggat aggatttagt ggttcaaatg ctaatgagta 9900
tttaaataga acttctatta ctaactcctg tgttgctaat tgtgaagaaa actcaaaaat 9960
tccacagata atattaaata tgtgcgaaat atcaaaaact tataatcctt caagatctga 10020
tgacatacta ttactaaaag aactttactc attattgtac gcacttatag aagaattccc 10080
aaaacctttt gaatacaaag ataaggaatt acacacatat attcaagatg ctcttaattt 10140
cattaattct aattacatgc atagcataac tgttcaagaa attgctgatt atgtgaactt 10200
aagtagaagt tatttatata aaatgttcat aaaaaacctt ggaatttctc ctcaaagata 10260
tttaataaac cttagaatgt acaaagccac ccttttatta aaaagcacta aacttcctat 10320
aggagaagtc gcaagtagtg taggttatag tgactccctg ttattttcaa aaactttttc 10380
aaaacatttt tcaatgtctc cactaaatta cagaaataat caagtaaata aaccaagtat 10440
ataaatttaa aatacagctt taaaacaaaa aaatttcaaa aataaaaagt ataacagagg 10500
cgtaaattaa aacctctgtt atactttttg agct 10534
<210> 24
<211> 5754
<212> DNA
<213> 人工序列
<220>
<223> pEC750S-uppHR
<400> 24
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg gtctagagct ggagatagat tatttggtac taagtaatta 540
gtaatctatt agaattaaaa gctatctaca taagtttctg aatgacccaa gataatttta 600
ctggggggaa tatagaaaat ggagagacga gataagaaaa attattactt ggatattgct 660
gaaacagttt tagagagagg aacctgtcta aggagaaact atggttctat aattgttaaa 720
aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780
gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840
tgtaggagtg tacatagtga agctaatgca ataataagcg cttcgagctc gaattcgtaa 900
tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 960
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1020
attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1080
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1140
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1200
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1260
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1320
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1380
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1440
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1500
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1560
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1620
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1680
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1740
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 1800
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 1860
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 1920
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 1980
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2040
atatatgagt aaacttggtc tgacagttac caaagctagc ttaatactag tatatactta 2100
atgtgataag tgtctgacag ctgaccggtc taaagaggtc cgccaatgaa atctataaat 2160
aaactaaatt aagtttattt aattaacaac tatggatata aaataggtac taatcaaaat 2220
agtgaggagg atatatttga atacatacga acaaattaat aaagtgaaaa aaatacttcg 2280
gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg 2340
actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca 2400
aagtaaagaa atacttatac aaaaaattag acctatttca aagaaaatag gagataaaag 2460
caacttacga tatattgaat taacaattat tattcagcaa gaaatggtac cgtggaatca 2520
tcctcccaaa caagaattta tttatggaga atggttacaa gagctttatg aacaaggata 2580
cattcctcag aaggaattaa attcagattt aaccataatg ctttaccaag caaaacgaaa 2640
aaataaaaga atatacggaa attatgactt agaggaatta ctacctgata ttccattttc 2700
tgatgtgaga agagccatta tggattcgtc agaggaatta atagataatt atcaggatga 2760
tgaaaccaac tctatattaa ctttatgccg tatgatttta actatggaca cgggtaaaat 2820
cataccaaaa gatattgcgg gaaatgcagt ggctgaatct tctccattag aacataggga 2880
gagaattttg ttagcagttc gtagttatct tggagagaat attgaatgga ctaatgaaaa 2940
tgtaaattta actataaact atttaaataa cagattaaaa aaattataaa aaaattgaaa 3000
aaatggtgga aacacttttt tcaatttttt tgttttatta tttaatattt gggaaatatt 3060
cattctaatt ggtaatcaga ttttagaagt tgttaacttc aggtttgtct gtaactaaaa 3120
actagtattt aacctaggat caaaaaaatt tccaataatc ccactctaag ccacaaacac 3180
gccctataaa atcccgcttt aatcccactt tgagacacat gtaatattac tttacgccct 3240
agtatagtga taatttttta cattcaatgc cacgcaaaaa aataaagggg cactataata 3300
aaagttcctt cggaactaac taaagtaaaa aattatcttt acaacctccc caaaaaaaag 3360
aacaggtaca aagtacccta taatacaagc gtaaaaaaaa tgagggtaaa aataaaaaaa 3420
taaaaaaata aaaaaataaa aaaataaaaa aataaaaaaa taaaaaaata taaaaataaa 3480
aaaatataaa aataaaaaaa tataaaaata aaaaaataaa aaaatataaa aataaaaaaa 3540
taaaaaaata taaaaatatt ttttatttaa agtttgaaaa aaattttttt atattatata 3600
atctttgaag aaaagaatat aaaaaatgag cctttataaa agcccatttt ttttcatata 3660
cgtaatatga cgttctaatg tttttattgg tacttctaac attagagtaa tttctttatt 3720
tttaaagcct ttttctttaa gggcttttat tttttttctt aatacattta attcctcttt 3780
ttttgttgct tttcctttag cttttaattg ctcttgataa ttttttttac ctctaatatt 3840
ttctcttctc ttatattcct ttttagaaat tattattgtc atatattttt gttcttcttc 3900
tgtaatttct aataactcta taagagtttc attcttatac ttatattgct tatttttatc 3960
taaataacat ctttcagcac ttctagttgc tcttataact tctctttcac ttaaatgttg 4020
tctaaacata ctattaagtt ctaaaacatc atttaatgcc ttctcaatgt cttctgtaaa 4080
gctacaaaga taatatctat ataaaaataa tataagctct ctgtgtcctt ttaaatcata 4140
ttctcttagt tcacaaagtt ttattatgtc ttgtattctt ccataatata aacttctttc 4200
tctataaata taatttattt tgcttggtct accctttttc ctttcatatg gttttaattc 4260
aggtaaaaat ccattttgta tttctcttaa gtcataaata tattcgtact catctaatat 4320
attgactact gtttttgatt tagagtttat acttcctgga actcttaata ttctcgttgc 4380
atctaaggct tgtctatctg ctccaaagta ttttaattga ttatataaat attcttgaac 4440
cgctttccat aatggtaatg ctttactagg tactgcattt attatccata ttaaatacat 4500
tcctcttcca ctatctatta catagtttgg tataggaata ctttgattaa aataattctt 4560
ttctaagtcc attaatacct ggtctttagt tttgccagtt ttataataat ccaagtctat 4620
aaacagtgta tttaactctt ttatattttc taatcgccta cacggcttat aaaaggtatt 4680
tagagttata tagatatttt catcactcat atctaaatct tttaattcag cgtatttata 4740
gtgccattgg ctatatcctt ttttatctat aacgctcctg gttatccacc ctttacttct 4800
actatgaata ttatctatat agttcttttt attcagcttt aatgcgtttc tcacttattc 4860
acctcccctt ctgtaaaact aagaaaatta tatcatattt tcaataatta ttaactattc 4920
ttaaactctt aataaaaaat agagtaagtc cccaattgaa acttaatcta ttttttatgt 4980
tttaatttat tatttttatt aaaatatttt aaactaaatt aaatgattct ttttaatttt 5040
ttactatttc attccataat atattactat aattatttac aaataatatt tcttcatttg 5100
taatatttag atgatttact aattttagtt tttatatatt aaataattaa tgtataattt 5160
atataaaaaa tcaaaggagc ttataaatta tgattatttc caaagatact aaagatttaa 5220
tttttttcaa ttttaacaat actttttgta atattatgtt taaatttaat tgtatttttt 5280
tcatataata aagccgttga agtaaaccaa tccattttcc ttatgatgtt attattaaat 5340
ttaagtttta taataatatc tttattatat ttattgtttt taaaaaaact agtgaaattt 5400
ctagtgaaat ttccggcttt attaaactta tttttaggaa ttttattttc attttcatct 5460
ttacaggatt tgattatatc tttaaatatg ttttatcaaa tattatcttt ttctaaattt 5520
atatatattt ttattatatt tattattata tatattttat ttttaagttt ctttctaaca 5580
gctattaaaa agaaacttaa aaataaaaac acgtactcta aaccaataaa taaaactatt 5640
tttattattg ctgccttgat tggaatagtt tttagtaaaa ttaatttcaa tattccacaa 5700
tattatatta taagctagca ggcctcgaga tctccatgga cgcgtgacgt cgac 5754
<210> 25
<211> 884
<212> DNA
<213> 人工序列
<220>
<223> 修复模板
<400> 25
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg gtctagagct ggagatagat tatttggtac taagtaatta 540
gtaatctatt agaattaaaa gctatctaca taagtttctg aatgacccaa gataatttta 600
ctggggggaa tatagaaaat ggagagacga gataagaaaa attattactt ggatattgct 660
gaaacagttt tagagagagg aacctgtcta aggagaaact atggttctat aattgttaaa 720
aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780
gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840
tgtaggagtg tacatagtga agctaatgca ataataagcg cttc 884
<210> 26
<211> 500
<212> DNA
<213> 人工序列
<220>
<223> upp基因上游片段
<400> 26
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg 500
<210> 27
<211> 377
<212> DNA
<213> 人工序列
<220>
<223> upp基因下游片段
<400> 27
gctggagata gattatttgg tactaagtaa ttagtaatct attagaatta aaagctatct 60
acataagttt ctgaatgacc caagataatt ttactggggg gaatatagaa aatggagaga 120
cgagataaga aaaattatta cttggatatt gctgaaacag ttttagagag aggaacctgt 180
ctaaggagaa actatggttc tataattgtt aaaaatgatg aaataatttc tactggatac 240
acaggagcac ctagaggtag aaaaaattgc atggatttga atagttgcat aagagaaaag 300
ttgaaagttc caagaggtac tcattatgag ttgtgtagga gtgtacatag tgaagctaat 360
gcaataataa gcgcttc 377
<210> 28
<211> 2666
<212> DNA
<213> 人工序列
<220>
<223> pEX-A2-gRNA-upp
<400> 28
ctcgagtatt tttgataaaa gcaatgatta acatggtttg acgtctgaga agagacgatt 60
ttctcaatag gagaaattaa ggtgcaaacc cttatcattc caccatgatc cacctgtagc 120
aagcatgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt atcaacttga 180
aaaagtggca ccgagtcggt gctttttttg ccatggacct gcttttgctc gcttggatcc 240
gaattcctgt gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca 300
taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct 360
cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac 420
gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc 480
tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt 540
tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg 600
ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg 660
agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat 720
accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta 780
ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct 840
gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc 900
ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa 960
gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg 1020
taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag 1080
tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt 1140
gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta 1200
cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc 1260
agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca 1320
cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa 1380
cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat 1440
ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct 1500
taccatctgg ccccagtgct gcaatgatac cgcgactccc acgctcaccg gctccagatt 1560
tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat 1620
ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta 1680
atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg 1740
gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt 1800
tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg 1860
cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg 1920
taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc 1980
ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa 2040
ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac 2100
cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt 2160
ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg 2220
gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa 2280
gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata 2340
aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc taagaaacca 2400
ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc 2460
gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg gtcacagctt 2520
gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg 2580
ggtgtcgggg ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccaat 2640
tgggtaccga gctcgcggcc gcaagc 2666
<210> 29
<211> 203
<212> DNA
<213> 人工序列
<220>
<223> gRNA表达盒
<400> 29
tatttttgat aaaagcaatg attaacatgg tttgacgtct gagaagagac gattttctca 60
ataggagaaa ttaaggtgca aacccttatc attccaccat gatccacctg tagcaagcat 120
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 180
ggcaccgagt cggtgctttt ttt 203
<210> 30
<211> 100
<212> DNA
<213> 人工序列
<220>
<223> 组成型启动子
<400> 30
tatttttgat aaaagcaatg attaacatgg tttgacgtct gagaagagac gattttctca 60
ataggagaaa ttaaggtgca aacccttatc attccaccat 100
<210> 31
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 靶向upp的前间区序列
<400> 31
gatccacctg tagcaagcat 20
<210> 32
<211> 5954
<212> DNA
<213> 人工序列
<220>
<223> pEC750S-Δupp
<400> 32
ataaggtacc aggaattaga gcagcgctat gttcagatac atttagtgct catgcaacaa 60
gagaacataa taatgctaat atattaacta tgggtcaaag ggttgttgga gcaggtcttg 120
ctttagatat agtaaaaaca tttatatcag ctaaatttga aggagatagg caccaaaaaa 180
gaatagataa gatttcagat attgaaaaaa agtatacaca ttagaaaaaa gcagctatgc 240
tgcaaataag atcaatttat attagaaaaa agcagctatg ctgcaaataa gatcaattta 300
tattagaaaa aagcagctat gctgcaaata agatcaattt atattagaaa aaagcagcta 360
tgctacaaat aagatcaatt tatattagaa aaaagtagct atgctgcaac aatattaatt 420
tatattacta gaaagctaaa tggggtatat aaatataaag ggctataaat actaaaagca 480
aacttggagg aataataatg gtctagagct ggagatagat tatttggtac taagtaatta 540
gtaatctatt agaattaaaa gctatctaca taagtttctg aatgacccaa gataatttta 600
ctggggggaa tatagaaaat ggagagacga gataagaaaa attattactt ggatattgct 660
gaaacagttt tagagagagg aacctgtcta aggagaaact atggttctat aattgttaaa 720
aatgatgaaa taatttctac tggatacaca ggagcaccta gaggtagaaa aaattgcatg 780
gatttgaata gttgcataag agaaaagttg aaagttccaa gaggtactca ttatgagttg 840
tgtaggagtg tacatagtga agctaatgca ataataagcg cttcgagctc gaattcgtaa 900
tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata 960
cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta 1020
attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa 1080
tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg 1140
ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag 1200
gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa 1260
ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 1320
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 1380
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 1440
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 1500
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 1560
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 1620
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 1680
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 1740
actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 1800
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 1860
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg 1920
gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca 1980
aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt 2040
atatatgagt aaacttggtc tgacagttac caaagctagc ttaatactag tatatactta 2100
atgtgataag tgtctgacag ctgaccggtc taaagaggtc cgccaatgaa atctataaat 2160
aaactaaatt aagtttattt aattaacaac tatggatata aaataggtac taatcaaaat 2220
agtgaggagg atatatttga atacatacga acaaattaat aaagtgaaaa aaatacttcg 2280
gaaacattta aaaaataacc ttattggtac ttacatgttt ggatcaggag ttgagagtgg 2340
actaaaacca aatagtgatc ttgacttttt agtcgtcgta tctgaaccat tgacagatca 2400
aagtaaagaa atacttatac aaaaaattag acctatttca aagaaaatag gagataaaag 2460
caacttacga tatattgaat taacaattat tattcagcaa gaaatggtac cgtggaatca 2520
tcctcccaaa caagaattta tttatggaga atggttacaa gagctttatg aacaaggata 2580
cattcctcag aaggaattaa attcagattt aaccataatg ctttaccaag caaaacgaaa 2640
aaataaaaga atatacggaa attatgactt agaggaatta ctacctgata ttccattttc 2700
tgatgtgaga agagccatta tggattcgtc agaggaatta atagataatt atcaggatga 2760
tgaaaccaac tctatattaa ctttatgccg tatgatttta actatggaca cgggtaaaat 2820
cataccaaaa gatattgcgg gaaatgcagt ggctgaatct tctccattag aacataggga 2880
gagaattttg ttagcagttc gtagttatct tggagagaat attgaatgga ctaatgaaaa 2940
tgtaaattta actataaact atttaaataa cagattaaaa aaattataaa aaaattgaaa 3000
aaatggtgga aacacttttt tcaatttttt tgttttatta tttaatattt gggaaatatt 3060
cattctaatt ggtaatcaga ttttagaagt tgttaacttc aggtttgtct gtaactaaaa 3120
actagtattt aacctaggat caaaaaaatt tccaataatc ccactctaag ccacaaacac 3180
gccctataaa atcccgcttt aatcccactt tgagacacat gtaatattac tttacgccct 3240
agtatagtga taatttttta cattcaatgc cacgcaaaaa aataaagggg cactataata 3300
aaagttcctt cggaactaac taaagtaaaa aattatcttt acaacctccc caaaaaaaag 3360
aacaggtaca aagtacccta taatacaagc gtaaaaaaaa tgagggtaaa aataaaaaaa 3420
taaaaaaata aaaaaataaa aaaataaaaa aataaaaaaa taaaaaaata taaaaataaa 3480
aaaatataaa aataaaaaaa tataaaaata aaaaaataaa aaaatataaa aataaaaaaa 3540
taaaaaaata taaaaatatt ttttatttaa agtttgaaaa aaattttttt atattatata 3600
atctttgaag aaaagaatat aaaaaatgag cctttataaa agcccatttt ttttcatata 3660
cgtaatatga cgttctaatg tttttattgg tacttctaac attagagtaa tttctttatt 3720
tttaaagcct ttttctttaa gggcttttat tttttttctt aatacattta attcctcttt 3780
ttttgttgct tttcctttag cttttaattg ctcttgataa ttttttttac ctctaatatt 3840
ttctcttctc ttatattcct ttttagaaat tattattgtc atatattttt gttcttcttc 3900
tgtaatttct aataactcta taagagtttc attcttatac ttatattgct tatttttatc 3960
taaataacat ctttcagcac ttctagttgc tcttataact tctctttcac ttaaatgttg 4020
tctaaacata ctattaagtt ctaaaacatc atttaatgcc ttctcaatgt cttctgtaaa 4080
gctacaaaga taatatctat ataaaaataa tataagctct ctgtgtcctt ttaaatcata 4140
ttctcttagt tcacaaagtt ttattatgtc ttgtattctt ccataatata aacttctttc 4200
tctataaata taatttattt tgcttggtct accctttttc ctttcatatg gttttaattc 4260
aggtaaaaat ccattttgta tttctcttaa gtcataaata tattcgtact catctaatat 4320
attgactact gtttttgatt tagagtttat acttcctgga actcttaata ttctcgttgc 4380
atctaaggct tgtctatctg ctccaaagta ttttaattga ttatataaat attcttgaac 4440
cgctttccat aatggtaatg ctttactagg tactgcattt attatccata ttaaatacat 4500
tcctcttcca ctatctatta catagtttgg tataggaata ctttgattaa aataattctt 4560
ttctaagtcc attaatacct ggtctttagt tttgccagtt ttataataat ccaagtctat 4620
aaacagtgta tttaactctt ttatattttc taatcgccta cacggcttat aaaaggtatt 4680
tagagttata tagatatttt catcactcat atctaaatct tttaattcag cgtatttata 4740
gtgccattgg ctatatcctt ttttatctat aacgctcctg gttatccacc ctttacttct 4800
actatgaata ttatctatat agttcttttt attcagcttt aatgcgtttc tcacttattc 4860
acctcccctt ctgtaaaact aagaaaatta tatcatattt tcaataatta ttaactattc 4920
ttaaactctt aataaaaaat agagtaagtc cccaattgaa acttaatcta ttttttatgt 4980
tttaatttat tatttttatt aaaatatttt aaactaaatt aaatgattct ttttaatttt 5040
ttactatttc attccataat atattactat aattatttac aaataatatt tcttcatttg 5100
taatatttag atgatttact aattttagtt tttatatatt aaataattaa tgtataattt 5160
atataaaaaa tcaaaggagc ttataaatta tgattatttc caaagatact aaagatttaa 5220
tttttttcaa ttttaacaat actttttgta atattatgtt taaatttaat tgtatttttt 5280
tcatataata aagccgttga agtaaaccaa tccattttcc ttatgatgtt attattaaat 5340
ttaagtttta taataatatc tttattatat ttattgtttt taaaaaaact agtgaaattt 5400
ctagtgaaat ttccggcttt attaaactta tttttaggaa ttttattttc attttcatct 5460
ttacaggatt tgattatatc tttaaatatg ttttatcaaa tattatcttt ttctaaattt 5520
atatatattt ttattatatt tattattata tatattttat ttttaagttt ctttctaaca 5580
gctattaaaa agaaacttaa aaataaaaac acgtactcta aaccaataaa taaaactatt 5640
tttattattg ctgccttgat tggaatagtt tttagtaaaa ttaatttcaa tattccacaa 5700
tattatatta taagctagca cgcctcgagt atttttgata aaagcaatga ttaacatggt 5760
ttgacgtctg agaagagacg attttctcaa taggagaaat taaggtgcaa acccttatca 5820
ttccaccatg atccacctgt agcaagcatg ttttagagct agaaatagca agttaaaata 5880
aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt ttgccatgga 5940
cgcgtgacgt cgac 5954
<210> 33
<211> 5853
<212> DNA
<213> 人工序列
<220>
<223> pEC750C-Δupp
<400> 33
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gtatttttga taaaagcaat gattaacatg gtttgacgtc tgagaagaga 2640
cgattttctc aataggagaa attaaggtgc aaacccttat cattccacca tgatccacct 2700
gtagcaagca tgttttagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa 2760
cttgaaaaag tggcaccgag tcggtgcttt ttttgccatg gacgcgtgac gtcgacataa 2820
ggtaccagga attagagcag cgctatgttc agatacattt agtgctcatg caacaagaga 2880
acataataat gctaatatat taactatggg tcaaagggtt gttggagcag gtcttgcttt 2940
agatatagta aaaacattta tatcagctaa atttgaagga gataggcacc aaaaaagaat 3000
agataagatt tcagatattg aaaaaaagta tacacattag aaaaaagcag ctatgctgca 3060
aataagatca atttatatta gaaaaaagca gctatgctgc aaataagatc aatttatatt 3120
agaaaaaagc agctatgctg caaataagat caatttatat tagaaaaaag cagctatgct 3180
acaaataaga tcaatttata ttagaaaaaa gtagctatgc tgcaacaata ttaatttata 3240
ttactagaaa gctaaatggg gtatataaat ataaagggct ataaatacta aaagcaaact 3300
tggaggaata ataatggtct agagctggag atagattatt tggtactaag taattagtaa 3360
tctattagaa ttaaaagcta tctacataag tttctgaatg acccaagata attttactgg 3420
ggggaatata gaaaatggag agacgagata agaaaaatta ttacttggat attgctgaaa 3480
cagttttaga gagaggaacc tgtctaagga gaaactatgg ttctataatt gttaaaaatg 3540
atgaaataat ttctactgga tacacaggag cacctagagg tagaaaaaat tgcatggatt 3600
tgaatagttg cataagagaa aagttgaaag ttccaagagg tactcattat gagttgtgta 3660
ggagtgtaca tagtgaagct aatgcaataa taagcgcttc gagctcgaat tcgtaatcat 3720
ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 3780
ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 3840
cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 3900
tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 3960
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4020
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4080
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4140
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4200
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4260
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4320
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4380
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4440
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 4500
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 4560
gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 4620
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 4680
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 4740
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 4800
ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 4860
atgagtaaac ttggtctgac agttaccaaa gctagcttaa tactagtata tacttaatgt 4920
gataagtgtc tgacagctga ccggtctaaa gaggtcccta gcgcctacgg ggaatttgta 4980
tcgataaggg gtacaaattc ccactaagcg ctcggccggg gatcgatccc cgggtacgta 5040
cccggcagtt tttctttttc ggcaagtgtt caagaagtta ttaagtcggg agtgcagtcg 5100
aagtgggcaa gttgaaaaat tcacaaaaat gtggtataat atctttgttc attagagcga 5160
taaacttgaa tttgagaggg aacttagatg gtatttgaaa aaattgataa aaatagttgg 5220
aacagaaaag agtattttga ccactacttt gcaagtgtac cttgtaccta cagcatgacc 5280
gttaaagtgg atatcacaca aataaaggaa aagggaatga aactatatcc tgcaatgctt 5340
tattatattg caatgattgt aaaccgccat tcagagttta ggacggcaat caatcaagat 5400
ggtgaattgg ggatatatga tgagatgata ccaagctata caatatttca caatgatact 5460
gaaacatttt ccagcctttg gactgagtgt aagtctgact ttaaatcatt tttagcagat 5520
tatgaaagtg atacgcaacg gtatggaaac aatcatagaa tggaaggaaa gccaaatgct 5580
ccggaaaaca tttttaatgt atctatgata ccgtggtcaa ccttcgatgg ctttaatctg 5640
aatttgcaga aaggatatga ttatttgatt cctattttta ctatggggaa atattataaa 5700
gaagataaca aaattatact tcctttggca attcaagttc atcacgcagt atgtgacgga 5760
tttcacattt gccgttttgt aaacgaattg caggaattga taaatagtta acttcaggtt 5820
tgtctgtaac taaaaactag tatttaacct agg 5853
<210> 34
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-pNF2
<400> 34
agctcggtac ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcg 60
tgctagctta taatataata ttgtggaata ttgaaattaa ttttactaaa aactattcca 120
atcaaggcag caataataaa aatagtttta tttattggtt tagagtacgt gtttttattt 180
ttaagtttct ttttaatagc tgttagaaag aaacttaaaa ataaaatata tataataata 240
aatataataa aaatatatat aaatttagaa aaagataata tttgataaaa catatttaaa 300
gatataatca aatcctgtaa agatgaaaat gaaaataaaa ttcctaaaaa taagtttaat 360
aaagccggaa atttcactag aaatttcact agttttttta aaaacaataa atataataaa 420
gatattatta taaaacttaa atttaataat aacatcataa ggaaaatgga ttggtttact 480
tcaacggctt tattatatga aaaaaataca attaaattta aacataatat tacaaaaagt 540
attgttaaaa ttgaaaaaaa ttaaatcttt agtatctttg gaaataatca taatttataa 600
gctcctttga ttttttatat aaattataca ttaattattt aatatataaa aactaaaatt 660
agtaaatcat ctaaatatta caaatgaaga aatattattt gtaaataatt atagtaatat 720
attatggaat gaaatagtaa aaaattaaaa agaatcattt aatttagttt aaaatatttt 780
aataaaaata ataaattaaa acataaaaaa tagattaagt ttcaattggg gacttactct 840
attttttatt aagagtttaa gaatagttaa taattattga aaatatgata taattttctt 900
agttttacag aaggggaggt gaataagtga gaaacgcatt aaagctgaat aaaaagaact 960
atatagataa tattcatagt agaagtaaag ggtggataac caggagcgtt atagataaaa 1020
aaggatatag ccaatggcac tataaatacg ctgaattaaa agatttagat atgagtgatg 1080
aaaatatcta tataactcta aatacctttt ataagccgtg taggcgatta gaaaatataa 1140
aagagttaaa tacactgttt atagacttgg attattataa aactggcaaa actaaagacc 1200
aggtattaat ggacttagaa aagaattatt ttaatcaaag tattcctata ccaaactatg 1260
taatagatag tggaagagga atgtatttaa tatggataat aaatgcagta cctagtaaag 1320
cattaccatt atggaaagcg gttcaagaat atttatataa tcaattaaaa tactttggag 1380
cagatagaca agccttagat gcaacgagaa tattaagagt tccaggaagt ataaactcta 1440
aatcaaaaac agtagtcaat atattagatg agtacgaata tatttatgac ttaagagaaa 1500
tacaaaatgg atttttacct gaattaaaac catatgaaag gaaaaagggt agaccaagca 1560
aaataaatta tatttataga gaaagaagtt tatattatgg aagaatacaa gacataataa 1620
aactttgtga actaagagaa tatgatttaa aaggacacag agagcttata ttatttttat 1680
atagatatta tctttgtagc tttacagaag acattgagaa ggcattaaat gatgttttag 1740
aacttaatag tatgtttaga caacatttaa gtgaaagaga agttataaga gcaactagaa 1800
gtgctgaaag atgttattta gataaaaata agcaatataa gtataagaat gaaactctta 1860
tagagttatt agaaattaca gaagaagaac aaaaatatat gacaataata atttctaaaa 1920
aggaatataa gagaagagaa aatattagag gtaaaaaaaa ttatcaagag caattaaaag 1980
ctaaaggaaa agcaacaaaa aaagaggaat taaatgtatt aagaaaaaaa ataaaagccc 2040
ttaaagaaaa aggctttaaa aataaagaaa ttactctaat gttagaagta ccaataaaaa 2100
cattagaacg tcatattacg tatatgaaaa aaaatgggct tttataaagg ctcatttttt 2160
atattctttt cttcaaagat tatataatat aaaaaaattt ttttcaaact ttaaataaaa 2220
aatattttta tattttttta tttttttatt tttatatttt tttatttttt tatttttata 2280
tttttttatt tttatatttt tttattttta tattttttta tttttttatt tttttatttt 2340
tttatttttt tattttttta tttttttatt tttaccctca ttttttttac gcttgtatta 2400
tagggtactt tgtacctgtt cttttttttg gggaggttgt aaagataatt ttttacttta 2460
gttagttccg aaggaacttt tattatagtg cccctttatt tttttgcgtg gcattgaatg 2520
taaaaaatta tcactatact agggcgtaaa gtaatattac atgtgtctca aagtgggatt 2580
aaagcgggat tttatagggc gtgtttgtgg cttagagtgg gattattgga aatttttttg 2640
atcctaggtt aaatactagt ttttagttac agacaaacct gaagttaact atttatcaat 2700
tcctgcaatt cgtttacaaa acggcaaatg tgaaatccgt cacatactgc gtgatgaact 2760
tgaattgcca aaggaagtat aattttgtta tcttctttat aatatttccc catagtaaaa 2820
ataggaatca aataatcata tcctttctgc aaattcagat taaagccatc gaaggttgac 2880
cacggtatca tagatacatt aaaaatgttt tccggagcat ttggctttcc ttccattcta 2940
tgattgtttc cataccgttg cgtatcactt tcataatctg ctaaaaatga tttaaagtca 3000
gacttacact cagtccaaag gctggaaaat gtttcagtat cattgtgaaa tattgtatag 3060
cttggtatca tctcatcata tatccccaat tcaccatctt gattgattgc cgtcctaaac 3120
tctgaatggc ggtttacaat cattgcaata taataaagca ttgcaggata tagtttcatt 3180
cccttttcct ttatttgtgt gatatccact ttaacggtca tgctgtaggt acaaggtaca 3240
cttgcaaagt agtggtcaaa atactctttt ctgttccaac tatttttatc aattttttca 3300
aataccatct aagttccctc tcaaattcaa gtttatcgct ctaatgaaca aagatattat 3360
accacatttt tgtgaatttt tcaacttgcc cacttcgact gcactcccga cttaataact 3420
tcttgaacac ttgccgaaaa agaaaaactg ccgggtacgt acccggggat cgatccccgg 3480
ccgagcgctt agtgggaatt tgtacccctt atcgatacaa attccccgta ggcgctaggg 3540
acctctttag accggtcagc tgtcagacac ttatcacatt aagtatatac tagtattaag 3600
ctagctttgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3660
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3720
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3780
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3840
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3900
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3960
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4020
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4080
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4140
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4200
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4260
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4320
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4380
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4440
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4500
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 4560
aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 4620
gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 4680
ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 4740
cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcactct 4800
atcattgata gagtttgaaa ctctatcatt gatagagtat aatatctttg ttcatttaag 4860
ccatctacta aacaagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 4920
tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttg 4966
<210> 35
<211> 400
<212> DNA
<213> 人工序列
<220>
<223> catB基因上游片段
<400> 35
gtctttacac ttttgcccat taatttttga gttccttatt tttagggagc ttttattatt 60
tttatcatga aaatttcata aaatactcat aaactaagga tgtcttcata atcagattag 120
tactccattt tcaatccatt taatctggga atatgatatt ttaattacgt attatttaag 180
atatattaac gtgtaatata ataccccgca aatattaatt atcacataca tatcccccct 240
ttattggggc attttttgta cccattattt tagtattgtg cagtacttaa ataaaaaaat 300
gccgcaaatt catttttatt gaataatgcg gtatttcttc tattctttat ttttattact 360
ctataaataa tgtaatcaag acatgactat ctaaatatat 400
<210> 36
<211> 400
<212> DNA
<213> 人工序列
<220>
<223> catB基因下游片段
<400> 36
aattcataat tcgggcctcc taaaaatttt cgtaattcta ttttagaagg cttttttccg 60
tgacctagcc atttcaatct cctttttaca atgatattta cgctttagtt tattatagca 120
cattctgtaa taccgaacta ttcaattttc agagaccatt ttttattgat tcataactta 180
agaatactac gaattactct aatattttac tttttcttat ctcttgttat tttaacatcg 240
gaattactac taatattaat ttttattttt ccatccgcat ttgctccaac atttttttaa 300
ctatactttc cttttgttaa taaattatgt tattgttgaa caatataaga aaagtgcgta 360
acatttttta ttaaaaataa ttaggtattt ctatctgtgg 400
<210> 37
<211> 218
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 37
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Lys
50 55 60
His Lys Glu Phe Arg Ile Cys Asp His Glu Gly Ser Leu Gly Tyr Trp
65 70 75 80
Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu Thr
85 90 95
Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe Tyr
100 105 110
Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys Phe
115 120 125
Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser Ile
130 135 140
Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu Gly
145 150 155 160
Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln Glu
165 170 175
Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile Cys
180 185 190
Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu Ala
195 200 205
Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 38
<211> 9113
<212> DNA
<213> 人工序列
<220>
<223> pCas9ind-gRNA_catB
<400> 38
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtatattgat aaaaataata 4200
atagtgggta taattaagtt gttaggaggt tagttagaat gatgtcaaga ttagataaaa 4260
gtaaagtgat taacagcgca ttagagctgc ttaatgaggt cggaatcgaa ggtttaacaa 4320
cccgtaaact cgcccagaag ctaggtgtag agcagcctac attgtattgg catgtaaaaa 4380
ataagcgggc tttgctcgac gccttagcca ttgagatgtt agataggcac catactcact 4440
tttgcccttt agaaggggaa agctggcaag attttttacg taataacgct aaaagtttta 4500
gatgtgcttt actaagtcat cgcgatggag caaaagtaca tttaggtaca cggcctacag 4560
aaaaacagta tgaaactctc gaaaatcaat tagccttttt atgccaacaa ggtttttcac 4620
tagagaatgc attatatgca ctcagcgctg tggggcattt tactttaggt tgcgtattgg 4680
aagatcaaga gcatcaagtc gctaaagaag aaagggaaac acctactact gatagtatgc 4740
cgccattatt acgacaagct atcgaattat ttgatcacca aggtgcagag ccagccttct 4800
tattcggcct tgaattgatc atatgcggat tagaaaaaca acttaaatgt gaaagtgggt 4860
cttaaaagca gcataacctt tttccgtgat ggtaacttca cggtaaccaa gatgtcgagt 4920
tgagctcgaa ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 4980
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 5040
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 5100
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 5160
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 5220
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 5280
agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg 5340
cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga 5400
ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg 5460
tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg 5520
gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc 5580
gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg 5640
gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca 5700
ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt 5760
ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag 5820
ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg 5880
gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc 5940
ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt 6000
tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt 6060
ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc 6120
gggcctcttg cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat 6180
ataatgggag ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa 6240
acagcaaaga atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt 6300
aagagtgtgt tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt 6360
agatgctaaa aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc 6420
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa 6480
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc 6540
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc 6600
agaaaaatta aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca 6660
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca 6720
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga 6780
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca 6840
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt 6900
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa 6960
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa 7020
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta 7080
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta 7140
tgagtcccta ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat 7200
tttttattaa gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa 7260
agaaaattat agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac 7320
aaaaaaaaat acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt 7380
taataaaaaa ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca 7440
aaacttaaat gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt 7500
caaatgtacc gacatacaag agaaacatta actatatata ttcaatttat gagattatct 7560
taacagatat aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat 7620
tggaagcagt acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc 7680
ataattaatt tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta 7740
tcaaataaca aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt 7800
atagtgcttg tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat 7860
agaattcata aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa 7920
agagtttatt aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac 7980
agtagcaacc tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca 8040
atgtattaat tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt 8100
tacagaggaa gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa 8160
aaaagctcat gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga 8220
gagtgccgac acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag 8280
gatagtcact cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc 8340
acgacgaaaa cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac 8400
taaacaatca agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt 8460
aatacatacg ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt 8520
tggatgtagt ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg 8580
ctaaaaagta tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat 8640
tttgaagtta ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt 8700
taataatagt atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt 8760
tatggattat aagcggctcg aggacgtcaa accatgttaa tcattgcttt tatcaaaaat 8820
aggatccact ctatcattga tagagtttga aactctatca ttgatagagt ataatatctt 8880
tgttcatgta catcatgcta tctgtgagtt ttagagctag aaatagcaag ttaaaataag 8940
gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttt gaagcttgtc 9000
tttacacttt tgcccctcga gtccctatca gtgatagatt gaaactctat cattgataga 9060
gtataatatc tttgttcatt agagcgataa acttgaattt gagagggaac ttc 9113
<210> 39
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pNF2
<400> 39
gggcgcactt atacaccacc 20
<210> 40
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pNF2
<400> 40
tgctacgcac cccctaaagg 20
<210> 41
<211> 50
<212> DNA
<213> 人工序列
<220>
<223> ΔcatB_gRNA_rev
<400> 41
aatctatcac tgatagggac tcgaggggca aaagtgtaaa gacaagcttc 50
<210> 42
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pCas9ind_fwd
<400> 42
agctcttgat ccggcaaaca 20
<210> 43
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物pCas9ind _rev
<400> 43
gcaaccctag tgttcggtga 20
<210> 44
<211> 219
<212> PRT
<213> 丁酸梭菌（Clostridium butyricum）
<400> 44
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 45
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 45
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ile Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 46
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 46
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Glu Glu Phe Arg Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Val Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 47
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 47
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Ser Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 48
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 梭菌属菌种（Clostridium sp.）2-1
<400> 48
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Gln Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 49
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 二醇梭菌（Clostridium diolis）
<400> 49
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 50
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 50
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ile Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Lys Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Gln Pro Asp Asn Thr Phe Ser Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Asn Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Lys Glu Trp Leu Glu Asn Lys
210 215
<210> 51
<211> 221
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 51
Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Asn Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Glu Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys Tyr Ile
210 215 220
<210> 52
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 52
Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Arg Glu Trp Leu Glu Asn Lys
210 215
<210> 53
<211> 219
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 53
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Thr Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Ile Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Ile Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 54
<211> 219
<212> PRT
<213> 糖乙酸多丁醇梭菌（Clostridium saccharoperbutylacetonicum）
<400> 54
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Thr Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe Tyr Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Ile Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Ile Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys
210 215
<210> 55
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 55
Met Asn Phe Asn Leu Ile Asp Ile Asn Asn Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Asn Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Arg Ser Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Gly Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Arg Glu Trp Leu Glu Asn Lys
210 215
<210> 56
<211> 221
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 56
Met Asn Phe Asn Leu Ile Asp Ile Asn His Trp Asn Arg Lys Pro Phe
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Lys Leu Lys Asn Ile
35 40 45
Lys Phe Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Ile Cys Phe Asp His Lys Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Ile Phe His Glu Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Leu Arg Phe
100 105 110
Tyr Ser Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Ser Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Cys Asn Glu
145 150 155 160
Gly Thr Tyr Leu Thr Pro Ile Phe Thr Ala Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Ile Phe Ile Pro Ile Ser Ile Gln Val His His Ser Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Glu Trp Leu Glu Asn Lys Tyr Ile
210 215 220
<210> 57
<211> 219
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 57
Met Asn Phe Asn Leu Ile Asp Ile Lys His Trp Ser Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Asn Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asp Leu Leu Tyr Glu Ile Arg Leu Lys Asn Ile
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Met Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp His Ser Gly Ser Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asn Glu Ser Phe Pro Arg Phe
100 105 110
Tyr Ser Asp Tyr Phe Asp Asp Ile Lys Asn Tyr Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Asp Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Glu
145 150 155 160
Gly Thr Tyr Leu Ile Pro Ile Phe Thr Thr Gly Lys Tyr Phe Lys Gln
165 170 175
Glu Asn Lys Met Phe Ile Pro Ile Ser Ile Gln Val His His Ala Ile
180 185 190
Cys Asp Gly Tyr His Ala Ser Arg Phe Ile Asn Glu Met Gln Glu Leu
195 200 205
Ala Phe Ser Phe Gln Asp Trp Leu Glu Asn Lys
210 215
<210> 58
<211> 219
<212> PRT
<213> 肉毒梭菌（Clostridium botulinum）
<400> 58
Met Lys Phe Asn Leu Ile Asp Ile Glu His Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu Tyr Tyr Leu His Ser Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Glu Ile Lys Leu Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Asp Tyr Asp Glu Ser Phe Ser Cys Phe
100 105 110
Tyr Asn Asp Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Ala Ile Met Lys
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asn
145 150 155 160
Gly Thr Tyr Leu Val Pro Ile Phe Thr Met Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ile Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Leu Asn Ser Gln Thr Trp Leu Lys His Lys
210 215
<210> 59
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> Anaerocolumna aminovalerica
<400> 59
Met Lys Phe Asn Leu Ile Asp Ile Glu Asn Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Glu Ile Lys Leu Lys Asp Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Leu Ala Thr Val Val Asn Asn
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Glu Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Ser Arg Phe
100 105 110
Tyr Thr Ala Tyr Leu Asp Asp Ile Lys Asn His Gly Asn Ile Met Lys
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Ser
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asp
145 150 155 160
Gly Lys Tyr Leu Leu Pro Ile Phe Thr Thr Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Ser Lys Ile Phe Ile Pro Met Ser Val Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ile Ser Arg Phe Ile Asn Glu Val Gln Glu Val
195 200 205
Ile Leu Asn Tyr Gln Thr Trp Leu Gly Asp Lys
210 215
<210> 60
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> Desnuesiella massiliensis
<400> 60
Met Lys Phe Asn Leu Ile Asp Ile Glu His Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Asp Ile Lys Leu Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn
50 55 60
His Glu Glu Phe Arg Thr Cys Phe Tyr Glu Asn Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Glu Ile Trp Ser Glu Tyr Asp Glu Ser Phe Ser Cys Phe
100 105 110
Tyr Ser Lys Tyr Leu Asp Asp Ile Lys Asn Tyr Gly Asp Ile Met Arg
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Val Tyr Asn Asp
145 150 155 160
Gly Arg Tyr Leu Val Pro Ile Phe Thr Ile Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Leu Asn Ser Gln Thr Trp Leu Arg His Lys
210 215
<210> 61
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 梭菌属菌种（Clostridium sp.）HMP27
<400> 61
Met Lys Phe Asn Leu Ile Asp Thr Glu His Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ser Val Arg Cys Thr Tyr Ser Ile Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu His Asp Ile Lys Gln Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Phe Ile Tyr Ile Ile Ala Thr Val Val Asn Thr
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Ser Gly Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Ile Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Lys Ser Phe Ser Cys Phe
100 105 110
Tyr Ser Lys Tyr Leu His Asp Ile Lys Asn Tyr Gly Asp Ile Met Ser
115 120 125
Phe Thr Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Gly Thr Tyr Leu Val Pro Ile Phe Thr Ile Gly Lys Tyr Phe Lys Gln
165 170 175
Ala Asp Lys Ile Leu Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ile Leu Asn Tyr Gln Thr Trp Leu Lys His Lys
210 215
<210> 62
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 德雷克氏梭菌（Clostridium drakei）
<400> 62
Met Lys Phe Asn Leu Ile Asp Ile Glu Asn Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Ala Val Ile Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Arg Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Val Phe His Lys Glu Asp Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Pro Arg Phe
100 105 110
Tyr Asp Asn Tyr Leu Asp Asp Ile Lys Ser Tyr Gly Asp Val Leu Lys
115 120 125
Phe Met Pro Lys Pro Asp Glu Pro Gly Asn Thr Phe Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Met Gly Lys Phe Phe His Gln
165 170 175
Asp Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Val Gln Glu Leu
195 200 205
Ser Ser Asn Phe Glu Thr Trp Leu Asp Glu Lys
210 215
<210> 63
<211> 219
<212> PRT
<213> 粪味梭菌（Clostridium scatologenes）
<400> 63
Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Ala Val Ile Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Glu Asn Arg Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Ser Tyr Thr Val Phe His Lys Glu Asp Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Ser Phe Pro Arg Phe
100 105 110
Tyr Asp Asn Tyr Leu Asp Asp Ile Lys Ser Tyr Gly Asp Val Leu Lys
115 120 125
Phe Met Pro Lys Pro Asp Glu Pro Gly Asn Thr Phe Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Met Gly Lys Phe Phe His Gln
165 170 175
Asp Asn Lys Ile Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Val Gln Glu Leu
195 200 205
Ser Ser Asn Phe Glu Thr Trp Leu Gly Glu Lys
210 215
<210> 64
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 突尼斯梭菌（Clostridium tunisiense）
<400> 64
Met Lys Phe Asn Leu Ile Asp Thr Glu His Trp Asp Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Phe Asn Ser Val Lys Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu Asn His Ile Arg Leu Lys Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Val Asn Asn
50 55 60
His Glu Glu Phe Arg Ile Cys Phe Asp Glu Asn Asn Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Asn Tyr Thr Ile Phe His Glu Asp Asn Lys
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Glu Glu Ser Phe Ser Gly Phe
100 105 110
Tyr Asn Lys Tyr Leu Glu Asp Ile Lys Thr Tyr Gly His Ile Met Ser
115 120 125
Phe Glu Pro Lys Leu Asn Glu Ser Thr Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Gln Asp Asp
145 150 155 160
Gly Thr Tyr Leu Thr Pro Ile Phe Thr Leu Gly Lys Tyr Phe Glu Gln
165 170 175
Asn Asn Lys Thr Phe Ile Pro Ile Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Ser Asp Phe Gln Ile Trp Leu Thr Tyr Lys
210 215
<210> 65
<211> 219
<212> PRT
<213> 人工序列
<220>
<223> 毛螺菌科（Lachnospiraceae）
<400> 65
Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Thr Val Val Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Gln Lys Gly Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Asn Phe Pro Arg Phe
100 105 110
Tyr Tyr Asn Tyr Leu Glu Asp Ile Arg Asn Tyr Ser Asp Val Leu Asn
115 120 125
Phe Met Pro Lys Thr Gly Glu Pro Ala Asn Thr Ile Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Leu Gly Lys Tyr Phe Gln Gln
165 170 175
Asp Asn Lys Ile Leu Leu Pro Met Ser Val Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Phe Asn Glu Ala Gln Glu Leu
195 200 205
Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys
210 215
<210> 66
<211> 219
<212> PRT
<213> 产气荚膜梭菌（Clostridium perfringens）
<400> 66
Met Lys Phe Asn Leu Ile Asp Ile Glu Asp Trp Asn Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Leu Asn Ala Val Arg Cys Thr Tyr Ser Met Thr Ala
20 25 30
Asn Ile Glu Ile Thr Gly Leu Leu Arg Glu Ile Lys Leu Lys Gly Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Thr Thr Val Val Asn Arg
50 55 60
His Lys Glu Phe Arg Thr Cys Phe Asp Gln Lys Gly Lys Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Asn Pro Ser Tyr Thr Val Phe His Lys Asp Asn Glu
85 90 95
Thr Phe Ser Ser Ile Trp Thr Glu Tyr Asp Glu Asn Phe Pro Arg Phe
100 105 110
Tyr Tyr Asn Tyr Leu Glu Asp Ile Arg Asn Tyr Ser Asp Val Leu Asn
115 120 125
Phe Met Pro Lys Thr Gly Glu Pro Ala Asn Thr Ile Asn Val Ser Ser
130 135 140
Ile Pro Trp Val Asn Phe Thr Gly Phe Asn Leu Asn Ile Tyr Asn Asp
145 150 155 160
Ala Thr Tyr Leu Ile Pro Ile Phe Thr Leu Gly Lys Tyr Phe Gln Gln
165 170 175
Asp Asn Lys Ile Leu Leu Pro Met Ser Val Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Ile Ser Arg Phe Phe Asn Glu Ala Gln Glu Leu
195 200 205
Ala Ser Asn Tyr Glu Thr Trp Leu Gly Glu Lys
210 215
<210> 67
<211> 218
<212> PRT
<213> 人工序列
<220>
<223> 梭菌属菌种（Clostrdium sp.）BL8
<400> 67
Met Lys Phe Asn Leu Ile Asp Ile Asp Gln Trp Asp Arg Lys Pro Tyr
1 5 10 15
Phe Glu His Tyr Phe Asn Ser Val Lys Cys Thr Tyr Ser Ile Thr Ala
20 25 30
Asn Ile Glu Ile Thr Asn Leu Leu Lys Asp Ile Lys Ile Thr Lys Leu
35 40 45
Lys Leu Tyr Pro Thr Leu Ile Tyr Ile Ile Ala Thr Val Ile Asn Asn
50 55 60
His Glu Glu Phe Arg Thr Cys Phe Asp Glu Asn Asn Asn Leu Gly Tyr
65 70 75 80
Trp Asp Ser Met Ser Pro Asn Tyr Thr Ile Phe His Glu Glu Thr Lys
85 90 95
Thr Phe Ser Asn Ile Trp Thr Glu Tyr Asp Lys Ser Phe Ser Gly Phe
100 105 110
Tyr Asn Lys Tyr Val Glu Asp Asn Lys Asn Tyr Gly Asn Ile Met Asn
115 120 125
Phe Asp Pro Lys Leu Asn Glu Pro Ala Asn Thr Phe Pro Ile Ser Cys
130 135 140
Ile Pro Trp Val Ser Phe Thr Gly Phe Asn Leu Asn Ile Gln Asp His
145 150 155 160
Gly Thr Tyr Leu Thr Pro Ile Phe Thr Leu Gly Lys Tyr Phe Glu Glu
165 170 175
Asn Asn Lys Val Phe Ile Pro Met Ser Ile Gln Val His His Ala Val
180 185 190
Cys Asp Gly Tyr His Thr Ser Arg Phe Ile Asn Glu Val Gln Glu Leu
195 200 205
Ala Ser Asn Ser Gln Ser Trp Leu Lys His
210 215
<210> 68
<211> 660
<212> DNA
<213> 产气荚膜梭菌（Clostridium perfringens）
<400> 68
atgaaattta atttgataga tattgaggat tggaatagaa agccatactt tgagcattat 60
ttaaatgcgg ttaggtgcac ttacagtatg actgcaaata tagagataac tggtttactg 120
cgtgaaatta aacttaaggg cctgaaactg taccctacgc ttatttatat catcacaact 180
gtggttaacc gtcacaagga gttccgcacc tgttttgatc aaaaaggtaa gttaggatac 240
tgggatagta tgaacccaag ttatactgtc tttcataagg ataacgaaac tttttcaagt 300
atttggacag agtatgacga gaacttccca cgtttttact ataattacct tgaggatatt 360
agaaactata gcgacgtttt gaatttcatg cctaagacag gtgaacctgc taatacaatt 420
aatgtgtcca gcattccttg ggtgaatttt accggattca acctgaatat atacaatgat 480
gcaacatatc taatccctat ttttactttg ggtaagtatt ttcagcagga taataaaatt 540
ttattaccta tgtctgtaca ggtgcatcat gcggtttgcg acggttatca tataagcaga 600
ttttttaatg aggcacagga attagcgtca aattatgaga catggttagg agaaaaataa 660
<210> 69
<211> 624
<212> DNA
<213> 艰难梭菌（Clostridium difficile）
<400> 69
atggtatttg aaaaaattga taaaaatagt tggaacagaa aagagtattt tgaccactac 60
tttgcaagtg taccttgtac atacagcatg accgttaaag tggatatcac acaaataaag 120
gaaaagggaa tgaaactata tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc 180
cattcagagt ttaggacggc aatcaatcaa gatggtgaat tggggatata tgatgagatg 240
ataccaagct atacaatatt tcacaatgat actgaaacat tttccagcct ttggactgag 300
tgtaagtctg actttaaatc atttttagca gattatgaaa gtgatacgca acggtatgga 360
aacaatcata gaatggaagg aaagccaaat gctccggaaa acatttttaa tgtatctatg 420
ataccgtggt caaccttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 480
attcctattt ttactatggg gaaatattat aaagaagata acaaaattat acttcctttg 540
gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 600
ttgcaggaat tgataaatag ttaa 624
<210> 70
<211> 624
<212> DNA
<213> 产气荚膜梭菌（Clostridium perfringens）
<400> 70
atggtatttg aaaaaattga taaaaatagt tggaacagaa aagagtattt tgaccactac 60
tttgcaagtg taccttgtac atacagcatg accgttaaag tggatatcac acaaataaag 120
gaaaagggaa tgaaactata tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc 180
cattcagagt ttaggacggc aatcaatcaa gatggtgaat tggggatata tgatgagatg 240
ataccaagct atacaatatt tcacaatgat actgaaacat tttccagcct ttggactgag 300
tgtaagtctg actttaaatc atttttagca gattatgaaa gtgatacgca acggtatgga 360
aacaatcata gaatggaagg aaagccaaat gctccggaaa acatttttaa tgtatctatg 420
ataccgtggt caaccttcga tggctttaat ctgaatttgc agaaaggata tgattatttg 480
attcctattt ttactatggg gaaatattat aaagaagata acaaaattat acttcctttg 540
gcaattcaag ttcatcacgc agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa 600
ttgcaggaat tgataaatag ttaa 624
<210> 71
<211> 3897
<212> DNA
<213> 人工序列
<220>
<223> 优化的MAD7
<400> 71
ctcgagtccc tatcagtgat agattgaaac tctatcattg atagagtata atatctttgt 60
tcattagagc gataaacttg aatttgagag ggaacttaga tgaacaacgg cacaaataat 120
tttcagaact tcatagggat atcaagtttg cagaaaacgt taagaaatgc tttaataccc 180
acggaaacca cgcaacagtt catagttaag aacggaataa ttaaagaaga tgagttaaga 240
ggcgagaaca gacagatttt aaaagatata atggatgact actacagagg attcatatct 300
gagactttaa gttctattga tgacatagat tggactagct tattcgaaaa aatggaaatt 360
cagttaaaaa atggtgataa taaagatacc ttaattaagg aacagacaga gtatagaaaa 420
gcaatacata aaaaatttgc gaacgacgat agatttaaga acatgtttag cgccaaatta 480
attagtgaca tattacctga atttgttata cacaacaata attattcggc atcagagaaa 540
gaggaaaaaa cccaggtgat aaaattgttt tcgagatttg cgactagctt taaagattac 600
ttcaagaaca gagcaaattg cttttcagcg gacgatattt catcaagcag ctgccataga 660
atagttaacg acaatgcaga gatattcttt tcaaatgcgt tagtttacag aagaatagta 720
aaatcgttaa gcaatgacga tataaacaaa atttcgggcg atatgaaaga ttcattaaaa 780
gaaatgagtt tagaagaaat atattcttac gagaagtatg gggaatttat tacccaggaa 840
ggcattagct tctataatga tatatgtggg aaagtgaatt cttttatgaa cttatattgt 900
cagaaaaata aagaaaacaa aaatttatac aaacttcaga aacttcacaa acagattcta 960
tgcattgcgg acactagcta tgaggttccg tataaatttg aaagtgacga ggaagtgtac 1020
caatcagtta acggcttcct tgataacatt agcagcaaac atatagttga aagattaaga 1080
aaaataggcg ataactataa cggctacaac ttagataaaa tttatatagt gtccaaattt 1140
tacgagagcg ttagccaaaa aacctacaga gactgggaaa caattaatac cgccttagaa 1200
attcattaca ataatatatt gccgggtaac ggtaaaagta aagccgacaa agtaaaaaaa 1260
gcggttaaga atgatttaca gaaatccata accgaaataa atgaactagt gtcaaactat 1320
aagttatgca gtgacgacaa cataaaagcg gagacttata tacatgagat tagccatata 1380
ttgaataact ttgaagcaca ggaattgaaa tacaatccgg aaattcacct agttgaatcc 1440
gagttaaaag cgagtgagct taaaaacgtg ttagacgtga taatgaatgc gtttcattgg 1500
tgttcggttt ttatgactga ggaacttgtt gataaagaca acaattttta tgcggaatta 1560
gaggagattt acgatgaaat ttatccagta attagtttat acaacttagt tagaaactac 1620
gttacccaga aaccgtacag cacgaaaaag attaaattga actttggaat accgacgtta 1680
gcagacggtt ggtcaaagtc caaagagtat tctaataacg ctataatatt aatgagagac 1740
aatttatatt atttaggcat atttaatgcg aagaataaac cggacaagaa gattatagag 1800
ggtaatacgt cagaaaataa gggtgactac aaaaagatga tttataattt gttaccgggt 1860
cccaacaaaa tgataccgaa agttttcttg agcagcaaga cgggggtgga aacgtataaa 1920
ccgagcgcct atatactaga ggggtataaa cagaataaac atataaagtc ttcaaaagac 1980
tttgatataa ctttctgtca tgatttaata gactacttca aaaactgtat tgcaattcat 2040
cccgagtgga aaaacttcgg ttttgatttt agcgacacca gtacttatga agacatttcc 2100
gggttttata gagaggtaga gttacaaggt tacaagattg attggacata cattagcgaa 2160
aaagacattg atttattaca ggaaaaaggt caattatatt tattccagat atataacaaa 2220
gatttttcga aaaaatcaac cgggaatgac aaccttcaca ccatgtactt aaaaaatctt 2280
ttctcagaag aaaatcttaa ggatatagtt ttaaaactta acggcgaagc ggaaatattc 2340
ttcaggaaga gcagcataaa gaacccaata attcataaaa aaggctcgat tttagttaac 2400
agaacctacg aagcagaaga aaaagaccag tttggcaaca ttcaaattgt gagaaaaaat 2460
attccggaaa acatttatca ggagttatac aaatacttca acgataaaag cgacaaagag 2520
ttatctgatg aagcagccaa attaaagaat gtagtgggac accacgaggc agcgacgaat 2580
atagttaagg actatagata cacgtatgat aaatacttcc ttcatatgcc tattacgata 2640
aatttcaaag ccaataaaac gggttttatt aatgatagga tattacagta tatagctaaa 2700
gaaaaagact tacatgtgat aggcattgat agaggcgaga gaaacttaat atacgtgtcc 2760
gtgattgata cttgtggtaa tatagttgaa cagaaaagct ttaacattgt aaacggctac 2820
gactatcaga taaaattaaa acaacaggag ggcgctagac agattgcgag aaaagaatgg 2880
aaagaaattg gtaaaattaa agagataaaa gagggctact taagcttagt aatacacgag 2940
atatctaaaa tggtaataaa atacaatgca attatagcga tggaggattt gtcttatggt 3000
tttaaaaaag ggagatttaa ggttgaaaga caagtttacc agaaatttga aaccatgtta 3060
ataaataaat taaactattt agtatttaaa gatatttcga ttaccgagaa tggcggttta 3120
ttaaaaggtt atcagttaac atacattcct gataaactta aaaacgtggg tcatcagtgc 3180
ggctgcattt tttatgtgcc tgctgcatac acgagcaaaa ttgatccgac caccggcttt 3240
gtgaatatat ttaaatttaa agacttaaca gtggacgcaa aaagagaatt cattaaaaaa 3300
tttgactcaa ttagatatga cagtgaaaaa aatttattct gctttacatt tgactacaat 3360
aactttatta cgcaaaacac ggttatgagc aaatcatcgt ggagtgtgta tacatacggc 3420
gtgagaataa aaagaagatt tgtgaacggc agattctcaa acgaaagtga taccattgac 3480
ataaccaaag atatggagaa aacgttggaa atgacggaca ttaactggag agatggccac 3540
gatcttagac aagacattat agattatgaa attgttcagc acatattcga aattttcaga 3600
ttaacagtgc aaatgagaaa ctccttgtct gaattagagg acagagatta cgatagatta 3660
atttcacctg tattaaacga aaataacatt ttttatgaca gcgcgaaagc gggggatgca 3720
cttcctaagg atgccgatgc aaatggtgcg tattgtattg cattaaaagg gttatatgaa 3780
attaaacaaa ttaccgaaaa ttggaaagaa gatggtaaat tttcgagaga taaattaaaa 3840
ataagcaata aagattggtt cgactttata cagaataaga gatatttata agtcgac 3897
<210> 72
<211> 1263
<212> PRT
<213> 人工序列
<220>
<223> MAD7
<400> 72
Met Asn Asn Gly Thr Asn Asn Phe Gln Asn Phe Ile Gly Ile Ser Ser
1 5 10 15
Leu Gln Lys Thr Leu Arg Asn Ala Leu Ile Pro Thr Glu Thr Thr Gln
20 25 30
Gln Phe Ile Val Lys Asn Gly Ile Ile Lys Glu Asp Glu Leu Arg Gly
35 40 45
Glu Asn Arg Gln Ile Leu Lys Asp Ile Met Asp Asp Tyr Tyr Arg Gly
50 55 60
Phe Ile Ser Glu Thr Leu Ser Ser Ile Asp Asp Ile Asp Trp Thr Ser
65 70 75 80
Leu Phe Glu Lys Met Glu Ile Gln Leu Lys Asn Gly Asp Asn Lys Asp
85 90 95
Thr Leu Ile Lys Glu Gln Thr Glu Tyr Arg Lys Ala Ile His Lys Lys
100 105 110
Phe Ala Asn Asp Asp Arg Phe Lys Asn Met Phe Ser Ala Lys Leu Ile
115 120 125
Ser Asp Ile Leu Pro Glu Phe Val Ile His Asn Asn Asn Tyr Ser Ala
130 135 140
Ser Glu Lys Glu Glu Lys Thr Gln Val Ile Lys Leu Phe Ser Arg Phe
145 150 155 160
Ala Thr Ser Phe Lys Asp Tyr Phe Lys Asn Arg Ala Asn Cys Phe Ser
165 170 175
Ala Asp Asp Ile Ser Ser Ser Ser Cys His Arg Ile Val Asn Asp Asn
180 185 190
Ala Glu Ile Phe Phe Ser Asn Ala Leu Val Tyr Arg Arg Ile Val Lys
195 200 205
Ser Leu Ser Asn Asp Asp Ile Asn Lys Ile Ser Gly Asp Met Lys Asp
210 215 220
Ser Leu Lys Glu Met Ser Leu Glu Glu Ile Tyr Ser Tyr Glu Lys Tyr
225 230 235 240
Gly Glu Phe Ile Thr Gln Glu Gly Ile Ser Phe Tyr Asn Asp Ile Cys
245 250 255
Gly Lys Val Asn Ser Phe Met Asn Leu Tyr Cys Gln Lys Asn Lys Glu
260 265 270
Asn Lys Asn Leu Tyr Lys Leu Gln Lys Leu His Lys Gln Ile Leu Cys
275 280 285
Ile Ala Asp Thr Ser Tyr Glu Val Pro Tyr Lys Phe Glu Ser Asp Glu
290 295 300
Glu Val Tyr Gln Ser Val Asn Gly Phe Leu Asp Asn Ile Ser Ser Lys
305 310 315 320
His Ile Val Glu Arg Leu Arg Lys Ile Gly Asp Asn Tyr Asn Gly Tyr
325 330 335
Asn Leu Asp Lys Ile Tyr Ile Val Ser Lys Phe Tyr Glu Ser Val Ser
340 345 350
Gln Lys Thr Tyr Arg Asp Trp Glu Thr Ile Asn Thr Ala Leu Glu Ile
355 360 365
His Tyr Asn Asn Ile Leu Pro Gly Asn Gly Lys Ser Lys Ala Asp Lys
370 375 380
Val Lys Lys Ala Val Lys Asn Asp Leu Gln Lys Ser Ile Thr Glu Ile
385 390 395 400
Asn Glu Leu Val Ser Asn Tyr Lys Leu Cys Ser Asp Asp Asn Ile Lys
405 410 415
Ala Glu Thr Tyr Ile His Glu Ile Ser His Ile Leu Asn Asn Phe Glu
420 425 430
Ala Gln Glu Leu Lys Tyr Asn Pro Glu Ile His Leu Val Glu Ser Glu
435 440 445
Leu Lys Ala Ser Glu Leu Lys Asn Val Leu Asp Val Ile Met Asn Ala
450 455 460
Phe His Trp Cys Ser Val Phe Met Thr Glu Glu Leu Val Asp Lys Asp
465 470 475 480
Asn Asn Phe Tyr Ala Glu Leu Glu Glu Ile Tyr Asp Glu Ile Tyr Pro
485 490 495
Val Ile Ser Leu Tyr Asn Leu Val Arg Asn Tyr Val Thr Gln Lys Pro
500 505 510
Tyr Ser Thr Lys Lys Ile Lys Leu Asn Phe Gly Ile Pro Thr Leu Ala
515 520 525
Asp Gly Trp Ser Lys Ser Lys Glu Tyr Ser Asn Asn Ala Ile Ile Leu
530 535 540
Met Arg Asp Asn Leu Tyr Tyr Leu Gly Ile Phe Asn Ala Lys Asn Lys
545 550 555 560
Pro Asp Lys Lys Ile Ile Glu Gly Asn Thr Ser Glu Asn Lys Gly Asp
565 570 575
Tyr Lys Lys Met Ile Tyr Asn Leu Leu Pro Gly Pro Asn Lys Met Ile
580 585 590
Pro Lys Val Phe Leu Ser Ser Lys Thr Gly Val Glu Thr Tyr Lys Pro
595 600 605
Ser Ala Tyr Ile Leu Glu Gly Tyr Lys Gln Asn Lys His Ile Lys Ser
610 615 620
Ser Lys Asp Phe Asp Ile Thr Phe Cys His Asp Leu Ile Asp Tyr Phe
625 630 635 640
Lys Asn Cys Ile Ala Ile His Pro Glu Trp Lys Asn Phe Gly Phe Asp
645 650 655
Phe Ser Asp Thr Ser Thr Tyr Glu Asp Ile Ser Gly Phe Tyr Arg Glu
660 665 670
Val Glu Leu Gln Gly Tyr Lys Ile Asp Trp Thr Tyr Ile Ser Glu Lys
675 680 685
Asp Ile Asp Leu Leu Gln Glu Lys Gly Gln Leu Tyr Leu Phe Gln Ile
690 695 700
Tyr Asn Lys Asp Phe Ser Lys Lys Ser Thr Gly Asn Asp Asn Leu His
705 710 715 720
Thr Met Tyr Leu Lys Asn Leu Phe Ser Glu Glu Asn Leu Lys Asp Ile
725 730 735
Val Leu Lys Leu Asn Gly Glu Ala Glu Ile Phe Phe Arg Lys Ser Ser
740 745 750
Ile Lys Asn Pro Ile Ile His Lys Lys Gly Ser Ile Leu Val Asn Arg
755 760 765
Thr Tyr Glu Ala Glu Glu Lys Asp Gln Phe Gly Asn Ile Gln Ile Val
770 775 780
Arg Lys Asn Ile Pro Glu Asn Ile Tyr Gln Glu Leu Tyr Lys Tyr Phe
785 790 795 800
Asn Asp Lys Ser Asp Lys Glu Leu Ser Asp Glu Ala Ala Lys Leu Lys
805 810 815
Asn Val Val Gly His His Glu Ala Ala Thr Asn Ile Val Lys Asp Tyr
820 825 830
Arg Tyr Thr Tyr Asp Lys Tyr Phe Leu His Met Pro Ile Thr Ile Asn
835 840 845
Phe Lys Ala Asn Lys Thr Gly Phe Ile Asn Asp Arg Ile Leu Gln Tyr
850 855 860
Ile Ala Lys Glu Lys Asp Leu His Val Ile Gly Ile Asp Arg Gly Glu
865 870 875 880
Arg Asn Leu Ile Tyr Val Ser Val Ile Asp Thr Cys Gly Asn Ile Val
885 890 895
Glu Gln Lys Ser Phe Asn Ile Val Asn Gly Tyr Asp Tyr Gln Ile Lys
900 905 910
Leu Lys Gln Gln Glu Gly Ala Arg Gln Ile Ala Arg Lys Glu Trp Lys
915 920 925
Glu Ile Gly Lys Ile Lys Glu Ile Lys Glu Gly Tyr Leu Ser Leu Val
930 935 940
Ile His Glu Ile Ser Lys Met Val Ile Lys Tyr Asn Ala Ile Ile Ala
945 950 955 960
Met Glu Asp Leu Ser Tyr Gly Phe Lys Lys Gly Arg Phe Lys Val Glu
965 970 975
Arg Gln Val Tyr Gln Lys Phe Glu Thr Met Leu Ile Asn Lys Leu Asn
980 985 990
Tyr Leu Val Phe Lys Asp Ile Ser Ile Thr Glu Asn Gly Gly Leu Leu
995 1000 1005
Lys Gly Tyr Gln Leu Thr Tyr Ile Pro Asp Lys Leu Lys Asn Val
1010 1015 1020
Gly His Gln Cys Gly Cys Ile Phe Tyr Val Pro Ala Ala Tyr Thr
1025 1030 1035
Ser Lys Ile Asp Pro Thr Thr Gly Phe Val Asn Ile Phe Lys Phe
1040 1045 1050
Lys Asp Leu Thr Val Asp Ala Lys Arg Glu Phe Ile Lys Lys Phe
1055 1060 1065
Asp Ser Ile Arg Tyr Asp Ser Glu Lys Asn Leu Phe Cys Phe Thr
1070 1075 1080
Phe Asp Tyr Asn Asn Phe Ile Thr Gln Asn Thr Val Met Ser Lys
1085 1090 1095
Ser Ser Trp Ser Val Tyr Thr Tyr Gly Val Arg Ile Lys Arg Arg
1100 1105 1110
Phe Val Asn Gly Arg Phe Ser Asn Glu Ser Asp Thr Ile Asp Ile
1115 1120 1125
Thr Lys Asp Met Glu Lys Thr Leu Glu Met Thr Asp Ile Asn Trp
1130 1135 1140
Arg Asp Gly His Asp Leu Arg Gln Asp Ile Ile Asp Tyr Glu Ile
1145 1150 1155
Val Gln His Ile Phe Glu Ile Phe Arg Leu Thr Val Gln Met Arg
1160 1165 1170
Asn Ser Leu Ser Glu Leu Glu Asp Arg Asp Tyr Asp Arg Leu Ile
1175 1180 1185
Ser Pro Val Leu Asn Glu Asn Asn Ile Phe Tyr Asp Ser Ala Lys
1190 1195 1200
Ala Gly Asp Ala Leu Pro Lys Asp Ala Asp Ala Asn Gly Ala Tyr
1205 1210 1215
Cys Ile Ala Leu Lys Gly Leu Tyr Glu Ile Lys Gln Ile Thr Glu
1220 1225 1230
Asn Trp Lys Glu Asp Gly Lys Phe Ser Arg Asp Lys Leu Lys Ile
1235 1240 1245
Ser Asn Lys Asp Trp Phe Asp Phe Ile Gln Asn Lys Arg Tyr Leu
1250 1255 1260
<210> 73
<211> 363
<212> DNA
<213> 人工序列
<220>
<223> CatB启动子
<400> 73
taaaaaatgt tacgcacttt tcttatattg ttcaacaata acataattta ttaacaaaag 60
gaaagtatag ttaaaaaaat gttggagcaa atgcggatgg aaaaataaaa attaatatta 120
gtagtaattc cgatgttaaa ataacaagag ataagaaaaa gtaaaatatt agagtaattc 180
gtagtattct taagttatga atcaataaaa aatggtctct gaaaattgaa tagttcggta 240
ttacagaatg tgctataata aactaaagcg taaatatcat tgtaaaaagg agattgaaat 300
ggctaggtca cggaaaaaag ccttctaaaa tagaattacg aaaattttta ggaggcccga 360
att 363
<210> 74
<211> 322
<212> DNA
<213> 人工序列
<220>
<223> CATQ启动子
<400> 74
ctgcgtacac atccagacat cgctttagag tatggtgaat taaagatgga gcgggcttat 60
cgattctcag aggatattga aggctactgc actggtaagg atgcatttgt aaagcaacta 120
gaaaaggatg ctttgcgatg gtggcaaact gtctgttagg aggttattct caaaggattg 180
caagaagcag ttgaggataa tccgtataac taactattac acattcttaa cattgctggt 240
ttgtatcggt agaataacac gaattaacaa aggatatatt ttgtagtagc aagtgtattt 300
gttttatatt ctatgaacct at 322
<210> 75
<211> 1368
<212> PRT
<213> 酿脓链球菌（Streptococcus pyogenes）
<400> 75
Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val
1 5 10 15
Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe
20 25 30
Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile
35 40 45
Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu
50 55 60
Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys
65 70 75 80
Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser
85 90 95
Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys
100 105 110
His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr
115 120 125
His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp
130 135 140
Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His
145 150 155 160
Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro
165 170 175
Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr
180 185 190
Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala
195 200 205
Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn
210 215 220
Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn
225 230 235 240
Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe
245 250 255
Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp
260 265 270
Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp
275 280 285
Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp
290 295 300
Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser
305 310 315 320
Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys
325 330 335
Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe
340 345 350
Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser
355 360 365
Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp
370 375 380
Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg
385 390 395 400
Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu
405 410 415
Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe
420 425 430
Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile
435 440 445
Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp
450 455 460
Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu
465 470 475 480
Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr
485 490 495
Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser
500 505 510
Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys
515 520 525
Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln
530 535 540
Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr
545 550 555 560
Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp
565 570 575
Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly
580 585 590
Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp
595 600 605
Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr
610 615 620
Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala
625 630 635 640
His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr
645 650 655
Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp
660 665 670
Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe
675 680 685
Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe
690 695 700
Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu
705 710 715 720
His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly
725 730 735
Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly
740 745 750
Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln
755 760 765
Thr Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile
770 775 780
Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro
785 790 795 800
Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu
805 810 815
Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg
820 825 830
Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys
835 840 845
Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg
850 855 860
Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys
865 870 875 880
Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys
885 890 895
Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp
900 905 910
Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr
915 920 925
Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp
930 935 940
Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser
945 950 955 960
Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg
965 970 975
Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val
980 985 990
Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe
995 1000 1005
Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala
1010 1015 1020
Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe
1025 1030 1035
Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala
1040 1045 1050
Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu
1055 1060 1065
Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val
1070 1075 1080
Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr
1085 1090 1095
Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys
1100 1105 1110
Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro
1115 1120 1125
Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val
1130 1135 1140
Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys
1145 1150 1155
Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser
1160 1165 1170
Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys
1175 1180 1185
Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu
1190 1195 1200
Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly
1205 1210 1215
Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val
1220 1225 1230
Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser
1235 1240 1245
Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys
1250 1255 1260
His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys
1265 1270 1275
Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala
1280 1285 1290
Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn
1295 1300 1305
Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala
1310 1315 1320
Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser
1325 1330 1335
Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr
1340 1345 1350
Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp
1355 1360 1365
<210> 76
<211> 4107
<212> DNA
<213> 酿脓链球菌（Streptococcus pyogenes）
<400> 76
atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg atgggcggtg 60
atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa tacagaccgc 120
cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga gacagcggaa 180
gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa tcgtatttgt 240
tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt ctttcatcga 300
cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc tatttttgga 360
aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca tctgcgaaaa 420
aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc cttagcgcat 480
atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga taatagtgat 540
gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga agaaaaccct 600
attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag taaatcaaga 660
cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt atttgggaat 720
ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga tttggcagaa 780
gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa tttattggcg 840
caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc agatgctatt 900
ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct atcagcttca 960
atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc tttagttcga 1020
caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa cggatatgca 1080
ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa accaatttta 1140
gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga tttgctgcgc 1200
aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg tgagctgcat 1260
gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg tgagaagatt 1320
gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg tggcaatagt 1380
cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa ttttgaagaa 1440
gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa ctttgataaa 1500
aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta ttttacggtt 1560
tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc agcatttctt 1620
tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg aaaagtaacc 1680
gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag tgttgaaatt 1740
tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt gctaaaaatt 1800
attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga ggatattgtt 1860
ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa aacatatgct 1920
cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac tggttgggga 1980
cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa aacaatatta 2040
gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat ccatgatgat 2100
agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg cgatagttta 2160
catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat tttacagact 2220
gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga aaatatcgtt 2280
attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc gcgagagcgt 2340
atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa agagcatcct 2400
gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca aaatggaaga 2460
gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga tgtcgatcac 2520
attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt aacgcgttct 2580
gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa aaagatgaaa 2640
aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt tgataattta 2700
acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat caaacgccaa 2760
ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag tcgcatgaat 2820
actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac cttaaaatct 2880
aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga gattaacaat 2940
taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt gattaagaaa 3000
tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga tgttcgtaaa 3060
atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt cttttactct 3120
aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat tcgcaaacgc 3180
cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg gcgagatttt 3240
gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa aacagaagta 3300
cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga caagcttatt 3360
gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc aacggtagct 3420
tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt aaaatccgtt 3480
aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa tccgattgac 3540
tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa actacctaaa 3600
tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc cggagaatta 3660
caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata tttagctagt 3720
cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt gtttgtggag 3780
cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc taagcgtgtt 3840
attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca tagagacaaa 3900
ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa tcttggagct 3960
cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac gtctacaaaa 4020
gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga aacacgcatt 4080
gatttgagtc agctaggagg tgactga 4107
<210> 77
<211> 1170
<212> DNA
<213> 人工序列
<220>
<223> bdhA
<400> 77
atgctaagtt ttgattattc aataccaact aaagtttttt ttggaaaagg aaaaatagac 60
gtaattggag aagaaattaa gaaatatggc tcaagagtgc ttatagttta tggcggagga 120
agtataaaaa ggaacggtat atatgataga gcaacagcta tattaaaaga aaacaatata 180
gctttctatg aactttcagg agtagagcca aatcctagga taacaacagt aaaaaaaggc 240
atagaaatat gtagagaaaa taatgtggat ttagtattag caataggggg aggaagtgca 300
atagactgtt ctaaggtaat tgcagctgga gtttattatg atggcgatac atgggacatg 360
gttaaagatc catctaaaat aactaaagtt cttccaattg caagtatact tactctttca 420
gcaacagggt ctgaaatgga tcaaattgca gtaatttcaa atatggagac taatgaaaag 480
cttggagtag gacatgatga tatgagacct aaattttcag tgttagatcc tacatatact 540
tttacagtac ctaaaaatca aacagcagcg ggaacagctg acattatgag tcacaccttt 600
gaatcttact ttagtggtgt tgaaggtgct tatgtgcagg acggtatagc agaagcaatc 660
ttaagaacat gtataaagta tggaaaaata gcaatggaga agactgatga ttacgaggct 720
agagctaatt tgatgtgggc ttcaagttta gctataaatg gtctattatc acttggtaag 780
gatagaaaat ggagttgtca tcctatggaa cacgagttaa gtgcatatta tgatataaca 840
catggtgtag gacttgcaat tttaacacct aattggatgg aatatattct aaatgacgat 900
acacttcata aatttgtttc ttatggaata aatgtttggg gaatagacaa gaacaaagat 960
aactatgaaa tagcacgaga ggctattaaa aatacgagag aatactttaa ttcattgggt 1020
attccttcaa agcttagaga agttggaata ggaaaagata aactagaact aatggcaaag 1080
caagctgtta gaaattctgg aggaacaata ggaagtttaa gaccaataaa tgcagaggat 1140
gttcttgaga tatttaaaaa atcttattaa 1170
<210> 78
<211> 1173
<212> DNA
<213> 人工序列
<220>
<223> bdhB
<400> 78
gtggttgatt tcgaatattc aataccaact agaatttttt tcggtaaaga taagataaat 60
gtacttggaa gagagcttaa aaaatatggt tctaaagtgc ttatagttta tggtggagga 120
agtataaaga gaaatggaat atatgataaa gctgtaagta tacttgaaaa aaacagtatt 180
aaattttatg aacttgcagg agtagagcca aatccaagag taactacagt tgaaaaagga 240
gttaaaatat gtagagaaaa tggagttgaa gtagtactag ctataggtgg aggaagtgca 300
atagattgcg caaaggttat agcagcagca tgtgaatatg atggaaatcc atgggatatt 360
gtgttagatg gctcaaaaat aaaaagggtg cttcctatag ctagtatatt aaccattgct 420
gcaacaggat cagaaatgga tacgtgggca gtaataaata atatggatac aaacgaaaaa 480
ctaattgcgg cacatccaga tatggctcct aagttttcta tattagatcc aacgtatacg 540
tataccgtac ctaccaatca aacagcagca ggaacagctg atattatgag tcatatattt 600
gaggtgtatt ttagtaatac aaaaacagca tatttgcagg atagaatggc agaagcgtta 660
ttaagaactt gtattaaata tggaggaata gctcttgaga agccggatga ttatgaggca 720
agagccaatc taatgtgggc ttcaagtctt gcgataaatg gacttttaac atatggtaaa 780
gacactaatt ggagtgtaca cttaatggaa catgaattaa gtgcttatta cgacataaca 840
cacggcgtag ggcttgcaat tttaacacct aattggatgg agtatatttt aaataatgat 900
acagtgtaca agtttgttga atatggtgta aatgtttggg gaatagacaa agaaaaaaat 960
cactatgaca tagcacatca agcaatacaa aaaacaagag attactttgt aaatgtacta 1020
ggtttaccat ctagactgag agatgttgga attgaagaag aaaaattgga cataatggca 1080
aaggaatcag taaagcttac aggaggaacc ataggaaacc taagaccagt aaacgcctcc 1140
gaagtcctac aaatattcaa aaaatctgtg taa 1173
<210> 79
<211> 6560
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-ΔbdhB
<400> 79
gatccccggg taccgagctc gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 960
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1020
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1080
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 1140
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200
caaagctagc ttaatactag tatatactta atgtgataag tgtctgacag ctgaccggtc 1260
taaagaggtc cctagcgcct acggggaatt tgtatcgata aggggtacaa attcccacta 1320
agcgctcggc cggggatcga tccccgggta cgtacccggc agtttttctt tttcggcaag 1380
tgttcaagaa gttattaagt cgggagtgca gtcgaagtgg gcaagttgaa aaattcacaa 1440
aaatgtggta taatatcttt gttcattaga gcgataaact tgaatttgag agggaactta 1500
gatggtattt gaaaaaattg ataaaaatag ttggaacaga aaagagtatt ttgaccacta 1560
ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacaaataaa 1620
ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaaccg 1680
ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 1740
gataccaagc tatacaatat ttcacaatga tactgaaaca ttttccagcc tttggactga 1800
gtgtaagtct gactttaaat catttttagc agattatgaa agtgatacgc aacggtatgg 1860
aaacaatcat agaatggaag gaaagccaaa tgctccggaa aacattttta atgtatctat 1920
gataccgtgg tcaaccttcg atggctttaa tctgaatttg cagaaaggat atgattattt 1980
gattcctatt tttactatgg ggaaatatta taaagaagat aacaaaatta tacttccttt 2040
ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 2100
attgcaggaa ttgataaata gttaacttca ggtttgtctg taactaaaaa ctagtattta 2160
acctaggatc aaaaaaattt ccaataatcc cactctaagc cacaaacacg ccctataaaa 2220
tcccgcttta atcccacttt gagacacatg taatattact ttacgcccta gtatagtgat 2280
aattttttac attcaatgcc acgcaaaaaa ataaaggggc actataataa aagttccttc 2340
ggaactaact aaagtaaaaa attatcttta caacctcccc aaaaaaaaga acaggtacaa 2400
agtaccctat aatacaagcg taaaaaaaat gagggtaaaa ataaaaaaat aaaaaaataa 2460
aaaaataaaa aaataaaaaa ataaaaaaat aaaaaaatat aaaaataaaa aaatataaaa 2520
ataaaaaaat ataaaaataa aaaaataaaa aaatataaaa ataaaaaaat aaaaaaatat 2580
aaaaatattt tttatttaaa gtttgaaaaa aattttttta tattatataa tctttgaaga 2640
aaagaatata aaaaatgagc ctttataaaa gcccattttt tttcatatac gtaatatgac 2700
gttctaatgt ttttattggt acttctaaca ttagagtaat ttctttattt ttaaagcctt 2760
tttctttaag ggcttttatt ttttttctta atacatttaa ttcctctttt tttgttgctt 2820
ttcctttagc ttttaattgc tcttgataat tttttttacc tctaatattt tctcttctct 2880
tatattcctt tttagaaatt attattgtca tatatttttg ttcttcttct gtaatttcta 2940
ataactctat aagagtttca ttcttatact tatattgctt atttttatct aaataacatc 3000
tttcagcact tctagttgct cttataactt ctctttcact taaatgttgt ctaaacatac 3060
tattaagttc taaaacatca tttaatgcct tctcaatgtc ttctgtaaag ctacaaagat 3120
aatatctata taaaaataat ataagctctc tgtgtccttt taaatcatat tctcttagtt 3180
cacaaagttt tattatgtct tgtattcttc cataatataa acttctttct ctataaatat 3240
aatttatttt gcttggtcta ccctttttcc tttcatatgg ttttaattca ggtaaaaatc 3300
cattttgtat ttctcttaag tcataaatat attcgtactc atctaatata ttgactactg 3360
tttttgattt agagtttata cttcctggaa ctcttaatat tctcgttgca tctaaggctt 3420
gtctatctgc tccaaagtat tttaattgat tatataaata ttcttgaacc gctttccata 3480
atggtaatgc tttactaggt actgcattta ttatccatat taaatacatt cctcttccac 3540
tatctattac atagtttggt ataggaatac tttgattaaa ataattcttt tctaagtcca 3600
ttaatacctg gtctttagtt ttgccagttt tataataatc caagtctata aacagtgtat 3660
ttaactcttt tatattttct aatcgcctac acggcttata aaaggtattt agagttatat 3720
agatattttc atcactcata tctaaatctt ttaattcagc gtatttatag tgccattggc 3780
tatatccttt tttatctata acgctcctgg ttatccaccc tttacttcta ctatgaatat 3840
tatctatata gttcttttta ttcagcttta atgcgtttct cacttattca cctccccttc 3900
tgtaaaacta agaaaattat atcatatttt caataattat taactattct taaactctta 3960
ataaaaaata gagtaagtcc ccaattgaaa cttaatctat tttttatgtt ttaatttatt 4020
atttttatta aaatatttta aactaaatta aatgattctt tttaattttt tactatttca 4080
ttccataata tattactata attatttaca aataatattt cttcatttgt aatatttaga 4140
tgatttacta attttagttt ttatatatta aataattaat gtataattta tataaaaaat 4200
caaaggagct tataaattat gattatttcc aaagatacta aagatttaat ttttttcaat 4260
tttaacaata ctttttgtaa tattatgttt aaatttaatt gtattttttt catataataa 4320
agccgttgaa gtaaaccaat ccattttcct tatgatgtta ttattaaatt taagttttat 4380
aataatatct ttattatatt tattgttttt aaaaaaacta gtgaaatttc tagtgaaatt 4440
tccggcttta ttaaacttat ttttaggaat tttattttca ttttcatctt tacaggattt 4500
gattatatct ttaaatatgt tttatcaaat attatctttt tctaaattta tatatatttt 4560
tattatattt attattatat atattttatt tttaagtttc tttctaacag ctattaaaaa 4620
gaaacttaaa aataaaaaca cgtactctaa accaataaat aaaactattt ttattattgc 4680
tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attatattat 4740
aagctagcac gcctcgagac tctatcattg atagagtttg aaactctatc attgatagag 4800
tataatatct ttgttcatgc ttattacgac ataacacagt tttagagcta gaaatagcaa 4860
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 4920
tgaagcttct cgagatctcc atggacgcgt gacgtcgact cttaagaaca tgtataaagt 4980
atggaaaaat agcaatggag aagactgatg attacgaggc tagagctaat ttgatgtggg 5040
cttcaagttt agctataaat ggtctattat cacttggtaa ggatagaaaa tggagttgtc 5100
atcctatgga acacgagtta agtgcatatt atgatataac acatggtgta ggacttgcaa 5160
ttttaacacc taattggatg gaatatattc taaatgacga tacacttcat aaatttgttt 5220
cttatggaat aaatgtttgg ggaatagaca agaacaaaga taactatgaa atagcacgag 5280
aggctattaa aaatacgaga gaatacttta attcattggg tattccttca aagcttagag 5340
aagttggaat aggaaaagat aaactagaac taatggcaaa gcaagctgtt agaaattctg 5400
gaggaacaat aggaagttta agaccaataa atgcagagga tgttcttgag atatttaaaa 5460
aatcttatta atagaaactg tagaggtatt tttataattt aaaagatgtt aaagagtgag 5520
gagtaatttt gttctaacgc ctcactcttt tcattttatg attaaatgta tgctgattta 5580
cgctaactta aatcctaaat aataacctaa tgttaatatt ttgtaacaaa tggataaaag 5640
cgtaaaaata ttattgtaat aattttaagt aggtttaaaa tatatataat gtagaagcat 5700
tcctacatta tattatttaa ataataatct aaacaggagg ggttaaagtg gttgatttca 5760
aatctgtgta aacctaccgg ggtttgggcg tagccattat attcatgaac tccaagaaag 5820
cagtatgcta gcaaagaaat aaaactcaaa gcagagagaa aatttagaca ttcaactata 5880
aataaaaaat accccccaaa gcattaatat cttggggagt attttttatt ttgaagtatt 5940
ctgttcagct aaatattctt ctaaggtaat acctctgttc ataatttctt gtgaggcagg 6000
aagaccgata tatcttacat gccatggctc aaaattatac tttgttatgt tttctttatc 6060
cttaggatat cttattatga aaccatattt accacaattt tgttgaagcc atttataaga 6120
atttgtattc ataaatccat catctaaaga agagtattcg gttgatagta agtccattgc 6180
caatccagtt tgatgctcac ttgtaccagg ttcagctaca tatttatcag cttcggcttt 6240
tccgtctcgt gctacttttt cattatataa tttttgctga tacgaataag gtctataacc 6300
tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat tttcaagtcc 6360
tgttgcagct tcgctctcca tttgatttac attaggatca gaactactaa taaatttaac 6420
gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480
aagtaggatg ttctgtttct ttacctcggg tttcttggct tgttttttag gtgtagaaac 6540
tttctttttg ggttcgtttg 6560
<210> 80
<211> 6560
<212> DNA
<213> 人工序列
<220>
<223> pGRNA_ΔbdhA_ΔbdhB
<400> 80
gatccccggg taccgagctc gaattcgtaa tcatggtcat agctgtttcc tgtgtgaaat 60
tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg 120
ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag 180
tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 240
ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 300
ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 360
gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 420
gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 480
cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 540
ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 600
tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 660
gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 720
tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 780
ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 840
ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 900
ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 960
accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 1020
tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 1080
cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 1140
taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 1200
caaagctagc ttaatactag tatatactta atgtgataag tgtctgacag ctgaccggtc 1260
taaagaggtc cctagcgcct acggggaatt tgtatcgata aggggtacaa attcccacta 1320
agcgctcggc cggggatcga tccccgggta cgtacccggc agtttttctt tttcggcaag 1380
tgttcaagaa gttattaagt cgggagtgca gtcgaagtgg gcaagttgaa aaattcacaa 1440
aaatgtggta taatatcttt gttcattaga gcgataaact tgaatttgag agggaactta 1500
gatggtattt gaaaaaattg ataaaaatag ttggaacaga aaagagtatt ttgaccacta 1560
ctttgcaagt gtaccttgta cctacagcat gaccgttaaa gtggatatca cacaaataaa 1620
ggaaaaggga atgaaactat atcctgcaat gctttattat attgcaatga ttgtaaaccg 1680
ccattcagag tttaggacgg caatcaatca agatggtgaa ttggggatat atgatgagat 1740
gataccaagc tatacaatat ttcacaatga tactgaaaca ttttccagcc tttggactga 1800
gtgtaagtct gactttaaat catttttagc agattatgaa agtgatacgc aacggtatgg 1860
aaacaatcat agaatggaag gaaagccaaa tgctccggaa aacattttta atgtatctat 1920
gataccgtgg tcaaccttcg atggctttaa tctgaatttg cagaaaggat atgattattt 1980
gattcctatt tttactatgg ggaaatatta taaagaagat aacaaaatta tacttccttt 2040
ggcaattcaa gttcatcacg cagtatgtga cggatttcac atttgccgtt ttgtaaacga 2100
attgcaggaa ttgataaata gttaacttca ggtttgtctg taactaaaaa ctagtattta 2160
acctaggatc aaaaaaattt ccaataatcc cactctaagc cacaaacacg ccctataaaa 2220
tcccgcttta atcccacttt gagacacatg taatattact ttacgcccta gtatagtgat 2280
aattttttac attcaatgcc acgcaaaaaa ataaaggggc actataataa aagttccttc 2340
ggaactaact aaagtaaaaa attatcttta caacctcccc aaaaaaaaga acaggtacaa 2400
agtaccctat aatacaagcg taaaaaaaat gagggtaaaa ataaaaaaat aaaaaaataa 2460
aaaaataaaa aaataaaaaa ataaaaaaat aaaaaaatat aaaaataaaa aaatataaaa 2520
ataaaaaaat ataaaaataa aaaaataaaa aaatataaaa ataaaaaaat aaaaaaatat 2580
aaaaatattt tttatttaaa gtttgaaaaa aattttttta tattatataa tctttgaaga 2640
aaagaatata aaaaatgagc ctttataaaa gcccattttt tttcatatac gtaatatgac 2700
gttctaatgt ttttattggt acttctaaca ttagagtaat ttctttattt ttaaagcctt 2760
tttctttaag ggcttttatt ttttttctta atacatttaa ttcctctttt tttgttgctt 2820
ttcctttagc ttttaattgc tcttgataat tttttttacc tctaatattt tctcttctct 2880
tatattcctt tttagaaatt attattgtca tatatttttg ttcttcttct gtaatttcta 2940
ataactctat aagagtttca ttcttatact tatattgctt atttttatct aaataacatc 3000
tttcagcact tctagttgct cttataactt ctctttcact taaatgttgt ctaaacatac 3060
tattaagttc taaaacatca tttaatgcct tctcaatgtc ttctgtaaag ctacaaagat 3120
aatatctata taaaaataat ataagctctc tgtgtccttt taaatcatat tctcttagtt 3180
cacaaagttt tattatgtct tgtattcttc cataatataa acttctttct ctataaatat 3240
aatttatttt gcttggtcta ccctttttcc tttcatatgg ttttaattca ggtaaaaatc 3300
cattttgtat ttctcttaag tcataaatat attcgtactc atctaatata ttgactactg 3360
tttttgattt agagtttata cttcctggaa ctcttaatat tctcgttgca tctaaggctt 3420
gtctatctgc tccaaagtat tttaattgat tatataaata ttcttgaacc gctttccata 3480
atggtaatgc tttactaggt actgcattta ttatccatat taaatacatt cctcttccac 3540
tatctattac atagtttggt ataggaatac tttgattaaa ataattcttt tctaagtcca 3600
ttaatacctg gtctttagtt ttgccagttt tataataatc caagtctata aacagtgtat 3660
ttaactcttt tatattttct aatcgcctac acggcttata aaaggtattt agagttatat 3720
agatattttc atcactcata tctaaatctt ttaattcagc gtatttatag tgccattggc 3780
tatatccttt tttatctata acgctcctgg ttatccaccc tttacttcta ctatgaatat 3840
tatctatata gttcttttta ttcagcttta atgcgtttct cacttattca cctccccttc 3900
tgtaaaacta agaaaattat atcatatttt caataattat taactattct taaactctta 3960
ataaaaaata gagtaagtcc ccaattgaaa cttaatctat tttttatgtt ttaatttatt 4020
atttttatta aaatatttta aactaaatta aatgattctt tttaattttt tactatttca 4080
ttccataata tattactata attatttaca aataatattt cttcatttgt aatatttaga 4140
tgatttacta attttagttt ttatatatta aataattaat gtataattta tataaaaaat 4200
caaaggagct tataaattat gattatttcc aaagatacta aagatttaat ttttttcaat 4260
tttaacaata ctttttgtaa tattatgttt aaatttaatt gtattttttt catataataa 4320
agccgttgaa gtaaaccaat ccattttcct tatgatgtta ttattaaatt taagttttat 4380
aataatatct ttattatatt tattgttttt aaaaaaacta gtgaaatttc tagtgaaatt 4440
tccggcttta ttaaacttat ttttaggaat tttattttca ttttcatctt tacaggattt 4500
gattatatct ttaaatatgt tttatcaaat attatctttt tctaaattta tatatatttt 4560
tattatattt attattatat atattttatt tttaagtttc tttctaacag ctattaaaaa 4620
gaaacttaaa aataaaaaca cgtactctaa accaataaat aaaactattt ttattattgc 4680
tgccttgatt ggaatagttt ttagtaaaat taatttcaat attccacaat attatattat 4740
aagctagcac gcctcgagac tctatcattg atagagtttg aaactctatc attgatagag 4800
tataatatct ttgttcatgc ttattacgac ataacacagt tttagagcta gaaatagcaa 4860
gttaaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt 4920
tgaagcttct cgagatctcc atggacgcgt gacgtcgacc ttctaatctc ctctactatt 4980
ttagggttag ctacattagc taaataggta atagctacag ttgtctttga attctcacct 5040
aaagtaagtt cttccacttt aaaatcagtg cttctaattt tttttcttaa aagggctaca 5100
tttgtggtta aagattcagt gaagccctct ctaggacctc ttattacagt ttcaacagtt 5160
ggttctgtta tagctctttc agggggtttt ccaatactta taataattgc tttactttca 5220
ccatctagga ataatgctat acttcctttt aaaatggaca atataacatc atccatgctt 5280
ttatatacat ttttatcatt aacagcaaaa attgattttg tatattcaaa tatgtttaaa 5340
tggggatggt tattgtaatc ttcttctata agttttttta taacagagga ttctattaca 5400
tcagattgga taagattatt tatgtagaca atcattgcag aaaaatttct attattagct 5460
attttaaatt ctctaatcgt taaatctgag caatttgtaa ataaggtttc tatagtatgt 5520
ttatttgttt taaggctagt tgaaaccgtc ttcgcgttat ttttagatgc ttcttcttta 5580
ttaaaaattt tattaaacaa cgaaaaattc accccctcaa tttatttata taatagtagt 5640
ttgcatgaaa tttcgttgtt tattcatatt agatgcttgt attaaaataa taaaatagta 5700
aaatataagt agacaaacta taaatctatt actaggaggt aagaagtatg ctaagtttta 5760
aatctgtgta aacctaccgg ggtttgggcg tagccattat attcatgaac tccaagaaag 5820
cagtatgcta gcaaagaaat aaaactcaaa gcagagagaa aatttagaca ttcaactata 5880
aataaaaaat accccccaaa gcattaatat cttggggagt attttttatt ttgaagtatt 5940
ctgttcagct aaatattctt ctaaggtaat acctctgttc ataatttctt gtgaggcagg 6000
aagaccgata tatcttacat gccatggctc aaaattatac tttgttatgt tttctttatc 6060
cttaggatat cttattatga aaccatattt accacaattt tgttgaagcc atttataaga 6120
atttgtattc ataaatccat catctaaaga agagtattcg gttgatagta agtccattgc 6180
caatccagtt tgatgctcac ttgtaccagg ttcagctaca tatttatcag cttcggcttt 6240
tccgtctcgt gctacttttt cattatataa tttttgctga tacgaataag gtctataacc 6300
tgaaacagct agaagtgtaa gaccatcctt tgatgctgca ttaaacatat tttcaagtcc 6360
tgttgcagct tcgctctcca tttgatttac attaggatca gaactactaa taaatttaac 6420
gttaggagtt ctcaaatttt gaggtatata gtttcctgat aatttacttt gcttgtttac 6480
aagtaggatg ttctgtttct ttacctcggg tttcttggct tgttttttag gtgtagaaac 6540
tttctttttg ggttcgtttg 6560
<210> 81
<211> 1654
<212> DNA
<213> 人工序列
<220>
<223> bgaR acrIIA4表达盒
<400> 81
aaaaagtata acagaggttt taatttacgc ctctgttata ctttttattt ttgaaatttt 60
tttgttttaa agctgtattt taaatttata tacttggttt atttacttga ttatttctgt 120
aatttagtgg agacattgaa aaatgttttg aaaaagtttt tgaaaataac agggagtcac 180
tataacctac actacttgcg acttctccta taggaagttt agtgcttttt aataaaaggg 240
tggctttgta cattctaagg tttattaaat atctttgagg agaaattcca aggtttttta 300
tgaacatttt atataaataa cttctactta agttcacata atcagcaatt tcttgaacag 360
ttatgctatg catgtaatta gaattaatga aattaagagc atcttgaata tatgtgtgta 420
attccttatc tttgtattca aaaggttttg ggaattcttc tataagtgcg tacaataatg 480
agtaaagttc ttttagtaat agtatgtcat cagatcttga aggattataa gtttttgata 540
tttcgcacat atttaatatt atctgtggaa tttttgagtt ttcttcacaa ttagcaacac 600
aggagttagt aatagaagtt ctatttaaat actcattagc atttgaacca ctaaatccta 660
tccagtagta ttcccaagga tcatcaatag aagccacata ctcaacttgc atacctttta 720
gtagtataaa aatatcacct tgttttaagt tatatacctt accattaaat ttaaaagttc 780
catatccctt agttacgtaa tgaataacag catttttcaa tacttcatag ttatatccta 840
atcctggtat accttgttct ataccacatt catctacatt catttcaaag ttttctttaa 900
catacttttt ccacaatatt tgcatttcta cctcctaacc tataaaatta gccaatttta 960
tagtagtctt atattaaaca tttacatgag agctttgcaa agcagtttat caacataaaa 1020
gctttttatt ttaaaataaa ttcttctaaa tataagaata ttttaaagaa atatctttat 1080
atattagtta ttaaaattta taagattata agaaacatta taacatattt tagaactttt 1140
taactattct aaaagattaa tttacatatt aacatttaat tatgggtaaa aactattttg 1200
aaaaatgatt tatatggaat tatgtttctt aaatatacaa tcatgtttca tgaatacata 1260
attattttaa atgtattggg agggtaaaat gatattaaaa aatgaatacc atgaagatac 1320
tgcagaatct agaatccgcg gtagtcgacg tggaattgtg agcggataac aatttcacag 1380
gagggctgaa atgaatatta atgacttaat tagagaaata aaaaacaaag attacacagt 1440
gaaattgagt ggtacggata gcaatagtat aacacagcta attattagag ttaataatga 1500
tggaaacgag tatgtaattt ctgaaagtga aaatgaatca atagttgaaa aattcatatc 1560
tgcatttaaa aacggttgga atcaagaata cgaggatgaa gaagaatttt ataatgacat 1620
gcaaacaatc accttaaaaa gtgagttgaa ctaa 1654
<210> 82
<211> 4984
<212> DNA
<213> 人工序列
<220>
<223> pGRNAind
<400> 82
caagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac ggactagcct 60
tattttaact tgctatttct agctctaaaa cagagaccgc tagcgatatc cccgggagat 120
ctggtctcaa tgaacaaaga tattatactc tatcaatgat agagtttcaa actctatcaa 180
tgatagagtg agctcgaatt cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 240
tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 300
ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 360
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 420
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 480
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 540
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 600
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 660
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 720
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 780
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 840
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 900
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 960
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1020
gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 1080
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 1140
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 1200
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 1260
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 1320
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaaag 1380
ctagcttaat actagtatat acttaatgtg ataagtgtct gacagctgac cggtctaaag 1440
aggtccctag cgcctacggg gaatttgtat cgataagggg tacaaattcc cactaagcgc 1500
tcggccgggg atcgatcccc gggtacgtac ccggcagttt ttctttttcg gcaagtgttc 1560
aagaagttat taagtcggga gtgcagtcga agtgggcaag ttgaaaaatt cacaaaaatg 1620
tggtataata tctttgttca ttagagcgat aaacttgaat ttgagaggga acttagatgg 1680
tatttgaaaa aattgataaa aatagttgga acagaaaaga gtattttgac cactactttg 1740
caagtgtacc ttgtacctac agcatgaccg ttaaagtgga tatcacacaa ataaaggaaa 1800
agggaatgaa actatatcct gcaatgcttt attatattgc aatgattgta aaccgccatt 1860
cagagtttag gacggcaatc aatcaagatg gtgaattggg gatatatgat gagatgatac 1920
caagctatac aatatttcac aatgatactg aaacattttc cagcctttgg actgagtgta 1980
agtctgactt taaatcattt ttagcagatt atgaaagtga tacgcaacgg tatggaaaca 2040
atcatagaat ggaaggaaag ccaaatgctc cggaaaacat ttttaatgta tctatgatac 2100
cgtggtcaac cttcgatggc tttaatctga atttgcagaa aggatatgat tatttgattc 2160
ctatttttac tatggggaaa tattataaag aagataacaa aattatactt cctttggcaa 2220
ttcaagttca tcacgcagta tgtgacggat ttcacatttg ccgttttgta aacgaattgc 2280
aggaattgat aaatagttaa cttcaggttt gtctgtaact aaaaactagt atttaaccta 2340
ggatcaaaaa aatttccaat aatcccactc taagccacaa acacgcccta taaaatcccg 2400
ctttaatccc actttgagac acatgtaata ttactttacg ccctagtata gtgataattt 2460
tttacattca atgccacgca aaaaaataaa ggggcactat aataaaagtt ccttcggaac 2520
taactaaagt aaaaaattat ctttacaacc tccccaaaaa aaagaacagg tacaaagtac 2580
cctataatac aagcgtaaaa aaaatgaggg taaaaataaa aaaataaaaa aataaaaaaa 2640
taaaaaaata aaaaaataaa aaaataaaaa aatataaaaa taaaaaaata taaaaataaa 2700
aaaatataaa aataaaaaaa taaaaaaata taaaaataaa aaaataaaaa aatataaaaa 2760
tattttttat ttaaagtttg aaaaaaattt ttttatatta tataatcttt gaagaaaaga 2820
atataaaaaa tgagccttta taaaagccca ttttttttca tatacgtaat atgacgttct 2880
aatgttttta ttggtacttc taacattaga gtaatttctt tatttttaaa gcctttttct 2940
ttaagggctt ttattttttt tcttaataca tttaattcct ctttttttgt tgcttttcct 3000
ttagctttta attgctcttg ataatttttt ttacctctaa tattttctct tctcttatat 3060
tcctttttag aaattattat tgtcatatat ttttgttctt cttctgtaat ttctaataac 3120
tctataagag tttcattctt atacttatat tgcttatttt tatctaaata acatctttca 3180
gcacttctag ttgctcttat aacttctctt tcacttaaat gttgtctaaa catactatta 3240
agttctaaaa catcatttaa tgccttctca atgtcttctg taaagctaca aagataatat 3300
ctatataaaa ataatataag ctctctgtgt ccttttaaat catattctct tagttcacaa 3360
agttttatta tgtcttgtat tcttccataa tataaacttc tttctctata aatataattt 3420
attttgcttg gtctaccctt tttcctttca tatggtttta attcaggtaa aaatccattt 3480
tgtatttctc ttaagtcata aatatattcg tactcatcta atatattgac tactgttttt 3540
gatttagagt ttatacttcc tggaactctt aatattctcg ttgcatctaa ggcttgtcta 3600
tctgctccaa agtattttaa ttgattatat aaatattctt gaaccgcttt ccataatggt 3660
aatgctttac taggtactgc atttattatc catattaaat acattcctct tccactatct 3720
attacatagt ttggtatagg aatactttga ttaaaataat tcttttctaa gtccattaat 3780
acctggtctt tagttttgcc agttttataa taatccaagt ctataaacag tgtatttaac 3840
tcttttatat tttctaatcg cctacacggc ttataaaagg tatttagagt tatatagata 3900
ttttcatcac tcatatctaa atcttttaat tcagcgtatt tatagtgcca ttggctatat 3960
ccttttttat ctataacgct cctggttatc caccctttac ttctactatg aatattatct 4020
atatagttct ttttattcag ctttaatgcg tttctcactt attcacctcc ccttctgtaa 4080
aactaagaaa attatatcat attttcaata attattaact attcttaaac tcttaataaa 4140
aaatagagta agtccccaat tgaaacttaa tctatttttt atgttttaat ttattatttt 4200
tattaaaata ttttaaacta aattaaatga ttctttttaa ttttttacta tttcattcca 4260
taatatatta ctataattat ttacaaataa tatttcttca tttgtaatat ttagatgatt 4320
tactaatttt agtttttata tattaaataa ttaatgtata atttatataa aaaatcaaag 4380
gagcttataa attatgatta tttccaaaga tactaaagat ttaatttttt tcaattttaa 4440
caatactttt tgtaatatta tgtttaaatt taattgtatt tttttcatat aataaagccg 4500
ttgaagtaaa ccaatccatt ttccttatga tgttattatt aaatttaagt tttataataa 4560
tatctttatt atatttattg tttttaaaaa aactagtgaa atttctagtg aaatttccgg 4620
ctttattaaa cttattttta ggaattttat tttcattttc atctttacag gatttgatta 4680
tatctttaaa tatgttttat caaatattat ctttttctaa atttatatat atttttatta 4740
tatttattat tatatatatt ttatttttaa gtttctttct aacagctatt aaaaagaaac 4800
ttaaaaataa aaacacgtac tctaaaccaa taaataaaac tatttttatt attgctgcct 4860
tgattggaat agtttttagt aaaattaatt tcaatattcc acaatattat attataagct 4920
agcacgcctc gagatctcca tggacgcgtg acgtcgactc tagaggatcc ccgggtaccg 4980
agct 4984
<210> 83
<211> 200
<212> DNA
<213> 人工序列
<220>
<223> gRNA表达盒
<400> 83
gagctcactc tatcattgat agagtttgaa actctatcat tgatagagta taatatcttt 60
gttcattgag accagatctc ccggggatat cgctagcggt ctctgtttta gagctagaaa 120
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 180
tttttttgaa gcttgagctc 200
<210> 84
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 84
tcatgatttc tccatattag ctag 24
<210> 85
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 85
aaacctagct aatatggaga aatc 24
<210> 86
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 86
tcatgttaca cttggaacag gcgt 24
<210> 87
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 87
aaacacgcct gttccaagtg taac 24
<210> 88
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 88
tcatttccgg cagtaggatc ccca 24
<210> 89
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 89
aaactgggga tcctactgcc ggaa 24
<210> 90
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 90
tcatgcttat tacgacataa caca 24
<210> 91
<211> 24
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 91
aaactgtgtt atgtcgtaat aagc 24
<210> 92
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 92
atgcatggat ccaaacgaac ccaaaaagaa agtttc 36
<210> 93
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 93
ggttgatttc aaatctgtgt aaacctaccg 30
<210> 94
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 94
acacagattt gaaatcaacc actttaaccc 30
<210> 95
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 95
atgcatgtcg actcttaaga acatgtataa agtatgg 37
<210> 96
<211> 36
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 96
atgcatggat ccaaacgaac ccaaaaagaa agtttc 36
<210> 97
<211> 30
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 97
gctaagtttt aaatctgtgt aaacctaccg 30
<210> 98
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 98
acacagattt aaaacttagc atacttctta cc 32
<210> 99
<211> 37
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 99
atgcatgtcg accttctaat ctcctctact attttag 37
<210> 100
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 100
acacattgaa gggagctttt 20
<210> 101
<211> 20
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 101
ggcaacaaca tcaggccttt 20
<210> 102
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-xylB
<400> 102
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gaagcttcaa aaaaagcacc gactcggtgc cactttttca agttgataac 2640
ggactagcct tattttaact tgctatttct agctctaaaa cctagctaat atggagaaat 2700
catgaacaaa gatattatac tctatcaatg atagagtttc aaactctatc aatgatagag 2760
tctcgagatc tccatggacg cgtgacgtcg actctagagg atccccgggt accgagctcg 2820
aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2880
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2940
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3000
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3060
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3120
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3180
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3240
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3300
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3360
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3420
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3480
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3540
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3600
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3660
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3840
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3900
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3960
atctaaagta tatatgagta aacttggtct gacagttacc aaagctagct taatactagt 4020
atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtcc ctagcgccta 4080
cggggaattt gtatcgataa ggggtacaaa ttcccactaa gcgctcggcc ggggatcgat 4140
ccccgggtac gtacccggca gtttttcttt ttcggcaagt gttcaagaag ttattaagtc 4200
gggagtgcag tcgaagtggg caagttgaaa aattcacaaa aatgtggtat aatatctttg 4260
ttcattagag cgataaactt gaatttgaga gggaacttag atggtatttg aaaaaattga 4320
taaaaatagt tggaacagaa aagagtattt tgaccactac tttgcaagtg taccttgtac 4380
ctacagcatg accgttaaag tggatatcac acaaataaag gaaaagggaa tgaaactata 4440
tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc 4500
aatcaatcaa gatggtgaat tggggatata tgatgagatg ataccaagct atacaatatt 4560
tcacaatgat actgaaacat tttccagcct ttggactgag tgtaagtctg actttaaatc 4620
atttttagca gattatgaaa gtgatacgca acggtatgga aacaatcata gaatggaagg 4680
aaagccaaat gctccggaaa acatttttaa tgtatctatg ataccgtggt caaccttcga 4740
tggctttaat ctgaatttgc agaaaggata tgattatttg attcctattt ttactatggg 4800
gaaatattat aaagaagata acaaaattat acttcctttg gcaattcaag ttcatcacgc 4860
agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag 4920
ttaacttcag gtttgtctgt aactaaaaac tagtatttaa cctagg 4966
<210> 103
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-xylR
<400> 103
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gactctatca ttgatagagt ttgaaactct atcattgata gagtataata 2640
tctttgttca tgttacactt ggaacaggcg tgttttagag ctagaaatag caagttaaaa 2700
taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt ttttgaagct 2760
tctcgagatc tccatggacg cgtgacgtcg actctagagg atccccgggt accgagctcg 2820
aattcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca 2880
cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa 2940
ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 3000
ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc 3060
gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 3120
cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 3180
tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 3240
cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 3300
aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 3360
cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 3420
gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 3480
ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 3540
cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 3600
aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 3660
tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc 3720
ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 3780
tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 3840
ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 3900
agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca 3960
atctaaagta tatatgagta aacttggtct gacagttacc aaagctagct taatactagt 4020
atatacttaa tgtgataagt gtctgacagc tgaccggtct aaagaggtcc ctagcgccta 4080
cggggaattt gtatcgataa ggggtacaaa ttcccactaa gcgctcggcc ggggatcgat 4140
ccccgggtac gtacccggca gtttttcttt ttcggcaagt gttcaagaag ttattaagtc 4200
gggagtgcag tcgaagtggg caagttgaaa aattcacaaa aatgtggtat aatatctttg 4260
ttcattagag cgataaactt gaatttgaga gggaacttag atggtatttg aaaaaattga 4320
taaaaatagt tggaacagaa aagagtattt tgaccactac tttgcaagtg taccttgtac 4380
ctacagcatg accgttaaag tggatatcac acaaataaag gaaaagggaa tgaaactata 4440
tcctgcaatg ctttattata ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc 4500
aatcaatcaa gatggtgaat tggggatata tgatgagatg ataccaagct atacaatatt 4560
tcacaatgat actgaaacat tttccagcct ttggactgag tgtaagtctg actttaaatc 4620
atttttagca gattatgaaa gtgatacgca acggtatgga aacaatcata gaatggaagg 4680
aaagccaaat gctccggaaa acatttttaa tgtatctatg ataccgtggt caaccttcga 4740
tggctttaat ctgaatttgc agaaaggata tgattatttg attcctattt ttactatggg 4800
gaaatattat aaagaagata acaaaattat acttcctttg gcaattcaag ttcatcacgc 4860
agtatgtgac ggatttcaca tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag 4920
ttaacttcag gtttgtctgt aactaaaaac tagtatttaa cctagg 4966
<210> 104
<211> 4966
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-glcG
<400> 104
agctcggtac ccggggatcc tctagagtcg acgtcacgcg tccatggaga tctcgaggcg 60
tgctagctta taatataata ttgtggaata ttgaaattaa ttttactaaa aactattcca 120
atcaaggcag caataataaa aatagtttta tttattggtt tagagtacgt gtttttattt 180
ttaagtttct ttttaatagc tgttagaaag aaacttaaaa ataaaatata tataataata 240
aatataataa aaatatatat aaatttagaa aaagataata tttgataaaa catatttaaa 300
gatataatca aatcctgtaa agatgaaaat gaaaataaaa ttcctaaaaa taagtttaat 360
aaagccggaa atttcactag aaatttcact agttttttta aaaacaataa atataataaa 420
gatattatta taaaacttaa atttaataat aacatcataa ggaaaatgga ttggtttact 480
tcaacggctt tattatatga aaaaaataca attaaattta aacataatat tacaaaaagt 540
attgttaaaa ttgaaaaaaa ttaaatcttt agtatctttg gaaataatca taatttataa 600
gctcctttga ttttttatat aaattataca ttaattattt aatatataaa aactaaaatt 660
agtaaatcat ctaaatatta caaatgaaga aatattattt gtaaataatt atagtaatat 720
attatggaat gaaatagtaa aaaattaaaa agaatcattt aatttagttt aaaatatttt 780
aataaaaata ataaattaaa acataaaaaa tagattaagt ttcaattggg gacttactct 840
attttttatt aagagtttaa gaatagttaa taattattga aaatatgata taattttctt 900
agttttacag aaggggaggt gaataagtga gaaacgcatt aaagctgaat aaaaagaact 960
atatagataa tattcatagt agaagtaaag ggtggataac caggagcgtt atagataaaa 1020
aaggatatag ccaatggcac tataaatacg ctgaattaaa agatttagat atgagtgatg 1080
aaaatatcta tataactcta aatacctttt ataagccgtg taggcgatta gaaaatataa 1140
aagagttaaa tacactgttt atagacttgg attattataa aactggcaaa actaaagacc 1200
aggtattaat ggacttagaa aagaattatt ttaatcaaag tattcctata ccaaactatg 1260
taatagatag tggaagagga atgtatttaa tatggataat aaatgcagta cctagtaaag 1320
cattaccatt atggaaagcg gttcaagaat atttatataa tcaattaaaa tactttggag 1380
cagatagaca agccttagat gcaacgagaa tattaagagt tccaggaagt ataaactcta 1440
aatcaaaaac agtagtcaat atattagatg agtacgaata tatttatgac ttaagagaaa 1500
tacaaaatgg atttttacct gaattaaaac catatgaaag gaaaaagggt agaccaagca 1560
aaataaatta tatttataga gaaagaagtt tatattatgg aagaatacaa gacataataa 1620
aactttgtga actaagagaa tatgatttaa aaggacacag agagcttata ttatttttat 1680
atagatatta tctttgtagc tttacagaag acattgagaa ggcattaaat gatgttttag 1740
aacttaatag tatgtttaga caacatttaa gtgaaagaga agttataaga gcaactagaa 1800
gtgctgaaag atgttattta gataaaaata agcaatataa gtataagaat gaaactctta 1860
tagagttatt agaaattaca gaagaagaac aaaaatatat gacaataata atttctaaaa 1920
aggaatataa gagaagagaa aatattagag gtaaaaaaaa ttatcaagag caattaaaag 1980
ctaaaggaaa agcaacaaaa aaagaggaat taaatgtatt aagaaaaaaa ataaaagccc 2040
ttaaagaaaa aggctttaaa aataaagaaa ttactctaat gttagaagta ccaataaaaa 2100
cattagaacg tcatattacg tatatgaaaa aaaatgggct tttataaagg ctcatttttt 2160
atattctttt cttcaaagat tatataatat aaaaaaattt ttttcaaact ttaaataaaa 2220
aatattttta tattttttta tttttttatt tttatatttt tttatttttt tatttttata 2280
tttttttatt tttatatttt tttattttta tattttttta tttttttatt tttttatttt 2340
tttatttttt tattttttta tttttttatt tttaccctca ttttttttac gcttgtatta 2400
tagggtactt tgtacctgtt cttttttttg gggaggttgt aaagataatt ttttacttta 2460
gttagttccg aaggaacttt tattatagtg cccctttatt tttttgcgtg gcattgaatg 2520
taaaaaatta tcactatact agggcgtaaa gtaatattac atgtgtctca aagtgggatt 2580
aaagcgggat tttatagggc gtgtttgtgg cttagagtgg gattattgga aatttttttg 2640
atcctaggtt aaatactagt ttttagttac agacaaacct gaagttaact atttatcaat 2700
tcctgcaatt cgtttacaaa acggcaaatg tgaaatccgt cacatactgc gtgatgaact 2760
tgaattgcca aaggaagtat aattttgtta tcttctttat aatatttccc catagtaaaa 2820
ataggaatca aataatcata tcctttctgc aaattcagat taaagccatc gaaggttgac 2880
cacggtatca tagatacatt aaaaatgttt tccggagcat ttggctttcc ttccattcta 2940
tgattgtttc cataccgttg cgtatcactt tcataatctg ctaaaaatga tttaaagtca 3000
gacttacact cagtccaaag gctggaaaat gtttcagtat cattgtgaaa tattgtatag 3060
cttggtatca tctcatcata tatccccaat tcaccatctt gattgattgc cgtcctaaac 3120
tctgaatggc ggtttacaat cattgcaata taataaagca ttgcaggata tagtttcatt 3180
cccttttcct ttatttgtgt gatatccact ttaacggtca tgctgtaggt acaaggtaca 3240
cttgcaaagt agtggtcaaa atactctttt ctgttccaac tatttttatc aattttttca 3300
aataccatct aagttccctc tcaaattcaa gtttatcgct ctaatgaaca aagatattat 3360
accacatttt tgtgaatttt tcaacttgcc cacttcgact gcactcccga cttaataact 3420
tcttgaacac ttgccgaaaa agaaaaactg ccgggtacgt acccggggat cgatccccgg 3480
ccgagcgctt agtgggaatt tgtacccctt atcgatacaa attccccgta ggcgctaggg 3540
acctctttag accggtcagc tgtcagacac ttatcacatt aagtatatac tagtattaag 3600
ctagctttgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact 3660
tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat 3720
cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc 3780
ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct 3840
accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg 3900
cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca 3960
cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc 4020
tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga 4080
taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac 4140
gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga 4200
agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag 4260
ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg 4320
acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag 4380
caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc 4440
tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc 4500
tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc 4560
aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag 4620
gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca 4680
ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag 4740
cggataacaa tttcacacag gaaacagcta tgaccatgat tacgaattcg agctcactct 4800
atcattgata gagtttgaaa ctctatcatt gatagagtat aatatctttg ttcatttccg 4860
gcagtaggat ccccagtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta 4920
tcaacttgaa aaagtggcac cgagtcggtg ctttttttga agcttg 4966
<210> 105
<211> 4938
<212> DNA
<213> 人工序列
<220>
<223> pGRNA-bdhB
<400> 105
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gtatattgat aaaaataata atagtgggta taattaagtt gttaggaggt 2640
tagttagagc ttattacgac ataacacagt tttagagcta gaaatagcaa gttaaaataa 2700
ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt tgaagcttgt 2760
cgactctaga ggatccccgg gtaccgagct cgaattcgta atcatggtca tagctgtttc 2820
ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt 2880
gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc 2940
ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg 3000
ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct 3060
cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca 3120
cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga 3180
accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc 3240
acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg 3300
cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat 3360
acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt 3420
atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc 3480
agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg 3540
acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg 3600
gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg 3660
gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg 3720
gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca 3780
gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga 3840
acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga 3900
tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt 3960
ctgacagtta ccaaagctag cttaatacta gtatatactt aatgtgataa gtgtctgaca 4020
gctgaccggt ctaaagaggt ccctagcgcc tacggggaat ttgtatcgat aaggggtaca 4080
aattcccact aagcgctcgg ccggggatcg atccccgggt acgtacccgg cagtttttct 4140
ttttcggcaa gtgttcaaga agttattaag tcgggagtgc agtcgaagtg ggcaagttga 4200
aaaattcaca aaaatgtggt ataatatctt tgttcattag agcgataaac ttgaatttga 4260
gagggaactt agatggtatt tgaaaaaatt gataaaaata gttggaacag aaaagagtat 4320
tttgaccact actttgcaag tgtaccttgt acctacagca tgaccgttaa agtggatatc 4380
acacaaataa aggaaaaggg aatgaaacta tatcctgcaa tgctttatta tattgcaatg 4440
attgtaaacc gccattcaga gtttaggacg gcaatcaatc aagatggtga attggggata 4500
tatgatgaga tgataccaag ctatacaata tttcacaatg atactgaaac attttccagc 4560
ctttggactg agtgtaagtc tgactttaaa tcatttttag cagattatga aagtgatacg 4620
caacggtatg gaaacaatca tagaatggaa ggaaagccaa atgctccgga aaacattttt 4680
aatgtatcta tgataccgtg gtcaaccttc gatggcttta atctgaattt gcagaaagga 4740
tatgattatt tgattcctat ttttactatg gggaaatatt ataaagaaga taacaaaatt 4800
atacttcctt tggcaattca agttcatcac gcagtatgtg acggatttca catttgccgt 4860
tttgtaaacg aattgcagga attgataaat agttaacttc aggtttgtct gtaactaaaa 4920
actagtattt aacctagg 4938
<210> 106
<211> 4790
<212> DNA
<213> 人工序列
<220>
<223> pEC750C
<400> 106
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gatctccatg gacgcgtgac gtcgactcta gaggatcccc gggtaccgag 2640
ctcgaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 2700
tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 2760
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2820
ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 2880
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2940
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 3000
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 3060
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 3120
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 3180
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 3240
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 3300
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3360
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3420
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3480
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 3540
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3600
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 3660
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3720
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 3780
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaaagct agcttaatac 3840
tagtatatac ttaatgtgat aagtgtctga cagctgaccg gtctaaagag gtccctagcg 3900
cctacgggga atttgtatcg ataaggggta caaattccca ctaagcgctc ggccggggat 3960
cgatccccgg gtacgtaccc ggcagttttt ctttttcggc aagtgttcaa gaagttatta 4020
agtcgggagt gcagtcgaag tgggcaagtt gaaaaattca caaaaatgtg gtataatatc 4080
tttgttcatt agagcgataa acttgaattt gagagggaac ttagatggta tttgaaaaaa 4140
ttgataaaaa tagttggaac agaaaagagt attttgacca ctactttgca agtgtacctt 4200
gtacctacag catgaccgtt aaagtggata tcacacaaat aaaggaaaag ggaatgaaac 4260
tatatcctgc aatgctttat tatattgcaa tgattgtaaa ccgccattca gagtttagga 4320
cggcaatcaa tcaagatggt gaattgggga tatatgatga gatgatacca agctatacaa 4380
tatttcacaa tgatactgaa acattttcca gcctttggac tgagtgtaag tctgacttta 4440
aatcattttt agcagattat gaaagtgata cgcaacggta tggaaacaat catagaatgg 4500
aaggaaagcc aaatgctccg gaaaacattt ttaatgtatc tatgataccg tggtcaacct 4560
tcgatggctt taatctgaat ttgcagaaag gatatgatta tttgattcct atttttacta 4620
tggggaaata ttataaagaa gataacaaaa ttatacttcc tttggcaatt caagttcatc 4680
acgcagtatg tgacggattt cacatttgcc gttttgtaaa cgaattgcag gaattgataa 4740
atagttaact tcaggtttgt ctgtaactaa aaactagtat ttaacctagg 4790
<210> 107
<211> 35
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 107
acttgggtcg accacgataa aacaaggttt taagg 35
<210> 108
<211> 42
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 108
taccagggat ccgtattaat gtaactatga tatcaattct tg 42
<210> 109
<211> 46
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 109
atgcatggtc ccaatgaata ggtttacact tactttagtt ttatgg 46
<210> 110
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 110
atgcgagtta acaacttcta aaatctgatt accaattag 39
<210> 111
<211> 47
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 111
atgcatggat cccaatgaat aggtttacac ttactttagt tttatgg 47
<210> 112
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 112
atgcgagagc tcaacttcta aaatctgatt accaattag 39
<210> 113
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 113
atgcatggat ccgtctgaca gttaccaggt cc 32
<210> 114
<211> 39
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 114
atgcgagagc tccaattgtt caaaaaaata atggcggag 39
<210> 115
<211> 32
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 115
atgcatggat cccggcagtt tttctttttc gg 32
<210> 116
<211> 40
<212> DNA
<213> 人工序列
<220>
<223> 引物
<400> 116
atgcgagagc tcggttaaat actagttttt agttacagac 40
<210> 117
<211> 2686
<212> DNA
<213> 人工序列
<220>
<223> pUC19
<400> 117
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgactcta gaggatcccc gggtaccgag ctcgaattca ctggccgtcg 300
ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac 360
atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac 420
agttgcgcag cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt 480
gcggtatttc acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt 540
taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc 600
cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt 660
caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg 720
ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc 780
gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac 840
aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt 900
tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag 960
aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg 1020
aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa 1080
tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc 1140
aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag 1200
tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa 1260
ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc 1320
taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg 1380
agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa 1440
caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa 1500
tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg 1560
gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag 1620
cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg 1680
caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt 1740
ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt 1800
aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac 1860
gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag 1920
atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg 1980
tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca 2040
gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga 2100
actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca 2160
gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc 2220
agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca 2280
ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa 2340
aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc 2400
cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc 2460
gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg 2520
cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat 2580
cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca 2640
gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaaga 2686
<210> 118
<211> 4282
<212> DNA
<213> 人工序列
<220>
<223> pNF2
<400> 118
ctggagagga ttgtccttat acttatcata agcatgaagg acttgttatt cctagataga 60
gaattaatta tgttaaagag atataataaa ctcattataa ttataatttt tagtataatt 120
attattgcaa ttttttcgta taaatatcta ataatgccaa aagagcatag aatagaaatt 180
tcaacattat caaacataga agtttttaaa tttaatagtt tttcaaagtt tagtaacgaa 240
aaaatgtata ctattaatga tagtgataag ttaataaaat tcaaaacact atttaataat 300
ttagataaat caaaagatat aaaaaagatt agtattccgg aaagtgaaaa tttaaatgca 360
tttaaatttt ctgcacatat aaaacttaac tttaactatg ttaataaaga tagccaaata 420
actgaaggtg cttttcttat gtatattttg gtagacaatt tagaagggaa gtcatatatg 480
acttttttag gacaagattc aagctatata ttagatagta atgaaactaa cattttaaga 540
gaaatattta tgaattcaga gattaattaa tttatgaatt cataaatatt atctaagcac 600
gataaaacaa ggttttaagg ataagaaaag tcatgagatt tatagtaaat cttgtgactt 660
tttttattga atagtagaga gagttcggaa gtataacacg ctatattctt gatattttta 720
gaatagcaag cattggattt gtcctgacac tttcccaaaa attaaggagt tattccttaa 780
accaaaaaga ttaatgtggg aacaaattta gtgtatccat ttttgaaggg cgcacttata 840
caccaccaaa atggtgtgtg cgaaatcttt aaaaaagatt tatcaaaaag cttttttaaa 900
gctgggacat ttagaaaatc aataatgttt tttgcccaat acgctagtct taaaatctgc 960
aaggttgata actatttagt cccaggtatt agaatggggc atatatatac aaagtatata 1020
tatgcgtaaa tatatgtggg actgtgggaa caaaattgcg tgctaaaatt gtattgaaaa 1080
ggtaatgaaa aggtcatgct ttggtattgc taacgtatag aaaaggtaat gaaaagctca 1140
tggttctata aaaaagatgt acccacgaaa ataataggct ttgcctattt ccccatgtaa 1200
tatgggggca gttttctctt atgctctttc ttaacatatt gaataaatac aaaatgcagc 1260
tttgtgggaa taaaaatatt tttgttttta ttcttatagt tagacaaaat tttaatcttt 1320
tttgtgctat aacaagatta aaatttgtgg gaacattaag aaatattgtt gtcacaaata 1380
aaaaggagag tgggaacaat tgctataaaa aacgcagaaa ttaagattag agttacaaaa 1440
gagcaaaaag aattatttaa gaaaattgca aaagctgaaa atatgagtat gagtgaattt 1500
attattgtga ccacagaata tttagccaga aaaaaagatg aaaatatgaa atcaaaagac 1560
atgatcgaga gaagagctgc gaagactgaa gaaaaaatta tgaagctaaa aaagaaacta 1620
aataaaaaca ggtaatatag attacagttt taagcttgtt ttccctatag actagagtaa 1680
atatataaat atacctgtca agggcttata agccccttta gggggtgcgt agcacccttg 1740
acaggtatat ttatatattt tagggtgcca ttaagggaaa caagctttaa aatgccttta 1800
aaggcatttt aaaataaata aaaaaaagat ggtttttacc atctttttta actcccgaaa 1860
gggagttctt tcttttcttg atactatacg taactatttc gatttgccct gaacctaatc 1920
aaagctagat aaattcagta ttagggcata aaaaaacttg ctttttcggg tggaaatctg 1980
tataatttaa attgcttaga taaaaattac caattccata cgaaaggagc aagttttaca 2040
taaggttaaa gccttatgtg aattctcatt taattacatg aataataata acacagaaag 2100
tgaagaatta aaagagcaaa gtcaactatt gcttgacaaa tgcacaaaaa agaaaaagaa 2160
aaatcctaaa tttagtagtt atatagaacc attagtaagc aagaaattat ctgaaagaat 2220
aaaggaatgt ggtgactttt tgcagatgtt atctgattta aaccttgaaa attcgaaact 2280
gcatagagca agtttttgtg gtaacagatt ttgtcctatg tgtagctggc gtattgcttg 2340
taaggatagt ttggaaatat ctattctcat ggagcattta cgcaaagagg aaagcaaaga 2400
atttatcttt ttgaccttaa caactccaaa tgtgaaaggt gcggaccttg ataattccat 2460
aaaagcatac aataaagcat ttaaaaagtt aatggaacgc aaagaggtca agagcatagt 2520
aaaaggctac ataagaaagc tagaagtaac ctataatttg gacaagagtt ccaaatcata 2580
taatacttat cacccacatt tccatgtggt actagcagtc aatagaagtt actttaaaaa 2640
gcaaaatcta tatataaacc atcatagatg gcttagtttg tggcaagagt caactggtga 2700
ttattcgata actcaagttg atgtaagaaa ggctaaaatt aacgattata aagaggttta 2760
tgagcttgct aagtattcgg ctaaggattc cgactattta atcaatagag aagtgtttac 2820
ggtattctac aaatctttaa agggtaaaca ggtacttgta tttagtggat tatttaaaga 2880
cgctcataaa atgtataaga atggagagct agatctgtat aagaagttgg atactatcga 2940
atatgcttat atggtaagtt ataactggct taaaaagaag tatgatactt caaatattag 3000
agaattaact gaggaagaaa agcagaaatt caataaaaat ttaatcgaag atgtggatat 3060
tgagtaggtg ggattatatc tcaccttttt tattgtcttt tcatgttgaa attttgacgc 3120
ttaatgcatg aagtattgac aagtttaaaa attacggttt ttaatcctta gttgattagc 3180
aggattatgg ccggaatgct ccgtccagtc ctgttaagga attaaaattc cctaaaaccc 3240
ttggctatga tttatagcga gaatcgtcaa ttaaaaattt aataggtgct atgaaagtcg 3300
attaataatt aattttaaaa tgcaatatga aacataatta caagaatttg acttttaata 3360
caagaattga tatcatagtt acattaatac atttattttg aagggggaaa atgttttatg 3420
aaaagactac ttaaactacc tattttatca ttattaggat tatttttaat tggatcaact 3480
ccaacattag ctttaactaa agataataat caaaatttag atactatgaa agtaaactta 3540
tatactgaaa cagtagatgt gtttgataaa gatgcattta aacaaacatt tactaataaa 3600
gatataaaat ttctagagga ttctttgaat gcaaaaataa attattcagg taaatctgtt 3660
acagtaacaa tgaaaaacaa aattaagcca tctactaaac aagggcttgt tttatatgta 3720
aatggaaaat cagttaatgt tgattcagat ggcagtataa aagtacctaa agatactaag 3780
aaaatttcta aattaaataa agataaatca atgatggatg gatcaatgat ggataaatca 3840
ttacatgatg agaattgtgt agtatcagat agtttttata atgctgatgt taataatata 3900
aattcaaaag aagcagaagc tgtatttaaa gtaagttctg gtgaattatt agctaaaatg 3960
gatgaaaaag aagatgatta catacaaaag aactcatcta aaattctagc agctgcttat 4020
cataagggat atggggacaa gtactatgaa ggagattggg ttcattgcaa taggtttaat 4080
ggtcaactta cagatgatgt tcactataat tggagaactg gaagtgtttc agaaaaagca 4140
gctgcaatga gaaattttta tggcagtgat tgtcatatag cattagttca agcaggtagt 4200
ggatgtacaa gtataggttc atgcgaatgc aatacagatc aaatagctgc gtattgttca 4260
ggtttcgtaa aagataaaaa ta 4282
<210> 119
<211> 5473
<212> DNA
<213> 人工序列
<220>
<223> pNF3
<400> 119
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatccccggg 3060
taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 3120
ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 3180
aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 3240
tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca 3300
gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg 3360
acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct 3420
ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg 3480
gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt 3540
caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 3600
attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 3660
aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 3720
tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 3780
agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 3840
gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 3900
cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 3960
agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 4020
taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 4080
tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 4140
taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 4200
acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 4260
ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 4320
cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 4380
agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 4440
tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 4500
agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 4560
tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 4620
ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 4680
tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 4740
aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 4800
tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 4860
agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 4920
taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 4980
caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 5040
agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 5100
aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 5160
gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 5220
tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 5280
gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 5340
ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 5400
ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 5460
aggaagcgga aga 5473
<210> 120
<211> 9128
<212> DNA
<213> 人工序列
<220>
<223> pMTL007S-E1
<400> 120
gatcgggccc cctgcagggt gtagtagcct gtgaaataag taaggaaaaa aaagaagtaa 60
gtgttatata tgatgattat tttgtagatg tagataggat aatagaatcc atagaaaata 120
taggttatac agttatataa aaattacttt aaaaattaat aaaaacatgg taaaatataa 180
atcgtataaa gttgtgtaat ttttaagctt gagctcataa caatttcaca caggaaacag 240
ctatgaccat gattacggat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc 300
ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata 360
gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc 420
gctaataaag atcttgtaca atctgtagga gaacctatgg gaacgaaacg aaagcgatgc 480
cgagaatctg aatttaccaa gacttaacac taactgggga taccctaaac aagaatgcct 540
aatagaaagg aggaaaaagg ctatagcact agagcttgaa aatcttgcaa gggtacggag 600
tactcgtagt agtctgagaa gggtaacgcc ctttacatgg caaaggggta cagttattgt 660
gtactaaaat taaaaattga ttagggagga aaacctcaaa atgaaaccaa caatggcaat 720
tttagaaaga atcagtaaaa attcacaaga aaatatagac gaagttttta caagacttta 780
tcgttatctt ttacgtccag atatttatta cgtggcgacg cgtgcgactc atagaattat 840
ttcctcccgt taaataatag ataactatta aaaatagaca atacttgctc ataagtaacg 900
gtacttaaat tgtttacttt ggcgtgtttc attgcttgat gaaactgatt tttagtaaac 960
agttgacgat attctcgatt gacccatttt gaaacaaagt acgtatatag cttccaatat 1020
ttatctggaa catctgtggt atggcgggta agttttatta agacactgtt tacttttggt 1080
ttaggatgaa agcattccgc tggcagctta agcaattgct gaatcgagac ttgagtgtgc 1140
aagagcaacc ctagtgttcg gtgaatatcc aaggtacgct tgtagaatcc ttcttcaaca 1200
atcagataga tgtcagacgc atggctttca aaaaccactt ttttaataat ttgtgtgctt 1260
aaatggtaag gaatactccc aacaatttta tacctctgtt tgttagggaa ttgaaactgt 1320
agaatatctt ggtgaattaa agtgacacga gtattcagtt ttaatttttc tgacgataag 1380
ttgaatagat gactgtctaa ttcaatagac gttacctgtt tacttatttt agccagtttc 1440
gtcgttaaat gccctttacc tgttccaatt tcgtaaacgg tatcggtttc ttttaaattc 1500
aattgtttta ttatttggtt gagtactttt tcactcgtta aaaagttttg agaatatttt 1560
atatttttgt tcataccagc accagaagca ccagcatctc ttgggttaat tgaggcctga 1620
gtataaggtg acttatactt gtaatctatc taaacgggga acctctctag tagacaatcc 1680
cgtgctaaat tgtaggactg ccctttaata aatacttcta tatttaaaga ggtatttatg 1740
aaaagcggaa tttatcagat taaaaatact ttctctagag aaaatttcgt ctggattagt 1800
tacttatcgt gtaaaatctg ataaatggaa ttggttctac ataaatgcct aacgactatc 1860
cctttgggga gtagggtcaa gtgactcgaa acgatagaca acttgcttta acaagttgga 1920
gatatagtct gctctgcatg gtgacatgca gctggatata attccggggt aagattaacg 1980
accttatctg aacataatgc catatgaatc cctcctaatt tatacgtttt ctctaacaac 2040
ttaattatac ccactattat tatttttatc aatataacgc gttgggaaat ggcaatgata 2100
gcgaaacaac gtaaaactct tgttgtatgc tttcattgtc atcgtcacgt gattcataaa 2160
cacaagtgaa tgtcgacagt gaatttttac gaacgaacaa taacagagcc gtatactccg 2220
agaggggtac gtacggttcc cgaagagggt ggtgcaaacc agtcacagta atgtgaacaa 2280
ggcggtacct ccctacttca ccatatcatt ttctgcagcc ccctagaaat aattttgttt 2340
aactttaaga aggagatata catatatggc tagatcgtcc attccgacag catcgccagt 2400
cactatggcg tgctgctagc gctatatgcg ttgatgcaat ttctatgcac tcgtagtagt 2460
ctgagaaggg taacgccctt tacatggcaa aggggtacag ttattgtgta ctaaaattaa 2520
aaattgatta gggaggaaaa cctcaaaatg aaaccaacaa tggcaatttt agaaagaatc 2580
agtaaaaatt cacaagaaaa tatagacgaa gtttttacaa gactttatcg ttatctttta 2640
cgtccagata tttattacgt ggcgtatcaa aatttatatt ccaataaagg agcttccaca 2700
aaaggaatat tagatgatac agcggatggc tttagtgaag aaaaaataaa aaagattatt 2760
caatctttaa aagacggaac ttactatcct caacctgtac gaagaatgta tattgcaaaa 2820
aagaattcta aaaagatgag acctttagga attccaactt tcacagataa attgatccaa 2880
gaagctgtga gaataattct tgaatctatc tatgaaccgg tattcgaaga tgtgtctcac 2940
ggttttagac ctcaacgaag ctgtcacaca gctttgaaaa caatcaaaag agagtttggc 3000
ggcgcaagat ggtttgtgga gggagatata aaaggctgct tcgataatat agaccacgtt 3060
acactcattg gactcatcaa tcttaaaatc aaagatatga aaatgagcca attgatttat 3120
aaatttctaa aagcaggtta tctggaaaac tggcagtatc acaaaactta cagcggaaca 3180
cctcaaggtg gaattctatc tcctcttttg gccaacatct atcttcatga attggataag 3240
tttgttttac aactcaaaat gaagtttgac cgagaaagtc cagaaagaat aacacctgaa 3300
tatcgggagc tccacaatga gataaaaaga atttctcacc gtctcaagaa gttggagggt 3360
gaagaaaaag ctaaagttct tttagaatat caagaaaaac gtaaaagatt acccacactc 3420
ccctgtacct cacagacaaa taaagtattg aaatacgtcc ggtatgcgga cgacttcatt 3480
atctctgtta aaggaagcaa agaggactgt caatggataa aagaacaatt aaaacttttt 3540
attcataaca agctaaaaat ggaattgagt gaagaaaaaa cactcatcac acatagcagt 3600
caacccgctc gttttctggg atatgatata cgagtaagga gatctggaac gataaaacga 3660
tctggtaaag tcaaaaagag aacactcaat gggagtgtag aactccttat tcctcttcaa 3720
gacaaaattc gtcaatttat ttttgacaag aaaatagcta tccaaaagaa agatagctca 3780
tggtttccag ttcacaggaa atatcttatt cgttcaacag acttagaaat catcacaatt 3840
tataattctg aactccgcgg gatttgtaat tactacggtc tagcaagtaa ttttaaccag 3900
ctcaattatt ttgcttatct tatggaatac agctgtctaa aaacgatagc ctccaaacat 3960
aagggaacac tttcaaaaac catttccatg tttaaagatg gaagtggttc gtgggggatc 4020
ccgtatgaga taaagcaagg taagcagcgc cgttattttg caaattttag tgaatgtaaa 4080
tccccttatc aatttacgga tgagataagt caagctcctg tattgtatgg ctatgcccgg 4140
aatactcttg aaaacaggtt aaaagctaaa tgttgtgaat tatgtgggac gtctgatgaa 4200
aatacttcct atgaaattca ccatgtcaat aaggtcaaaa atcttaaagg caaagaaaaa 4260
tgggaaatgg caatgatagc gaaacaacgt aaaactcttg ttgtatgctt tcattgtcat 4320
cgtcacgtga ttcataaaca caagtgaatg tcgagcaccc gttctcggag cactgtccga 4380
ccgctttggc cgccgcccag tcctgctcgc ttcgctactt ggagccacta tcgactacgc 4440
gatcatggcg accacacccg tcctgtggat cgccaagccg ccgatggtag tgtggggtct 4500
ccccatgcga gagtagggaa ctgccaggca tcaaataaaa cgaaaggctc agtcgaaaga 4560
ctgggccttt cgttttatct gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc 4620
gccgggagcg gatttgaacg ttgcgaagca acggcccgga gggtggcggg caggacgccc 4680
gccataaact gccaggcatc aaattaagca gaaggccatc ctgacggatg gcctttttgc 4740
gtttctacaa actcttcctg tcgtcatatc tacaagccat ccccccacag atacgggcgc 4800
gccgccatta tttttttgaa caattgacaa ttcatttctt attttttatt aagtgatagt 4860
caaaaggcat aacagtgctg aatagaaaga aatttacaga aaagaaaatt atagaattta 4920
gtatgattaa ttatactcat ttatgaatgt ttaattgaat acaaaaaaaa atacttgtta 4980
tgtattcaat tacgggttaa aatatagaca agttgaaaaa tttaataaaa aaataagtcc 5040
tcagctctta tatattaagc taccaactta gtatataagc caaaacttaa atgtgctacc 5100
aacacatcaa gccgttagag aactctatct atagcaatat ttcaaatgta ccgacataca 5160
agagaaacat taactatata tattcaattt atgagattat cttaacagat ataaatgtaa 5220
attgcaataa gtaagattta gaagtttata gcctttgtgt attggaagca gtacgcaaag 5280
gcttttttat ttgataaaaa ttagaagtat atttattttt tcataattaa tttatgaaaa 5340
tgaaaggggg tgagcaaagt gacagaggaa agcagtatct tatcaaataa caaggtatta 5400
gcaatatcat tattgacttt agcagtaaac attatgactt ttatagtgct tgtagctaag 5460
tagtacgaaa gggggagctt taaaaagctc cttggaatac atagaattca taaattaatt 5520
tatgaaaaga agggcgtata tgaaaacttg taaaaattgc aaagagttta ttaaagatac 5580
tgaaatatgc aaaatacatt cgttgatgat tcatgataaa acagtagcaa cctattgcag 5640
taaatacaat gagtcaagat gtttacataa agggaaagtc caatgtatta attgttcaaa 5700
gatgaaccga tatggatggt gtgccataaa aatgagatgt tttacagagg aagaacagaa 5760
aaaagaacgt acatgcatta aatattatgc aaggagcttt aaaaaagctc atgtaaagaa 5820
gagtaaaaag aaaaaataat ttatttatta atttaatatt gagagtgccg acacagtatg 5880
cactaaaaaa tatatctgtg gtgtagtgag ccgatacaaa aggatagtca ctcgcatttt 5940
cataatacat cttatgttat gattatgtgt cggtgggact tcacgacgaa aacccacaat 6000
aaaaaaagag ttcggggtag ggttaagcat agttgaggca actaaacaat caagctagga 6060
tatgcagtag cagaccgtaa ggtcgttgtt taggtgtgtt gtaatacata cgctattaag 6120
atgtaaaaat acggatacca atgaagggaa aagtataatt tttggatgta gtttgtttgt 6180
tcatctatgg gcaaactacg tccaaagccg tttccaaatc tgctaaaaag tatatccttt 6240
ctaaaatcaa agtcaagtat gaaatcataa ataaagttta attttgaagt tattatgata 6300
ttatgttttt ctattaaaat aaattaagta tatagaatag tttaataata gtatatactt 6360
aatgtgataa gtgtctgaca gtgtcacaga aaggatgatt gttatggatt ataagcggcc 6420
ggcccaatga ataggtttac acttacttta gttttatgga aatgaaagat catatcatat 6480
ataatctaga ataaaattaa ctaaaataat tattatctag ataaaaaatt tagaagccaa 6540
tgaaatctat aaataaacta aattaagttt atttaattaa caactatgga tataaaatag 6600
gtactaatca aaatagtgag gaggatatat ttgaatacat acgaacaaat taataaagtg 6660
aaaaaaatac ttcggaaaca tttaaaaaat aaccttattg gtacttacat gtttggatca 6720
ggagttgaga gtggactaaa accaaatagt gatcttgact ttttagtcgt cgtatctgaa 6780
ccattgacag atcaaagtaa agaaatactt atacaaaaaa ttagacctat ttcaaagaaa 6840
ataggagata aaagcaactt acgatatatt gaattaacaa ttattattca gcaagaaatg 6900
gtaccgtgga atcatcctcc caaacaagaa tttatttatg gagaatggtt acaagagctt 6960
tatgaacaag gatacattcc tcagaaggaa ttaaattcag atttaaccat aatgctttac 7020
caagcaaaac gaaaaaataa aagaatatac ggaaattatg acttagagga attactacct 7080
gatattccat tttctgatgt gagaagagcc attatggatt cgtcagagga attaatagat 7140
aattatcagg atgatgaaac caactctata ttaactttat gccgtatgat tttaactatg 7200
gacacgggta aaatcatacc aaaagatatt gcgggaaatg cagtggctga atcttctcca 7260
ttagaacata gggagagaat tttgttagca gttcgtagtt atcttggaga gaatattgaa 7320
tggactaatg aaaatgtaaa tttaactata aactatttaa ataacagatt aaaaaaatta 7380
taaaaaaatt gaaaaaatgg tggaaacact tttttcaatt tttttgtttt attatttaat 7440
atttgggaaa tattcattct aattggtaat cagattttag aagtttaaac tcctttttga 7500
taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt 7560
agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca 7620
aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct 7680
ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta 7740
gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct 7800
aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc 7860
aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca 7920
gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga 7980
aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg 8040
aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt 8100
cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag 8160
cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt 8220
tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt 8280
tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga 8340
ggaagcggaa gagcgcccaa tacgcagggc cccctgcttc ggggtcatta tagcgatttt 8400
ttcggtatat ccatcctttt tcgcacgata tacaggattt tgccaaaggg ttcgtgtaga 8460
ctttccttgg tgtatccaac ggcgtcagcc gggcaggata ggtgaagtag gcccacccgc 8520
gagcgggtgt tccttcttca ctgtccctta ttcgcacctg gcggtgctca acgggaatcc 8580
tgctctgcga ggctggccgg ctaccgccgg cgtaacagat gagggcaagc ggatggctga 8640
tgaaaccaag ccaaccagga agggcagccc acctatcaag gtgtactgcc ttccagacga 8700
acgaagagcg attgaggaaa aggcggcggc ggccggcatg agcctgtcgg cctacctgct 8760
ggccgtcggc cagggctaca aaatcacggg cgtcgtggac tatgagcacg tccgcgagct 8820
ggcccgcatc aatggcgacc tgggccgcct gggcggcctg ctgaaactct ggctcaccga 8880
cgacccgcgc acggcgcggt tcggtgatgc cacgatcctc gccctgctgg cgaagatcga 8940
agagaagcag gacgagcttg gcaaggtcat gatgggcgtg gtccgcccga gggcagagcc 9000
atgacttttt tagccgctaa aacggccggg gggtgcgcgt gattgccaag cacgtcccca 9060
tgcgctccat caagaagagc gacttcgcgg agctggtgaa gtacatcacc gacgagcaag 9120
gcaagacc 9128
<210> 121
<211> 5002
<212> DNA
<213> 人工序列
<220>
<223> pEC751S
<400> 121
atcaaaaaaa tttccaataa tcccactcta agccacaaac acgccctata aaatcccgct 60
ttaatcccac tttgagacac atgtaatatt actttacgcc ctagtatagt gataattttt 120
tacattcaat gccacgcaaa aaaataaagg ggcactataa taaaagttcc ttcggaacta 180
actaaagtaa aaaattatct ttacaacctc cccaaaaaaa agaacaggta caaagtaccc 240
tataatacaa gcgtaaaaaa aatgagggta aaaataaaaa aataaaaaaa taaaaaaata 300
aaaaaataaa aaaataaaaa aataaaaaaa tataaaaata aaaaaatata aaaataaaaa 360
aatataaaaa taaaaaaata aaaaaatata aaaataaaaa aataaaaaaa tataaaaata 420
ttttttattt aaagtttgaa aaaaattttt ttatattata taatctttga agaaaagaat 480
ataaaaaatg agcctttata aaagcccatt ttttttcata tacgtaatat gacgttctaa 540
tgtttttatt ggtacttcta acattagagt aatttcttta tttttaaagc ctttttcttt 600
aagggctttt attttttttc ttaatacatt taattcctct ttttttgttg cttttccttt 660
agcttttaat tgctcttgat aatttttttt acctctaata ttttctcttc tcttatattc 720
ctttttagaa attattattg tcatatattt ttgttcttct tctgtaattt ctaataactc 780
tataagagtt tcattcttat acttatattg cttattttta tctaaataac atctttcagc 840
acttctagtt gctcttataa cttctctttc acttaaatgt tgtctaaaca tactattaag 900
ttctaaaaca tcatttaatg ccttctcaat gtcttctgta aagctacaaa gataatatct 960
atataaaaat aatataagct ctctgtgtcc ttttaaatca tattctctta gttcacaaag 1020
ttttattatg tcttgtattc ttccataata taaacttctt tctctataaa tataatttat 1080
tttgcttggt ctaccctttt tcctttcata tggttttaat tcaggtaaaa atccattttg 1140
tatttctctt aagtcataaa tatattcgta ctcatctaat atattgacta ctgtttttga 1200
tttagagttt atacttcctg gaactcttaa tattctcgtt gcatctaagg cttgtctatc 1260
tgctccaaag tattttaatt gattatataa atattcttga accgctttcc ataatggtaa 1320
tgctttacta ggtactgcat ttattatcca tattaaatac attcctcttc cactatctat 1380
tacatagttt ggtataggaa tactttgatt aaaataattc ttttctaagt ccattaatac 1440
ctggtcttta gttttgccag ttttataata atccaagtct ataaacagtg tatttaactc 1500
ttttatattt tctaatcgcc tacacggctt ataaaaggta tttagagtta tatagatatt 1560
ttcatcactc atatctaaat cttttaattc agcgtattta tagtgccatt ggctatatcc 1620
ttttttatct ataacgctcc tggttatcca ccctttactt ctactatgaa tattatctat 1680
atagttcttt ttattcagct ttaatgcgtt tctcacttat tcacctcccc ttctgtaaaa 1740
ctaagaaaat tatatcatat tttcaataat tattaactat tcttaaactc ttaataaaaa 1800
atagagtaag tccccaattg aaacttaatc tattttttat gttttaattt attattttta 1860
ttaaaatatt ttaaactaaa ttaaatgatt ctttttaatt ttttactatt tcattccata 1920
atatattact ataattattt acaaataata tttcttcatt tgtaatattt agatgattta 1980
ctaattttag tttttatata ttaaataatt aatgtataat ttatataaaa aatcaaagga 2040
gcttataaat tatgattatt tccaaagata ctaaagattt aatttttttc aattttaaca 2100
atactttttg taatattatg tttaaattta attgtatttt tttcatataa taaagccgtt 2160
gaagtaaacc aatccatttt ccttatgatg ttattattaa atttaagttt tataataata 2220
tctttattat atttattgtt tttaaaaaaa ctagtgaaat ttctagtgaa atttccggct 2280
ttattaaact tatttttagg aattttattt tcattttcat ctttacagga tttgattata 2340
tctttaaata tgttttatca aatattatct ttttctaaat ttatatatat ttttattata 2400
tttattatta tatatatttt atttttaagt ttctttctaa cagctattaa aaagaaactt 2460
aaaaataaaa acacgtactc taaaccaata aataaaacta tttttattat tgctgccttg 2520
attggaatag tttttagtaa aattaatttc aatattccac aatattatat tataagctag 2580
cacgcctcga gatctccatg gacgcgtgac gtcgactcta gaggatcccc gggtaccgag 2640
ctcgaattcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat 2700
tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag 2760
ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg 2820
ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc 2880
ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc 2940
agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa 3000
catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt 3060
tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg 3120
gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg 3180
ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag 3240
cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc 3300
caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa 3360
ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg 3420
taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc 3480
taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac 3540
cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg 3600
tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt 3660
gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt 3720
catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa 3780
atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaaagct agcttaatac 3840
tagtatatac ttaatgtgat aagtgtctga cagctgaccg gtctaaagag gtcccaatga 3900
ataggtttac acttacttta gttttatgga aatgaaagat catatcatat ataatctaga 3960
ataaaattaa ctaaaataat tattatctag ataaaaaatt tagaagccaa tgaaatctat 4020
aaataaacta aattaagttt atttaattaa caactatgga tataaaatag gtactaatca 4080
aaatagtgag gaggatatat ttgaatacat acgaacaaat taataaagtg aaaaaaatac 4140
ttcggaaaca tttaaaaaat aaccttattg gtacttacat gtttggatca ggagttgaga 4200
gtggactaaa accaaatagt gatcttgact ttttagtcgt cgtatctgaa ccattgacag 4260
atcaaagtaa agaaatactt atacaaaaaa ttagacctat ttcaaagaaa ataggagata 4320
aaagcaactt acgatatatt gaattaacaa ttattattca gcaagaaatg gtaccgtgga 4380
atcatcctcc caaacaagaa tttatttatg gagaatggtt acaagagctt tatgaacaag 4440
gatacattcc tcagaaggaa ttaaattcag atttaaccat aatgctttac caagcaaaac 4500
gaaaaaataa aagaatatac ggaaattatg acttagagga attactacct gatattccat 4560
tttctgatgt gagaagagcc attatggatt cgtcagagga attaatagat aattatcagg 4620
atgatgaaac caactctata ttaactttat gccgtatgat tttaactatg gacacgggta 4680
aaatcatacc aaaagatatt gcgggaaatg cagtggctga atcttctcca ttagaacata 4740
gggagagaat tttgttagca gttcgtagtt atcttggaga gaatattgaa tggactaatg 4800
aaaatgtaaa tttaactata aactatttaa ataacagatt aaaaaaatta taaaaaaatt 4860
gaaaaaatgg tggaaacact tttttcaatt tttttgtttt attatttaat atttgggaaa 4920
tattcattct aattggtaat cagattttag aagttgttaa cttcaggttt gtctgtaact 4980
aaaaactagt atttaaccta gg 5002
<210> 122
<211> 3907
<212> DNA
<213> 人工序列
<220>
<223> pFW01
<400> 122
tcgagatctc catggacgcg tgacgtcgac tctagaggat ccccgggtac cgagctcgaa 60
ttcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 120
caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 180
cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 240
gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 300
ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 360
ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 420
agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 480
taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 540
cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 600
tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 660
gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 720
gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 780
tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 840
gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 900
cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 960
aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt 1020
tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt 1080
ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag 1140
attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat 1200
ctaaagtata tatgagtaaa cttggtctga cagttaccag gtccactgcc gggcctcttg 1260
cgggatcaaa agaaaaacga aatgatacac caatcagtgc aaaaaaagat ataatgggag 1320
ataagacggt tcgtgttcgt gctgacttgc accatatcat aaaaatcgaa acagcaaaga 1380
atggcggaaa cgtaaaagaa gttatggaaa taagacttag aagcaaactt aagagtgtgt 1440
tgatagtgca gtatcttaaa attttgtata ataggaattg aagttaaatt agatgctaaa 1500
aatttgtaat taagaaggag tgattacatg aacaaaaata taaaatattc tcaaaacttt 1560
ttaacgagtg aaaaagtact caaccaaata ataaaacaat tgaatttaaa agaaaccgat 1620
accgtttacg aaattggaac aggtaaaggg catttaacga cgaaactggc taaaataagt 1680
aaacaggtaa cgtctattga attagacagt catctattca acttatcgtc agaaaaatta 1740
aaactgaata ctcgtgtcac tttaattcac caagatattc tacagtttca attccctaac 1800
aaacagaggt ataaaattgt tgggagtatt ccttaccatt taagcacaca aattattaaa 1860
aaagtggttt ttgaaagcca tgcgtctgac atctatctga ttgttgaaga aggattctac 1920
aagcgtacct tggatattca ccgaacacta gggttgctct tgcacactca agtctcgatt 1980
cagcaattgc ttaagctgcc agcggaatgc tttcatccta aaccaaaagt aaacagtgtc 2040
ttaataaaac ttacccgcca taccacagat gttccagata aatattggaa gctatatacg 2100
tactttgttt caaaatgggt caatcgagaa tatcgtcaac tgtttactaa aaatcagttt 2160
catcaagcaa tgaaacacgc caaagtaaac aatttaagta ccgttactta tgagcaagta 2220
ttgtctattt ttaatagtta tctattattt aacgggagga aataattcta tgagtcccta 2280
ggcaggcctc cgccattatt tttttgaaca attgacaatt catttcttat tttttattaa 2340
gtgatagtca aaaggcataa cagtgctgaa tagaaagaaa tttacagaaa agaaaattat 2400
agaatttagt atgattaatt atactcattt atgaatgttt aattgaatac aaaaaaaaat 2460
acttgttatg tattcaatta cgggttaaaa tatagacaag ttgaaaaatt taataaaaaa 2520
ataagtcctc agctcttata tattaagcta ccaacttagt atataagcca aaacttaaat 2580
gtgctaccaa cacatcaagc cgttagagaa ctctatctat agcaatattt caaatgtacc 2640
gacatacaag agaaacatta actatatata ttcaatttat gagattatct taacagatat 2700
aaatgtaaat tgcaataagt aagatttaga agtttatagc ctttgtgtat tggaagcagt 2760
acgcaaaggc ttttttattt gataaaaatt agaagtatat ttattttttc ataattaatt 2820
tatgaaaatg aaagggggtg agcaaagtga cagaggaaag cagtatctta tcaaataaca 2880
aggtattagc aatatcatta ttgactttag cagtaaacat tatgactttt atagtgcttg 2940
tagctaagta gtacgaaagg gggagcttta aaaagctcct tggaatacat agaattcata 3000
aattaattta tgaaaagaag ggcgtatatg aaaacttgta aaaattgcaa agagtttatt 3060
aaagatactg aaatatgcaa aatacattcg ttgatgattc atgataaaac agtagcaacc 3120
tattgcagta aatacaatga gtcaagatgt ttacataaag ggaaagtcca atgtattaat 3180
tgttcaaaga tgaaccgata tggatggtgt gccataaaaa tgagatgttt tacagaggaa 3240
gaacagaaaa aagaacgtac atgcattaaa tattatgcaa ggagctttaa aaaagctcat 3300
gtaaagaaga gtaaaaagaa aaaataattt atttattaat ttaatattga gagtgccgac 3360
acagtatgca ctaaaaaata tatctgtggt gtagtgagcc gatacaaaag gatagtcact 3420
cgcattttca taatacatct tatgttatga ttatgtgtcg gtgggacttc acgacgaaaa 3480
cccacaataa aaaaagagtt cggggtaggg ttaagcatag ttgaggcaac taaacaatca 3540
agctaggata tgcagtagca gaccgtaagg tcgttgttta ggtgtgttgt aatacatacg 3600
ctattaagat gtaaaaatac ggataccaat gaagggaaaa gtataatttt tggatgtagt 3660
ttgtttgttc atctatgggc aaactacgtc caaagccgtt tccaaatctg ctaaaaagta 3720
tatcctttct aaaatcaaag tcaagtatga aatcataaat aaagtttaat tttgaagtta 3780
ttatgatatt atgtttttct attaaaataa attaagtata tagaatagtt taataatagt 3840
atatacttaa tgtgataagt gtctgacagt gtcacagaaa ggatgattgt tatggattat 3900
aagcggc 3907
<210> 123
<211> 6525
<212> DNA
<213> 人工序列
<220>
<223> pNF3S
<400> 123
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatcccaatg 3060
aataggttta cacttacttt agttttatgg aaatgaaaga tcatatcata tataatctag 3120
aataaaatta actaaaataa ttattatcta gataaaaaat ttagaagcca atgaaatcta 3180
taaataaact aaattaagtt tatttaatta acaactatgg atataaaata ggtactaatc 3240
aaaatagtga ggaggatata tttgaataca tacgaacaaa ttaataaagt gaaaaaaata 3300
cttcggaaac atttaaaaaa taaccttatt ggtacttaca tgtttggatc aggagttgag 3360
agtggactaa aaccaaatag tgatcttgac tttttagtcg tcgtatctga accattgaca 3420
gatcaaagta aagaaatact tatacaaaaa attagaccta tttcaaagaa aataggagat 3480
aaaagcaact tacgatatat tgaattaaca attattattc agcaagaaat ggtaccgtgg 3540
aatcatcctc ccaaacaaga atttatttat ggagaatggt tacaagagct ttatgaacaa 3600
ggatacattc ctcagaagga attaaattca gatttaacca taatgcttta ccaagcaaaa 3660
cgaaaaaata aaagaatata cggaaattat gacttagagg aattactacc tgatattcca 3720
ttttctgatg tgagaagagc cattatggat tcgtcagagg aattaataga taattatcag 3780
gatgatgaaa ccaactctat attaacttta tgccgtatga ttttaactat ggacacgggt 3840
aaaatcatac caaaagatat tgcgggaaat gcagtggctg aatcttctcc attagaacat 3900
agggagagaa ttttgttagc agttcgtagt tatcttggag agaatattga atggactaat 3960
gaaaatgtaa atttaactat aaactattta aataacagat taaaaaaatt ataaaaaaat 4020
tgaaaaaatg gtggaaacac ttttttcaat ttttttgttt tattatttaa tatttgggaa 4080
atattcattc taattggtaa tcagatttta gaagttgagc tcgaattcac tggccgtcgt 4140
tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 4200
tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 4260
gttgcgcagc ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg 4320
cggtatttca caccgcatat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt 4380
aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt gtctgctccc 4440
ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc agaggttttc 4500
accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat ttttataggt 4560
taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg gaaatgtgcg 4620
cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 4680
ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta ttcaacattt 4740
ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg ctcacccaga 4800
aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg gttacatcga 4860
actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac gttttccaat 4920
gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg acgccgggca 4980
agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt actcaccagt 5040
cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg ctgccataac 5100
catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac cgaaggagct 5160
aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt gggaaccgga 5220
gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag caatggcaac 5280
aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc aacaattaat 5340
agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc ttccggctgg 5400
ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta tcattgcagc 5460
actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg ggagtcaggc 5520
aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga ttaagcattg 5580
gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac ttcattttta 5640
atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg 5700
tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga 5760
tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt 5820
ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag 5880
agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa 5940
ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag 6000
tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca 6060
gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac 6120
cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa 6180
ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc 6240
agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg 6300
tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc 6360
ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc 6420
ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag 6480
ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaaga 6525
<210> 124
<211> 6554
<212> DNA
<213> 人工序列
<220>
<223> pNF3E
<400> 124
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatccgtctg 3060
acagttacca ggtccactgc cgggcctctt gcgggatcaa aagaaaaacg aaatgataca 3120
ccaatcagtg caaaaaaaga tataatggga gataagacgg ttcgtgttcg tgctgacttg 3180
caccatatca taaaaatcga aacagcaaag aatggcggaa acgtaaaaga agttatggaa 3240
ataagactta gaagcaaact taagagtgtg ttgatagtgc agtatcttaa aattttgtat 3300
aataggaatt gaagttaaat tagatgctaa aaatttgtaa ttaagaagga gtgattacat 3360
gaacaaaaat ataaaatatt ctcaaaactt tttaacgagt gaaaaagtac tcaaccaaat 3420
aataaaacaa ttgaatttaa aagaaaccga taccgtttac gaaattggaa caggtaaagg 3480
gcatttaacg acgaaactgg ctaaaataag taaacaggta acgtctattg aattagacag 3540
tcatctattc aacttatcgt cagaaaaatt aaaactgaat actcgtgtca ctttaattca 3600
ccaagatatt ctacagtttc aattccctaa caaacagagg tataaaattg ttgggagtat 3660
tccttaccat ttaagcacac aaattattaa aaaagtggtt tttgaaagcc atgcgtctga 3720
catctatctg attgttgaag aaggattcta caagcgtacc ttggatattc accgaacact 3780
agggttgctc ttgcacactc aagtctcgat tcagcaattg cttaagctgc cagcggaatg 3840
ctttcatcct aaaccaaaag taaacagtgt cttaataaaa cttacccgcc ataccacaga 3900
tgttccagat aaatattgga agctatatac gtactttgtt tcaaaatggg tcaatcgaga 3960
atatcgtcaa ctgtttacta aaaatcagtt tcatcaagca atgaaacacg ccaaagtaaa 4020
caatttaagt accgttactt atgagcaagt attgtctatt tttaatagtt atctattatt 4080
taacgggagg aaataattct atgagtccct aggcaggcct ccgccattat ttttttgaac 4140
aattggagct cgaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 4200
gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 4260
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg 4320
atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc 4380
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 4440
gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc 4500
tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag 4560
ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg 4620
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata 4680
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga 4740
aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca 4800
ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat 4860
cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag 4920
agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc 4980
gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct 5040
cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca 5100
gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt 5160
ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat 5220
gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt 5280
gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta 5340
cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga 5400
ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt 5460
gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc 5520
gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct 5580
gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata 5640
ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt 5700
gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc 5760
gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg 5820
caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact 5880
ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg 5940
tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg 6000
ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac 6060
tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca 6120
cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga 6180
gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc 6240
ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct 6300
gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg 6360
agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct 6420
tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc 6480
tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc 6540
gaggaagcgg aaga 6554
<210> 125
<211> 6271
<212> DNA
<213> 人工序列
<220>
<223> pNF3C
<400> 125
gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca 60
cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct 120
cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat 180
tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg ccaagcttgc 240
atgcctgcag gtcgaccacg ataaaacaag gttttaagga taagaaaagt catgagattt 300
atagtaaatc ttgtgacttt ttttattgaa tagtagagag agttcggaag tataacacgc 360
tatattcttg atatttttag aatagcaagc attggatttg tcctgacact ttcccaaaaa 420
ttaaggagtt attccttaaa ccaaaaagat taatgtggga acaaatttag tgtatccatt 480
tttgaagggc gcacttatac accaccaaaa tggtgtgtgc gaaatcttta aaaaagattt 540
atcaaaaagc ttttttaaag ctgggacatt tagaaaatca ataatgtttt ttgcccaata 600
cgctagtctt aaaatctgca aggttgataa ctatttagtc ccaggtatta gaatggggca 660
tatatataca aagtatatat atgcgtaaat atatgtggga ctgtgggaac aaaattgcgt 720
gctaaaattg tattgaaaag gtaatgaaaa ggtcatgctt tggtattgct aacgtataga 780
aaaggtaatg aaaagctcat ggttctataa aaaagatgta cccacgaaaa taataggctt 840
tgcctatttc cccatgtaat atgggggcag ttttctctta tgctctttct taacatattg 900
aataaataca aaatgcagct ttgtgggaat aaaaatattt ttgtttttat tcttatagtt 960
agacaaaatt ttaatctttt ttgtgctata acaagattaa aatttgtggg aacattaaga 1020
aatattgttg tcacaaataa aaaggagagt gggaacaatt gctataaaaa acgcagaaat 1080
taagattaga gttacaaaag agcaaaaaga attatttaag aaaattgcaa aagctgaaaa 1140
tatgagtatg agtgaattta ttattgtgac cacagaatat ttagccagaa aaaaagatga 1200
aaatatgaaa tcaaaagaca tgatcgagag aagagctgcg aagactgaag aaaaaattat 1260
gaagctaaaa aagaaactaa ataaaaacag gtaatataga ttacagtttt aagcttgttt 1320
tccctataga ctagagtaaa tatataaata tacctgtcaa gggcttataa gcccctttag 1380
ggggtgcgta gcacccttga caggtatatt tatatatttt agggtgccat taagggaaac 1440
aagctttaaa atgcctttaa aggcatttta aaataaataa aaaaaagatg gtttttacca 1500
tcttttttaa ctcccgaaag ggagttcttt cttttcttga tactatacgt aactatttcg 1560
atttgccctg aacctaatca aagctagata aattcagtat tagggcataa aaaaacttgc 1620
tttttcgggt ggaaatctgt ataatttaaa ttgcttagat aaaaattacc aattccatac 1680
gaaaggagca agttttacat aaggttaaag ccttatgtga attctcattt aattacatga 1740
ataataataa cacagaaagt gaagaattaa aagagcaaag tcaactattg cttgacaaat 1800
gcacaaaaaa gaaaaagaaa aatcctaaat ttagtagtta tatagaacca ttagtaagca 1860
agaaattatc tgaaagaata aaggaatgtg gtgacttttt gcagatgtta tctgatttaa 1920
accttgaaaa ttcgaaactg catagagcaa gtttttgtgg taacagattt tgtcctatgt 1980
gtagctggcg tattgcttgt aaggatagtt tggaaatatc tattctcatg gagcatttac 2040
gcaaagagga aagcaaagaa tttatctttt tgaccttaac aactccaaat gtgaaaggtg 2100
cggaccttga taattccata aaagcataca ataaagcatt taaaaagtta atggaacgca 2160
aagaggtcaa gagcatagta aaaggctaca taagaaagct agaagtaacc tataatttgg 2220
acaagagttc caaatcatat aatacttatc acccacattt ccatgtggta ctagcagtca 2280
atagaagtta ctttaaaaag caaaatctat atataaacca tcatagatgg cttagtttgt 2340
ggcaagagtc aactggtgat tattcgataa ctcaagttga tgtaagaaag gctaaaatta 2400
acgattataa agaggtttat gagcttgcta agtattcggc taaggattcc gactatttaa 2460
tcaatagaga agtgtttacg gtattctaca aatctttaaa gggtaaacag gtacttgtat 2520
ttagtggatt atttaaagac gctcataaaa tgtataagaa tggagagcta gatctgtata 2580
agaagttgga tactatcgaa tatgcttata tggtaagtta taactggctt aaaaagaagt 2640
atgatacttc aaatattaga gaattaactg aggaagaaaa gcagaaattc aataaaaatt 2700
taatcgaaga tgtggatatt gagtaggtgg gattatatct cacctttttt attgtctttt 2760
catgttgaaa ttttgacgct taatgcatga agtattgaca agtttaaaaa ttacggtttt 2820
taatccttag ttgattagca ggattatggc cggaatgctc cgtccagtcc tgttaaggaa 2880
ttaaaattcc ctaaaaccct tggctatgat ttatagcgag aatcgtcaat taaaaattta 2940
ataggtgcta tgaaagtcga ttaataatta attttaaaat gcaatatgaa acataattac 3000
aagaatttga cttttaatac aagaattgat atcatagtta cattaatacg gatcccggca 3060
gtttttcttt ttcggcaagt gttcaagaag ttattaagtc gggagtgcag tcgaagtggg 3120
caagttgaaa aattcacaaa aatgtggtat aatatctttg ttcattagag cgataaactt 3180
gaatttgaga gggaacttag atggtatttg aaaaaattga taaaaatagt tggaacagaa 3240
aagagtattt tgaccactac tttgcaagtg taccttgtac ctacagcatg accgttaaag 3300
tggatatcac acaaataaag gaaaagggaa tgaaactata tcctgcaatg ctttattata 3360
ttgcaatgat tgtaaaccgc cattcagagt ttaggacggc aatcaatcaa gatggtgaat 3420
tggggatata tgatgagatg ataccaagct atacaatatt tcacaatgat actgaaacat 3480
tttccagcct ttggactgag tgtaagtctg actttaaatc atttttagca gattatgaaa 3540
gtgatacgca acggtatgga aacaatcata gaatggaagg aaagccaaat gctccggaaa 3600
acatttttaa tgtatctatg ataccgtggt caaccttcga tggctttaat ctgaatttgc 3660
agaaaggata tgattatttg attcctattt ttactatggg gaaatattat aaagaagata 3720
acaaaattat acttcctttg gcaattcaag ttcatcacgc agtatgtgac ggatttcaca 3780
tttgccgttt tgtaaacgaa ttgcaggaat tgataaatag ttaacttcag gtttgtctgt 3840
aactaaaaac tagtatttaa ccgagctcga attcactggc cgtcgtttta caacgtcgtg 3900
actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 3960
gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 4020
atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 4080
gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac 4140
acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca 4200
gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga 4260
aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa 4320
taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt 4380
gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa 4440
tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta 4500
ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag 4560
taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca 4620
gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta 4680
aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc 4740
gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc 4800
ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca 4860
ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc 4920
acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca 4980
taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac 5040
tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg 5100
cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg 5160
ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg 5220
gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac 5280
gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc 5340
aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct 5400
aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc 5460
actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc 5520
gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg 5580
atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa 5640
atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc 5700
ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt 5760
gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa 5820
cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc 5880
tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc 5940
cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct 6000
ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat 6060
gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc 6120
tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg 6180
ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc 6240
gcagcgagtc agtgagcgag gaagcggaag a 6271
<210> 126
<211> 2793
<212> DNA
<213> 人工序列
<220>
<223> OREP
<400> 126
cacgataaaa caaggtttta aggataagaa aagtcatgag atttatagta aatcttgtga 60
ctttttttat tgaatagtag agagagttcg gaagtataac acgctatatt cttgatattt 120
ttagaatagc aagcattgga tttgtcctga cactttccca aaaattaagg agttattcct 180
taaaccaaaa agattaatgt gggaacaaat ttagtgtatc catttttgaa gggcgcactt 240
atacaccacc aaaatggtgt gtgcgaaatc tttaaaaaag atttatcaaa aagctttttt 300
aaagctggga catttagaaa atcaataatg ttttttgccc aatacgctag tcttaaaatc 360
tgcaaggttg ataactattt agtcccaggt attagaatgg ggcatatata tacaaagtat 420
atatatgcgt aaatatatgt gggactgtgg gaacaaaatt gcgtgctaaa attgtattga 480
aaaggtaatg aaaaggtcat gctttggtat tgctaacgta tagaaaaggt aatgaaaagc 540
tcatggttct ataaaaaaga tgtacccacg aaaataatag gctttgccta tttccccatg 600
taatatgggg gcagttttct cttatgctct ttcttaacat attgaataaa tacaaaatgc 660
agctttgtgg gaataaaaat atttttgttt ttattcttat agttagacaa aattttaatc 720
ttttttgtgc tataacaaga ttaaaatttg tgggaacatt aagaaatatt gttgtcacaa 780
ataaaaagga gagtgggaac aattgctata aaaaacgcag aaattaagat tagagttaca 840
aaagagcaaa aagaattatt taagaaaatt gcaaaagctg aaaatatgag tatgagtgaa 900
tttattattg tgaccacaga atatttagcc agaaaaaaag atgaaaatat gaaatcaaaa 960
gacatgatcg agagaagagc tgcgaagact gaagaaaaaa ttatgaagct aaaaaagaaa 1020
ctaaataaaa acaggtaata tagattacag ttttaagctt gttttcccta tagactagag 1080
taaatatata aatatacctg tcaagggctt ataagcccct ttagggggtg cgtagcaccc 1140
ttgacaggta tatttatata ttttagggtg ccattaaggg aaacaagctt taaaatgcct 1200
ttaaaggcat tttaaaataa ataaaaaaaa gatggttttt accatctttt ttaactcccg 1260
aaagggagtt ctttcttttc ttgatactat acgtaactat ttcgatttgc cctgaaccta 1320
atcaaagcta gataaattca gtattagggc ataaaaaaac ttgctttttc gggtggaaat 1380
ctgtataatt taaattgctt agataaaaat taccaattcc atacgaaagg agcaagtttt 1440
acataaggtt aaagccttat gtgaattctc atttaattac atgaataata ataacacaga 1500
aagtgaagaa ttaaaagagc aaagtcaact attgcttgac aaatgcacaa aaaagaaaaa 1560
gaaaaatcct aaatttagta gttatataga accattagta agcaagaaat tatctgaaag 1620
aataaaggaa tgtggtgact ttttgcagat gttatctgat ttaaaccttg aaaattcgaa 1680
actgcataga gcaagttttt gtggtaacag attttgtcct atgtgtagct ggcgtattgc 1740
ttgtaaggat agtttggaaa tatctattct catggagcat ttacgcaaag aggaaagcaa 1800
agaatttatc tttttgacct taacaactcc aaatgtgaaa ggtgcggacc ttgataattc 1860
cataaaagca tacaataaag catttaaaaa gttaatggaa cgcaaagagg tcaagagcat 1920
agtaaaaggc tacataagaa agctagaagt aacctataat ttggacaaga gttccaaatc 1980
atataatact tatcacccac atttccatgt ggtactagca gtcaatagaa gttactttaa 2040
aaagcaaaat ctatatataa accatcatag atggcttagt ttgtggcaag agtcaactgg 2100
tgattattcg ataactcaag ttgatgtaag aaaggctaaa attaacgatt ataaagaggt 2160
ttatgagctt gctaagtatt cggctaagga ttccgactat ttaatcaata gagaagtgtt 2220
tacggtattc tacaaatctt taaagggtaa acaggtactt gtatttagtg gattatttaa 2280
agacgctcat aaaatgtata agaatggaga gctagatctg tataagaagt tggatactat 2340
cgaatatgct tatatggtaa gttataactg gcttaaaaag aagtatgata cttcaaatat 2400
tagagaatta actgaggaag aaaagcagaa attcaataaa aatttaatcg aagatgtgga 2460
tattgagtag gtgggattat atctcacctt ttttattgtc ttttcatgtt gaaattttga 2520
cgcttaatgc atgaagtatt gacaagttta aaaattacgg tttttaatcc ttagttgatt 2580
agcaggatta tggccggaat gctccgtcca gtcctgttaa ggaattaaaa ttccctaaaa 2640
cccttggcta tgatttatag cgagaatcgt caattaaaaa tttaataggt gctatgaaag 2700
tcgattaata attaatttta aaatgcaata tgaaacataa ttacaagaat ttgactttta 2760
atacaagaat tgatatcata gttacattaa tac 2793
<210> 127
<211> 2793
<212> DNA
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 127
cacgataaaa caaggtttta aggataagaa aagtcatgag atttatagta aatcttgtga 60
ctttttttat tgaatagtag agagagttcg gaagtataac acgctatatt cttgatattt 120
ttagaatagc aagcattgga tttgtcctga cactttccca aaaattaagg agttattcct 180
taaaccaaaa agattaatgt gggaacaaat ttagtgtatc catttttgaa gggcgcactt 240
atacaccacc aaaatggtgt gtgcgaaatc tttaaaaaag atttatcaaa aagctttttt 300
aaagctggga catttagaaa atcaataatg ttttttgccc aatacgctag tcttaaaatc 360
tgcaaggttg ataactattt agtcccaggt attagaatgg ggcatatata tacaaagtat 420
atatatgcgt aaatatatgt gggactgtgg gaacaaaatt gcgtgctaaa attgtattga 480
aaaggtaatg aaaaggtcat gctttggtat tgctaacgta tagaaaaggt aatgaaaagc 540
tcatggttct ataaaaaaga tgtacccacg aaaataatag gctttgccta tttccccatg 600
taatatgggg gcagttttct cttatgctct ttcttaacat attgaataaa tacaaaatgc 660
agctttgtgg gaataaaaat atttttgttt ttattcttat agttagacaa aattttaatc 720
ttttttgtgc tataacaaga ttaaaatttg tgggaacatt aagaaatatt gttgtcacaa 780
ataaaaagga gagtgggaac aattgctata aaaaacgcag aaattaagat tagagttaca 840
aaagagcaaa aagaattatt taagaaaatt gcaaaagctg aaaatatgag tatgagtgaa 900
tttattattg tgaccacaga atatttagcc agaaaaaaag atgaaaatat gaaatcaaaa 960
gacatgatcg agagaagagc tgcgaagact gaagaaaaaa ttatgaagct aaaaaagaaa 1020
ctaaataaaa acaggtaata tagattacag ttttaagctt gttttcccta tagactagag 1080
taaatatata aatatacctg tcaagggctt ataagcccct ttagggggtg cgtagcaccc 1140
ttgacaggta tatttatata ttttagggtg ccattaaggg aaacaagctt taaaatgcct 1200
ttaaaggcat tttaaaataa ataaaaaaaa gatggttttt accatctttt ttaactcccg 1260
aaagggagtt ctttcttttc ttgatactat acgtaactat ttcgatttgc cctgaaccta 1320
atcaaagcta gataaattca gtattagggc ataaaaaaac ttgctttttc gggtggaaat 1380
ctgtataatt taaattgctt agataaaaat taccaattcc atacgaaagg agcaagtttt 1440
acataaggtt aaagccttat gtgaattctc atttaattac atgaataata ataacacaga 1500
aagtgaagaa ttaaaagagc aaagtcaact attgcttgac aaatgcacaa aaaagaaaaa 1560
gaaaaatcct aaatttagta gttatataga accattagta agcaagaaat tatctgaaag 1620
aataaaggaa tgtggtgact ttttgcagat gttatctgat ttaaaccttg aaaattcgaa 1680
actgcataga gcaagttttt gtggtaacag attttgtcct atgtgtagct ggcgtattgc 1740
ttgtaaggat agtttggaaa tatctattct catggagcat ttacgcaaag aggaaagcaa 1800
agaatttatc tttttgacct taacaactcc aaatgtgaaa ggtgcggacc ttgataattc 1860
cataaaagca tacaataaag catttaaaaa gttaatggaa cgcaaagagg tcaagagcat 1920
agtaaaaggc tacataagaa agctagaagt aacctataat ttggacaaga gttccaaatc 1980
atataatact tatcacccac atttccatgt ggtactagca gtcaatagaa gttactttaa 2040
aaagcaaaat ctatatataa accatcatag atggcttagt ttgtggcaag agtcaactgg 2100
tgattattcg ataactcaag ttgatgtaag aaaggctaaa attaacgatt ataaagaggt 2160
ttatgagctt gctaagtatt cggctaagga ttccgactat ttaatcaata gagaagtgtt 2220
tacggtattc tacaaatctt taaagggtaa acaggtactt gtatttagtg gattatttaa 2280
agacgctcat aaaatgtata agaatggaga gctagatctg tataagaagt tggatactat 2340
cgaatatgct tatatggtaa gttataactg gcttaaaaag aagtatgata cttcaaatat 2400
tagagaatta actgaggaag aaaagcagaa attcaataaa aatttaatcg aagatgtgga 2460
tattgagtag gtgggattat atctcacctt ttttattgtc ttttcatgtt gaaattttga 2520
cgcttaatgc atgaagtatt gacaagttta aaaattacgg tttttaatcc ttagttgatt 2580
agcaggatta tggccggaat gctccgtcca gtcctgttaa ggaattaaaa ttccctaaaa 2640
cccttggcta tgatttatag cgagaatcgt caattaaaaa tttaataggt gctatgaaag 2700
tcgattaata attaatttta aaatgcaata tgaaacataa ttacaagaat ttgactttta 2760
atacaagaat tgatatcata gttacattaa tac 2793
<210> 128
<211> 329
<212> PRT
<213> 拜氏梭菌（Clostridium beijerinckii）
<400> 128
Met Asn Asn Asn Asn Thr Glu Ser Glu Glu Leu Lys Glu Gln Ser Gln
1 5 10 15
Leu Leu Leu Asp Lys Cys Thr Lys Lys Lys Lys Lys Asn Pro Lys Phe
20 25 30
Ser Ser Tyr Ile Glu Pro Leu Val Ser Lys Lys Leu Ser Glu Arg Ile
35 40 45
Lys Glu Cys Gly Asp Phe Leu Gln Met Leu Ser Asp Leu Asn Leu Glu
50 55 60
Asn Ser Lys Leu His Arg Ala Ser Phe Cys Gly Asn Arg Phe Cys Pro
65 70 75 80
Met Cys Ser Trp Arg Ile Ala Cys Lys Asp Ser Leu Glu Ile Ser Ile
85 90 95
Leu Met Glu His Leu Arg Lys Glu Glu Ser Lys Glu Phe Ile Phe Leu
100 105 110
Thr Leu Thr Thr Pro Asn Val Lys Gly Ala Asp Leu Asp Asn Ser Ile
115 120 125
Lys Ala Tyr Asn Lys Ala Phe Lys Lys Leu Met Glu Arg Lys Glu Val
130 135 140
Lys Ser Ile Val Lys Gly Tyr Ile Arg Lys Leu Glu Val Thr Tyr Asn
145 150 155 160
Leu Asp Lys Ser Ser Lys Ser Tyr Asn Thr Tyr His Pro His Phe His
165 170 175
Val Val Leu Ala Val Asn Arg Ser Tyr Phe Lys Lys Gln Asn Leu Tyr
180 185 190
Ile Asn His His Arg Trp Leu Ser Leu Trp Gln Glu Ser Thr Gly Asp
195 200 205
Tyr Ser Ile Thr Gln Val Asp Val Arg Lys Ala Lys Ile Asn Asp Tyr
210 215 220
Lys Glu Val Tyr Glu Leu Ala Lys Tyr Ser Ala Lys Asp Ser Asp Tyr
225 230 235 240
Leu Ile Asn Arg Glu Val Phe Thr Val Phe Tyr Lys Ser Leu Lys Gly
245 250 255
Lys Gln Val Leu Val Phe Ser Gly Leu Phe Lys Asp Ala His Lys Met
260 265 270
Tyr Lys Asn Gly Glu Leu Asp Leu Tyr Lys Lys Leu Asp Thr Ile Glu
275 280 285
Tyr Ala Tyr Met Val Ser Tyr Asn Trp Leu Lys Lys Lys Tyr Asp Thr
290 295 300
Ser Asn Ile Arg Glu Leu Thr Glu Glu Glu Lys Gln Lys Phe Asn Lys
305 310 315 320
Asn Leu Ile Glu Asp Val Asp Ile Glu
325
<210> 129
<211> 256
<212> PRT
<213> 人工序列
<220>
<223> 共有COG5655
<400> 129
Met Cys Gln Lys Arg Ser Asp Tyr Ser Asp Glu Lys Ala Trp Leu Lys
1 5 10 15
Asp Lys Ser Lys Asp Gly Lys Val Glu Pro Trp Arg Glu Lys Lys Glu
20 25 30
Ala Asn Val Lys Tyr Phe Glu Leu Leu Lys Ile Leu Met Phe Lys Lys
35 40 45
Ala Glu Arg Val Tyr Arg Cys Asn Glu Leu Leu Glu Leu Gln Lys Val
50 55 60
Asn Glu Thr Gly Glu Asn Lys Leu Cys Pro Asn Trp Phe Cys Lys Ser
65 70 75 80
Leu Leu Cys Pro Met Cys Asn Trp Arg Lys Pro Met Lys Ser Asp Leu
85 90 95
Gln Asp Gly Leu Tyr Val Lys Arg Val Ile Ser Tyr Gly Pro Leu Leu
100 105 110
Lys Trp Lys His Leu Lys Leu Asn Leu Lys Asn Val Glu Asp Gly Asp
115 120 125
Leu Leu Asn Lys Ser Leu Asp Glu Met Ala Leu Gly Phe Lys Arg Thr
130 135 140
Met Gly Phe Lys Lys Ile Ala Lys Asn Phe Val Gly Phe Met Lys Ser
145 150 155 160
Thr Glu Ile Thr Tyr Asn Glu Lys Asp Asn Ser Tyr Asn Gln His Met
165 170 175
His Val Leu Phe Cys Ser Glu Gln Thr Tyr Phe Lys Asn Phe Ile Asn
180 185 190
Asn Thr Pro Gln Glu Phe Trp Asn Lys Arg Trp Ser Lys Ala Met Lys
195 200 205
Leu Asp Tyr Asp Pro Gln Val Met Lys Leu Trp Thr Met Tyr Lys Lys
210 215 220
Glu Ile Lys Asn Tyr Ile Gln Thr Ala Leu Gln Glu Thr Ala Lys Tyr
225 230 235 240
Asp Val Lys Asp Met Asp Ser Ala Thr Ile Asp Asp Glu Lys Ser Leu
245 250 255
<210> 130
<211> 768
<212> DNA
<213> 粪肠球菌（Enterococcus faecalis）
<400> 130
gtgaggagga tatatttgaa tacatacgaa caaattaata aagtgaaaaa aatacttcgg 60
aaacatttaa aaaataacct tattggtact tacatgtttg gatcaggagt tgagagtgga 120
ctaaaaccaa atagtgatct tgacttttta gtcgtcgtat ctgaaccatt gacagatcaa 180
agtaaagaaa tacttataca aaaaattaga cctatttcaa agaaaatagg agataaaagc 240
aacttacgat atattgaatt aacaattatt attcagcaag aaatggtacc gtggaatcat 300
cctcccaaac aagaatttat ttatggagaa tggttacaag agctttatga acaaggatac 360
attcctcaga aggaattaaa ttcagattta accataatgc tttaccaagc aaaacgaaaa 420
aataaaagaa tatacggaaa ttatgactta gaggaattac tacctgatat tccattttct 480
gatgtgagaa gagccattat ggattcgtca gaggaattaa tagataatta tcaggatgat 540
gaaaccaact ctatattaac tttatgccgt atgattttaa ctatggacac gggtaaaatc 600
ataccaaaag atattgcggg aaatgcagtg gctgaatctt ctccattaga acatagggag 660
agaattttgt tagcagttcg tagttatctt ggagagaata ttgaatggac taatgaaaat 720
gtaaatttaa ctataaacta tttaaataac agattaaaaa aattataa 768
<210> 131
<211> 738
<212> DNA
<213> 艰难梭菌（Clostridium difficile）
<400> 131
atgaacaaaa atataaaata ttctcaaaac tttttaacga gtgaaaaagt actcaaccaa 60
ataataaaac aattgaattt aaaagaaacc gataccgttt acgaaattgg aacaggtaaa 120
gggcatttaa cgacgaaact ggctaaaata agtaaacagg taacgtctat tgaattagac 180
agtcatctat tcaacttatc gtcagaaaaa ttaaaactga atactcgtgt cactttaatt 240
caccaagata ttctacagtt tcaattccct aacaaacaga ggtataaaat tgttgggagt 300
attccttacc atttaagcac acaaattatt aaaaaagtgg tttttgaaag ccatgcgtct 360
gacatctatc tgattgttga agaaggattc tacaagcgta ccttggatat tcaccgaaca 420
ctagggttgc tcttgcacac tcaagtctcg attcagcaat tgcttaagct gccagcggaa 480
tgctttcatc ctaaaccaaa agtaaacagt gtcttaataa aacttacccg ccataccaca 540
gatgttccag ataaatattg gaagctatat acgtactttg tttcaaaatg ggtcaatcga 600
gaatatcgtc aactgtttac taaaaatcag tttcatcaag caatgaaaca cgccaaagta 660
aacaatttaa gtaccgttac ttatgagcaa gtattgtcta tttttaatag ttatctatta 720
tttaacggga ggaaataa 738
<210> 132
<211> 3792
<212> DNA
<213> 人工序列
<220>
<223> 枯草芽孢杆菌（B. subtilis）的优化的Mad7 CDS
<400> 132
atgaacaacg gcacaaataa ttttcagaac tttattggca tttcatcatt gcagaaaacg 60
ttaagaaatg ctttaattcc gacggaaaca acgcaacagt ttattgttaa aaacggaatt 120
attaaagaag atgaattaag aggcgaaaac agacagattt taaaagatat tatggatgac 180
tactacagag gatttatttc tgaaacatta tcatctattg atgacattga ttggacaagc 240
ttatttgaaa aaatggaaat tcagttaaaa aatggtgata ataaagatac attaattaaa 300
gaacagacag aatatagaaa agcaattcat aaaaaatttg cgaacgacga tagatttaaa 360
aacatgttta gcgccaaatt aatttcagac attttacctg aatttgttat tcataacaat 420
aattattcag catcagaaaa agaagaaaaa acacaggtga ttaaattgtt ttcaagattt 480
gcgacaagct ttaaagatta ctttaaaaac agagcaaatt gcttttcagc ggacgatatt 540
tcatcaagca gctgccatag aattgttaac gacaatgcag aaattttttt ttcaaatgcg 600
ttagtttaca gaagaattgt aaaatcatta agcaatgacg atattaacaa aatttcaggc 660
gatatgaaag attcattaaa agaaatgtca ttagaagaaa tttattctta cgaaaaatat 720
ggcgaattta ttacacagga aggcattagc ttttataatg atatttgtgg caaagtgaat 780
tcttttatga acttatattg tcagaaaaat aaagaaaaca aaaatttata caaacttcag 840
aaacttcata aacagattct gtgcattgcg gacacaagct atgaagttcc gtataaattt 900
gaatcagacg aagaagtgta ccaatcagtt aacggctttc ttgataacat tagcagcaaa 960
catattgttg aaagattaag aaaaattggc gataactata acggctacaa cttagataaa 1020
atttatattg tgtccaaatt ttacgaaagc gttagccaaa aaacatacag agactgggaa 1080
acaattaata cagccttaga aattcattac aataatattt tgccgggtaa cggtaaatca 1140
aaagccgaca aagtaaaaaa agcggttaaa aatgatttac agaaatccat tacagaaatt 1200
aatgaactgg tgtcaaacta taaattatgc tcagacgaca acattaaagc ggaaacatat 1260
attcatgaaa ttagccatat tttgaataac tttgaagcac aggaattgaa atacaatccg 1320
gaaattcatc tggttgaatc cgaattaaaa gcgtcagaac ttaaaaacgt gttagacgtg 1380
attatgaatg cgtttcattg gtgttcagtt tttatgacag aagaacttgt tgataaagac 1440
aacaattttt atgcggaatt agaagaaatt tacgatgaaa tttatccggt aatttcatta 1500
tacaacttag ttagaaacta cgttacacag aaaccgtaca gcacgaaaaa aattaaattg 1560
aactttggaa ttccgacgtt agcagacggt tggtcaaaat ccaaagaata ttctaataac 1620
gctattattt taatgagaga caatttatat tatttaggca tttttaatgc gaaaaataaa 1680
ccggacaaaa aaattattga aggtaatacg tcagaaaata aaggtgacta caaaaaaatg 1740
atttataatt tgttaccggg tccgaacaaa atgattccga aagttttttt gagcagcaaa 1800
acgggcgtgg aaacgtataa accgagcgcc tatattctgg aaggctataa acagaataaa 1860
catattaaat cttcaaaaga ctttgatatt acattttgtc atgatttaat tgactacttt 1920
aaaaactgta ttgcaattca tccggaatgg aaaaactttg gttttgattt tagcgacaca 1980
tcaacatatg aagacatttc cggcttttat agagaagtag aattacaagg ttacaaaatt 2040
gattggacat acattagcga aaaagacatt gatttattac aggaaaaagg tcaattatat 2100
ttatttcaga tttataacaa agatttttca aaaaaatcaa caggcaatga caaccttcat 2160
acaatgtact taaaaaatct tttttcagaa gaaaatctta aagatattgt tttaaaactt 2220
aacggcgaag cggaaatttt ttttagaaaa agcagcatta aaaacccgat tattcataaa 2280
aaaggctcaa ttttagttaa cagaacatac gaagcagaag aaaaagacca gtttggcaac 2340
attcaaattg tgagaaaaaa tattccggaa aacatttatc aggaattata caaatacttt 2400
aacgataaaa gcgacaaaga attatctgat gaagcagcca aattaaaaaa tgtagtggga 2460
catcatgaag cagcgacgaa tattgttaaa gactatagat acacgtatga taaatacttt 2520
cttcatatgc ctattacgat taattttaaa gccaataaaa cgggttttat taatgataga 2580
attttacagt atattgctaa agaaaaagac ttacatgtga ttggcattga tagaggcgaa 2640
agaaacttaa tttacgtgtc cgtgattgat acatgtggta atattgttga acagaaaagc 2700
tttaacattg taaacggcta cgactatcag attaaattaa aacaacagga aggcgctaga 2760
cagattgcga gaaaagaatg gaaagaaatt ggtaaaatta aagaaattaa agaaggctac 2820
ttaagcttag taattcatga aatttctaaa atggtaatta aatacaatgc aattattgcg 2880
atggaagatt tgtcttatgg ttttaaaaaa ggcagattta aagttgaaag acaagtttac 2940
cagaaatttg aaacaatgtt aattaataaa ttaaactatt tagtatttaa agatatttca 3000
attacagaaa atggcggttt attaaaaggt tatcagttaa catacattcc tgataaactt 3060
aaaaacgtgg gtcatcagtg cggctgcatt ttttatgtgc ctgctgcata cacgagcaaa 3120
attgatccga caacaggctt tgtgaatatt tttaaattta aagacttaac agtggacgca 3180
aaaagagaat ttattaaaaa atttgactca attagatatg actcagaaaa aaatttattt 3240
tgctttacat ttgactacaa taactttatt acgcaaaaca cggttatgag caaatcatca 3300
tggtcagtgt atacatacgg cgtgagaatt aaaagaagat ttgtgaacgg cagattttca 3360
aacgaatcag atacaattga cattacaaaa gatatggaaa aaacgttgga aatgacggac 3420
attaactgga gagatggcca tgatcttaga caagacatta ttgattatga aattgttcag 3480
catatttttg aaatttttag attaacagtg caaatgagaa actccttgtc tgaattagaa 3540
gacagagatt acgatagatt aatttcacct gtattaaacg aaaataacat tttttatgac 3600
agcgcgaaag cgggcgatgc acttcctaaa gatgccgatg caaatggtgc gtattgtatt 3660
gcattaaaag gcttatatga aattaaacaa attacagaaa attggaaaga agatggtaaa 3720
ttttcaagag ataaattaaa aattagcaat aaagattggt ttgactttat tcagaataaa 3780
agatatttat aa 3792
<210> 133
<211> 10469
<212> DNA
<213> 人工序列
<220>
<223> pCas9cond
<400> 133
catggataaa aagtacagta ttggtctaga cataggaact aactctgttg ggtgggctgt 60
tataacagat gaatataaag ttccatcaaa aaaatttaaa gtattaggaa acactgatag 120
acattcaata aaaaaaaact tgataggtgc tttattattc gattcaggag agactgctga 180
agctacacgt ttaaaaagaa cagctagacg tagatataca agaagaaaaa ataggatatg 240
ttatcttcaa gaaattttta gtaatgaaat ggcaaaagtt gatgattcat tctttcacag 300
actagaagaa agtttcttag ttgaagaaga taagaagcat gaaagacacc ctatttttgg 360
taatatcgta gatgaagtag catatcatga gaagtatcca actatctatc atttaagaaa 420
gaaattagtt gattctacag ataaagctga tctgagatta atatatttag ctttagctca 480
tatgattaaa tttagaggac attttttaat agaaggtgat ttaaacccag acaacagcga 540
tgtagataaa ttatttatcc aattagttca aacttataat caattattcg aagagaatcc 600
aattaatgca agtggtgtag acgctaaggc tatattatca gctagattat caaaatctag 660
aagattagaa aatctaatag ctcaacttcc tggagaaaag aaaaatggac tttttgggaa 720
cctaatagct ctctcactcg gactaacacc aaattttaaa agcaattttg atcttgctga 780
agacgcaaag ttacaactat caaaggatac atacgatgat gatttagata atttgttagc 840
tcaaataggt gatcaatatg ctgatttgtt tcttgcagca aaaaacttaa gtgatgcaat 900
tttactatca gatatactta gagtaaatac agaaataaca aaggctcctt tatcagcaag 960
tatgattaaa cgatatgatg agcatcatca agatttaaca ttattaaagg cacttgtaag 1020
acaacaatta ccagaaaaat ataaagaaat tttctttgat caatctaaaa atggatatgc 1080
tggatatata gacggtggag caagtcaaga agagttttat aaatttataa agcctatttt 1140
agaaaaaatg gatggaactg aagaattact tgttaaactt aacagagaag atttacttag 1200
aaaacaaaga acttttgata atggttcaat tcctcaccaa attcatttag gagaattaca 1260
tgctatacta agaagacaag aagattttta tccatttctt aaagataata gagaaaaaat 1320
tgaaaaaatt ttaactttta gaataccata ttatgtagga ccacttgcaa ggggaaattc 1380
aagatttgca tggatgacta gaaaatcaga agaaactata accccgtgga attttgaaga 1440
agtagtagat aaaggagcta gtgctcaatc atttatagaa agaatgacaa attttgataa 1500
gaatcttcct aacgaaaagg ttttgccaaa gcatagcctt ctttatgagt attttacagt 1560
ttataatgag cttactaaag taaaatacgt tacagaagga atgagaaaac cagcattttt 1620
gtctggtgaa caaaagaaag caatagtaga cctattattt aaaacaaata ggaaggttac 1680
cgtaaagcaa cttaaagaag attacttcaa aaaaattgaa tgctttgata gtgttgaaat 1740
atcaggagtt gaagatagat ttaatgcttc acttggtaca tatcacgatc tcttaaaaat 1800
tataaaagat aaggattttt tagataatga agaaaatgaa gatattcttg aagatatagt 1860
attaacattg acactttttg aagatagaga aatgatagaa gaaagattaa aaacatatgc 1920
acatcttttt gatgataagg ttatgaagca acttaaaaga agaagatata caggttgggg 1980
acgtttgtca agaaagctaa ttaatggtat tagagataaa caatcaggaa agactattct 2040
cgattttctt aaatcagatg gatttgctaa tagaaacttt atgcaattaa ttcatgatga 2100
ttctcttact ttcaaagagg atattcaaaa ggctcaagtt tctggacaag gcgatagctt 2160
acacgaacac attgctaacc ttgcagggag ccccgctatc aaaaaaggaa ttttacaaac 2220
agttaaagtt gtagatgaac ttgttaaagt tatgggaaga cacaaacctg agaatatagt 2280
tatagaaatg gccagagaaa atcaaacaac acaaaaagga caaaaaaatt ctagagagag 2340
aatgaagaga attgaagaag gaataaaaga gctaggatca caaatattaa aagaacatcc 2400
agttgaaaat actcaattgc aaaatgaaaa gttatatttg tattacttac aaaatggaag 2460
agatatgtat gttgatcaag aactcgatat taatagatta agtgactatg atgttgatca 2520
tattgttcct caatcatttt taaaagatga ttcaatcgat aacaaagtat taactagatc 2580
agataaaaat agaggaaagt cagataatgt accatctgaa gaagttgtta aaaaaatgaa 2640
gaactattgg agacaacttt taaatgcaaa gctaattaca caaagaaaat ttgacaattt 2700
aacaaaagca gaaagaggag gattaagcga attagacaaa gctggattta taaaaagaca 2760
acttgttgag acaagacaaa taactaagca tgttgctcaa atacttgatt caagaatgaa 2820
tacaaaatat gatgaaaatg ataaattaat cagagaagta aaagtaataa cattaaagtc 2880
aaaattagta tcagatttca gaaaggattt tcaattttac aaagttcgtg aaataaataa 2940
ctatcatcat gctcatgatg catacttaaa tgctgttgta ggaactgctc ttattaagaa 3000
atatcctaaa ctagaaagcg aatttgttta tggagattat aaagtttatg atgtgcgcaa 3060
aatgatcgcg aaatccgaac aagaaatcgg taaggctaca gcaaaatatt tcttttatag 3120
taatataatg aattttttta agacagaaat aactttggct aatggtgaaa tcagaaaaag 3180
accacttatc gaaacaaatg gagagacagg agaaatagta tgggataaag gaagagattt 3240
tgctactgtt agaaaagtac taagtatgcc acaagtaaat atcgtaaaga aaactgaagt 3300
tcaaactgga ggtttctcta aggaatcaat tttacctaag agaaattcag ataagttaat 3360
tgcaaggaaa aaagattggg acccaaaaaa atacggtggt tttgatagtc caacagttgc 3420
ctatagtgtt cttgtagtag cgaaagttga gaaaggtaag tcaaaaaagt tgaaaagcgt 3480
aaaagaactt cttggtatca caattatgga aagatcttca tttgaaaaaa atccaattga 3540
ctttttagaa gctaagggtt ataaagaagt taaaaaggat ttaatcataa aactaccaaa 3600
gtatagtcta tttgaactcg aaaacggaag aaaacgaatg ctcgctagcg caggagaact 3660
tcaaaaagga aatgaacttg cgctgccatc aaagtatgta aatttcttat atttagcttc 3720
tcattatgag aaattaaaag gatcaccaga ggataatgaa caaaagcaac tatttgtaga 3780
acaacacaaa cattatttag atgaaataat agaacaaata tctgaatttt ctaaaagagt 3840
tatacttgcc gacgcaaatc tagataaggt gctttcagcg tataataaac acagagataa 3900
accaataaga gaacaagcag aaaacattat ccatcttttt acattaacta atcttggtgc 3960
accagctgca tttaagtact ttgatacaac aatagataga aaaagataca catctactaa 4020
agaagtatta gacgcaactt taatacatca atctattaca gggctttatg aaacaagaat 4080
tgatttaagt caactaggcg gagattaagt cgacaaagta ttgttaaaaa taactctgta 4140
gaattataaa ttagttctac agagttattt tttgacccgg gtaccgagct cgaattcgta 4200
atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat 4260
acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt 4320
aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta 4380
atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc 4440
gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa 4500
ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa 4560
aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct 4620
ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac 4680
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc 4740
gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc 4800
tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg 4860
tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga 4920
gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag 4980
cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta 5040
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag 5100
agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg 5160
caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac 5220
ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc 5280
aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag 5340
tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc 5400
agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac 5460
gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc 5520
accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg 5580
tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag 5640
tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc 5700
acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac 5760
atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag 5820
aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac 5880
tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg 5940
agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc 6000
gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact 6060
ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg 6120
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa 6180
tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt 6240
tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg 6300
tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga 6360
ctgccgggcc tcttgcggga tcaaaagaaa aacgaaatga tacaccaatc agtgcaaaaa 6420
aagatataat gggagataag acggttcgtg ttcgtgctga cttgcaccat atcataaaaa 6480
tcgaaacagc aaagaatggc ggaaacgtaa aagaagttat ggaaataaga cttagaagca 6540
aacttaagag tgtgttgata gtgcagtatc ttaaaatttt gtataatagg aattgaagtt 6600
aaattagatg ctaaaaattt gtaattaaga aggagtgatt acatgaacaa aaatataaaa 6660
tattctcaaa actttttaac gagtgaaaaa gtactcaacc aaataataaa acaattgaat 6720
ttaaaagaaa ccgataccgt ttacgaaatt ggaacaggta aagggcattt aacgacgaaa 6780
ctggctaaaa taagtaaaca ggtaacgtct attgaattag acagtcatct attcaactta 6840
tcgtcagaaa aattaaaact gaatactcgt gtcactttaa ttcaccaaga tattctacag 6900
tttcaattcc ctaacaaaca gaggtataaa attgttggga gtattcctta ccatttaagc 6960
acacaaatta ttaaaaaagt ggtttttgaa agccatgcgt ctgacatcta tctgattgtt 7020
gaagaaggat tctacaagcg taccttggat attcaccgaa cactagggtt gctcttgcac 7080
actcaagtct cgattcagca attgcttaag ctgccagcgg aatgctttca tcctaaacca 7140
aaagtaaaca gtgtcttaat aaaacttacc cgccatacca cagatgttcc agataaatat 7200
tggaagctat atacgtactt tgtttcaaaa tgggtcaatc gagaatatcg tcaactgttt 7260
actaaaaatc agtttcatca agcaatgaaa cacgccaaag taaacaattt aagtaccgtt 7320
acttatgagc aagtattgtc tatttttaat agttatctat tatttaacgg gaggaaataa 7380
ttctatgagt ccctaggccc aactaactca acgctagtag tggatttaat cccaaatgag 7440
ccaacagaac cagaaccaga aacagaatca gaacaagtaa cattggattt agaaatggaa 7500
gaagaaaaaa gcaatgactt cgtgtgaata atgcacgaaa tcgttgctta ttttttttta 7560
aaagcggtat actagatata acgaaacaac gaactgaata gaaacgaaaa aagagccatg 7620
acacatttat aaaatgtttg acgacatttt ataaatgcat agcccgataa gattgccaaa 7680
ccaacgctta tcagttagtc agatgaactc ttccctcgta agaagttatt taattaactt 7740
tgtttgaaga cggtatataa ccgtactatc attatatagg gaaatcagag agttttcaag 7800
tatctaagct actgaattta agaattgtta agcaatcaat cggaaatcgt ttgattgctt 7860
tttttgtatt catttataga aggtggagtt tgtatgaatc atgatgaatg taaaacttat 7920
ataaaaaata gtttattgga gataagaaaa ttagcaaata tctatacact agaaacgttt 7980
aagaaagagt tagaaaagag aaatatctac ttagaaacaa aatcagataa gtatttttct 8040
tcggaggggg aagattatat atataagtta atagaaaata acaaaataat ttattcgatt 8100
agtggaaaaa aattgactta taaaggaaaa aaatcttttt caaaacatgc aatattgaaa 8160
cagttgaatg aaaaagcaaa ccaagttaat taaacaacct attttatagg atttatagga 8220
aaggagaaca gctgaatgaa tatccctttt gttgtagaaa ctgtgcttca tgacggcttg 8280
ttaaagtaca aatttaaaaa tagtaaaatt cgctcaatca ctaccaagcc aggtaaaagc 8340
aaaggggcta tttttgcgta tcgctcaaaa tcaagcatga ttggcggtcg tggtgttgtt 8400
ctgacttccg aggaagcgat tcaagaaaat caagatacat ttacacattg gacacccaac 8460
gtttatcgtt atggaacgta tgcagacgaa aaccgttcat acacgaaagg acattctgaa 8520
aacaatttaa gacaaatcaa taccttcttt attgattttg atattcacac ggcaaaagaa 8580
actatttcag caagcgatat tttaacaacc gctattgatt taggttttat gcctactatg 8640
attatcaaat ctgataaagg ttatcaagca tattttgttt tagaaacgcc agtctatgtg 8700
acttcaaaat cagaatttaa atctgtcaaa gcagccaaaa taatttcgca aaatatccga 8760
gaatattttg gaaagtcttt gccagttgat ctaacgtgta atcattttgg tattgctcgc 8820
ataccaagaa cggacaatgt agaatttttt gatcctaatt accgttattc tttcaaagaa 8880
tggcaagatt ggtctttcaa acaaacagat aataagggct ttactcgttc aagtctaacg 8940
gttttaagcg gtacagaagg caaaaaacaa gtagatgaac cctggtttaa tctcttattg 9000
cacgaaacga aattttcagg agaaaagggt ttaatagggc gtaataacgt catgtttacc 9060
ctctctttag cctactttag ttcaggctat tcaatcgaaa cgtgcgaata taatatgttt 9120
gagtttaata atcgattaga tcaaccctta gaagaaaaag aagtaatcaa aattgttaga 9180
agtgcctatt cagaaaacta tcaaggggct aatagggaat acattaccat tctttgcaaa 9240
gcttgggtat caagtgattt aaccagtaaa gatttatttg tccgtcaagg gtggtttaaa 9300
ttcaagaaaa aaagaagcga acgtcaacgt gttcatttgt cagaatggaa agaagattta 9360
atggcttata ttagcgaaaa aagcgatgta tacaagcctt atttagtgac gaccaaaaaa 9420
gagattagag aagtgctagg cattcctgaa cggacattag ataaattgct gaaggtactg 9480
aaggcgaatc aggaaatttt ctttaagatt aaaccaggaa gaaatggtgg cattcaactt 9540
gctagtgtta aatcattgtt gctatcgatc attaaagtaa aaaaagaaga aaaagaaagc 9600
tatataaagg cgctgacaaa ttcttttgac ttagagcata cattcattca agagacttta 9660
aacaagctag cagaacgccc taaaacggac acacaactcg atttgtttag ctatgataca 9720
ggctgaaaat aaaacccgca ctatgccatt acatttatat ctatgatacg tgtttgtttt 9780
ttctttgctg tttagcgaat gattagcaga aatatacaga gtaagatttt aattaattat 9840
tagggggaga aggagagagt agcccgaaaa cttttagttg gcttggactg aacgaagtga 9900
gggaaaggct actaaaacgt cgaggggcag tgagagcgaa gcgaacactt gattttttaa 9960
ttttctatct tttataggtc attagagtat acttatttgt cctataaact atttagcagc 10020
ataatagatt tattgaatag gtcatttaag ttgagcatat tagaggagga aaatcttgga 10080
gaaatatttg aagaacccga ttacatggat tggattagtt cttgtggtta cgtggttttt 10140
aactaaaagt agtgaatttt tgatttttgg tgtgtgtgtc ttgttgttag tatttgctag 10200
tcaaagtgat taaatagaat tctagcgcca ttcgccattc aggctgcgca actgttggga 10260
agggcgatcg gtgcgggcct cttcgctatt acgccagctg gcgaaagggg gatgtgctgc 10320
aaggcgatta agttgggtaa cgccagggtt ttcccagtca cgacgttgta aaacgacggc 10380
cagtgccaag cttgcatgcc tgcaggcctc gagtatattg ataaaaataa taatagtggg 10440
tataattaag ttgttaggag gttagttac 10469
<210> 134
<211> 8559
<212> DNA
<213> 人工序列
<220>
<223> pMAD7
<400> 134
tcgagtccct atcagtgata gattgaaact ctatcattga tagagtataa tatctttgtt 60
cattagagcg ataaacttga atttgagagg gaacttagat gaacaacggc acaaataatt 120
ttcagaactt catagggata tcaagtttgc agaaaacgtt aagaaatgct ttaataccca 180
cggaaaccac gcaacagttc atagttaaga acggaataat taaagaagat gagttaagag 240
gcgagaacag acagatttta aaagatataa tggatgacta ctacagagga ttcatatctg 300
agactttaag ttctattgat gacatagatt ggactagctt attcgaaaaa atggaaattc 360
agttaaaaaa tggtgataat aaagatacct taattaagga acagacagag tatagaaaag 420
caatacataa aaaatttgcg aacgacgata gatttaagaa catgtttagc gccaaattaa 480
ttagtgacat attacctgaa tttgttatac acaacaataa ttattcggca tcagagaaag 540
aggaaaaaac ccaggtgata aaattgtttt cgagatttgc gactagcttt aaagattact 600
tcaagaacag agcaaattgc ttttcagcgg acgatatttc atcaagcagc tgccatagaa 660
tagttaacga caatgcagag atattctttt caaatgcgtt agtttacaga agaatagtaa 720
aatcgttaag caatgacgat ataaacaaaa tttcgggcga tatgaaagat tcattaaaag 780
aaatgagttt agaagaaata tattcttacg agaagtatgg ggaatttatt acccaggaag 840
gcattagctt ctataatgat atatgtggga aagtgaattc ttttatgaac ttatattgtc 900
agaaaaataa agaaaacaaa aatttataca aacttcagaa acttcacaaa cagattctat 960
gcattgcgga cactagctat gaggttccgt ataaatttga aagtgacgag gaagtgtacc 1020
aatcagttaa cggcttcctt gataacatta gcagcaaaca tatagttgaa agattaagaa 1080
aaataggcga taactataac ggctacaact tagataaaat ttatatagtg tccaaatttt 1140
acgagagcgt tagccaaaaa acctacagag actgggaaac aattaatacc gccttagaaa 1200
ttcattacaa taatatattg ccgggtaacg gtaaaagtaa agccgacaaa gtaaaaaaag 1260
cggttaagaa tgatttacag aaatccataa ccgaaataaa tgaactagtg tcaaactata 1320
agttatgcag tgacgacaac ataaaagcgg agacttatat acatgagatt agccatatat 1380
tgaataactt tgaagcacag gaattgaaat acaatccgga aattcaccta gttgaatccg 1440
agttaaaagc gagtgagctt aaaaacgtgt tagacgtgat aatgaatgcg tttcattggt 1500
gttcggtttt tatgactgag gaacttgttg ataaagacaa caatttttat gcggaattag 1560
aggagattta cgatgaaatt tatccagtaa ttagtttata caacttagtt agaaactacg 1620
ttacccagaa accgtacagc acgaaaaaga ttaaattgaa ctttggaata ccgacgttag 1680
cagacggttg gtcaaagtcc aaagagtatt ctaataacgc tataatatta atgagagaca 1740
atttatatta tttaggcata tttaatgcga agaataaacc ggacaagaag attatagagg 1800
gtaatacgtc agaaaataag ggtgactaca aaaagatgat ttataatttg ttaccgggtc 1860
ccaacaaaat gataccgaaa gttttcttga gcagcaagac gggggtggaa acgtataaac 1920
cgagcgccta tatactagag gggtataaac agaataaaca tataaagtct tcaaaagact 1980
ttgatataac tttctgtcat gatttaatag actacttcaa aaactgtatt gcaattcatc 2040
ccgagtggaa aaacttcggt tttgatttta gcgacaccag tacttatgaa gacatttccg 2100
ggttttatag agaggtagag ttacaaggtt acaagattga ttggacatac attagcgaaa 2160
aagacattga tttattacag gaaaaaggtc aattatattt attccagata tataacaaag 2220
atttttcgaa aaaatcaacc gggaatgaca accttcacac catgtactta aaaaatcttt 2280
tctcagaaga aaatcttaag gatatagttt taaaacttaa cggcgaagcg gaaatattct 2340
tcaggaagag cagcataaag aacccaataa ttcataaaaa aggctcgatt ttagttaaca 2400
gaacctacga agcagaagaa aaagaccagt ttggcaacat tcaaattgtg agaaaaaata 2460
ttccggaaaa catttatcag gagttataca aatacttcaa cgataaaagc gacaaagagt 2520
tatctgatga agcagccaaa ttaaagaatg tagtgggaca ccacgaggca gcgacgaata 2580
tagttaagga ctatagatac acgtatgata aatacttcct tcatatgcct attacgataa 2640
atttcaaagc caataaaacg ggttttatta atgataggat attacagtat atagctaaag 2700
aaaaagactt acatgtgata ggcattgata gaggcgagag aaacttaata tacgtgtccg 2760
tgattgatac ttgtggtaat atagttgaac agaaaagctt taacattgta aacggctacg 2820
actatcagat aaaattaaaa caacaggagg gcgctagaca gattgcgaga aaagaatgga 2880
aagaaattgg taaaattaaa gagataaaag agggctactt aagcttagta atacacgaga 2940
tatctaaaat ggtaataaaa tacaatgcaa ttatagcgat ggaggatttg tcttatggtt 3000
ttaaaaaagg gagatttaag gttgaaagac aagtttacca gaaatttgaa accatgttaa 3060
taaataaatt aaactattta gtatttaaag atatttcgat taccgagaat ggcggtttat 3120
taaaaggtta tcagttaaca tacattcctg ataaacttaa aaacgtgggt catcagtgcg 3180
gctgcatttt ttatgtgcct gctgcataca cgagcaaaat tgatccgacc accggctttg 3240
tgaatatatt taaatttaaa gacttaacag tggacgcaaa aagagaattc attaaaaaat 3300
ttgactcaat tagatatgac agtgaaaaaa atttattctg ctttacattt gactacaata 3360
actttattac gcaaaacacg gttatgagca aatcatcgtg gagtgtgtat acatacggcg 3420
tgagaataaa aagaagattt gtgaacggca gattctcaaa cgaaagtgat accattgaca 3480
taaccaaaga tatggagaaa acgttggaaa tgacggacat taactggaga gatggccacg 3540
atcttagaca agacattata gattatgaaa ttgttcagca catattcgaa attttcagat 3600
taacagtgca aatgagaaac tccttgtctg aattagagga cagagattac gatagattaa 3660
tttcacctgt attaaacgaa aataacattt tttatgacag cgcgaaagcg ggggatgcac 3720
ttcctaagga tgccgatgca aatggtgcgt attgtattgc attaaaaggg ttatatgaaa 3780
ttaaacaaat taccgaaaat tggaaagaag atggtaaatt ttcgagagat aaattaaaaa 3840
taagcaataa agattggttc gactttatac agaataagag atatttataa gtcgacaaag 3900
tattgttaaa aataactctg tagaattata aattagttct acagagttat tttttgaccc 3960
gggtatattg ataaaaataa taatagtggg tataattaag ttgttaggag gttagttaga 4020
atgatgtcaa gattagataa aagtaaagtg attaacagcg cattagagct gcttaatgag 4080
gtcggaatcg aaggtttaac aacccgtaaa ctcgcccaga agctaggtgt agagcagcct 4140
acattgtatt ggcatgtaaa aaataagcgg gctttgctcg acgccttagc cattgagatg 4200
ttagataggc accatactca cttttgccct ttagaagggg aaagctggca agatttttta 4260
cgtaataacg ctaaaagttt tagatgtgct ttactaagtc atcgcgatgg agcaaaagta 4320
catttaggta cacggcctac agaaaaacag tatgaaactc tcgaaaatca attagccttt 4380
ttatgccaac aaggtttttc actagagaat gcattatatg cactcagcgc tgtggggcat 4440
tttactttag gttgcgtatt ggaagatcaa gagcatcaag tcgctaaaga agaaagggaa 4500
acacctacta ctgatagtat gccgccatta ttacgacaag ctatcgaatt atttgatcac 4560
caaggtgcag agccagcctt cttattcggc cttgaattga tcatatgcgg attagaaaaa 4620
caacttaaat gtgaaagtgg gtcttaaaag cagcataacc tttttccgtg atggtaactt 4680
cacggtaacc aagatgtcga gttgagctcg aattcgtaat catggtcata gctgtttcct 4740
gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt 4800
aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc 4860
gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg 4920
agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg 4980
gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca 5040
gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac 5100
cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac 5160
aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg 5220
tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac 5280
ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat 5340
ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag 5400
cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac 5460
ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt 5520
gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaaggac agtatttggt 5580
atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc 5640
aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga 5700
aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac 5760
gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc 5820
cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct 5880
gacagttacc aggtccactg ccgggcctct tgcgggatca aaagaaaaac gaaatgatac 5940
accaatcagt gcaaaaaaag atataatggg agataagacg gttcgtgttc gtgctgactt 6000
gcaccatatc ataaaaatcg aaacagcaaa gaatggcgga aacgtaaaag aagttatgga 6060
aataagactt agaagcaaac ttaagagtgt gttgatagtg cagtatctta aaattttgta 6120
taataggaat tgaagttaaa ttagatgcta aaaatttgta attaagaagg agtgattaca 6180
tgaacaaaaa tataaaatat tctcaaaact ttttaacgag tgaaaaagta ctcaaccaaa 6240
taataaaaca attgaattta aaagaaaccg ataccgttta cgaaattgga acaggtaaag 6300
ggcatttaac gacgaaactg gctaaaataa gtaaacaggt aacgtctatt gaattagaca 6360
gtcatctatt caacttatcg tcagaaaaat taaaactgaa tactcgtgtc actttaattc 6420
accaagatat tctacagttt caattcccta acaaacagag gtataaaatt gttgggagta 6480
ttccttacca tttaagcaca caaattatta aaaaagtggt ttttgaaagc catgcgtctg 6540
acatctatct gattgttgaa gaaggattct acaagcgtac cttggatatt caccgaacac 6600
tagggttgct cttgcacact caagtctcga ttcagcaatt gcttaagctg ccagcggaat 6660
gctttcatcc taaaccaaaa gtaaacagtg tcttaataaa acttacccgc cataccacag 6720
atgttccaga taaatattgg aagctatata cgtactttgt ttcaaaatgg gtcaatcgag 6780
aatatcgtca actgtttact aaaaatcagt ttcatcaagc aatgaaacac gccaaagtaa 6840
acaatttaag taccgttact tatgagcaag tattgtctat ttttaatagt tatctattat 6900
ttaacgggag gaaataattc tatgagtccc taggcaggcc tccgccatta tttttttgaa 6960
caattgacaa ttcatttctt attttttatt aagtgatagt caaaaggcat aacagtgctg 7020
aatagaaaga aatttacaga aaagaaaatt atagaattta gtatgattaa ttatactcat 7080
ttatgaatgt ttaattgaat acaaaaaaaa atacttgtta tgtattcaat tacgggttaa 7140
aatatagaca agttgaaaaa tttaataaaa aaataagtcc tcagctctta tatattaagc 7200
taccaactta gtatataagc caaaacttaa atgtgctacc aacacatcaa gccgttagag 7260
aactctatct atagcaatat ttcaaatgta ccgacataca agagaaacat taactatata 7320
tattcaattt atgagattat cttaacagat ataaatgtaa attgcaataa gtaagattta 7380
gaagtttata gcctttgtgt attggaagca gtacgcaaag gcttttttat ttgataaaaa 7440
ttagaagtat atttattttt tcataattaa tttatgaaaa tgaaaggggg tgagcaaagt 7500
gacagaggaa agcagtatct tatcaaataa caaggtatta gcaatatcat tattgacttt 7560
agcagtaaac attatgactt ttatagtgct tgtagctaag tagtacgaaa gggggagctt 7620
taaaaagctc cttggaatac atagaattca taaattaatt tatgaaaaga agggcgtata 7680
tgaaaacttg taaaaattgc aaagagttta ttaaagatac tgaaatatgc aaaatacatt 7740
cgttgatgat tcatgataaa acagtagcaa cctattgcag taaatacaat gagtcaagat 7800
gtttacataa agggaaagtc caatgtatta attgttcaaa gatgaaccga tatggatggt 7860
gtgccataaa aatgagatgt tttacagagg aagaacagaa aaaagaacgt acatgcatta 7920
aatattatgc aaggagcttt aaaaaagctc atgtaaagaa gagtaaaaag aaaaaataat 7980
ttatttatta atttaatatt gagagtgccg acacagtatg cactaaaaaa tatatctgtg 8040
gtgtagtgag ccgatacaaa aggatagtca ctcgcatttt cataatacat cttatgttat 8100
gattatgtgt cggtgggact tcacgacgaa aacccacaat aaaaaaagag ttcggggtag 8160
ggttaagcat agttgaggca actaaacaat caagctagga tatgcagtag cagaccgtaa 8220
ggtcgttgtt taggtgtgtt gtaatacata cgctattaag atgtaaaaat acggatacca 8280
atgaagggaa aagtataatt tttggatgta gtttgtttgt tcatctatgg gcaaactacg 8340
tccaaagccg tttccaaatc tgctaaaaag tatatccttt ctaaaatcaa agtcaagtat 8400
gaaatcataa ataaagttta attttgaagt tattatgata ttatgttttt ctattaaaat 8460
aaattaagta tatagaatag tttaataata gtatatactt aatgtgataa gtgtctgaca 8520
gtgtcacaga aaggatgatt gttatggatt ataagcggc 8559

Claims (15)

1.一种核酸，其识别梭菌属(Clostridium)细菌的基因组内序列SEQ ID NO：18的catB基因或与其具有至少70％同一性的序列。
2.根据权利要求1所述的核酸，其特征在于所述核酸选自表达盒和载体，优选为质粒。
3.根据权利要求1或2所述的核酸，其特征在于所述核酸包含向导RNA(gRNA)和/或修饰模板。
4.根据权利要求1至3中的任一项所述的核酸，其特征在于所述梭菌属细菌是能够在野生型中产生异丙醇的细菌。
5.根据权利要求1至4中的任一项所述的核酸，其特征在于所述梭菌属细菌是拜氏梭菌(C.beijerinckii)细菌，所述细菌的进化分枝选自DSM 6423、LMG 7814、LMG 7815、NRRL B–593、NCCB 27006和与菌株DSM6423具有至少95％同一性的进化分枝。
6.根据权利要求2至5中的任一项所述的核酸，其特征在于它是序列SEQ ID NO：21的质粒pCas9ind–ΔcatB或序列SEQ ID NO：38的质粒pCas9ind–gRNA_catB。
7.根据权利要求2至6中的任一项所述的核酸的用途，其用于转化和/或遗传修饰能够在野生型中产生异丙醇的梭菌属细菌。
8.一种识别拜氏梭菌DSM 6423的基因组内序列SEQ ID NO：18的catB基因或与其具有至少70％同一性的序列的核酸的用途，其用于转化和/或遗传修饰拜氏梭菌DSM 6423细菌。
9.根据权利要求1至6中的任一项所述的核酸的用途，所述核酸在被Dam–和Dcm–型甲基转移酶识别的基序处不表现出甲基化，所述核酸用于转化选自DSM 6423、LMG 7814、LMG7815、NRRL B–593、NCCB 27006和与菌株DSM 6423表现出至少95％、优选地97％的同一性的进化分枝的拜氏梭菌进化分枝。
10.一种利用遗传修饰工具转化并优选地遗传修饰梭菌属细菌的方法，其特征在于所述方法包括通过在所述细菌中引入根据权利要求1至6中的任一项所述的核酸来转化所述细菌的步骤。
11.根据权利要求10所述的方法，其特征在于使用负责切开编码或控制酰胺醇–O–乙酰转移酶转录的靶序列的至少一条链的酶，用CRISPR工具转化所述细菌。
12.一种遗传修饰的梭菌属细菌，其通过根据权利要求10或11所述的方法获得。
13.一种在编号LMG P–31151下保藏的拜氏梭菌DSM6423ΔcatB细菌。
14.根据权利要求12所述的遗传修饰的细菌或根据权利要求13所述的在编号LMG P–31151下保藏的拜氏梭菌DSM6423ΔcatB细菌的用途，其用于优选地在工业规模上生产溶剂、优选为异丙醇，或溶剂的混合物。
15.一种试剂盒，其包含(i)根据权利要求2至6中的任一项所述的核酸和(ii)至少一种工具，所述工具选自遗传修饰工具的元件、作为gRNA的核酸、作为修复模板的核酸、至少一个引物对和允许由所述工具编码的蛋白质表达的诱导物。
CN201980088931.2A 2018-12-20 2019-12-20 遗传修饰的梭菌属细菌、其制备和用途 Pending CN113614229A (zh)

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