CN117255855A

CN117255855A - 来自宏基因组的新颖的CRISPR-Cas核酸酶

Info

Publication number: CN117255855A
Application number: CN202280018475.6A
Authority: CN
Inventors: P·肖尔茨; C·苏瑞克; R·贝克
Original assignee: Brain Biotechnology Co ltd
Current assignee: Brain Biotechnology Co ltd
Priority date: 2021-03-02
Filing date: 2022-03-02
Publication date: 2023-12-19
Also published as: IL305545A; WO2022184765A1; JP2024509139A; KR20230156365A; AU2022228662A1; CA3210899A1; EP4301852A1

Abstract

本发明涉及编码RNA引导的DNA核酸内切酶的核酸分子，其是(a)编码包含SEQ ID NO:9、1至5、7、8和10至15中任一项的氨基酸序列或由SEQ ID NO:9、1至5、7、8和10至15中任一项的氨基酸序列组成的RNA引导的DNA核酸内切酶的核酸分子；(b)包含SEQ ID NO:24、16至20、22、23和25至30中任一项的核苷酸序列或由SEQ ID NO:24、16至20、22、23和25至30中任一项的核苷酸序列组成的核酸分子；(c)编码氨基酸序列与(a)的氨基酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的RNA引导的DNA核酸内切酶的核酸分子；(d)包含与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列或由与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列组成的核酸分子；(e)相对于(d)的核酸分子简并的核酸分子；(f)对应于(a)至(d)中任一项的核酸分子的核酸分子，其中T被U替代。

Description

来自宏基因组的新颖的CRISPR-Cas核酸酶

技术领域

本发明涉及编码RNA引导的DNA核酸内切酶(RNA-guided DNA endonuclease)的核酸分子，其是(a)编码包含SEQ ID NO:9、1至5、7、8和10至15中任一项的氨基酸序列或由SEQID NO:9、1至5、7、8和10至15中任一项的氨基酸序列组成的RNA引导的DNA核酸内切酶的核酸分子；(b)包含SEQ ID NO:24、16至20、22、23和25至30中任一项的核苷酸序列或由SEQ IDNO:24、16至20、22、23和25至30中任一项的核苷酸序列组成的核酸分子；(c)编码氨基酸序列与(a)的氨基酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的RNA引导的DNA核酸内切酶的核酸分子；(d)包含与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列或由与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列组成的核酸分子；(e)相对于(d)的核酸分子简并的核酸分子；或者(f)对应于(a)至(d)中任一项的核酸分子的核酸分子，其中T被U替代。

背景技术

在本说明书中，引用了包括专利申请和制造商手册的多个文献。这些文献的公开虽然被认为与本发明的可专利性无关，但通过引用以其整体并入本文。更具体地，所有引用的文献被引用并入的程度与每个单独的文献被具体地和单独地指明被引用并入一样。

CRISPR-Cas系统是原核生物针对侵入的外来核酸的广泛适应性免疫系统。到目前为止，已经鉴别了超过30种不同的CRISPR-Cas系统，它们的基因座结构、数量和编码Cas(CRISPR相关)蛋白的基因的特性(identity)各不相同。

CRISPR系统在原核基因组中的典型特征是存在短的(30-45bp)重复序列(重复段，repeats)，这些序列插入有相似长度的可变序列(间隔子(spacer))。Cas蛋白位于重复段-间隔子簇的上游或下游。根据它们的基因组成和机制差异，这些亚型分为两个CRISPR类(1类和2类)。它们的主要区别之一是1类CRISPR系统需要多个Cas蛋白的复合物来降解DNA，而2类Cas蛋白是单独的大的多结构域核酸酶。2类Cas蛋白的序列特异性可以通过合成的CRISPR RNA(crRNA)简单地进行修饰，以引入靶向的双链DNA断裂。此类2类Cas蛋白的最著名成员是Cas9、Cpf1(Cas12a)和Cms1，它们被用于基因组编辑，并被成功应用于许多真核生物，包括真菌、植物和哺乳动物细胞。Cas9及其同源物是2类II型CRISPR核酸酶，而Cpf1(WO2016/205711BROAD Inst.；WO2017/141173Benson Hill)和Cms1(WO2019/030695BensonHill)属于2类V型核酸酶。Cms1和Cpf1 CRISPR核酸酶是一类与其它CRISPR核酸酶(例如II型核酸酶)相比具有某些理想特性的CRISPR核酸酶。例如，与Cas9核酸酶相比，Cms1和Cpf1不需要反式激活crRNA(trans-activating crRNA，tracrRNA)，它与前体crRNA(pre-crRNA)部分互补(Deltcheva等人(2011),Nature,471(7340):602-607)。tracrRNA和pre-crRNA的碱基配对形成Cas9结合的RNA：RNA双链体，由RNase III和其它未鉴别的核酸酶处理。这种成熟的tracrRNA：crRNA双链体介导Cas9对靶DNA的识别和切割。相反，V型核酸酶无需tracrRNA或细胞核酸酶(如RNase III)即可处理pre-crRNA，这显著简化了V型核酸酶用于(多重)基因组编辑的应用。

已在培养的细菌的基因组或公共可用的宏基因组数据集(例如肠道宏基因组)中鉴别出多种新的2类蛋白，如C2c1(Cas12b)、C2c2(Cas13a)和C2c3(Cas12c)(Shmakov等人(2015),Mol Cell,60(3):385-97)。根据最近的CRISPR-Cas系统分类，2类包含3个型和17个亚型(Makarova等人(2020),Nat Rev Microbiol,18(2):67-83)。

此外，在最近发表的一篇文章中，通过宏基因组测序在未培养的原核生物中发现了两种新的2类蛋白(CasX(Cas12a)和CasY(Cas12d))(Burstein等人(2017),Nature,542:237-241)，表明尚未培养和/或鉴别的生物体中存在未开发的Cas蛋白。

因此，发现新颖的2类Cas蛋白的一种有前途的方法是通过对选择的环境DNA(例如1cm³森林土壤含有～2.5×10¹⁰bp DNA或～2000万个基因)下一代测序(next generationsequencing)来获取宏基因组资源，并计算鉴别CRISPR-Cas系统(例如，Lei和Sun(2016)，Bioinformatics,32(17):i520-i528)。

如所讨论的，已知的CRISPR-Cas系统在其作用模式上表现出某些差异。这些分子差异不仅扩展了在广泛的不同遗传背景下使用CRISPR-Cas系统用于基因组编辑的可能性，而且在应用于某些生物体时规避了特定Cas核酸酶的问题，例如在人中预先存在的对Cas9的免疫响应(Charlesworth等人(2019),Nat Med,25(2):249-254)。因此，从与高等真核生物较少直接接触的细菌物种中鉴别Cas核酸酶尤为重要。可以假设存在多种具有未知特征的、未发现的CRISPR-Cas系统，尤其是在宏基因组资源中。因此，尽管从现有技术中已知几种不同的CRISPR-Cas系统，但仍然需要鉴别进一步的CRISPR-Cas系统，尤其是这些系统的RNA引导的DNA核酸内切酶。本发明解决了这种需要。

发明内容

相应地，本发明在第一方面涉及编码RNA引导的DNA核酸内切酶的核酸分子，其是(a)编码包含SEQ ID NO:1-15中任一项、优选SEQ ID NO:9、1至5、7、8和10至15中任一项或SEQ ID NO:6的氨基酸序列或由SEQ ID NO:1-15中任一项、优选SEQ ID NO:9、1至5、7、8和10至15中任一项或SEQ ID NO:6的氨基酸序列组成的RNA引导的DNA核酸内切酶的核酸分子；(b)包含SEQ ID NO:16-30中任一项、优选SEQ ID NO:9、1至5、7、8和10至15中任一项或SEQ ID NO:22的核苷酸序列或由SEQ ID NO:16-30中任一项、优选SEQ ID NO:9、1至5、7、8和10至15中任一项或SEQ ID NO:22的核苷酸序列组成的核酸分子；(c)编码氨基酸序列与(a)的氨基酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的RNA引导的DNA核酸内切酶的核酸分子；(d)包含与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列或由与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列组成的核酸分子；(e)相对于(d)的核酸分子简并的核酸分子；或者(f)对应于(a)至(d)中任一项的核酸分子的核酸分子，其中T被U替代。

SEQ ID NO:1-15分别为新颖的CRISPR-Cas核酸内切酶BMC01-BMC15的氨基酸序列，其中BMC是BRAIN Metagenome Cas的缩写。新颖的CRISPR-Cas核酸内切酶BMC01-BMC15分别由SEQ ID NO:16-30的核苷酸序列编码。BMC01-BMC15的氨基酸序列的无根树(unrooted tree)如本申请的图1所示。可以看出，BMC01至BMC05和BMC07至BMC15彼此之间，比与本申请知晓的任何现有技术CRISPR-Cas核酸内切酶之间更密切相关，并且比与BMC06之间更密切相关。为此理由，SEQ ID NO:1-15优选为SEQ ID NO:9、1至5、7、8和10至15或为SEQ ID NO:6，以及SEQ ID NO:16至30优选为SEQ ID NO:24、16至20、22、23和25至30或为SEQ ID NO:22。在SEQ ID NO:9、1至5、7、8和10至15中，首先提到SEQ ID NO:9，因为相应的新颖的CRISPR-Cas核酸内切酶BMC09在所附实施例中经过了最广泛的测试。为此理由，最优选氨基酸序列SEQ ID NO:9以及相应的核苷酸序列SEQ ID NO:24。

根据本发明，术语“核酸分子”定义为核苷酸的线性分子链。根据本发明的核酸分子由至少2327个核苷酸组成。在本文中称为“核酸分子”的分子组也包含完整基因。术语“核酸分子”在本文中可与术语“多核苷酸”互换使用。

根据本发明的术语“核酸分子”包括DNA，例如cDNA或者双链或单链基因组DNA和RNA。在这方面，“DNA”(脱氧核糖核酸)是指称为核苷酸碱基的化学结构单元(chemicalbuilding block)腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)和胸腺嘧啶(T)在脱氧核糖主链上连接在一起的任何链或序列。DNA可以有一条核苷酸碱基链，或两条可以形成双螺旋结构的互补链。“RNA”(核糖核酸)是指称为核苷酸碱基的化学结构单元腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)和尿嘧啶(U)在核糖主链上连接在一起的任何链或序列。RNA典型地具有一条核苷酸碱基链。还包括的是单链和双链杂交分子，即DNA-DNA、DNA-RNA和RNA-RNA。核酸分子也可以通过本领域已知的许多手段进行修饰。此类修饰的非限制性实例包括甲基化、“加帽”、用类似物取代一个或多个天然存在的核苷酸，以及核苷酸间修饰，例如具有不带电荷的连接(linkage)(例如甲基膦酸酯、磷酸三酯、氨基磷酸酯、氨基甲酸酯等)和带电荷的连接(例如，硫代磷酸酯、二硫代磷酸酯等)的修饰。多核苷酸可含有一个或多个额外的共价连接部分，例如蛋白质(例如核酸酶、毒素、抗体、信号肽、聚-L-赖氨酸等)、嵌入剂(例如吖啶、补骨脂素等)、螯合剂(例如金属、放射性金属、铁、氧化性金属等)和烷化剂等。多核苷酸可以通过形成甲基或乙基磷酸三酯或烷基磷酸氨基酯键(alkyl phosphorarnidate linkage)来衍生化。进一步包括的是本领域已知的核酸模拟分子，例如DNA或RNA的合成或半合成衍生物和混合的聚合物。根据本发明的此类核酸模拟分子或核酸衍生物包括硫代磷酸酯核酸、氨基磷酸酯核酸、2’-O-甲氧基乙基核糖核酸、吗啉代核酸、己糖醇核酸(HNA)、肽核酸(PNA)和锁核酸(LNA)(参见Braasch和Corey，Chem Biol2001,8:1)。LNA是一种RNA衍生物，其中核糖环受2’-氧和4’-碳之间的亚甲基键限制。还包括的是含有修饰的碱基的核酸，例如硫代尿嘧啶、硫代鸟嘌呤和氟尿嘧啶。核酸分子典型地携带遗传信息，包括通过细胞机构(cellular machinery)用来制造蛋白质和/或多肽的信息。本发明的核酸分子可以额外包含启动子、增强子、响应元件、信号序列、聚腺苷酸序列、内含子、5’-和3’-非编码区等。

本文中与术语“蛋白质”互换使用的术语“多肽”描述氨基酸的线性分子链，包括单链蛋白质或它们的片段。根据本发明的多肽/蛋白质含有至少775个氨基酸。多肽可进一步形成由至少两个相同或不同分子组成的寡聚体。此类多聚体的相应高级结构相应地称为同源或异源二聚体、同源或异源三聚体等。本发明的多肽可以形成异源多聚体或同源多聚体，例如异源二聚体或同源二聚体。另外，其中一个(或多个)氨基酸和/或一个(或多个)肽键已被功能类似物替代的此类蛋白质/多肽的拟肽也涵盖在本发明中。此类功能类似物包括除20种基因编码氨基酸以外的所有已知氨基酸，例如硒代半胱氨酸。术语“多肽”和“蛋白质”也指天然修饰的多肽和蛋白质，其中修饰受到例如糖基化、乙酰化、磷酸化、泛素化和本领域熟知的类似修饰的影响。

术语“RNA引导的DNA核酸内切酶”或“CRISPR(-Cas)核酸内切酶”描述了一种酶，其具有切割脱氧核糖核苷酸(DNA)链中的磷酸二酯键从而产生双链断裂(DSB)的能力。BMC01-15被归类为新颖的V型2类CRISPR核酸酶，已知其引入带有5’突出的交错切口。因此，RNA引导的DNA核酸内切酶包含核酸内切酶结构域，特别是RuvC结构域。BMC01-15的RuvC结构域被认为包含三个分割型(split)RuvC基序。RNA引导的DNA核酸内切酶也包含能够与crRNA结合的结构域，crRNA也称为向导RNA(guide RNA,gRNA；在本文中也称为靶向DNA的RNA)。

RNA引导的DNA核酸内切酶的切割位点由向导RNA引导。gRNA赋予RNA引导的DNA核酸内切酶以靶序列特异性。此类gRNA是非编码短RNA序列，其与互补的靶DNA序列结合。gRNA首先通过可与RNA引导的DNA核酸内切酶相互作用的结合结构域与RNA引导的DNA核酸内切酶结合。可与RNA引导的DNA核酸内切酶相互作用的结合结构域典型地包含具有茎环结构的区域。该茎环优选包含序列NATTTCTACTNTTGTAGAT(SEQ ID NO:31)，其中，N表示在该位置可以出现任何碱基。茎环最优选分别包含BMC01-BMC15的茎环正向重复序列(stem loopdirect repeat sequence)(SEQ ID NO:32)，但以RNA的形式(即，其中T被U代替)。gRNA序列通过配对到DNA链上的特定位置引导复合物(称为gRNA和RNA引导的DNA核酸内切酶的CRISPR核糖核蛋白(RNP)复合物)，其中RNA引导的DNA核酸内切酶通过切割靶位点的DNA链来执行其核酸内切酶活性。gRNA的基因组靶位点可以是任何约20个(典型地是17至26个)核苷酸的DNA序列，前提是其满足两个条件：(i)该序列与基因组的其余部分相比是唯一的，并且(ii)该靶紧邻前间隔子邻近基序(Protospacer Adjacent Motif，PAM)。

因此，RNA引导的DNA核酸内切酶的切割位点进一步由PAM限定。PAM是短的DNA序列(通常长度为2-6个碱基对)，其位于用于由CRISPR系统切割的靶向的DNA区域之后。确切的序列取决于使用哪种CRISPR核酸内切酶。CRISPR核酸内切酶及其各自的PAM序列是本领域已知的(参见https://www.addgene.org/crispr/guide/#pam-table)。例如，由第一个鉴别出的RNA引导的DNA核酸内切酶Cas9识别的PAM是5’-NGG-3’(其中“N”可以是任何核苷酸碱基)。PAM是RNA引导的DNA核酸内切酶切割所必需的。在Cas9中，发现在由向导RNA靶向的DNA序列的下游约2-6个核苷酸和自切割位点的下游约3-6个核苷酸。在V型系统(包括BMC01-BMC15)中，PAM位于靶序列和切割位点这两者的上游。RNA引导的DNA核酸内切酶和向导RNA的复合物包含所谓的PAM相互作用结构域(Andres等人(2014),Nature,513(7519):569-573)。因此，可被靶向的、用于由RNA引导的DNA核酸内切酶编辑的基因组位置受限于核酸酶特异性PAM序列的存在和位置。由于BMC01-BMC15属于V型2类CRISPR核酸酶的组，预测了富含T的PAM位点(TTTN，其中N可以是A、T、C和G中的任何核苷酸)。

术语“序列同一性百分比(％)”描述了与构成模板核酸或氨基酸序列总长度的核苷酸或氨基酸残基的数量相比，两个或更多个比对的核酸或氨基酸序列的相同核苷酸/氨基酸的匹配数(“命中(hits)”)。换言之，使用比对，对于两个或更多个序列或子序列，当在比较窗口上、或在使用本领域已知的序列比较算法测量的指定区域上、或在手动比对和目视检查时，在比较和比对(子)序列以获得最大对应性时，可以确定相同的氨基酸残基或核苷酸的百分比(例如，70％同一性)。该定义也适用于任何待比对序列的互补序列。

与本发明相关的氨基酸序列以及核苷酸序列分析和比对优选使用NCBI BLAST算法进行(Stephen F.Altschul、Thomas L.Madden、Alejandro A.Jinghui Zhang、Zheng Zhang、Webb Miller和David J.Lipman(1997)，“Gapped BLAST and PSI-BLAST:anew generation of protein database search programs”，Nucleic Acids Res.25:3389-3402)。技术人员知晓用于比对核酸序列的其它合适的程序。

如上文所定义，本发明预期了至少70％、至少80％、至少90％和至少95％的氨基酸序列和核苷酸序列同一性。此外，本发明预期了至少99.5％、至少99.8％和99.9％同一性的增加偏好的氨基酸序列同一性。

关于这些氨基酸序列和由这些核苷酸序列编码的氨基酸序列，优选它们维持或基本上维持本发明的SEQ ID NO:1-16的RNA引导的DNA核酸内切酶的活性。因此，维持或基本上维持的是与gRNA结合以形成能够与感兴趣的DNA靶位点结合的复合物的能力，其中核酸内切酶活性诱导DSB。

术语“简并”表示遗传密码的简并性。众所周知，编码一个氨基酸的密码子可能在其三个位置中的任何一个有差异；但是，这种差异往往排在第二或第三位。例如，氨基酸谷氨酸由GAA和GAG密码子指定(第三位的差异)；氨基酸亮氨酸由UUA、UUG、CUU、CUC、CUA、CUG密码子指定(第一位或第三位的差异)；氨基酸丝氨酸由UCA、UCG、UCC、UCU、AGU、AGC指定(第一位、第二位或第三位的差异)。

正如V型CRISPR核酸内切酶Cms1和Cpf1一样，BMC01-BMC15不需要反式激活crRNA(tracrRNA)。此外，本申请中鉴别的含有CRISPR系统的BMC01-BMC15含有在各自重复段的3’端有RNA茎环的CRISPR重复序列，该重复段在Cpf1和Cms1蛋白家族的crRNA中是保守的，并且在已知的CRISPR-Cas核酸内切酶中，BMC01-BMC15的“最近邻”是Cpf1多肽的成员。为此理由，BMC01-BMC15可归类为新颖的2类V型核酸酶，其与1类和2类CRISPR-Cas核酸内切酶的已知集合总体上没有显著序列同一性，并且相对于单独的Cpf1型核酸内切酶，总体上序列同一性低。

由于BMC01-BMC15是新颖的CRISPR-Cas核酸内切酶，其与CRISPR-Cas核酸内切酶的已知集合明显不同，BMC01-BMC15扩展了适用于不同生物技术和制药领域的基因组编辑、基因调控和核酸富集/纯化的CRISPR-Cas核酸内切酶的已知集合，从而有利地扩展了分子基因组编辑的选择。

BMC01-BMC15至少在两个方面与已知CRISPR-Cas核酸内切酶的已知集合明显不同。

首先，它们在结构上是不同的。例如，WO 2021/154866中的SEQ ID NO:20仅与CMS09(SEQ ID NO:9)具有57.1％的序列相似性，与来自Candidatus Roizmanbacteria细菌的假设蛋白US54 C0016G0015具有55.26％的序列同一性。BMC核酸内切酶在结构上也与已知的CRISPR-Cas核酸内切酶明显不同。BMC核酸内切酶是通过如实施例1中所示的创新筛选方法发现的，其中通过下一代测序获取万亿碱基级(terabase scale)的宏基因组资源，并且富集了含有几乎未触及的新颖的序列空间的候选门级辐射类群(Candidata PhylaRadiation,CPR)物种。令人惊讶的是，尽管与已知的CRISPR-Cas核酸内切酶相比，其序列在结构上明显不同，但BMC01-BMC15仍然非常适合基因组编辑，这由实施例3至5证明。

其次，BMC01-BMC15是从环境样本中分离出来的，尤其是从水生、沉积物和土壤生活环境(habitats)中分离出来的。另一方面，大多数已知的CRISPR-Cas核酸内切酶是从病原细菌中分离出来的。例如，Cas9在酿脓链球菌(Streptococcus pyogenes)M1中发现，Cas12a(Cpf1)来自普雷沃菌属(Prevotella)和弗朗西斯氏菌属1(Francisella 1)，Cas13a(C2c2)来自沙希纤毛菌(Leptotrichia shahii)。为此理由，当应用于哺乳动物、优选人、尤其是用于治疗应用的基因组编辑时，BMC01-BMC15的免疫原性将低于大多数已知的CRISPR-Cas核酸内切酶。

根据本发明的第一方面的优选实施方案，核酸分子可操作地连接至启动子，该启动子对于核酸分子是天然的或异源的。

启动子是导致特定基因转录起始的DNA区域。启动子通常位于基因转录起始位点附近、DNA上游(朝向有义链的5’区域)。启动子典型地为100～1000个碱基对长。为了进行转录，合成RNA的酶(称为RNA聚合酶)必须附着在基因附近的DNA上。启动子包含特定的DNA序列，例如为RNA聚合酶和招募RNA聚合酶的被称为转录因子的蛋白质提供安全初始结合位点的响应元件。因此，RNA聚合酶和转录因子与启动子位点的结合确保了基因的转录。

就此而言，术语“可操作地连接”定义了启动子与相同DNA链的基因连接，使得在RNA聚合酶和转录因子结合后启动基因的转录。通常每个基因在其活生物体基因组的自然环境中可操作地连接至启动子。该启动子在本文中称为“天然启动子”或“野生型启动子”。异源启动子不同于天然启动子或野生型启动子。因此，可操作地连接至与核酸分子异源的启动子的核酸分子在自然界中不存在。

可用于表达所期望的基因的异源启动子是本领域已知的并且可以例如从EPD(eukaryotic promoter database，真核启动子数据库)或EDPnew(https://epd.epfl.ch//index.php)获得。在该数据库中可以找到包括动物、植物和酵母启动子的真核启动子。

例如，启动子可以是组成型活性的、可诱导的、组织特异性的或发育阶段特异性的启动子。通过使用此类启动子，可以调节所期望的表达时间和位点。

酵母中的AOX1或GAL1启动子或CMV-(巨细胞病毒)、SV40-、RSV-启动子(劳斯肉瘤病毒)、鸡β-肌动蛋白启动子、CAG-启动子(鸡β-肌动蛋白启动子和巨细胞病毒立即早期增强子的组合)、gai10启动子、人延伸因子1α启动子、CaM-激酶启动子和苜蓿银纹夜蛾多核型多角体病毒(AcMNPV)多角体启动子是组成型活性启动子的实例。

诱导型启动子的实例是可由缺氧或冷应激诱导的Adhl启动子、可由热应激诱导的Hsp70启动子、均可由光诱导的PPDK启动子和pep羧化酶(pepcarboxylase)启动子。化学诱导型启动子也是有用的，例如安全剂(safener)诱导的In2-2启动子(US5,364,780)、雌激素诱导的ERE启动子、以及生长素诱导的且绒毡层(tapetum)特异性但也在愈伤组织中有活性的Axigl启动子(WO03060123 A1)。

组织特异性启动子是仅在特定组织中起始转录的启动子。发育阶段特异性启动子是仅在特定发育阶段起始转录的启动子。

根据本发明第一方面的进一步优选实施方案，核酸分子连接至编码核定位信号(NLS)的核酸序列。

关于NLS的进一步细节将在下文中提供。

根据本发明第一方面的另一个优选实施方案，所述核酸分子针对在真核细胞，优选植物细胞或动物细胞中的表达进行了密码子优化。

如前所述，BMC01-BMC15是从细菌宏基因组样本中分离的。因此，如果BMC01-BMC15在真核细胞中表达，它们在异源宿主细胞或生物体中表达。

分别编码BMC01-BMC15多肽的基因可以针对在靶细胞中的表达进行了密码子优化，并且可以任选地包括编码NLS和/或肽标签(例如纯化标签)的序列。关于标签的进一步细节将在下文中提供。

密码子优化是用于通过适应宿主细胞的密码子偏倚来改善基因表达和提高感兴趣基因的翻译效率的过程。因此，“密码子优化的基因”是其密码子使用频率被设计为模仿宿主细胞的优选密码子使用频率的基因。核酸分子可以全部或部分密码子优化。由于任何一种氨基酸(甲硫氨酸和色氨酸除外)均由多种密码子编码，因此可以在不改变编码氨基酸的情况下改变核酸分子的序列。密码子优化是当一个或多个密码子在核酸水平上发生改变时，使得氨基酸不变但在特定宿主生物体中的表达增加。本领域普通技术人员将认识到密码子表和提供广泛生物体偏好信息的其它参考文献在本领域是可获得的(参见，例如，Zhang等人(1991)Gene 105:61-72；Murray等人(1989)Nucl.Acids Res.17:477-508)。用于优化针对表达的核苷酸序列的方法在例如美国专利第6,015,891号中提供。用于密码子优化的程序在本领域中是可获得的(例如，在genomes.urv.es/OPTIMIZER的OPTIMIZER；来自在www.genscript.com/codon_opt.html的GenScript的OptimumGene.TM.)。

真核细胞优选是中国仓鼠卵巢(CHO)细胞，因此密码子优化优选是针对CHO细胞中的表达的优化。CHO细胞具有特别的商业利益，因为它们是重组蛋白治疗剂工业生产中常用的哺乳动物宿主。

下文将提供关于合适的真核细胞(包括植物和动物细胞)的进一步细节。

本发明在第二方面涉及编码第一方面的核酸分子的载体。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第二方面。

根据本发明的载体通常并且优选地能够指导本发明的核酸分子的复制和/或表达和/或由其编码的多肽的表达。

优选地，载体是质粒、粘粒、病毒、噬菌体或例如在基因工程中常规使用的另一种载体。

示例性质粒和载体列于例如Studier和同事(Studier，W.F.；Rosenberg A.H.；Dunn J.J.；Dubendroff J.W.,1990,Use of the T7 RNA polymerase to directexpression of cloned genes,Methods Enzymol.185、61-89)或Novagen、Promega、NewEngland Biolabs、Clontech和Gibco BRL公司提供的手册。其它优选的质粒和载体可见于：Glover,D.M.,1985,DNA cloning:a practical approach,Vol.I-III，IRL Press Ltd.,Oxford；Rodriguez,R.L.和Denhardt,D.T.(eds),1988,Vectors:a survey of molecularcloning vectors and their uses,179-204,Butterworth,Stoneham；Goedeel,D.V.,1990,Systems for heterologous gene expression,Methods Enzymol.185,3-7；Sambrook,J.；Russell,D.W.,2001,Molecular cloning:a laboratory manual,第3版,Cold Spring Harbor Laboratory Press,New York。

特别优选的载体是可用于CRISPR基因组编辑的载体，特别是仅表达编码RNA引导的DNA核酸内切酶的本发明核酸分子的载体或表达编码RNA引导的DNA内切酶的本发明核酸分子和向导RNA二者的载体(所谓的“一体式载体”)。DNA核酸内切酶和。在前一种情况下，第二载体被用于向导RNA的表达。CRISPR基因组编辑载体是可商购的，例如，从OriGene、Vector Builder或ThermoFisher。

上述本发明的核酸分子也可以插入到载体中，从而生成与另一核酸分子的翻译融合物。为此目的，可以应用重叠延伸PCR(例如Wurch,T.,Lestienne,F.和Pauwels,P.J.,Amodified overlap extension PCR method to create chimeric genes in the absenceof restriction enzymes,Biotechn.Techn.12,9,Sept.1998,653-657)。由此产生的产物称为融合蛋白，将在下文进一步描述。其它核酸分子可以编码如下的蛋白质，其可以例如增加溶解度和/或促进由本发明的核酸分子编码的蛋白质的纯化。非限制性实例包括pET32、pET41、pET43。载体还可以包含额外的可表达核酸，其编码一种或多种伴侣蛋白以促进正确的蛋白质折叠。合适的细菌表达宿主包括例如源自BL21(例如BL21(DE3)、BL21(DE3)PlysS、BL21(DE3)RIL、BL21(DE3)PRARE)或的菌株。

有关载体修饰技术，参见J.F.Sambrook和D.W.Russell,编,Cold Spring HarborLaboratory Press,2001,ISBN-10 0-87969-577-3。通常，载体可含有一个或多个复制起点(ori)和用于克隆或表达的遗传系统、一个或多个用于在宿主中选择的标记(例如抗生素抗性)、以及一个或多个表达盒。合适的复制起点包括，例如，Col E1、SV40病毒和M13复制起点。

插入载体中的编码序列可以是例如通过标准方法合成的，或从天然来源分离的。编码序列与转录调控元件和/或其它氨基酸编码序列的连接可以使用已建立的方法进行。确保在原核生物或真核细胞中表达的转录调控元件(表达盒的部分)是本领域技术人员所熟知的。这些元件包含确保转录起始的调控序列(例如，翻译起始密码子、转录终止序列、启动子、增强子和/或绝缘子)、内部核糖体进入位点(IRES)(Owens等人，(2001)，PNAS.98(4)1471-1476)和确保转录的终止和转录物稳定的任选的poly-A信号。额外的调控元件可包括转录和翻译增强子，和/或天然相关的或异源的启动子区域。调控元件可以是对于本发明核酸内切酶为天然的或者为异源调控元件。优选地，本发明的核酸分子可操作地连接至允许在原核生物或真核细胞中表达的此类表达控制序列。该载体还可以包含编码分泌信号的核苷酸序列作为进一步的调控元件。这样的序列是本领域技术人员所熟知的。此外，根据所使用的表达系统，可以将能够将表达的多肽引导至细胞区室的前导序列添加到本发明的核酸分子的编码序列。这样的前导序列是本领域所熟知的。专门设计的载体允许DNA在不同宿主例如细菌-真菌细胞或细菌-动物细胞之间穿梭。

此外，杆状病毒系统或基于牛痘病毒或塞姆利基森林病毒的系统可用作本发明核酸分子在真核表达系统中的载体。源自病毒如逆转录病毒、牛痘病毒、腺相关病毒、疱疹病毒或牛乳头瘤病毒的表达载体可用于将核酸或载体递送到靶细胞群中。本领域技术人员熟知的方法可用于构建重组病毒载体；参见，例如，在Sambrook和D.W.Russell编,ColdSpring Harbor Laboratory Press,2001中描述的技术。

允许在真核宿主细胞中表达的调控元件的实例是启动子，包括如上文所述的启动子。除了负责转录起始的元件外，此类调控元件还可包含转录终止信号，例如核酸下游的SV40-poly-A位点或tk-poly-A位点或SV40、lacZ和AcMNPV多角体多聚腺苷酸化信号。

与用于在大肠杆菌(E.coli)和其它细菌中培养的选择性标记物例如卡那霉素或氨苄青霉素抗性基因的共转染允许转染细胞的鉴别和分离。哺乳动物细胞培养的选择性标记物是dhfr、gpt、新霉素、潮霉素抗性基因。也可以扩增转染的核酸以表达大量编码的(多)肽。DHFR(二氢叶酸还原酶)标记物可用于开发携带数百甚至数千拷贝的目的基因的细胞系。另一种有用的选择性标记物是谷氨酰胺合酶(GS)。使用此类标记物，细胞在选择性培养基中生长，并选择具有最高抗性的细胞。

然而，如上文所述的本发明的核酸分子也可以设计用于直接引入或用于通过脂质体、噬菌体载体或病毒载体(例如腺病毒或逆转录病毒)引入细胞。

本发明在第三方面涉及包含第一方面的核酸分子或转化、转导或转染有第二方面的载体的宿主细胞。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第三方面。

所述宿主细胞可产生大量的RNA引导的DNA核酸内切酶，其中编码RNA引导的DNA核酸内切酶的分离的核苷酸序列在插入宿主之前插入合适的载体或表达载体。将载体或表达载体引入合适的宿主细胞中，该宿主细胞优选可以大量生长，并且从宿主细胞或培养基中纯化RNA引导的DNA核酸内切酶。

宿主细胞也可用于提供本发明的RNA引导的DNA核酸内切酶，而不需要纯化RNA引导的DNA核酸内切酶(参见Yuan,Y.；Wang,S.；Song,Z.；和Gao,R.,Immobilization of anL-aminoacylase-producing strain of Aspergillus oryzae into gelatin pelletsand its application in the resolution of D,L-methionine,BiotechnolAppl.Biochem.(2002).35:107-113)。本发明的RNA引导的DNA核酸内切酶可以由宿主细胞分泌。分子生物学领域的技术人员将理解可以使用多种表达系统中的任意者来提供RNA引导的DNA核酸内切酶。所使用的精确宿主细胞对本发明并不重要，只要宿主细胞在合适的生长条件下生长时产生RNA引导的DNA核酸内切酶即可。

含有本发明核酸分子的载体可以被克隆到其中的宿主细胞用于复制和分离足量的重组酶。用于此目的的方法是技术人员所熟知的(Sambrook和D.W.Russell,编,ColdSpring Harbor Laboratory Press,2001)。

RNA引导的DNA核酸内切酶的表达不仅可用于在宿主细胞中产生RNA引导的DNA核酸内切酶，而且其表达还可用于编辑宿主细胞的基因组。在这种情况下，宿主细胞还包含向导RNA。已在上文讨论了可用于CRISPR基因组编辑的载体。

根据本发明的第三方面的优选方面，宿主细胞是真核细胞或原核细胞，并且优选是植物细胞或动物细胞。

宿主细胞可以是不天然包括编码BMC01-BMC15中任一项的基因的真核细胞，可以是例如是真菌、藻类、植物或动物的细胞，其中动物可以是鸟类、爬行类、两栖类、鱼类、头足类、甲壳类、昆虫类、蛛形类、有袋类或哺乳类。编码相对于宿主细胞为非天然的BMC01-BMC15中任一项的基因可以可操作地连接至调控元件，例如启动子。启动子可以是宿主生物的天然启动子，也可以是另一物种的启动子。用于在异源宿主细胞如真核细胞中表达BMC01-BMC15中任一项的构建体可以任选地进一步包括转录终止子。编码BMC01-BMC15中任一项的基因可以任选地针对宿主物种进行密码子优化，可以任选地包括一个或多个内含子，并且可以任选地包括一个或多个肽标签序列、一个或多个核定位序列(NLS)和/或一个或多个连接子或工程化切割位点(例如2a序列)。在不同的实施方案中，宿主细胞可以包括上文公开的工程化的BMC01-BMC15 CRISPR系统中的任意者，其中编码效应物(effector)的核酸序列在向导RNA引入之前存在于细胞中。在其它实施方案中，经工程化以包括用于表达BMC01-BMC15多肽的基因的细胞还可包括编码可操作地连接至调控元件的向导RNA(例如，向导RNA)的多核苷酸。

细胞或生物体可以是不天然包括编码BMC01-BMC15中任一项的基因的原核细胞。合适的原核宿主细胞包括例如埃希氏菌属(Escherichia)的细菌物种，例如源自大肠杆菌BL21的菌株(例如BL21(DE3)、BL21(DE3)PlysS、BL21(DE3)RIL、BL21(DE3)PRARE、BL21codon plus、BL21(DE3)codon plus)、XL1Blue、NM522、JM101、JM109、JM105、RR1、DH5α、TOP 10、HB101或MM294。进一步的合适的细菌宿主细胞是但不限于链霉菌属(Streptomyces)；假单胞菌属(Pseudomonas)，如恶臭假单胞菌(Pseudomonas putida)；棒状杆菌属(Corynebacterium)，如谷氨酸棒状杆菌(C.glutamicum)；乳杆菌属(Lactobacillus)，如唾液乳杆菌(L.salivarius)或芽孢杆菌属(Bacillus)，如枯草芽孢杆菌(Bacillus subtilis)。

通常，真核宿主细胞优于原核宿主细胞。

真核细胞可以是酵母、真菌、阿米巴、昆虫、脊椎动物(例如哺乳动物)或植物细胞。

酵母细胞可以是例如来自酿酒酵母，多形欧加泰酵母(Ogataea angusta)，克鲁维酵母属菌种(Kluyveromyces sp.)如马克斯克鲁维酵母(K.marxianus)或乳酸克鲁维酵母(K.lactis)，或毕赤酵母属菌种(Pichia sp.)如巴斯德毕赤酵母(P.pastoris)，昆虫细胞如果蝇S2或贪夜蛾Sf9细胞，植物细胞，或真菌细胞，优选毛霉科(Trichocomaceae)的细胞，更优选曲霉属(Aspergillus)、青霉属(Penicillium)或木霉属(Trichoderma)的细胞，或黑粉菌科(Ustilaginaceae)，最优选黑粉菌菌种(Ustilago sp.)的细胞。

可以使用的植物宿主细胞包括单子叶植物和双子叶植物(即分别为单子叶植物的和双子叶植物的)，优选农作物细胞和烟草细胞。农作物包括谷类(例如玉米、小麦、稻)、豆类(例如大豆、豌豆和苜蓿)、水果和蔬菜(例如生菜、番茄和马铃薯)。感兴趣的植物物种的实例包括但不限于玉米(Zea mays)、芸苔属(Brassica sp.)(例如，甘蓝型油菜(B.napus)、白菜型油菜(B.rapa)、芥菜型油菜(B.juncea))，特别是那些可用作种子油来源的芸苔属物种、苜蓿(Medicago sativa)、稻(Oryza sativa)、黑麦(rye)(Secale cereale)、高粱(sorghum)(Sorghum bicolor、Sorghum vulgare)、亚麻荠(Camelina sativa)、粟(millet)(例如珍珠粟(pearl millet)(Pennisetum glaucum)、糜子(proso millet)(Panicummiliaceum)、狐尾粟(foxtail millet)(Setaria italica)、子(finger millet)(Eleusine coracana))、葵花(Helianthus annuus)、藜麦(Chenopodium quinoa)、菊苣(Cichoriumintybus)、生菜(Lactuca sativa)、红花(Carthamus tinctorius)、小麦(Triticum aestivum)、大豆(Glycine max)、烟草(Nicotiana tabacum)、马铃薯(Solanumtuberosum)、花生(Arachis hypogaea)、棉花(Gossypium barbadense、Gossypiumhirsutum)、甘薯(Ipomoea batatus)、木薯(Manihot esculenta)、咖啡(Coffea spp.)、椰子(Cocos nucifera)、菠萝(Ananas comosus)、柑橘树(Citrus spp.)、可可(Theobromacacao)、茶(Camellia sinensis)、香蕉(Musa spp.)、鳄梨(Persea americana)、无花果(Ficus casica)、番石榴(Psidium guajava)、芒果(Mangifera indica)、橄榄(Oleaeuropaea)、木瓜(Carica papaya)、腰果(Anacardium occidentale)、夏威夷果(Macadamiaintegrifolia)、杏仁(Prunus amygdalus)、甜菜(Beta vulgaris)、甘蔗(Saccharumspp.)、油棕(Elaeis guineensis)、白杨(Populus spp.)、桉树(Eucalyptus spp.)、燕麦(Avena sativa)、大麦(Hordeum vulgare)、蔬菜、观赏植物和针叶树。

可以使用的哺乳动物宿主细胞包括人Hela、HEK293、H9和Jurkat细胞、小鼠NIH3T3和C127细胞、COS1、COS 7和CV1、鹌鹑QC1-3细胞、小鼠L细胞、Bowes黑色素瘤细胞和中国仓鼠卵巢(CHO)细胞。

本发明在第四方面涉及植物、种子或植物的一部分或者动物，其包含第一方面的核酸分子或转化、转导或转染有第二方面的载体，所述植物的一部分不是单个植物细胞。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第四方面。

植物或其部分可以是单子叶植物的植物或其部分或者双子叶植物的植物或其部分。双子叶植物的植物优选是烟草植物或其部分。同样，种子可以是单子叶植物的或双子叶植物的植物种子，并且优选是烟草植物种子。农作物的优选实例已在上文中描述。植物的部分的非限制性实例是叶、茎或根。

动物优选是哺乳动物并且最优选是非人类哺乳动物。哺乳动物可以是例如小鼠、大鼠、仓鼠、猫、犬、马、猪、牛、猴、猿等。

通过在植物、种子或植物的一部分或者动物中表达第一方面的核酸分子以及向导RNA，可以编辑宿主的基因组。基因组可以被编辑，例如，为了引入靶向的基因突变、用于基因治疗、用于创建染色体重排、用于研究基因功能、用于转基因生物的生产、用于内源基因标记或用于靶向的转基因添加。

本发明在第五方面涉及产生RNA引导的DNA核酸内切酶的方法，包括培养第三方面的宿主细胞并分离所产生的RNA引导的DNA核酸内切酶。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第五方面。

用于培养原核或真核宿主的合适条件是本领域技术人员所熟知的。一般来说，培养细菌的适合条件是在Luria Bertani(LB)培养基中通气生长。为了增加表达产物的产量和溶解度，可以用已知增强或促进两者的合适添加剂缓冲或补充培养基。大肠杆菌可在4至约37℃培养，确切的温度或温度的次序取决于将要过表达的分子。

一般来说，曲霉菌属菌种(Aspergillus sp.)可以在约10℃至约40℃、优选约25℃在沙氏葡萄糖琼脂或马铃薯葡萄糖琼脂上生长。酵母培养的合适条件是例如从Guthrie和Fink,“Guide to Yeast Genetics and Molecular Cell Biology”(2002)；AcademicPress Inc.已知的。本领域技术人员也知晓所有这些条件并且可以进一步使这些条件适应特定宿主物种的需要和所表达的多肽的要求。如果诱导型启动子控制存在于宿主细胞中的载体中的本发明的核酸，则可以通过添加合适的诱导剂来诱导多肽的表达。合适的表达方案和策略是技术人员已知的。

根据细胞类型及其具体要求，哺乳动物细胞培养可以，例如在含有10％(v/v)FCS、2mM L-谷氨酰胺和100U/ml青霉素/链霉素的RPMI或DMEM培养基中进行。细胞可以在5％CO₂、水饱和的气氛中保持在37℃。用于真核细胞的合适的表达方案是技术人员熟知的并且可以例如从Sambrook,2001检索到。

用于分离所产生的RNA引导的DNA核酸内切酶的方法是本领域所熟知的并且包括但不限于方法步骤例如离子交换层析、凝胶过滤层析(尺寸排阻层析)、亲和层析、高压液相层析(HPLC)、反相HPLC、圆盘凝胶电泳或免疫沉淀，参见，例如Sambrook,2001。

蛋白质分离步骤优选为蛋白质纯化步骤。根据本发明的蛋白质纯化指定了旨在进一步从复杂混合物中分离本发明的多肽的过程或一系列过程，优选达到同质程度(homogeneity)。例如，纯化步骤利用蛋白质大小、物理化学性质和结合亲和力的差异。例如，蛋白质可以根据它们的等电点通过pH分级凝胶或离子交换柱进行纯化。此外，可以通过尺寸排阻色谱法或通过SDS-PAGE(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)分析根据其大小或分子量分离蛋白质。在本领域中，通常使用2D-PAGE纯化蛋白质，然后通过肽质量指纹图谱进一步分析以确定蛋白质身份。这对于科学目的很有用，蛋白质的检测限非常低，纳克量的蛋白质足以进行它们的分析。蛋白质也可以通过高效液相色谱或反相色谱的极性/疏水性进行纯化。因此，用于蛋白质纯化的方法是技术人员熟知的。

本发明第六方面涉及由第一方面的核酸分子编码的RNA引导的DNA核酸内切酶。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第六方面。

SEQ ID NO:1-15的氨基酸序列是本发明的RNA引导的DNA核酸内切酶的特别优选的实例。

本发明第六方面的RNA引导的DNA核酸内切酶也可以是融合蛋白，其中RNA引导的DNA核酸内切酶的氨基酸序列与融合配偶体融合。融合可以是直接融合或通过连接子的融合。连接子优选是肽，例如GS-连接子。

融合配偶体可以位于N-末端、C-末端、两个末端，或位于RNA引导的DNA核酸内切酶多肽的内部位置，优选位于N-末端或C-末端。

融合配偶体优选是核定位信号(NLS)、细胞穿透结构域、质体靶向信号、线粒体靶向信号肽、同时靶向质体和线粒体的信号肽、标记物结构域、标签(例如纯化标签)、DNA修饰酶或反式激活结构域。

DNA修饰酶可以通过磷酸化、平端化DNA的去磷酸化来修饰DNA，其中平端化是指消化单链突出。去磷酸化酶的非限制性实例是虾碱性磷酸酶(rSAP)、快速CIP磷酸酶和南极磷酸酶。磷酸化酶的非限制性实例是多核苷酸激酶，例如T4 PNK。平端化酶的非限制性实例是DNA聚合酶I大(Klenow)片段、T4 DNA聚合酶或绿豆核酸酶。

反式激活结构域(Transactivation domains或trans-activating domains,TAD)))是转录因子支架结构域，其含有其它蛋白质例如转录共调节因子的结合位点。非限制性实例是九氨基酸反式激活结构域(9aaTAD)和富含谷氨酰胺(Q)的TAD。

通常，NLS包含碱性氨基酸段(stretch)。核定位信号是本领域已知的。NLS可以位于根据本发明的RNA引导的DNA核酸内切酶多肽的N末端、C末端或两者。例如，根据本发明的RNA引导的DNA核酸内切酶多肽可以包含约或多于约1、2、3、4、5、6、7、8、9、10个或更多个位于或靠近氨基-末端的NLS，约或多于约1、2、3、4、5、6、7、8、9、10个或更多个位于或靠近羧基末端的NLS，或这些的组合(例如，位于氨基末端的零个或至少一个或多个NLS，以及位于羧基末端的零个或一个或多个NLS)。当存在多于一个NLS时，可以独立于其它NLS选择每一个，使得单个NLS可以多于一个拷贝存在和/或与一个或多个其它NLS组合存在于一个或多个拷贝中。在一些实施方案中，当NLS的最邻近氨基酸沿多肽链在距离N或C末端约1、2、3、4、5、10、15、20、25、30、40、50个或更多个氨基酸内时，NLS被认为靠近N或C末端。RNA引导的DNA核酸内切酶多肽序列和NLS在一些实施方案中可以与长度在1至约20个氨基酸之间的连接子融合。

NLS的非限制性实例包括源自以下的NLS序列：SV40病毒大T抗原的NLS；来自核质蛋白的NLS(例如，核质蛋白二分NLS)；c-myc NLS；hRNPAl M9 NLS；来自核输入蛋白α(importin-alpha)的IBB域；肌瘤T蛋白、p53蛋白；c-abl IV蛋白，或流感病毒NS1；肝炎病毒δ抗原的NLS，Mxl蛋白；聚(ADP-核糖)聚合酶；和类固醇激素受体(人)糖皮质激素的NLS序列。一般而言，一种或多种NLS的强度足以驱动根据本发明的RNA引导的DNA核酸内切酶多肽在真核细胞的细胞核中以可检测的量积累。

质体、线粒体和双靶向信号肽定位信号也是本领域已知的(参见，例如，Nassoury和Morse(2005)Biochim Biophys Acta 1743:5-19；Kunze和Berger(2015)Front Physiol6:259；Herrmann and Neupert(2003)IUBMB Life55:219-225；Soil(2002)Curr OpinPlant Biol 5:529-535；Carrie和Small(2013)Biochim Biophys Acta 1833:253-259；Carrie等人(2009)FEBS J 276:1187-1195；Silva-Filho(2003)Curr Opin Plant Biol 6:589-595；Peeters和Small(2001)Biochim Biophys Acta 1541:54-63；Murcha等人(2014)Exp Bot 65:6301-6335；Mackenzie(2005)Trends Cell Biol 15:548-554；Glaser等人(1998)Plant Mol Biol 38:311-338)。

标记物结构域的非限制性实例包括荧光蛋白、纯化标签和表位标签。在某些实施方案中，标记物结构域可以是荧光蛋白。合适的荧光蛋白的非限制性实例包括绿色荧光蛋白(例如，GFP、GFP-2、tagGFP、turboGFP、EGFP、Emerald、Azami Green、Monomeric AzamiGreen、CopGFP、AceGFP、ZsGreenl)、黄色荧光蛋白(例如YFP、EYFP、Citrine、Venus、YPet、PhiYFP、ZsYellowl)、蓝色荧光蛋白(例如EBFP、EBFP2、Azurite、mKalamal、GFPuv、Sapphire、T-sapphire)、青色荧光蛋白(例如ECFP、Cerulean、CyPet、AmCyanl、Midoriishi-Cyan)、红色荧光蛋白(mKate、mKate2、mPlum、DsRed单体、mCherry、mRFP1、DsRed-Express、DsRed2、DsRed-Monomer、HcRed-Tandem、HcRedl、AsRed2、eqFP611、mRasberry、mStrawberry、Jred)和橙色荧光蛋白(mOrange、mKO、Kusabira-Orange)。

标签是允许在多肽混合物中鉴别根据本发明的RNA引导的DNA核酸内切酶多肽的短氨基酸序列。因此，标签优选是纯化标签。纯化标签的非限制性实例是His-标签(例如His-6-标签)、GST标签、DHFR标签和CBP标签，对已知纯化标签的综述可以在Kimple等人(2015),Curr Protoc Protein Sci.2013；73:Unit-9.9中找到。

本发明在第七方面涉及一种组合物，其包含第一方面的核酸分子，第二方面的载体，第三方面的宿主细胞，第四方面的植物、种子、细胞的一部分或动物，第六方面的RNA引导的DNA核酸内切酶，或它们的组合。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第七方面。

如本文所用，术语“组合物”指包含如下的组合物：第一方面的核酸分子，第二方面的载体，第三方面的宿主细胞，第四方面的植物、种子、细胞的一部分或动物，第六方面的RNA引导的DNA核酸内切酶，或它们的组合中的至少一种，该组合物在下文中也统称为化合物。

根据第七方面的优选实施方案，该组合物是药物组合物或诊断组合物。

根据本发明，术语“药物组合物”涉及用于施用于患者、优选人类患者的组合物。本发明的药物组合物包含至少一种上述化合物。它可以任选地包含能够改变本发明化合物的特性从而例如稳定、调节和/或激活它们的功能的其它分子。该组合物可以是固体、液体或气体形式，并且尤其可以是粉末(一种或多种)、片剂(一种或多种)、溶液(一种或多种)或气雾剂(一种或多种)的形式。本发明的药物组合物可以任选地并且额外地包含药学上可接受的载体。合适的药物载体的实例是本领域所熟知的，包括磷酸盐缓冲盐溶液、水、乳剂，例如油/水乳剂、各种类型的润湿剂、无菌溶液、包括DMSO等的有机溶剂。包含此类载体的组合物可以按常规方法配制。这些药物组合物可以以合适的剂量施用于受试者。剂量方案将由主治医师和临床因素决定。正如医学领域所熟知的那样，任何一位患者的剂量取决于许多因素，包括患者的体型、体表面积、年龄、待施用的特定化合物、性别、施用时间和途径、一般健康状况和其它同时施用的药物。对于给定情况的治疗有效量将容易地通过常规实验确定并且在普通临床医生或医师的技能和判断范围内。通常，药物组合物的常规施用方案应在每天1μg至5g活性化合物的范围内。然而，更优选的剂量可在每天0.01mg至100mg的范围内，甚至更优选0.01mg至50mg并且最优选0.01mg至10mg。观察变化所需的治疗时间长短以及治疗后出现反应的时间间隔因所期望的效果而异。具体的量可以通过本领域技术人员熟知的常规试验来确定。

该药物组合物可用于例如治疗或预防病原体疾病，例如病毒或细菌疾病。例如，第六方面的RNA引导的DNA核酸内切酶可以与靶向病原体基因组的gRNA一起使用，从而修饰病原体的基因组，从而预防或治疗由病原体引起的疾病。

该药物组合物还可以用于例如治疗或预防微生物组失衡。例如，由于过度使用抗生素，可能会导致微生物组失衡，这可能会导致致病菌和酵母过度生长。

“诊断组合物”涉及适用于检测包括传染性和非传染性疾病两者的受试者疾病的组合物。诊断组合物可以特别地包含如上文所述的标记物部分，其与连接到ssDNA链的本发明的融合蛋白相关，使得当根据本发明的RNA引导的DNA核酸内切酶多肽切割ssDNA时，它激活报告物(reporter)，使其发出荧光或改变颜色，从而能够对特定疾病的核酸标记物进行视觉检测。诊断组合物可应用于体液样本，例如血液、尿液或唾液。

本发明在第八方面涉及第一方面的核酸分子，第二方面的载体，第三方面的宿主细胞，第四方面的植物、种子、细胞的一部分或动物，第六方面的RNA引导的DNA核酸内切酶或它们的组合用于通过修饰受试者或植物基因组中靶位点的核苷酸序列来治疗受试者或植物的疾病的用途。

还描述了一种治疗或预防受试者或植物疾病的方法，包括通过第一方面的核酸分子，第二方面的载体，第三方面的宿主细胞，第四方面的植物、种子、细胞的一部分或动物，第六方面的RNA引导的DNA核酸内切酶或它们的组合，来修饰受试者或植物基因组中靶位点的核苷酸序列。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第八方面。

根据本发明，受试者或植物基因组中靶位点核苷酸序列的修饰是通过CRISPR技术进行的基因组编辑，特别是通过本文提供的新颖的RNA引导的DNA核酸内切酶进行的基因组编辑，其将与如下文所述的适当的gRNA和任选的修复底物组合使用以便确定基因组修饰的靶位点。

基因组编辑(也称为基因组工程)是一种基因工程，其中靶位点、优选感兴趣的基因，在细胞的基因组中被插入、删除、修饰或替换。靶位点、优选感兴趣的基因，可以在基因组中，但也可以在线粒体DNA(动物细胞)或叶绿体DNA(植物细胞)中。基因组编辑可导致细胞的基因组中的功能缺失突变或功能获得突变。功能缺失突变(也称为失活突变)导致感兴趣的基因功能较少或没有功能(部分或全部失活)。当等位基因完全丧失功能(完全失活)时，这在本文中也称为(基因)敲除。基因敲除可通过插入、删除、修饰或替换基因的一个或多个核苷酸来实现。功能获得性突变(也称为激活突变)可以改变感兴趣的基因，使其效应变得更强(激活增强)，或甚至被不同的(例如异常)功能所取代。功能获得性突变还可将细胞以前没有的新功能或效果引入细胞。在这种情况下，新基因可以添加到细胞的基因组(插入)或可以替换基因组内的基因。引入这种新功能或效果的功能获得性突变也称为基因敲入。基因组编辑也可导致一种或多种基因的上调或下调。通过靶向负责调节基因表达的DNA位点(例如启动子区域或编码转录因子的基因)，可以通过CRISPR技术上调或下调基因的表达。下面将提供关于CRISPR技术的作用模式的更多细节。

自发现以来，CRISPR技术已越来越多地应用于治疗性基因组编辑。多种病毒和非病毒载体的使用使得CRISPR系统能够有效地递送至靶细胞或组织。此外，CRISPR系统能够以多种方式例如诱变、基因整合、表观基因组调控、染色体重排、碱基编辑和mRNA编辑调节靶基因的表达(综述Le and Kim(2019),Hum Genet；138(6):563-590)。

受试者基因组中靶位点核苷酸序列的修饰优选是基因治疗。基因治疗基于在靶位点对核苷酸序列进行遗传操作的原理，用于治疗和预防疾病，特别是人类疾病。

在CRISPR技术的临床试验中，科学家们正在使用CRISPR技术来对抗人类的癌症和血液疾病。在这些试验中，从待治疗的受试者取出一些细胞，对DNA进行基因组编辑，然后将经过基因组编辑的细胞放回受试者体内，所述细胞现在被武装起来与待治疗的疾病作斗争。

本发明在第九方面涉及一种修饰细胞的基因组中靶位点的核苷酸序列的方法，包括将如下引入所述细胞：(i)靶向DNA的RNA或编码靶向DNA的RNA的DNA多核苷酸，其中靶向DNA的RNA包含(a)第一片段，其包含与靶DNA中的序列互补的核苷酸序列；(b)与第六方面的RNA引导的DNA核酸内切酶相互作用的第二片段；(ii)第六方面的RNA引导的DNA核酸内切酶，或第一方面的编码RNA引导的DNA核酸内切酶的核酸分子，或第二方面的载体，其中RNA引导的DNA核酸内切酶包含(a)与靶向DNA的RNA相互作用的RNA结合部分，和(b)显示定点酶活性的活性部分。

因此，本发明还涉及包含以下的组合物(例如药物或诊断组合物)：(i)靶向DNA的RNA或编码靶向DNA的RNA的DNA多核苷酸，其中靶向DNA的RNA包含：(a)第一片段，其包含与靶DNA中的序列互补的核苷酸序列；(b)与第六方面的RNA引导的DNA核酸内切酶相互作用的第二片段；和(ii)第六方面的RNA引导的DNA核酸内切酶，或第一方面的编码RNA引导的DNA核酸内切酶的核酸分子，或第二方面的载体，其中RNA引导的DNA核酸内切酶包含(a)与靶向DNA的RNA相互作用的RNA结合部分和(b)显示定点酶活性的活性部分。

如果适用，如上文所述的定义和优选实施方案经必要修正后适用于第九方面。

靶向DNA的RNA包括包含与靶DNA中的序列互补的核苷酸序列的第一片段和与RNA引导的DNA核酸内切酶相互作用的第二片段。如上文所讨论的，与靶DNA中的序列互补的核苷酸序列定义了RNA引导的DNA核酸内切酶的靶特异性。同样如上文所讨论的，靶向DNA的RNA与RNA引导的DNA核酸内切酶结合，由此形成复合物。第二片段与RNA引导的DNA核酸内切酶相互作用，并负责复合物的形成。与第六方面的RNA引导的DNA核酸内切酶相互作用的第二片段优选包含SEQ ID NO:31或由SEQ ID NO:31组成，更优选包含SEQ ID NO:32或由SEQID NO:32组成。SEQ ID NO:31是V型2类CRISPR核酸酶的第二片段的共有序列。在V型2类CRISPR核酸酶中，第二片段也称为5’柄(handle)。SEQ ID NO:32是BMC01-BMC15中任一项的第二片段。

RNA引导的DNA核酸内切酶包含作为与靶向DNA的RNA相互作用的RNA结合部分的第一片段和作为表现出定点酶活性的活性部分的第二片段。第一段与靶向DNA的RNA相互作用，并负责所讨论的复合物的形成。第二片段携带核酸内切酶结构域，其优选包含RuvC结构域。

同样如上文所讨论的，靶向DNA的RNA是向导RNA。向导RNA可以直接引入细胞或以编码靶向DNA的RNA的DNA多核苷酸的形式引入细胞。在后一种情况下，编码向导RNA的DNA通常可操作地连接至一个或多个用于表达向导RNA的启动子序列。例如，RNA编码序列可以可操作地连接至由RNA聚合酶III(Pol III)或RNA聚合酶II(Pol II)识别的启动子序列。编码靶向DNA的RNA的DNA多核苷酸优选是载体。许多单个gRNA空载体(带有和不带有CRISPR核酸内切酶)在本领域是可获得的。并且，几种空的多重gRNA载体是可获得的，可用于从单个质粒(有或没有CRISPR核酸内切酶的表达)表达多个gRNA。DNA多核苷酸以可表达的形式编码靶向DNA的RNA。

同样，RNA引导的DNA核酸内切酶可以直接引入细胞或以编码RNA引导的DNA核酸内切酶的核酸分子的形式引入细胞，后者优选是第二方面的载体。DNA多核苷酸以可表达的形式编码RNA引导的DNA核酸内切酶。

如上文更详细讨论的，RNA引导的DNA核酸内切酶和靶向DNA的RNA也可以由相同的DNA多核苷酸编码，例如一体式CRISPR-cas载体。

术语“以可表达的形式”是指编码RNA引导的DNA核酸内切酶和靶向DNA的RNA的一种或多种DNA多核苷酸处于确保靶向DNA的RNA被转录并且RNA引导的DNA核酸内切酶被转录并翻译成细胞中的活性酶的形式。

根据本发明第九方面的优选实施方案，在RNA引导的DNA核酸内切酶和靶向DNA的RNA直接引入细胞的情况下，它们以核糖核蛋白复合物(RNP)的形式引入。

RNP在体外组装并可通过本领域已知的方法递送至细胞，例如电穿孔或脂质转染。RNP能够以与基于核酸(例如基于载体)的RNA引导的DNA核酸内切酶相当的功效切割靶位点(Kim等人(2014),Genome Research24(6):1012-1019)。

将蛋白质(或肽)或RNP引入活细胞的手段是本领域已知的，包括但不限于显微注射、电穿孔、脂质转染(使用脂质体)、基于纳米颗粒的递送和蛋白质转导。可以使用这些方法中的任何一种。

用于脂质转染的脂质体是一种小囊泡，由与细胞膜相同的材料组成(即通常例如由磷脂类制成的脂质双层)，其可以填充有一种或多种蛋白质(例如Torchilin VP.(2006),Adv Drug Deliv Rev.,58(14):1532-55)。为了将蛋白质或RNP递送到细胞中，脂质体的脂质双层可与细胞膜的脂质双层融合，从而将所含的蛋白质递送到细胞中。优选根据本发明使用的脂质体由阳离子脂质组成。阳离子脂质体策略已成功应用于蛋白质递送(Zelphati等人(2001).J.Biol.Chem.276,35103-35110)。如本领域已知的，可以改变所用阳离子脂质的确切组成和/或混合物，这取决于感兴趣的蛋白质和所用的细胞类型(Felgner等人.(1994).J.Biol.Chem.269、2550-2561)。用于诱导同源定向DNA修复的、基于纳米粒子的Cas9核糖核酸蛋白和供体DNA的递送是例如在Lee et al.(2017),Nature BiomedicalEngineering,1:889-90中所描述的。

蛋白质转导指定了蛋白质从外部环境内化到细胞中(Ford等人(2001),GeneTherapy,8:1-4)。这种方法依赖于少量蛋白质和肽(优选10至16个氨基酸长)穿透细胞膜的固有特性。这些分子的转导特性可以赋予与之融合表达的蛋白质，因此提供了例如基因治疗的替代方案，用于将治疗性蛋白质递送到靶细胞中。例如，常用的能够穿透细胞膜的蛋白质或肽是：例如触角肽(antennapedia peptide)、单纯疱疹病毒VP22蛋白、HIV TAT蛋白转导结构域、源自神经递质或激素的肽、或9×Arg标签。

显微注射和电穿孔是本领域所熟知的并且技术人员知道如何进行这些方法。显微注射是指使用玻璃微量移液器将微观或临界宏观水平的物质引入单个活细胞的过程。电穿孔是由外部施加的电场引起的细胞质膜的电导率和渗透性的显著增加。通过增加渗透性，可以将蛋白质(或肽或核酸序列)引入活细胞中。

RNA引导的DNA核酸内切酶可以作为活性酶或酶原引入细胞。在后一种情况下，RNA引导的DNA核酸内切酶在细胞内发生生化变化(例如通过水解反应显示活性位点或改变构型以显示活性位点)，从而使酶原成为活性酶。

将核酸分子(一种或多种)和靶向DNA的RNA引入细胞的手段和方法同样是本领域已知的，并且这些方法包括转导或转染细胞。

转导是通过病毒或病毒载体将外来DNA引入细胞的过程。转导是分子生物学家用来将外来基因稳定引入宿主细胞的基因组的常用工具。通常，构建质粒，其中待转移的基因两侧是病毒序列，病毒蛋白使用这些病毒序列识别病毒基因组并将其包装到病毒颗粒中。该质粒与携带形成感染性病毒颗粒所需的病毒基因的其它质粒(DNA构建体)一起插入(通常通过转染)到生产者细胞中。在这些生产者细胞中，由这些包装构建体表达的病毒蛋白结合待转移的DNA/RNA(取决于病毒载体的类型)上的序列，并将其插入病毒颗粒中。为了安全，所使用的质粒都不包含病毒形成所需的所有序列，以至于需要同时转染多个质粒才能获得感染性病毒颗粒。此外，只有携带待转移的序列的质粒含有允许将遗传物质包装在病毒颗粒中的信号，以便没有编码病毒蛋白的基因被包装。然后将从这些细胞中收集的病毒应用于待改变的细胞。这些感染的初始阶段模仿自然病毒的感染，并导致转移的基因的表达和(在慢病毒/逆转录病毒载体的情况下)待转移到细胞的基因组中的DNA的插入。然而，由于转移的遗传物质不编码任何病毒基因，这些感染不会生成新病毒(病毒是“复制缺陷的”)。在本案中，转导可用于生成在其基因组中以可表达形式包含RNA引导的DNA核酸内切酶的细胞。

转染是故意将裸的或纯化的核酸或纯化的蛋白质或组装的核糖核蛋白复合物引入细胞的过程。转染通常是基于非病毒的方法。

转染可以是基于化学的转染。基于化学的转染可分为几种类型：使用环糊精、聚合物、脂质体或纳米颗粒的转染。最便宜的方法之一是使用磷酸钙。含有磷酸根离子的HEPES缓冲盐溶液(HeBS)与含有待转染DNA的氯化钙溶液结合。当两者结合时，带正电荷的钙和带负电荷的磷酸盐会形成细小的沉淀物，将待转染的DNA结合到其表面。然后将沉淀物的悬浮液添加到待转染的细胞(通常是单层生长的细胞培养物)。通过尚未完全了解的过程，细胞吸收了一些沉淀物，并随之吸收了DNA。该过程已成为识别许多致癌基因的优选方法。其它方法使用高度支化的有机化合物，即所谓的树枝状聚合物，来结合DNA并将其转移到细胞中。另一种方法是使用阳离子聚合物，例如DEAE-葡聚糖或聚乙烯亚胺(PEI)。带负电荷的DNA与聚阳离子结合，复合物通过内吞作用被细胞摄取。如上所述，脂质转染(或脂质体转染)是一种用于通过脂质体将遗传物质注入细胞的技术，脂质体是可容易与细胞膜融合的囊泡，因为它们都是由磷脂双层构成的。脂质转染通常使用带正电荷(阳离子)的脂质(阳离子脂质体或混合物)，以与带负电荷(阴离子)的遗传物质形成聚集体。这种转染技术在转移到细胞方面与其它利用聚合物、DEAE-葡聚糖、磷酸钙和电穿孔的生化程序执行相同的任务。可以通过用温和的热休克处理转染的细胞来提高脂质转染的效率。Fugene是一系列广泛使用的专有非脂质体转染试剂，能够高效且低毒地直接转染多种细胞。

转染也可以是非化学方法。电穿孔(基因电转移)是一种流行的方法，当细胞暴露于强电场的短脉冲时，实现细胞膜的渗透性的瞬时增加。细胞挤压能够通过细胞膜变形将分子递送到细胞中。声致穿孔(Sonoporation)使用高强度超声波来诱导细胞膜中的孔形成。这种孔形成主要归因于气泡与附近细胞膜相互作用的空化作用，因为它通过添加超声造影剂(空化核的来源)而得到增强。光学转染是一种使用高度聚焦的激光在细胞的质膜中瞬时产生微小(直径约1μm)的孔的方法。原生质体融合是一种用溶菌酶处理转化的细菌细胞以去除细胞壁的技术。在此之后，融合剂(例如仙台病毒、PEG、电穿孔)被用于将携带感兴趣基因的原生质体与受体靶细胞融合。

最后，转染可以是一种基于粒子的方法。直接的转染方法是基因枪，其中DNA与惰性固体(通常是金)的纳米粒子偶联，然后将其直接“发射”(或粒子轰击)到靶细胞的细胞核中。因此，核酸以高速度通过膜穿透递送，通常与微弹相连。磁转染或磁辅助转染是一种利用磁力将DNA递送至靶细胞内的转染方法。穿刺感染是通过用细长的纳米结构和此类纳米结构阵列(例如已用质粒DNA功能化的碳纳米纤维或硅纳米线)穿刺细胞来进行的。

本发明第九方面的方法涉及用本发明的RNA引导的DNA核酸内切酶编辑(即“突变”)细胞的基因组中靶位点的核苷酸序列的方法。这基本上需要三个连续的先决条件：(1)将RNA引导的DNA核酸内切酶编码基因或RNA引导的DNA核酸内切酶本身有效地递送到靶细胞中；(2)靶细胞中CRISPR组件的有效表达或存在(第六方面的靶向DNA的RNA和RNA引导的DNA核酸内切酶)；(3)通过CRISPR核糖核蛋白复合物靶向感兴趣的基因组位点，并通过细胞自身的修复途径修复DNA。步骤(3)是在CRISPR组件在待编辑的基因组的细胞中表达后自动进行的。

通过基因组编辑，可以在细胞的基因组中插入、删除、修饰(包括单核苷酸多态性(SNP))或替换靶位点。靶位点可以在基因的编码区、基因的内含子、基因的控制区、基因之间的非编码区等。该基因可以是蛋白质编码基因或RNA编码基因。该基因可以是任何感兴趣的基因。

在这方面，基因组编辑使用细胞自身的修复途径，包括非同源末端连接(NHEJ)或同源定向重组(HDR)途径。一旦DNA被RNA引导的DNA核酸内切酶切割，细胞自身的DNA修复机制(NHEJ或HDR)就会添加或删除遗传物质的(piece)碎片，或者通过用定制的DNA序列替换现有片段(segment)来改变DNA。因此，在CRISPR-Cas系统中，CRISPR核酸酶在由短的(约20个核苷酸)gRNA确定的位点处在DNA中造成双链断裂，然后通过NHEJ或HDR在细胞内修复该断裂。优选基因组编辑使用NHEJ。在不同的实施方案中，优选基因组编辑使用HDR。

NHEJ使用各种酶直接连接双链断裂处的DNA末端。相反，在HDR中，同源序列被用作模板，用于在断点处再生缺失的DNA序列。NHEJ是规范的同源独立途径，因为它最多只涉及一个到几个互补碱基的比对以重新连接两端，而HDR使用更长的序列同源性来修复DNA损伤。

正是这些途径的自然特性形成了基于RNA引导的DNA核酸内切酶的基因组编辑的基础。NHEJ是易错的，并已显示会在修复位点引起突变。因此，如果能够在多个样本中的所需基因处创建双链断裂(DSB)，则很可能由于NHEJ失真造成的错误，在某些处理中该位点会产生突变。另一方面，可以通过在与DSB侧翼序列同源的序列中插入所需序列来利用HDR对修复DSB的同源序列的依赖性，当HDR系统将其用作模板时，会导致在感兴趣的基因组区域内创建期望的变化。尽管机制不同，但基于HDR的基因编辑的概念在某种程度上类似于基于同源重组的基因靶向。因此，根据这些原则，如果能够在基因组内的特定位置创建DSB，那么细胞自身的修复系统将有助于创建期望的突变。

用于HDR的同源序列模板在本文中也称为“修复模板”。

因此，根据本发明的第九方面，通过修饰细胞的基因组中靶位点的核苷酸序列，可以敲除(通过引入过早终止密码子)或敲入(通过修复底物)基因。同样可以通过本发明第九方面的方法改变基因的表达。例如，基因组中的靶位点可以是启动子区域，改变启动子区域可以增加或减少通过靶启动子区域控制的基因的表达。

因此，根据第九方面的优选实施方案，该方法还包括将修复底物引入所述细胞中。

修复底物可以直接引入细胞或作为核酸分子，优选以可表达形式编码修复底物的载体。实施例3中说明了使用修复底物用于校正小麦胚中的突变型β-葡萄糖醛酸酶。

适用于HDR的修复模板的设计和结构是本领域已知的。如果修复模板与双链断裂(DSB)处的原始DNA序列相同，或者它可以在DNA中引入非常特定的突变，HDR是无差错的。HDR途径的三个核心步骤是：(1)5’末端的DNA链在断裂处被切除以创建3’突出。这将充当链侵入所需蛋白质的底物和DNA修复合成的引物。(2)然后侵入链可以取代同源DNA双链中的一条链并与另一条配对。这导致称为置换环(D环)的杂交DNA的形成。(3)然后可以将重组中间体分解，以完成DNA修复过程。

例如，用于向基因中引入突变或插入新核苷酸或核苷酸序列的HDR模板需要在将被修饰的靶序列周围具有一定量的同源性。可以使用在CRISPR诱导的DSB处起始的同源臂。一般来说，修饰的插入位点应该非常靠近DSB，如果可能，最好距离小于10bp。需要注意的重要一点是，一旦DSB被引入和修复，CRISPR酶可能会继续切割DNA。只要gRNA靶位点/PAM位点保持完整，CRISPR核酸酶就将继续切割和修复DNA。如果要将非常特定的突变或序列引入感兴趣的基因，这种重复编辑可能会出现问题。为了解决这个问题，可以设计修复模板，使其在初始DSB修复后最终阻止进一步的CRISPR核酸酶靶向。阻止进一步编辑的两种常见方式是突变PAM序列或gRNA种子序列。在设计修复模板时，要考虑预期编辑的大小。ssDNA模板(也称为ssODN)通常用于较小的修饰。对于每个同源臂，小的插入/编辑可能需要少至30-50个碱基，并且最佳确切数量可能会因感兴趣的基因而异。通常使用50-80个碱基的同源臂。例如，理查森等人,(2016).Nat Biotechnol.34(3):339-44)发现不对称同源臂(PAM远端36个碱基和PAM近端91个碱基)支持高达60％的HDR效率。由于创建长度超过200个碱基的ssODN可能遇到困难，因此最好使用dsDNA质粒修复模板将荧光蛋白或选择盒等较大的插入物插入到感兴趣的基因中。这些模板可以具有至少800bp的同源臂。为了提高基于质粒修复模板的HDR编辑的频率，可以使用在模板两侧包含gRNA靶位点的自切割质粒。当存在CRISPR核酸酶和适当的gRNA(一种或多种)时，模板就会从载体中释放出来。为避免质粒克隆，可以使用PCR生成的长dsDNA模板。此外，Quadros等人(2017)Genome Biol.17；18(1):92)开发了Easi-CRISPR，这是一种允许进行大的突变并利用ssODN优势的技术。为了创建长度超过200个碱基的ssODN，编码修复模板的RNA在体外进行转录，然后使用逆转录酶创建互补的ssDNA。Easi-CRISPR在小鼠敲入模型中运行良好，将编辑效率从dsDNA的1-10％提高到ssODN的25-50％。尽管HDR的效率因基因座和实验系统而异，但ssODN模板通常提供最高频率的HDR编辑。

根据第九方面的优选实施方案，细胞不是编码所述RNA引导的DNA核酸内切酶的基因的天然宿主。

如上文所讨论，从包含不能应用标准微生物条件培养的细菌的宏基因组文库中分离SEQ ID NO:1-15的RNA引导的DNA内切核酸酶。为此理由，从中分离出SEQ ID NO 1-15的细菌的确切性质是未知的。

因此，不是SEQ ID NO:1-15中任一项的天然宿主的细胞可以是来自可培养的细菌菌株的任何细菌细胞或任何真核细胞。这种被操纵的细胞在自然界中并不存在。

根据第九方面的另一个优选实施方案，细胞是真核细胞，优选植物细胞或动物细胞。

真核细胞、植物细胞和动物细胞以及可从中获得细胞的真核生物、植物和动物，包括其优选实例，已在上文中结合本发明的第三和第四方面进行了描述。

这些细胞同样可以与本发明的第九方面结合使用。

根据第九方面的一个更优选的实施方案，该方法还包括培养植物细胞或动物细胞，以便在表达RNA引导的DNA核酸内切酶并在靶位点切割核苷酸序列以产生修饰的核苷酸序列的条件下产生植物或动物；以及选择包含所述修饰的核苷酸序列的植物或动物。

在这方面，待引入CRISPR-Cas系统的组分的细胞必须是全能干细胞或生殖系细胞(卵母细胞和/或精子)或能够发育成完整的植物或动物的干细胞集合。培养此类细胞以产生植物或动物的手段和方法是本领域已知的(参见例如，https://www.stembook.org/node/720)。

除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常理解的相同的含义。在冲突的情况下，将以包括定义的专利说明书为准。

本发明在第十方面涉及已通过根据本发明第九方面的方法产生修饰细胞，用于治疗受试者疾病的用途。

修饰细胞优选是修饰的T淋巴细胞，待治疗的疾病优选是癌症(Stadtmauer等人,Science 28Feb 2020:Vol.367,Issue 6481,eaba7365)。

待通过本发明第九方面的方法修饰的细胞优选从待治疗的受试者获得，然后根据本发明的第十方面使用修饰的细胞。

关于在本说明书中表征的实施方案，特别是在权利要求中，旨在将从属权利要求中提及的每个实施方案与所述从属权利要求所从属的每个权利要求(独立或从属)的每个实施方案结合。例如，如果独立权利要求1引用了3个备选方案A、B和C，从属权利要求2引用了3个备选方案D、E、F，而权利要求3从属于权利要求1和2，并引用了3个备选方案G、H和I，应当理解，本说明书明确公开了对应于组合A、D、G；A、D、H；A、D、I；A、E、G；A、E、H；A、E、I；A、F、G；A、F、H；A、F、I；B、D、G；B、D、H；B、D、I；B、E、G；B、E、H；B、E、I；B、F、G；B、F、H；B、F、I；C、D、G；C、D、H；C、D、I；C、E、G；C、E、H；C、E、I；C、F、G；C、F、H；C、F、I的实施方案，除非另有特别说明。

类似地，并且在独立和/或从属权利要求未提及备选方案的那些情况下，应当理解，如果从属权利要求回溯引用多个在先权利要求，则认为明确公开了由此涵盖的主题的任何组合。例如，在独立权利要求1、从属权利要求2回溯引用权利要求1、并且从属权利要求3回溯引用权利要求2和1的情况下，可知权利要求3和1的主题的组合与权利要求3、2和1的主题的组合一样被清楚和明确地公开。如果存在涉及权利要求1至3中任一项的、进一步的从属权利要求4，则可知权利要求4和1，权利要求4、2和1，权利要求4、3和1，以及权利要求4、3、2和1的主题的组合被清楚和明确地公开。

附图说明

附图示出了：

图1：氨基酸序列BMC01-BMC15的无根树。

图2：显示转化后48小时(在30℃孵育)的大肠杆菌菌落的示例性培养板，以可视化使用BMC01、BMC02、BMC03、BMC04、BMC05、BMC07或BMC08核酸酶引导DNA靶向malQ基因后的菌落耗竭以及由导致NHEJ介导的malQ基因敲除的Ku-LigD蛋白的共转化诱导的菌落生长的部分恢复(对于BMC01、BMC03和BMC04)。

图3：显示转化后48小时(在30℃孵育)的大肠杆菌菌落的示例性培养板，以观察使用BMC09、BMC10、BMC11、BMC12、BMC13、BMC14或BMC15核酸酶引导DNA靶向malQ基因后的菌落耗竭以及由导致NHEJ介导的malQ基因敲除的Ku-LigD蛋白的共转化诱导的菌落生长的部分恢复(对于BMC09和BMC13)。

图4：BMC表征流程(pipeline)

使用BMC09核酸酶和Ku-LigD NHEJ策略在大肠杆菌中的细胞耗竭和malQ基因敲除。malQ基因的节选显示，与BMC09核酸酶的靶区域直接相关的71bp的删除。

具体实施方式

实施例说明了本发明。

实施例

实施例1-生活环境选择和DNA制备

发现新颖的Cas蛋白的方法是通过对选择的环境DNA(例如1cm³森林土壤含有～2.5×10¹⁰bp DNA或～2000万个基因)的下一代测序来获取万亿碱基级的宏基因组资源，并计算鉴别CRISPR-Cas系统。

为此目的，从德国的不同地点选择了宏基因组生活环境。在DNA分离之前，对水生、沉积物和土壤生活环境采样和预处理。使用各种不同孔径(0.1-20μm)的过滤器的交错过滤过程被用来富集含有几乎未触及的新颖的序列空间的候选门级辐射类群(CPR)物种(Hug等人(2016)Nature Microbiology 1,16048)。

从0.2和0.1μm过滤器中提取DNA，并使用PowerWater DNA分离试剂盒(Qiagen)分离。

实施例2-下一代测序和序列评估

使用TruSeq PCR free Library Prep Kit(Illumina)制备用于下一代测序的DNA文库，并使用HiSeq 2500(Illumina)在快速模式(2×250Bp)下对宏基因组文库测序，产生每个样本的平均50Gbp的输出。

对产生的配对端宏基因组序列文库组装、注释和评估，通过鉴别CRISPR重复段来鉴别潜在的CRISPR操纵子。含有已鉴别的CRISPR重复段-间隔子单元的重叠群(contigs)用于分析周围区域Cas蛋白的存在。通过将紧邻CRISPR重复段的开放阅读框与基因组和蛋白质数据库比较，分析其模式和序列，以鉴别潜在的新颖的Cas蛋白。

BLAST核苷酸搜索可以用BLASTN程序执行，得分(score)＝100，字长(wordlength)＝12，以获得与编码本发明蛋白质的核苷酸序列同源的核苷酸序列。BLAST蛋白搜索可以用BLASTX程序执行，得分＝50，字长＝3，以获得与本发明的蛋白质或多肽同源的氨基酸序列。为了获得用于比较目的的空位比对，可以如Altschul等人(1997)Nucleic Acids Res.25:3389中描述的利用Gapped BLAST(在BLAST 2.0中)。或者，PSI-BLAST(BLAST 2.0中)可用于执行迭代搜索，检测分子之间的远距离关系。参见Altschul等人(1997)同上。当利用BLAST、Gapped BLAST、PSI-BLAST时，可以使用各自程序(例如，对于核苷酸序列为BLASTN，对于蛋白质为BLASTX)的默认参数。参见网站www.ncbi.nlm.nih.gov。也可以通过检查手动执行比对。

实施例3：使用选择的BMC序列构建大肠杆菌功能基因组编辑系统

3.1大肠杆菌BW25113中基因组编辑的CRISPR/BMC-Ec载体系统

用于BMC01、BMC02、BMC03、BMC04、BMC05、BMC07、BMC08、BMC09、BMC10、BMC11、BMC12、BMC13、BMC14或BMC15核酸酶诱导型表达，用于引导RNA(gRNA)转录的组成型表达和用于Ku-LigD蛋白的表达所需的遗传元件在16种独立的载体上提供(CRISPR/BMC01-Ec、CRISPR/BMC02-Ec、CRISPR/BMC03-Ec、CRISPR/BMC04-Ec、CRISPR/BMC05-Ec、CRISPR/BMC07-Ec、CRISPR/BMC08-Ec、CRISPR/BMC09-Ec、CRISPR/BMC10-Ec、CRISPR/BMC11-Ec,CRISPR/BMC12-Ec、CRISPR/BMC13-Ec、CRISPR/BMC14-Ec、CRISPR/BMC15-Ec、CRISPR/gRNA-Ec和CRISPR/Ku-LigD-Ec)。

在下面，描述了CRISPR/BMC01-Ec、CRISPR/gRNA-Ec和CRISPR/Ku-LigD-Ec载体系统的构建。CRISPR/BMC02-Ec、CRISPR/BMC03-Ec、CRISPR/BMC04-Ec、CRISPR/BMC05-Ec、CRISPR/BMC07-Ec、CRISPR/BMC08-Ec、CRISPR/BMC09-Ec、CRISPR/BMC10-Ec、CRISPR/BMC11-Ec、CRISPR/BMC12-Ec、CRISPR/BMC13-Ec、CRISPR/BMC14-Ec和CRISPR/BMC15-Ec载体系统以与CRISPR/BMC01-Ec载体系统类似的方法构建。

BMC01_大肠杆菌蛋白表达载体的设计

使用由基因合成提供商GeneArt(Thermo Fisher Scientific，雷根斯堡，德国)提供的生物信息学应用程序，对合成的3888bp BMC01核苷酸序列密码子优化，以在大肠杆菌BW25113中表达，SEQ ID NO:33。用于蛋白质表达，将所得合成基因融合至诱导型araC-ParaBAD诱导型启动子系统(SEQ ID NO:34)和fdT终止子(SEQ ID NO:35)(Otsuka&Kunisawa,Journal of Theoretical Biology 97(1982),415-436)。最终的BMC01_大肠杆菌蛋白表达盒通过Gibson Assembly Cloning(NEB，法兰克福，德国)插入大肠杆菌穿梭载体中，所述大肠杆菌穿梭载体含有用于附加型繁殖(episomal propagation)和重组大肠杆菌细胞的选择所有所需的遗传元件。

CRISPR/BMC01-Ec载体系统

提供构建的CRISPR/BMC01-Ec载体系统的完整核苷酸序列如SEQ ID NO:36。

CRISPR/BMC02-Ec载体系统

提供构建的CRISPR/BMC02-Ec载体系统的完整核苷酸序列如SEQ ID NO:57。

CRISPR/BMC03-Ec载体系统

提供构建的CRISPR/BMC03-Ec载体系统的完整核苷酸序列如SEQ ID NO:37。

CRISPR/BMC04-Ec载体系统

提供构建的CRISPR/BMC04-Ec载体系统的完整核苷酸序列如SEQ ID NO:38。

CRISPR/BMC05-Ec载体系统

提供构建的CRISPR/BMC05-Ec载体系统的完整核苷酸序列如SEQ ID NO:58。

CRISPR/BMC07-Ec载体系统

提供构建的CRISPR/BMC07-Ec载体系统的完整核苷酸序列如SEQ ID NO:59。

CRISPR/BMC08-Ec载体系统

提供构建的CRISPR/BMC08-Ec载体系统的完整核苷酸序列如SEQ ID NO:60。

CRISPR/BMC09-Ec载体系统

提供构建的CRISPR/BMC09-Ec载体系统的完整核苷酸序列如SEQ ID NO:39。

CRISPR/BMC10-Ec载体系统

提供构建的CRISPR/BMC10-Ec载体系统的完整核苷酸序列如SEQ ID NO:61。

CRISPR/BMC11-Ec载体系统

提供构建的CRISPR/BMC11-Ec载体系统的完整核苷酸序列如SEQ ID NO:62。

CRISPR/BMC12-Ec载体系统

提供构建的CRISPR/BMC12-Ec载体系统的完整核苷酸序列如SEQ ID NO:63。

CRISPR/BMC13-Ec载体系统

提供构建的CRISPR/BMC13-Ec载体系统的完整核苷酸序列如SEQ ID NO:40。

CRISPR/BMC14-Ec载体系统

提供构建的CRISPR/BMC14-Ec载体系统的完整核苷酸序列如SEQ ID NO:64。

CRISPR/BMC15-Ec载体系统

提供构建的CRISPR/BMC15-Ec载体系统的完整核苷酸序列如SEQ ID NO:65。

引导RNA(gRNA)表达载体的设计

用于由BMC01、BMC02、BMC03、BMC04、BMC05、BMC07、BMC08、BMC09、BMC10、BMC11、BMC12、BMC13、BMC14或BMC15核酸酶靶向特异性malQ基因的嵌合gRNA的表达由来自巨大芽孢杆菌(Bacillus megaterium)的SacB RNA聚合酶II启动子(SEQ ID NO:41)驱动(Richhardt等人,Applied Microbiology Biotechnology 86(2010),1959-1965)，并使用大肠杆菌rrnB基因的转录T1和T2终止区(SEQ ID NO:42)终止(Orosz等人，EuropeanJournal of Biochemistry 201(1991),653-659)。嵌合gRNA由与位于大肠杆菌基因组的malQ基因内部的malQ靶特异性24bp间隔子序列(SEQ ID NO:43)融合的恒定的19bp BMC家族茎环序列(SEQ ID NO:32)组成。

最终的gRNA表达盒通过Gibson Assembly Cloning(NEB，法兰克福，德国)插入大肠杆菌穿梭载体中，所述大肠杆菌穿梭载体含有用于附加型繁殖和重组大肠杆菌细胞的选择所有所需的遗传元件。

最终的CRISPR/gRNA-Ec载体系统的构建由Gibson Assembly Cloning(NEB，法兰克福，德国)介导。

提供构建的CRISPR/gRNA-Ec载体系统的完整核苷酸序列如SEQ ID NO:44。

Ku-LigD表达载体的设计

重组大肠杆菌细胞的基因组中CRISPR/BMC引入的双链断裂，通过来自结核分枝杆菌(Mycobacterium tuberculosis)的细菌蛋白Ku和LigD的共表达修复(Della等人,Science 306(2004)，683-685)，在缺乏同源修复模板的情况下，通过靶向DNA的易错非同源末端连接(NHEJ)实现无标记基因敲除(Yang等人,Biotechnology Letters 43(2021),2273-2281)。与产生的附加型BMC表达载体相比，分枝杆菌双组分NHEJ修复器异位提供在含有用于复制起始和标记选择的不同遗传元件的质粒上。Ku-LigD和BMC表达所使用的质粒之间的兼容性导致重组大肠杆菌细胞中稳定共存。为此，Ku-LigD表达质粒具有源自pUC的高拷贝复制起点(SEQ ID NO:45)以及额外来自pRO1614的核苷酸片段(SEQ ID NO:46)，其允许假单胞菌属中基于pUC的克隆载体的稳定维持(West等人,Gene 148(1)(1994)，81-86)。埃希氏菌属/假单胞菌属穿梭载体含有诺尔丝菌素乙酰转移酶基因(nat1)(SEQ ID NO:47)作为用于转化细胞的选择的显性标记(Krügel等人,Gene 127(1)(1993),127-131)。在其N末端，表达的乙酰转移酶蛋白与来自枯草芽孢杆菌的Veg家族蛋白的残基1至30(SEQ IDNO:48)融合，如在质粒pHN15中产生的(Kück和Hoff,Fungal Genetics Reports53(2006),article 3.https://doi.org/10.4148/1941-4765.1106)。融合蛋白的基因表达(SEQ IDNO:49)在来自枯草芽孢杆菌的组成型veg启动子(SEQ ID NO:50)的控制下。来自结核分枝杆菌的ku基因的822bp核苷酸序列(SEQ ID NO:51)和ligD的2280bp核苷酸序列(SEQ IDNO:52)两者由GeneArt(Thermo Fisher Scientific,雷根斯堡,德国)作为合成DNA片段提供。两者蛋白质编码序列的转录和翻译通过使用两种不同的组成型启动子独立控制。ligD的基因表达由来自枯草芽孢杆菌的Veg启动子RNA聚合酶II启动子(SEQ ID NO:53)调节。T7终止子序列(SEQ ID NO:54)用于转录终止。ku的基因表达在来自巨大芽孢杆菌的SacB RNA聚合酶II启动子(SEQ ID NO:55)。的控制下对于转录终止，将fdT终止子(SEQ ID NO:35)(Otsuka&Kunisawa,Journal of Theoretical Biology 97(1982),415-436)融合至编码序列的下游。

提供构建的CRISPR/Ku-LigD-Ec载体系统的完整核苷酸序列如SEQ ID NO:56。

所有克隆DNA元件的鉴别由LGC Genomics(柏林,德国)的桑格测序(Sanger-Sequencing)证实。

3.2大肠杆菌培养与转化

感受态大肠杆菌BW25113细胞的转化

简而言之，将带有用于偶联表达的(CRISPR/BMC(NN)-Ec(实施例4)表达质粒或携带CRISPR/BMC(NN)-Ec和CRISPR/Ku-LigD-Ec(实施例5))质粒的重组大肠杆菌BW25113宿主细胞的单菌落接种至5ml LB-卡那霉素(25μg/ml)培养基(实施例4)或LB-卡那霉素(25μg/ml)/诺尔丝菌素(50μg/ml)培养基(实施例5)，并在水平摇床上以250rpm、在37℃孵育12至14小时。将过夜生长的预培养物稀释到新鲜的60ml LB-卡那霉素(25μg/ml)(实施例4)或LB-卡那霉素(25μg/ml)/诺尔丝菌素(50μg/ml)培养基中(实施例5)，以获得600nm处的光密度(OD600)为0.06。将接种的培养基在水平摇床上以250rpm在30℃孵育，直到培养物在OD600处达到0.2的光密度。为了诱导BMC(NN)核酸酶表达，将600μl 20％L-(+)-阿拉伯糖(w/v)添加到培养物中，终浓度为0.2％(v/v)。继续培养直到培养物在OD600处达到0.5的光密度。随后将50ml培养物转移至一个50ml锥形管中，并通过在4℃以4000×g离心5分钟来收获。将沉淀的细胞重悬于50ml超纯水(冰冷)中，并在4℃以4000×g离心5分钟。

该洗涤程序重复两次。将洗涤的细胞重悬于25ml 10％(w/w)甘油中，并在4℃以4000×g重新离心5分钟。将沉淀的细胞重悬于5ml 10％(w/w)甘油中。在4℃以4000×g离心5分钟的最终的离心步骤后，将细胞重悬于125μl10％(w/w)甘油中。将25μl感受态细胞的等分试样储存在-80℃直至使用。对于转化程序，将感受态细胞的等分试样在冰上解冻，并添加50-100ng质粒DNA(CRISPR/gRNA-Ec)。使用Bio-Rad Gene Pulser Xcell电穿孔系统，在1mm间隙尺寸的电穿孔比色皿中，在1800V、25μF和200Ω对制备的细胞电穿孔。随后，在脉冲后立即向转化的细胞中添加975μL10-β/稳定生长培养基。再生在30℃、以250rpm的水平摇床上完成。最后，将25-100μl细胞悬浮液涂在补充有卡那霉素(25μg/ml)/氨比西林(100μg/ml)(实施例4)或卡那霉素(25μg/ml)/氨比西林(100μg/ml)/诺尔丝菌素(50μg/ml)(实施例5)的选择性M9-葡萄糖琼脂板上。转化的大肠杆菌细胞在选择性琼脂板上的生长在30℃筛选48小时。

实施例4：大肠杆菌耗竭分析，以证明新颖的BMC核酸酶的DNA靶向活性。

为了评估和可视化BMC家族核酸酶的DNA靶向活性，进行了所谓的耗竭分析，其中与阴性对照相比，监测核酸酶靶向后大肠杆菌细胞的存活率。较低的存活率意味着更好的核酸酶活性，因为大肠杆菌细胞不能使用非同源末端连接(NHEJ)修复DNA双链断裂，使用CRISPR核酸酶靶向基因组DNA导致细胞死亡。

对于该实验方法，CRISPR/BMC01-Ec、CRISPR/BMC02-Ec、CRISPR/BMC03-Ec、CRISPR/BMC04-Ec、CRISPR/BMC05-Ec、CRISPR/BMC07-Ec、CRISPR/BMC08-Ec、CRISPR/BMC09-Ec、CRISPR/BMC10-Ec、CRISPR/BMC11-Ec、CRISPR/BMC12-Ec、CRISPR/BMC13-Ec、CRISPR/BMC14-Ec或CRISPR/BMC15-Ec载体系统与含有靶向大肠杆菌malQ基因的间隔子序列的CRISPR/gRNA-Ec载体系统一起共转化。MalQ编码4-α-葡聚糖转移酶，是淀粉代谢的必需基因，但在培养于含有葡萄糖作为碳源的培养基上时对于大肠杆菌细胞的存活不是必需的。

同时，执行使用CRISPR/BMC01-Ec、CRISPR/BMC02-Ec、CRISPR/BMC03-Ec、CRISPR/BMC04-Ec、CRISPR/BMC05-Ec、CRISPR/BMC07-Ec、CRISPR/BMC08-Ec、CRISPR/BMC09-Ec、CRISPR/BMC10-Ec、CRISPR/BMC11-Ec、CRISPR/BMC12-Ec、CRISPR/BMC13-Ec、CRISPR/BMC14-Ec或CRISPR/BMC15-Ec载体系统与缺少靶向大肠杆菌基因组的间隔子序列的CRISPR/gRNA-Ec载体一起共转化的阴性对照实验，以证明Cas蛋白通过特异性间隔子被引导至靶DNA区域的依赖性。

转化并在30℃孵育48小时后，通过计数生长的菌落数量来分析培养板。

结果

所有实验进行5次生物学重复，并结合这些重复获得的结果来评估BMC01、BMC02、BMC03、BMC04、BMC05、BMC07、BMC08、BMC09、BMC10、BMC11、BMC12、BMC13、BMC14和BMC15核酸酶的DNA靶向(示例性板如图2和图3所示)。

如上所述，将BMC01、BMC02、BMC03、BMC04、BMC05、BMC07、BMC08、BMC09、BMC10、BMC11、BMC12、BMC13、BMC14或BMC15核酸酶中的每一种与靶向大肠杆菌malQ基因的gRNA共转化，可视BMC核酸酶的a)活性和b)DNA靶向效率。共转化并将板在30℃孵育48小时后，与阴性对照相比，所有BMC核酸酶(BMC01、BMC02、BMC03、BMC04、BMC05、BMC07、BMC08、BMC09、BMC10、BMC11、BMC12、BMC13、BMC14和BMC15)显示出强烈的菌落减少(>99.9％)，证明BMC核酸酶具有高效的gRNA依赖性DNA靶向。

实施例5：使用选择的BMC核酸酶和Ku-LigD介导的策略在大肠杆菌中NHEJ介导的基因组编辑

为了进一步评估和可视化BMC核酸酶的基因组编辑活性，选择了五种代表性核酸酶(BMC01、BMC03、BMC04、BMC09和BMC13)，并使用Ku-LigD介导的策略敲除位于大肠杆菌基因组上的malQ基因。由于大肠杆菌细胞天然地不能使用非同源末端连接(NHEJ)修复DNA双链断裂，使用CRISPR核酸酶靶向基因组DNA会导致细胞死亡。

为了防止细胞死亡并监测源自NHEJ的malQ基因敲除(malQ是非必需基因，当在这种实验方法所用条件下培养时，malQ的敲除不会对大肠杆菌细胞的表型产生任何影响)，将蛋白质Ku-LigD(两者在DNA修复机制中均发挥重要作用)共转化到大肠杆菌细胞中，为细胞提供使用NHEJ修复DNA双链断裂的能力(参见例如WO 2017/109167)。

对于该实验方法，分别将CRISPR/BMC01-Ec、CRISPR/BMC03-Ec、CRISPR/BMC04-Ec、CRISPR/BMC09-Ec或CRISPR/BMC13-Ec载体与CRISPR/Ku-LigD-Ec载体系统和含有靶向大肠杆菌malQ基因的间隔子序列的CRISPR/gRNA-Ec载体系统一起共转化。

同时，执行分别使用CRISPR/BMC01-Ec、CRISPR/BMC03-Ec、CRISPR/BMC04-Ec、CRISPR/BMC09-Ec或CRISPR/BMC13-Ec载体与CRISPR/Ku-LigD-Ec载体系统和缺少靶向大肠杆菌基因组的间隔子序列的CRISPR/gRNA-Ec载体一起共转化的阴性对照实验，以证明Cas蛋白通过特异性间隔子引导至靶DNA区域的依赖性。

结果

所有实验均进行5次生物学重复，并结合这些重复获得的结果来评估BMC01、BMC03、BMC04、BMC09和BMC13核酸酶的基因组编辑活性。示例性板如图2和图3所示。

为了可视化BMC核酸酶的基因组编辑活性，使用上述Ku-LigD介导的NHEJ策略进行实验。首先，在各自的BMC核酸酶、gRNA(靶向malQ基因)和Ku-LigD表达系统共转化48小时后，基于与阴性对照相比较生长的菌落以及细胞耗竭分析(实施例4)中获得的结果对板评估。总体而言，与阴性对照相比，所有测试的BMC核酸酶显示出强烈的菌落减少(≈98％)，但与实施例4中获得的结果(>99.9％)相比，菌落减少较弱。这些结果可以用以下事实来解释：Ku-LigD蛋白的表达使大肠杆菌能够使用NHEJ DNA修复机制修复DNA双链断裂。尽管从DNA双链断裂的修复来看，已知NHEJ介导的DNA修复可以是易错的，导致基因组内靶位置处的indels(插入/删除)和移码突变。BMC介导的DNA双链断裂通过NHEJ机制修复，但基因组序列由于引入indels而改变的那些细胞能够在BMC处理后存活下来，因为PAM和/或间隔子序列由于indels突变而改变，BMC核酸酶不能再次靶向基因组。

为了证明malQ基因被敲除，从每个平板(每种BMC核酸酶10个菌落)中分离出两个菌落，并使用桑格测序对感兴趣的基因组基因座(malQ基因内部)测序。

该实验方法的结果显示，对于所有选择的BMC核酸酶，在序列水平上均可检测到NHEJ介导的malQ基因敲除。

BMC01：10个评估的菌落中有9个显示出NHEJ介导的malQ基因敲除

BMC03：10个评估的菌落中有7个显示NHEJ介导的malQ基因敲除

BMC04：10个评估的菌落中有7个显示NHEJ介导的malQ基因敲除

BMC09：10个评估的菌落中有10个显示出NHEJ介导的malQ基因敲除

BMC13：10个评估的菌落中有8个显示出NHEJ介导的malQ基因敲除

为了证明BMC核酸酶表征的完整工作流程，图4显示了BMC09核酸酶的表征：阴性对照、细胞耗竭分析、Ku-LigD共表达的菌落生长以及一个大肠杆菌菌落的靶基因座的测序(malQ基因内部71bp删除)。

概括

总之，实施例4和5中获得的结果证明本文公开的所有BMC蛋白都是新颖的CRISPR核酸酶，具有高DNA靶向和基因组编辑效率并且与迄今为止描述的CRISPR核酸酶具有低相似性。

序列表

<110> BRAIN生物技术有限公司(BRAIN Biotech AG)

<120> 来自宏基因组的新颖的CRISPR-Cas核酸酶

<130> PA-1696/019-001

<150> EP 21 000 063.4

<151> 2021-03-02

<160> 65

<170> BiSSAP 1.3.6

<210> 1

<211> 1295

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC01的AA序列

<400> 1

Met Ile Phe Asn Asn Phe Thr Gln Lys Phe Ser Leu Ser Lys Thr Leu

1 5 10 15

Arg Phe Glu Leu Arg Pro Val Asp Ala Gly Gly Asn Val Ile Thr Asp

20 25 30

Leu Thr Ile Phe Glu Glu Thr Ile Lys Asn Asp Gln Lys Arg Tyr Glu

35 40 45

Ala Tyr Leu Ala Ile Lys Pro Leu Val Asp Glu Thr His Lys His Phe

50 55 60

Ile Gln Thr Val Leu Ser Gly Leu Thr Asp Leu Ile Lys Ser Asp Glu

65 70 75 80

Met Lys Asn Tyr Leu Glu His Lys Asn Leu Ile Arg Gln Lys Asp Val

85 90 95

Glu Glu Lys Val Lys Thr Lys Ser Ile Asp Val Ile Asn Lys Ile Glu

100 105 110

Lys Asp Trp Arg Lys Arg Val Ser Asp Ser Phe Thr Lys His Pro Gln

115 120 125

Tyr Lys Lys Met Phe Asp Lys Thr Leu Phe Ala Asp Glu Ser Pro Leu

130 135 140

Tyr Lys Leu Ala Glu Asn Asp Phe Gln Arg Ser Gln Ile Lys Ile Phe

145 150 155 160

Glu Lys Phe Thr Gly Tyr Phe Asn Gly Phe His Glu Asn Arg Lys Asn

165 170 175

Leu Tyr Val Ala Glu Lys Gln Gly Thr Ala Ile Ala Asn Arg Val Ile

180 185 190

Asn Glu Asn Leu Pro Lys Phe Ile Glu Asn Ala Asn Lys Leu Lys Arg

195 200 205

Ala Phe Glu Lys Tyr Pro Glu Phe Leu Ser Lys Ile Ser Glu Asp Lys

210 215 220

Ser Phe Gln Ala Leu Leu Ile Lys Asn Gln Leu Ser Leu Glu Lys Leu

225 230 235 240

Leu Gln Pro Leu Thr Phe Asn Leu Leu Ile Ser Gln Thr Gly Ile Asp

245 250 255

Ser Tyr Asn Glu Val Leu Gly Gly Tyr Thr Pro Glu Asn Ser Glu Pro

260 265 270

Ile Lys Gly Leu Asn Gln Leu Ile Asn Leu Tyr Arg Gln Lys Ile Asn

275 280 285

Leu Ala Arg Asn Asp Phe Pro Asn Leu Ala Pro Leu Tyr Lys Gln Leu

290 295 300

Leu Ser Asp Arg Glu Thr Asn Ser Val Val Tyr Lys Pro Leu Glu Asn

305 310 315 320

Val Ala Asp Val Tyr Ser Ser Val Phe Glu Leu Cys Gln Asn Leu Leu

325 330 335

Ser Lys Gln Ser Asp Ile Asn Lys Trp Ile Glu Asp Ile Asn Ile Ser

340 345 350

Ser Gly Gln Ile Trp Ile Tyr Lys Ser His Leu Ser Gly Leu Ser Val

355 360 365

Met Leu Phe Gly Glu Ser Gly Trp Gly Leu Ile Pro Arg Ile Leu Asn

370 375 380

Ile Ser Glu Asp Asp Glu Glu Glu Ile Ile Lys Ser Lys Ser Lys Lys

385 390 395 400

Ser Gln Gln Glu Tyr Phe Ser Phe Ala Glu Ile Gly Asn Ala Ile Asn

405 410 415

Asn Tyr Ser Phe Glu Asp Val Asn Ile Lys Ala Leu Ala Lys Gln Gly

420 425 430

Leu Cys Leu Trp Gln Lys Gln Gly Asn Glu Arg Leu Ile Lys Phe Gly

435 440 445

Lys Leu Phe Ser Gln Met Gln Asn Glu Leu Gln Ser Pro Lys Glu Lys

450 455 460

Trp Asp Ser Thr Glu Lys Glu Lys Ile Lys Glu Leu Leu Asp Thr Gly

465 470 475 480

Leu Glu Phe Val His Trp Leu Lys Val Ile Ser Asn Gln Pro Glu Asp

485 490 495

Lys Asp Glu Val Phe Tyr Ala Glu Trp Gln Ala Leu Thr Asp Thr Trp

500 505 510

Arg Gly Leu Pro Lys Leu Tyr Asp Arg Val Arg Asn Phe Ala Thr Lys

515 520 525

Lys Asp Tyr Ser Gln Asn Lys Leu Lys Ile Asn Phe Asp Lys Gly Thr

530 535 540

Leu Leu Asn Gly Trp Asp Thr Asn Lys Glu Thr Asp Asn Leu Gly Ile

545 550 555 560

Leu Leu Glu Asn Lys Gly Gln Tyr Tyr Leu Gly Ile Met Lys Asp Ser

565 570 575

Ser Ile Phe Asp Tyr Gln Trp Asp Ile Asp Asn Phe Gln Asn Pro Asn

580 585 590

Ser Lys Gln Ser Val Ala Lys Lys Asn Leu His Glu Ala Ile Val Ser

595 600 605

Asp Asn Thr Gln Asp Cys Trp Ser Lys Ile Val Tyr Lys Leu Leu Pro

610 615 620

Gly Pro Asn Lys Met Leu Pro Lys Val Phe Phe Ser Asp Lys Arg Gln

625 630 635 640

Lys Tyr Phe Gly Ala Asp Glu Lys Val Ile Asp Ile Asn Glu Asn Gly

645 650 655

Arg His Lys Lys Gly Asp Asn Phe Asn Ile Ser Asp Cys His Tyr Leu

660 665 670

Ile Asp Phe Tyr Lys Thr Ala Ile Asn Lys His Pro Glu Trp Ser Gln

675 680 685

Phe Asn Phe Lys Phe Ser Ala Thr Lys Ser Tyr Glu Asp Ile Ser Gln

690 695 700

Phe Tyr His Glu Val Gln Asn Gln Gly Tyr Arg Ile Glu Phe Asp His

705 710 715 720

Ile Arg Lys Asp Tyr Ile Gln Lys Met Val Ser Glu Gly Lys Leu Phe

725 730 735

Leu Phe Lys Ile His Ser Lys Asp Phe Ser Ser Tyr Ala Lys Gly Arg

740 745 750

Pro Asn Met His Thr Ile Tyr Trp Arg Ala Ile Phe Asn Pro Glu Asn

755 760 765

Leu Ala Asn Val Val Val Lys Leu Asn Gly Glu Ala Glu Phe Phe Tyr

770 775 780

Arg Lys Ser Ser Lys Asp Arg Ile Ile Ser His Pro Gln Gly Leu Glu

785 790 795 800

Val Ser Asn Lys Asn Pro Ser Asn Pro Lys Lys Thr Ser Arg Phe Ala

805 810 815

Tyr Asp Leu Ile Lys Asp Lys Arg Phe Thr Gln Asp Lys Phe Phe Phe

820 825 830

His Val Pro Ile Thr Leu Asn Phe Arg Glu Gly Glu Gly Tyr Arg Phe

835 840 845

Asn Gln Ser Val Ile Arg Glu Leu Lys Lys Tyr Tyr Gln Thr Asp Lys

850 855 860

Ala Asn Leu His Ile Ile Gly Ile Asp Arg Gly Glu Arg His Leu Leu

865 870 875 880

Tyr Tyr Cys Val Ile Asn Val Ala Ser Gly Lys Ile Val Glu Gln Gly

885 890 895

Ser Phe Asn Gln Ile Ser Thr Asn Tyr Thr Pro Glu Gln Ile Thr Asp

900 905 910

Asp Gly Glu Ile Ile Lys Gly Glu Thr Val Asn Lys Thr Thr Asp Tyr

915 920 925

His Asn Leu Leu Asn Thr Lys Glu Gly Asp Arg Gln Lys Ala Arg Lys

930 935 940

Asn Trp Gln Thr Ile Glu Asn Ile Lys Glu Leu Lys Ala Gly Tyr Leu

945 950 955 960

Ser Asn Val Ile His Lys Ile Ser Gln Leu Met Val Lys Tyr Asn Ala

965 970 975

Phe Val Val Leu Glu Glu Leu Lys Tyr Gly Phe Lys Arg Gly Arg Phe

980 985 990

Lys Val Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys Ala Leu Ile Asp

995 1000 1005

Lys Leu Asn Tyr Leu Val Phe Lys Asp Arg Ala Pro Ala Glu Val Gly

1010 1015 1020

Gly Val Leu Asn Ala Leu Gln Leu Ala Pro Pro Val Ala Ser Tyr Ile

1025 1030 1035 1040

Asp Ile Gly Lys Gln Ala Gly Phe Leu Phe Tyr Val Pro Ala His His

1045 1050 1055

Thr Ser Lys Ile Cys Pro Trp Thr Gly Phe Val Asp Trp Leu Lys Pro

1060 1065 1070

Arg Tyr Asp Gly Ile Asp Lys Ala Lys Ala Phe Phe Thr Cys Phe Glu

1075 1080 1085

Ser Ile His Phe Asn Thr Gln Lys Asn Tyr Phe Glu Phe Ala Phe Asp

1090 1095 1100

Tyr Glu Lys Phe Arg Gly Asn Ile Asn His Leu Pro Glu Gly Leu Lys

1105 1110 1115 1120

Arg Thr Ser Trp Thr Leu Cys Ser His Asn Ser Leu Arg Asp Ile Ala

1125 1130 1135

Thr Lys Asp Lys Asn Gly Asn Trp Pro Tyr Lys Gln Ile Asn Leu Thr

1140 1145 1150

Ala Glu Leu Leu Glu Ile Leu Lys Thr Leu Asn Pro Arg Asn Gly Glu

1155 1160 1165

Asn Leu Val Glu Arg Ile Ile Glu Met Asn Asp Lys Lys Phe Phe Glu

1170 1175 1180

Ser Leu Met Trp Ala Leu Arg Val Leu Leu Gln Leu Arg Tyr Gly Tyr

1185 1190 1195 1200

Ile Lys Arg Asn Asn Glu Gly Ile Ile Ile Glu Glu Val Asp Tyr Ile

1205 1210 1215

Leu Ser Pro Val Ala Asn Glu Asn Gly Glu Phe Phe Asp Ser Arg Asn

1220 1225 1230

Phe Val Asn Ile Glu Lys Ala Asp Phe Pro Lys Asp Ala Asp Ala Asn

1235 1240 1245

Gly Ala Tyr Asn Ile Ala Arg Lys Gly Leu Leu Leu Ile Ala Gln Asn

1250 1255 1260

Ile Asn Asn Ala Lys Ile Asn Asp Lys Gly Glu Val Lys Cys Asp Leu

1265 1270 1275 1280

Gln Ile Asp Lys Thr Thr Trp Phe Asn Trp Val Gln Ser Lys Ser

1285 1290 1295

<210> 2

<211> 1107

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC02的AA序列

<400> 2

Met Pro Lys Glu Asp Phe Asp Lys Ala Cys Ile Tyr Leu Ser Asn Phe

1 5 10 15

Asp Lys Phe Ser Thr Tyr Phe Val Gly Phe Asn Gln Asn Arg Glu Asn

20 25 30

Leu Tyr Thr Asp Glu Glu Gln Ala Thr Ala Ile Pro Tyr Arg Ile Ile

35 40 45

Asn Asp Asn Met Val Arg His Phe Asp Asn Cys Arg Lys Phe Glu Lys

50 55 60

Ile Val Lys Lys Tyr Gly Asp Ile Ser Asn Val Leu Ser Thr Tyr Lys

65 70 75 80

Glu Phe Phe Ala Pro Asp Cys Phe Lys Asn Lys Leu Asn Gln Ser Gln

85 90 95

Ile Asp His Tyr Asn Asn Thr Ile Gly His Thr Ala Asp Asp Ile Tyr

100 105 110

Gly Val Gly Ile Asn Gln Ile Leu Ser Lys Tyr Lys Gln Asp Asn Lys

115 120 125

Leu Asn Ser Ser Asp Leu Pro Leu Ile Ser Lys Leu Tyr Lys Gln Ile

130 135 140

Leu Ser Asp Thr Glu Ser Tyr Ala Ile Glu Asn Phe Ala Asp Asp Lys

145 150 155 160

Met Met Leu Asn Ala Val Asp Lys Glu Tyr Ser Arg Ile Lys Glu Asn

165 170 175

Asp Val Phe Ile Asn Ile Glu Thr Cys Met Asn Glu Tyr Leu Thr Leu

180 185 190

Glu Asn Ser His Met Ile Tyr Leu Lys Asn Asp Ser Ser Leu Thr Asp

195 200 205

Ile Ser Asn Lys Leu Trp Glu Asp Trp Ala Phe Val Lys Asn Ala Ile

210 215 220

Gln Lys Tyr Ser Lys Glu Ile Leu Cys Leu Ser Asp Lys Lys Ile Glu

225 230 235 240

Asp Met Leu Lys Met Ser His Tyr Ser Ile Ser Phe Val Gln Asn Ser

245 250 255

Val Tyr Tyr Tyr Val Asp Asn Tyr Met Glu Ser Cys Glu Asp Lys Arg

260 265 270

Lys Ser Ile Ile Asp Tyr Ile Lys Thr Phe Tyr Ser Ile Lys Tyr Asn

275 280 285

Asn Val Phe Ser Cys Tyr Lys Glu Ala Glu Ala Val Leu Arg Leu Asp

290 295 300

Ser Ile His Lys Asn Arg Arg Ser Pro Val Asp Lys Asn Gly Ile Gly

305 310 315 320

Gly Glu Gly Phe Ala Gln Ile Glu Lys Ile Lys Asn Phe Leu Asp Ser

325 330 335

Ile Leu Glu Val Lys Asn Phe Leu Asn Pro Leu Tyr Leu Ile Lys Ser

340 345 350

Gly Lys Met Ala Glu Ile Glu Asp Lys Ser Glu Glu Phe Tyr Asn Arg

355 360 365

Phe Asn Glu Leu Tyr Asn Ser Leu Ser Asp Thr Thr Tyr Leu Tyr Asn

370 375 380

Lys Val Arg Asn Tyr Leu Thr Lys Lys Pro Tyr Lys Lys Glu Lys Phe

385 390 395 400

Lys Met Asn Phe Glu Asn Ser Thr Leu Leu Ser Gly Trp Asp Val Asn

405 410 415

Lys Glu Asn Cys Ser Asn Ser Ile Ile Leu Ile Arg Asn Gly Lys Tyr

420 425 430

Tyr Leu Gly Ile Ile Asp Lys Gln Cys Gly Asn Met Phe Asn Phe Lys

435 440 445

Ile Asp Ala Glu Asp Asn Glu Lys Lys Arg Lys Glu Lys Glu Asp Leu

450 455 460

Ala Glu Asp Ile Leu Ser Asp Gly Ser Asp Ser Tyr Tyr Glu Lys Met

465 470 475 480

Val Tyr Lys Leu Leu Pro Asp Pro Ser Lys Met Leu Pro Lys Val Phe

485 490 495

Phe Ser Asn Lys Ser Ile Asp Phe Tyr Ala Pro Ser Glu Asp Ile Lys

500 505 510

Tyr Ile Arg Glu Asn Gly Leu Phe Lys Lys Asp Ala Lys Asn Lys Lys

515 520 525

Ala Leu Tyr Ile Trp Ile Glu Phe Met Gln Asn Ser Leu Lys Lys His

530 535 540

Pro Glu Trp Ser Asn Tyr Phe Asn Phe Asn Phe Lys Pro Ser Thr Glu

545 550 555 560

Tyr Ala Asp Val Ser Glu Phe Tyr Lys Gln Val Ser Asp Gln Gly Tyr

565 570 575

Ser Leu Ser Phe Asp Lys Ile Lys Asp Ser Tyr Ile Glu Ser Lys Ile

580 585 590

Lys Ser Gly Glu Leu Phe Leu Phe Glu Ile Tyr Asn Lys Asp Phe Ser

595 600 605

Pro Tyr Ser Lys Gly Asn Pro Asn Leu His Thr Ile Tyr Trp Lys Ser

610 615 620

Ile Phe Asp Lys Glu Asn Leu Ser Asn Val Val Ile Lys Leu Asn Gly

625 630 635 640

Gln Ala Glu Ile Phe Phe Arg Pro Ala Ser Leu Lys Arg Asn Glu Val

645 650 655

Val Val His Arg Ala Lys Glu Asn Ile Leu Asn Lys Asn Pro Leu Asn

660 665 670

Pro Lys Lys Glu Ser Met Phe Glu Tyr Asp Ile Val Lys Asp Lys Arg

675 680 685

Tyr Thr Gln Asp Lys Phe Phe Phe His Cys Pro Ile Thr Leu Asn Phe

690 695 700

Lys Ser Gly Asn Val Gly Lys Phe Asn Asp Lys Val Asn Gln Phe Leu

705 710 715 720

Lys Asn Asn Pro Asp Val Asn Val Ile Gly Phe Asp Arg Gly Glu Arg

725 730 735

His Leu Leu Tyr Cys Asn Val Leu Asn Gln Lys Gly Glu Ile Ile Glu

740 745 750

Gln Lys Ser Phe Asn Val Ile Glu Asn Lys Asn Asn Gly Ile Thr Gln

755 760 765

Lys Val Asp Tyr His Asn Leu Leu Asp Arg Lys Glu Lys Glu Arg Asp

770 775 780

Ala Ser Arg Lys Ser Trp Ser Thr Ile Glu Asn Ile Lys Glu Leu Lys

785 790 795 800

Glu Gly Tyr Leu Ser Asn Val Val His Glu Ile Ser Glu Leu Ile Ile

805 810 815

Lys Tyr Asn Ala Ile Leu Val Leu Glu Asp Leu Asn Phe Glu Phe Lys

820 825 830

Lys Gly Arg Phe Lys Ile Glu Lys Gln Val Tyr Gln Lys Phe Glu Lys

835 840 845

Ala Leu Ile Asp Lys Leu Ser Tyr Met Val Phe Lys Lys Glu Glu Ser

850 855 860

Asn Lys Pro Gly His Ser Leu Met Ala Tyr Gln Leu Ala Ser Pro Phe

865 870 875 880

Glu Ser Phe Gln Lys Leu Gly Lys Gln Cys Gly Phe Ile Phe Tyr Val

885 890 895

Asn Ser Asn Tyr Thr Ser Lys Ile Asp Pro Val Thr Gly Phe Val Asn

900 905 910

Leu Leu Lys Ile Lys Tyr Glu Ser Val Asp Lys Ser Cys Lys Phe Ile

915 920 925

Asn Asp Lys Phe Asp Asp Ile Arg Tyr Asn Ala Asp Arg Glu Tyr Phe

930 935 940

Glu Phe Thr Phe Asp Asn Gly Lys Trp Thr Ala Cys Ser His Gly Lys

945 950 955 960

Glu Arg Tyr Arg Tyr Asn Arg Asn Asp Lys Lys Tyr Asn Cys Phe Asp

965 970 975

Val Thr Glu Glu Leu Lys Ser Leu Phe Asn Lys Tyr Glu Ile Asp Phe

980 985 990

Lys Ala Gly Thr Asp Ile Lys Lys Ser Ile Cys Gln Val Gln Asp Lys

995 1000 1005

Asn Phe His Ser Glu Leu Leu Phe Asn Leu Ser Leu Ile Val Gln Leu

1010 1015 1020

Arg His Thr Tyr Lys Asn Gly Asp Ile Glu Lys Asp Phe Ile Leu Ser

1025 1030 1035 1040

Pro Ile Met Asp Lys Glu Thr Gly Lys Phe Phe Asp Ser Arg Glu Tyr

1045 1050 1055

Glu Asn Leu Glu Asn Ser Leu Leu Pro Thr Asn Ala Asp Ser Asn Gly

1060 1065 1070

Ala Tyr Asn Ile Ala Arg Lys Gly Leu Leu Thr Leu Arg Gln Ile Asp

1075 1080 1085

Lys Asp Gly Lys Pro Ser Asn Ile Ser Asn Lys Glu Trp Phe Asp Phe

1090 1095 1100

Val Gln Lys

1105

<210> 3

<211> 1323

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC03的AA序列

<400> 3

Met Glu Lys Asn Leu Asn Tyr Leu Glu Arg Phe Thr Lys His Tyr Asn

1 5 10 15

Thr Lys Lys Thr Leu Lys Asn Lys Leu Ile Pro Tyr Gly Asn Thr Ala

20 25 30

Glu Asn Met Ile Lys Asn Asn Ile Ile Ser Asn Glu Lys Gln Ile Ile

35 40 45

Leu Ser Ala Lys Lys Gln Lys Gln Ser Ile Asp Phe Leu Gln Lys Glu

50 55 60

Tyr Ile Glu Asn Lys Leu Ser Glu Ile Thr Leu Pro Tyr Leu Asn Asp

65 70 75 80

Tyr Tyr Asn Glu Phe Ile Lys Asn Lys Lys Glu Arg Asp Thr Asp Val

85 90 95

Ile Asp Asn Ile Glu Ile Ala Met Arg Lys His Ile Ser Lys Ser Leu

100 105 110

Thr Glu Asn Gly Asn His Lys Lys Tyr Leu Asn Lys Glu Val Phe Asp

115 120 125

Ile Ile Ser Glu Lys Lys Glu Leu Tyr Tyr Asp Val Thr Phe Lys Arg

130 135 140

Asn Ala Thr Tyr Leu Ser Asp Tyr Phe Gln Ser Arg Val Asn Leu Tyr

145 150 155 160

Lys Asp Ser Asn Lys Ser Ser Thr Ile Ala Ser Arg Cys Ile Asn Ile

165 170 175

Asn Leu Pro Ile Phe Ala Lys Asn Ile Val Leu Phe Asn Phe Ile Lys

180 185 190

Asn Lys Ala Asn Ile Ile Phe Asp Asp Leu Lys Glu Ile Thr Asp Asp

195 200 205

Glu Tyr Thr Leu Asp Ser Ile Phe Ser Ile Asp Phe Phe Asn Met Val

210 215 220

Leu Ser Gln Lys Gly Ile Asp Tyr Tyr Asn Thr Ile Leu Gly Gly Met

225 230 235 240

Thr Lys Glu Asp Gly Lys Lys Ile Lys Gly Ile Asn Glu Tyr Ile Asn

245 250 255

Leu Tyr Asn Gln Asn Val Lys Asp Glu Lys Asn Lys Leu Pro Tyr Pro

260 265 270

Lys Lys Leu Lys Lys Gln Leu Leu Ser Asp Ile Asn Ser Tyr Ser Ala

275 280 285

Arg Phe Glu Lys Phe Asp Thr Glu Gln Glu Met Val Lys Ser Ile Lys

290 295 300

Ser Leu Val Glu Asn Asp Leu Phe Gln Gly Glu Leu Phe Asp Lys Lys

305 310 315 320

Val Asp Ile Leu Lys Glu Thr Glu Arg Leu Leu Glu Arg Ile Ser Glu

325 330 335

Tyr Asp Ser Asn Ala Leu Phe Ile Thr Glu Lys Asn Ile Ser Tyr Ile

340 345 350

Ser Ile Asp Ile Phe Asn Asp Lys Phe Phe Ile Lys Thr Ala Ile Glu

355 360 365

Tyr Phe Tyr Glu Asn Asn Ile Cys Pro Asp Tyr Arg Lys Ile Tyr Asp

370 375 380

Asn Ala Ser Lys Asn Lys Arg Lys Gln Leu Gly Lys Glu Lys Asn Lys

385 390 395 400

Val Ile Lys Gln Lys Ser Phe Ser Ile Ser Phe Leu Gln Asp Ala Ile

405 410 415

Thr Phe Tyr Ile Lys Asp Ser Gly Ile Asn Lys Ile Ser Glu Asn Cys

420 425 430

Ile Ile Asn Tyr Phe Lys Lys His Thr Ile Lys Leu Thr Glu Leu Phe

435 440 445

Gly Lys Val Tyr Glu Asp Tyr Asn Val Ile Lys Pro Ile Leu Glu Gln

450 455 460

His Leu Val Glu Tyr Glu Gly Lys Ser Ile Ser Lys Asp Ser Ile Lys

465 470 475 480

Arg Ser Lys Ile Lys Leu Phe Ser Glu Asn Leu Lys Asn Ile Phe Tyr

485 490 495

Phe Ile Arg Pro Leu Asn Ile Ile Glu Glu Ala Leu Asn Tyr Asp Thr

500 505 510

Ser Phe Tyr Thr Pro Phe Asn Ile Leu Phe Glu Glu Ile Lys Lys Phe

515 520 525

Asn Lys Leu Tyr Asp Lys Ile Arg Asn Phe Ile Thr Lys Lys Pro Phe

530 535 540

Asn Asp Glu Glu Ile Asn Leu Tyr Phe Gly Ile Pro Asn Leu Gly Gly

545 550 555 560

Gly Phe Ile Asp Ser Gln Thr Asp Lys Ser Asn Asn Gly Thr Gln Tyr

565 570 575

Cys Thr Tyr Leu Phe Arg Lys Lys Asn Gln Leu Leu Asn Trp Glu Tyr

580 585 590

Phe Val Gly Ile Ser Lys Asn Lys His Leu Phe Arg Glu Lys Glu Asn

595 600 605

Ile Glu Leu Asn Ser Asp Glu Thr Ser Phe Gln Arg Tyr Ser Phe Tyr

610 615 620

Thr Pro Lys Asp Lys Ser Ile Tyr Gly Ser Ser Tyr Phe Ser Ala Asn

625 630 635 640

Glu Lys Asn Tyr Lys Asp Asp Lys Gln Glu Phe Ile Asn Ile Ile Asn

645 650 655

Asn Ile Val Asn Asn Ser Gly Asn Glu Leu Ala Ile Lys Glu Leu Lys

660 665 670

Lys Tyr Ile Asn Asn Ser Thr Glu Asn Ser Glu Thr Pro Asn Gly Cys

675 680 685

Leu Ser Val Leu Lys Asn Lys Cys Asn Glu Ile Tyr Asn Leu Val Ile

690 695 700

Asn His Asp Asp Phe Lys Glu Lys Asn Glu Asp Ile Ile Asn Lys Leu

705 710 715 720

Lys Asn Thr Leu Ser Lys Leu Ser Lys Val Pro Gln Ala Lys Glu Leu

725 730 735

Ile Asn Lys Lys Tyr Asn Leu Phe Ser Glu Ile Ile Ser Asp Ile Ser

740 745 750

Glu Ile Cys Leu Thr Ser Thr Gln Arg Tyr Tyr Pro Ile Asp Asp Glu

755 760 765

Glu Leu Asn Ser Ala Leu Asn Asp Glu Asn Lys Pro Leu Tyr Phe Phe

770 775 780

Lys Ile Ser Asn Lys Asp Leu Ser Ala Asp Glu Asn Ile Leu Asn Gly

785 790 795 800

Lys Arg Lys Ser Lys Gly Lys Asp Asn Ile His Thr Met Ile Leu Arg

805 810 815

Ala Met Met Asp Asp Asn Val Thr Asn Ile Ile Pro Thr Ser Cys Lys

820 825 830

Ile Ser Met Arg Glu Ala Ser Ile Lys Lys Asp Asp Leu Val Ile His

835 840 845

Lys Ala Asn Glu Pro Ile Lys Leu Lys Asn Ser Leu Ala Asn Lys Lys

850 855 860

Glu Ser Thr Phe Ser Tyr Asp Ile Thr Lys Asp Arg Arg Tyr Ser Arg

865 870 875 880

Asp Glu Phe Phe Phe Ser Ile Thr Ala Ser Ile Asn Ser Asp Cys Lys

885 890 895

Glu Asn Asp Tyr Tyr Phe Asn Gln Lys Val Asn Glu Tyr Leu Lys Asn

900 905 910

Asn Ser Lys Ile Asn Leu Leu Ala Val Asp Leu Gly Glu Thr Asn Ile

915 920 925

Ile Thr Ile Ser Val Ile Asp Gln Lys Gly Asn Ile Ile Leu Gln Lys

930 935 940

Asp Leu Asp Lys Phe Ile Asn Lys Glu Lys Asn Ile Ile Thr Asp Phe

945 950 955 960

Asn Leu Leu Leu Ser Asn Arg Ser Lys Glu Arg Asp Ile Ala Lys Arg

965 970 975

Asp Trp Gln Glu Gln Gln Gln Ile Lys Asn Leu Lys Glu Gly Met Ile

980 985 990

Ser Cys Ile Ile His Glu Ile Cys Lys Leu Met Ile Glu His Asn Ala

995 1000 1005

Ile Leu Ile Met Glu Asp Leu Asp Ala Asn Phe Lys Asn Arg Lys Lys

1010 1015 1020

Arg Ile Glu Lys Ala Ile Tyr Gln Lys Phe Glu Ile Ala Ile Leu Glu

1025 1030 1035 1040

Lys Leu Asn Asn Leu Val Phe Lys Asp Ile Pro Ile Asn Glu Val Gly

1045 1050 1055

Ser Val Thr Lys Pro Leu Gln Leu Ser Asp Lys Phe Glu Thr Tyr Glu

1060 1065 1070

Lys Val Gly Asn Gln Ser Gly Phe Val Phe Lys Val Ser Pro Phe Tyr

1075 1080 1085

Thr Ser Ile Ile Asp Pro Thr Thr Gly Phe Ile Asn Leu Phe Lys Lys

1090 1095 1100

Asn Phe Glu Ser Val Lys Tyr Ser Ile Glu Phe Phe Ser Lys Phe Glu

1105 1110 1115 1120

Ser Ile Arg Tyr Asn Thr Lys Glu Lys Tyr Phe Glu Phe Ala Phe Asp

1125 1130 1135

Tyr Lys Asn Phe Lys Glu Ile Lys Tyr Thr Glu Asn Ile Lys Thr Asp

1140 1145 1150

Trp Val Ala Cys Thr Thr Asn Ile Asp Arg Tyr Glu Tyr Asp Lys Lys

1155 1160 1165

Asn Lys Ile Tyr Lys Lys Tyr Asp Val Thr Thr Asp Leu Lys Asn Leu

1170 1175 1180

Phe Glu Asn Glu Glu Ile Tyr Tyr Gln Lys Gly Glu Asn Ile Leu Asp

1185 1190 1195 1200

Val Ile Leu Lys Lys Asn Asn Arg Glu Phe Phe Glu Lys Leu Thr Asn

1205 1210 1215

Leu Leu Lys Ile Thr Met Leu Phe Arg Tyr Arg Asn Ser His Leu Lys

1220 1225 1230

Leu Asp Tyr Ile Ser Ser Pro Val Lys Asn Ser Asn Gly Glu Phe Phe

1235 1240 1245

Ser Thr Glu Asn Gly Leu Glu Asn Tyr Pro Ile Asp Ser Asp Thr Asn

1250 1255 1260

Gly Ala Tyr His Ile Ala Leu Lys Gly Lys Met Ile Leu Asp Arg Ile

1265 1270 1275 1280

Asn Ser Asn Ser Ser Glu Lys Leu Asp Thr Tyr Ile Ser Ile Glu Asp

1285 1290 1295

Trp Leu Lys Phe Ile Gln Lys Phe Ser Val Asn Lys Ile Thr Glu Thr

1300 1305 1310

Lys Lys Asn Lys Lys Ile Asn Ile Lys Tyr Val

1315 1320

<210> 4

<211> 1324

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC04的AA序列

<400> 4

Met Lys Asn Leu Thr Glu Phe Thr Gly Leu Tyr Pro Val Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Lys Pro Thr Asp Asp Phe Asn Trp Glu Thr Phe

20 25 30

Leu Glu Ser Thr Ile Phe Lys His Asp Gln Glu Arg Ala Glu Ala Tyr

35 40 45

Pro Ile Val Lys Val Ile Val Asp Gln Phe His Lys Trp Phe Ile Glu

50 55 60

Asp Ala Leu Asn Lys Ser Thr Ile Asn Trp Asn Ser Leu Tyr Asp Ala

65 70 75 80

Tyr Phe Ala Pro Lys Asn Glu Asn Ser Val Glu Asn Leu Arg Lys Glu

85 90 95

Gln Asp Lys Ile Arg Lys Glu Ile Val Asp Thr Tyr Phe Lys Lys His

100 105 110

Asp Trp Trp Lys Tyr Val Ser Lys Asp His Ser Lys Leu Phe Lys Ile

115 120 125

Glu Leu Pro Ala Leu Leu Ser Asp Asp Ala Phe Ile Tyr Glu Ile Asn

130 135 140

Asp Lys Tyr Pro Asn Tyr Thr Gln Glu Ile Leu Ile Asp Ala Leu Ala

145 150 155 160

Lys Phe Gln Asn Phe Ser Val Tyr Phe Gly Gly Tyr Phe Lys Asn Arg

165 170 175

Asp Asn Met Tyr Lys Ser Asp Ala Gln Ser Thr Ser Ile Ala Asn Arg

180 185 190

Ile Val Asn Glu Asn Phe Thr Lys Phe Ala Asp Asn Ile Lys Ile Tyr

195 200 205

Asn Arg Leu Lys Glu Asn Cys Leu Ser Glu Leu Gln Lys Val Glu Leu

210 215 220

Asp Phe Thr Asp Glu Leu Thr Gly Leu Thr Phe Asp Asp Ile Phe Ser

225 230 235 240

Pro Ser Tyr Phe Asn Lys Cys Leu Thr Gln Lys Gly Ile Glu Lys Leu

245 250 255

Asn Leu Tyr Ile Gly Gly Lys Thr Gly Lys Asn Lys Glu Asp Lys Val

260 265 270

Phe Gly Ile Asn Arg Val Gly Asn Glu Phe Leu Gln Phe Asn Lys Glu

275 280 285

Ser Lys Leu Lys Leu Lys Asp Leu Lys Met Val Lys Leu Tyr Lys Gln

290 295 300

Ile Leu Ser Asp Arg Glu Gln Pro Ser Phe Leu Pro Glu Gln Phe Arg

305 310 315 320

Asn Glu Asp Glu Leu Ile Lys Ser Ile Glu Asp Phe His Asn Leu Ile

325 330 335

Thr Glu Gln Lys Leu Phe Glu Arg Leu Leu Lys Leu Met Gly Arg Leu

340 345 350

Lys Asn Gly Glu Cys Glu Asp Leu Asn Lys Ile His Val Val Gly Ser

355 360 365

Ser Leu Thr Gln Leu Ser Lys Val Leu Tyr Gly Asn Trp Glu Val Leu

370 375 380

Gly Thr Ala Leu Arg Asn Lys Phe Gln Thr Asn Lys Thr Lys Lys Asp

385 390 395 400

Lys Leu Glu Ser Glu Lys Asp Ile Gln Glu Trp Met Glu Arg Lys Ser

405 410 415

Phe Ser Leu Ala Gln Ile Ile Glu Val Glu Ser Ser Leu Gln Asp Asp

420 425 430

Lys Ser Ile Lys Val Ile Asp Leu Phe Thr Thr Phe Asn Ala Trp Gln

435 440 445

Lys Val Asn Glu Lys Pro Gln Leu Val Asp Leu Ile Lys Leu Cys Lys

450 455 460

Asp Asp Phe Gln Thr Arg Phe Arg Ala Val Lys Asp Leu Ile Glu Lys

465 470 475 480

Gly Glu Gln Ile Gln Gly Asn Glu Ser Ala Lys Glu Glu Ile Lys Ala

485 490 495

Val Leu Asp Asn Tyr Gln Asn Leu Leu His Val Val Lys Leu Leu Asn

500 505 510

Leu Gly Lys Lys Glu Ser Tyr Leu Asp Lys Asp Glu Thr Phe Tyr Asn

515 520 525

Glu Tyr Lys Glu Ile Leu Ser Ser Thr Glu Ser Asp Asn Val Cys Leu

530 535 540

Glu Asp Ile Ile Pro Leu Tyr Asn Lys Val Arg Ser Phe Leu Thr Arg

545 550 555 560

Lys Leu Gly Asp Glu Gly Lys Met Leu Leu Lys Phe Asp Cys Ser Thr

565 570 575

Leu Ala Asp Gly Trp Asp Val Gly Lys Glu Ser Ala Asn Asn Ser Thr

580 585 590

Ile Leu Ile Asp Asn Ser Lys Tyr Tyr Leu Ile Ile Thr Asn Pro Glu

595 600 605

Asn Lys Pro Asp Leu Ser Thr Ala Ile Thr Ser Asn Thr Asp Asn Val

610 615 620

Tyr Lys Lys Ile Val Tyr Arg Gln Ile Ala Asp Pro Thr Lys Asp Leu

625 630 635 640

Pro Asn Leu Met Val Ile Asp Gly Lys Thr Gln Arg Lys Thr Gly Asn

645 650 655

Lys Asp Asp Asp Gly Ile Asn Arg Val Leu Asp Gln Leu Lys Asp Lys

660 665 670

Tyr Leu Pro Gln Glu Val Asn Arg Ile Arg Lys Leu Gly Ser Tyr Leu

675 680 685

Lys Thr Ser Glu His Phe Asn Lys Lys Asp Ser Gln Val Tyr Leu Ala

690 695 700

Tyr Tyr Met Gln Arg Leu Ile Glu Tyr Lys Gln Gly Glu Met Glu Phe

705 710 715 720

Ser Phe Lys Asn Ser Glu Glu Tyr Asp Ser Tyr Ser Asp Phe Leu Asp

725 730 735

Asp Ile Thr Lys Gln Lys Tyr Ser Leu Ser Phe Val Asn Val Ser Lys

740 745 750

Glu Ile Ile Thr Gln Trp Ile Ser Glu Gly Lys Ile Phe Leu Phe Gln

755 760 765

Ile Tyr Asn Lys Asp Phe Glu Glu Lys Ala Thr Gly Thr Pro Asn Leu

770 775 780

His Thr Leu Tyr Trp Lys Glu Leu Phe Ser Glu Glu Asn Leu Lys Asp

785 790 795 800

Ile Val Tyr Lys Leu Asn Gly Glu Ala Glu Leu Phe Tyr Arg Lys Lys

805 810 815

Met Asp Gly Lys Pro Phe Thr His Asn Lys Gly Ala Val Leu Val Asn

820 825 830

Lys Thr Phe Ala Asp Gly Ser Pro Val Glu Pro Glu His Tyr Lys Glu

835 840 845

Tyr Val Glu Tyr Ile Thr Gly Lys Val Ile Glu Lys Gln Leu Ser Lys

850 855 860

Glu Ala Lys Asp Lys Leu His Leu Val Lys Thr Asn Lys Ala Lys Leu

865 870 875 880

Asp Ile Ile Lys Asp Lys Arg Tyr Phe Gln His Lys Leu Leu Phe His

885 890 895

Val Pro Ile Thr Ile Asn Phe Lys Ser Glu Gly Val Pro Lys Phe Asn

900 905 910

Asp Tyr Thr Leu Asn Tyr Leu Arg Glu Asn Lys Lys Asp Ile Asn Ile

915 920 925

Ile Gly Ile Asp Arg Gly Glu Arg Asn Leu Ile Tyr Val Ser Val Ile

930 935 940

Asn Gln Lys Gly Glu Asn Ile Ile Pro Pro Lys His Phe Asn Ile Val

945 950 955 960

Glu Ser Asp Met Phe Gly Met Glu Asp Lys Arg Lys Phe Asn Tyr Leu

965 970 975

Glu Lys Leu Ile Gln Lys Glu Gly Asn Arg Asp Asp Ala Arg Lys Asn

980 985 990

Trp Ser Lys Ile Glu Thr Ile Lys Asp Leu Lys Thr Gly Tyr Leu Ser

995 1000 1005

Leu Val Val His Glu Ile Ala Lys Leu Val Val Glu His His Ala Ile

1010 1015 1020

Val Val Leu Glu Asp Leu Asn Tyr Gly Phe Lys Arg Gly Arg Phe Asn

1025 1030 1035 1040

Val Glu Arg Gln Ile Tyr Gln Asn Phe Glu Lys Met Leu Ile Glu Lys

1045 1050 1055

Leu Asn Leu Leu Val Phe Lys Asn Asn Ser Asn Ser Pro Asp Tyr Gly

1060 1065 1070

Asn Ile Leu Asn Gly Leu Gln Leu Thr Ala Pro Phe Gly Ser Phe Lys

1075 1080 1085

Glu Leu Gly Lys Gln Ser Gly Trp Leu Phe Tyr Val Asn Ala Ser Tyr

1090 1095 1100

Thr Ser Lys Ile Asp Pro Gln Thr Gly Phe Ala Asn Leu Phe Asn Met

1105 1110 1115 1120

Lys Asp Ala Lys Lys Asp Thr Lys Ser Phe Phe Glu Lys Ile Thr Glu

1125 1130 1135

Ile Lys Tyr Asp Asp Gly Met Phe Lys Phe Thr Phe Asp Tyr Arg Asn

1140 1145 1150

Gly Phe Ser Ile Val Gln Thr Asp Tyr Lys Asn Ile Trp Thr Val Cys

1155 1160 1165

Thr Asn Asp Lys Arg Ile Leu Val Ser Lys Asp Asn Ile Ser Gly Lys

1170 1175 1180

Phe Lys His Glu Tyr Val Asp Ile Thr Glu Ser Ile Lys Asn Leu Phe

1185 1190 1195 1200

Ile Asn Asn Asn Ile Asn Asp Tyr His Ser Ile Ser Lys Glu Thr Ile

1205 1210 1215

Leu Ser Ile Lys Glu Lys Lys Phe Phe Asp Asp Leu Phe Phe Tyr Phe

1220 1225 1230

Lys Leu Ser Leu Gln Met Arg Asn Ser Ile Pro Asn Ser Asp Ile Asp

1235 1240 1245

Tyr Leu Ile Ser Pro Val Gln Ile Lys Gly Lys Pro Phe Phe Asp Ser

1250 1255 1260

Arg Ile Pro Asn Asn Ile Asn Ile Val Asp Ala Asp Ala Asn Gly Ala

1265 1270 1275 1280

Tyr His Ile Ala Leu Lys Gly Leu Tyr Leu Val Ile Asn Asp Phe Pro

1285 1290 1295

Thr Glu Lys Lys Gly Lys Ser Glu Tyr Leu Lys Lys Ile Thr Asn Glu

1300 1305 1310

Asp Trp Phe Glu Phe Ala Gln Arg Arg Ser Leu Lys

1315 1320

<210> 5

<211> 1230

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC05的AA序列

<400> 5

Met Arg Trp Asp Glu Lys Leu Gln Thr Phe Leu Asn Asp Gln Glu Ile

1 5 10 15

Glu Asp Ala Tyr Gln Val Leu Lys Pro Val Phe Asp Lys Leu His Glu

20 25 30

Asn Phe Ile Ile Gly Ser Leu Glu Asn Thr Asn Asn Lys Lys Leu Phe

35 40 45

Ser Phe Asp Lys Tyr Leu Lys Leu Lys Asn Asp Leu Leu His Val Asn

50 55 60

Lys Lys Glu Gln Glu Ser Asp Tyr Lys Lys Lys Glu Lys Glu Phe Glu

65 70 75 80

Thr Glu Gly Lys Leu Leu Arg Asn Thr Phe Ala Thr Val Trp Ile Asn

85 90 95

Glu Gly Lys Asn Phe Lys Asn Thr Ile Val Gly Gly Glu Asn Asp Arg

100 105 110

Glu Ile Leu Lys Glu Gly Gly Tyr Lys Ile Leu Thr Glu Ala Gly Ile

115 120 125

Leu Lys Tyr Ile Lys Met Asn Ile Asp Lys Phe Val Glu Leu Lys Leu

130 135 140

Lys Thr Arg Glu Asp Ile Leu Trp Lys Lys Glu Asn Arg Asn Leu Val

145 150 155 160

Glu Met Ala Asp Leu Glu Lys Ser Leu Gly Thr Ile Glu Ser Trp Gly

165 170 175

Val Phe Glu Gly Phe Phe Thr Tyr Phe Ser Gly Phe Asn Gln Asn Arg

180 185 190

Glu Asn Tyr Tyr Ser Thr Asp Glu Lys Ala Thr Ala Val Ala Ser Arg

195 200 205

Val Ile Asp Glu Asn Leu Pro Lys Phe Ser Asp Asn Val Leu Glu Phe

210 215 220

Asn Lys Lys Asn Asp Val Tyr Ile Gly Ile Phe Ser Phe Leu Lys Gly

225 230 235 240

Lys Asn Ile Val Leu Lys Gly Lys Ser Gly Asn Gly Glu Glu Gln Asp

245 250 255

Leu Leu Pro Ile Thr Glu Lys Ile Phe Glu Ile Glu Tyr Phe Lys Asn

260 265 270

Cys Leu Ser Glu Gly Glu Ile Glu Arg Tyr Asn Ser Asp Ile Gly Asn

275 280 285

Ala Asn Phe Leu Ile Asn Leu Tyr Asn Gln Gln Gln Asp Lys Lys Glu

290 295 300

Asn Lys Leu Arg Ile Phe Lys Thr Leu Tyr Lys Gln Ile Gly Cys Gly

305 310 315 320

Ile Lys Gly Asp Phe Ile Gln Leu Ile Lys Thr Asp Asp Glu Leu Lys

325 330 335

Lys Ile Phe Glu Asp Leu Lys Ile Thr Gly Asp Asn Phe Phe Lys Asn

340 345 350

Thr Gln Asn Leu Lys Glu Ile Ile Leu Ser Leu Glu Asn Phe Ser Gly

355 360 365

Ile Tyr Trp Ser Asp Lys Ala Leu Asn Thr Val Ser Gly Lys Tyr Phe

370 375 380

Ala Asn Trp Ala Ser Leu Lys Glu Leu Leu Lys Asn Ala Lys Ile Phe

385 390 395 400

Lys Lys Glu Lys Asp Glu Ile Lys Ile Pro Gln Thr Ile Glu Leu Ser

405 410 415

Asp Leu Phe Gly Val Leu Asp Ser Asn Glu Leu Ile Phe Lys Glu Ser

420 425 430

Phe Asn Glu Asn Asp Glu Leu Lys Gln Ile Ile Leu Lys Ser Tyr Glu

435 440 445

Lys Asn Ser Ile Lys Leu Leu Lys Met Ile Phe Val Asp Val Glu Glu

450 455 460

Asn Gln Lys Ile Phe Gly Asn Leu Lys Asp Gly Leu Pro Ile Asn Asp

465 470 475 480

Phe Lys Lys Asp Glu Asn Thr Gln Ile Ile Lys Thr Trp Leu Asp Gly

485 490 495

Leu Leu Asn Thr Asn Gln Ile Leu Lys Tyr Phe Lys Val Arg Glu Ser

500 505 510

Lys Ile Lys Gly Ala Pro Leu Asn Pro Glu Val Ser Glu Arg Leu Asn

515 520 525

Lys Ile Leu Asn Val Glu Asn Pro Thr Val Ile Tyr Asp Val Val Arg

530 535 540

Asn Tyr Leu Thr Lys Lys Pro Thr Glu Gly Leu Asn Lys Leu Lys Leu

545 550 555 560

Asn Phe Asp Asn Ala Val Leu Ala Ala Gly Trp Asp Val Asn Lys Glu

565 570 575

Ser Glu Arg Gly Cys Leu Ile Leu Lys Asp Gly Asp Asn Lys Lys Tyr

580 585 590

Leu Ala Ile Leu Thr Asn Lys Thr Gln Lys Phe Phe Gly Glu Lys Val

595 600 605

Lys Tyr Lys Glu Phe Val Gly Asp Glu Asn Trp Gln Lys Met Asp Tyr

610 615 620

Lys Leu Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Val Leu Leu Pro

625 630 635 640

Lys Ser Asp Arg Tyr Lys Phe Gly Ala Thr Asp Glu Ile Leu Lys Ile

645 650 655

Tyr Asn Glu Gly Gly Phe Lys Lys Asn Glu Pro Thr Phe Thr Lys Ala

660 665 670

Lys Leu Ala Lys Ile Val Asp Phe Phe Lys Asp Gly Leu Lys Asn Tyr

675 680 685

Pro Ser Ala Lys Ser Ser Trp Tyr Asn Leu Phe Ala Phe Asp Phe Ser

690 695 700

Asp Thr Glu Lys Tyr Glu Ser Ile Asp Arg Phe Tyr Thr Glu Val Glu

705 710 715 720

Lys Gln Gly Tyr Lys Leu Ser Trp Ser Ala Ile Ser Lys Asn Phe Ile

725 730 735

Phe Glu Lys Val Asp Ala Gly Asp Met Tyr Leu Phe Glu Ile Arg Asn

740 745 750

Lys Asp Asn Asn Leu Lys Asn Gly Lys Ala Lys Thr Gly Ala Lys Asn

755 760 765

Leu His Thr Ile Tyr Trp Gly Thr Ile Phe Gly Glu Ser Glu Asn Lys

770 775 780

Pro Lys Leu Asn Gly Glu Ala Glu Ile Phe Tyr Arg Pro Val Val Lys

785 790 795 800

Asp Leu Ile Lys Asp Lys Asp Lys Asn Gly Asp Ile Ile Lys Ala Ser

805 810 815

Glu Lys Arg Phe Glu Gln Glu Lys Phe Val Phe His Cys Pro Ile Thr

820 825 830

Leu Asn Phe Cys Leu Lys Ser Thr Arg Leu Asn Asp Val Ile Asn Gln

835 840 845

Ile Met Ile Glu Asn Lys Lys Asp Val Cys Phe Ile Gly Ile Asp Arg

850 855 860

Gly Glu Lys His Leu Ala Tyr Tyr Ser Val Val Asn Gln Lys Gly Glu

865 870 875 880

Ile Leu Glu Gln Gly Ser Phe Asn Glu Ile Asn Gly Gln Asn Tyr Ala

885 890 895

Lys Lys Leu Glu Glu Lys Ala Gly His Arg Asp Glu Ala Arg Lys Asn

900 905 910

Trp Lys Thr Ile Gly Thr Ile Lys Glu Leu Lys Asn Gly Tyr Ile Ser

915 920 925

Gln Val Val Arg Arg Ile Val Asp Leu Ala Val Lys Tyr Asn Ala Tyr

930 935 940

Ile Val Leu Glu Asp Leu Asn Ser Gly Phe Lys Arg Gly Arg Gln Lys

945 950 955 960

Ile Glu Lys Ser Val Tyr Gln Lys Leu Glu Leu Ala Leu Ala Lys Lys

965 970 975

Leu Asn Phe Leu Val Asp Lys Ser Lys Lys Asp Gly Glu Ile Gly Ser

980 985 990

Val Gln Lys Ala Leu Gln Leu Thr Pro Pro Ala Thr Asn Phe Ala Asp

995 1000 1005

Ile Glu Lys Ala Lys Gln Phe Gly Ile Met Leu Tyr Val Arg Ala Asn

1010 1015 1020

Tyr Thr Ser Gln Thr Asp Pro Val Thr Gly Trp Arg Lys Thr Ile Tyr

1025 1030 1035 1040

Phe Lys Ser Thr Thr Gln Glu Asn Leu Lys Lys Glu Ile Cys Glu Lys

1045 1050 1055

Phe Ser Glu Ile Gly Phe Asp Gly Asn Asp Tyr Tyr Phe Glu Tyr Lys

1060 1065 1070

Asp Glu Asn Ala Glu Lys Lys Trp Thr Met Tyr Ser Gly Val Ser Gly

1075 1080 1085

Lys Ser Leu Asp Arg Phe Arg Gly Lys Lys Asp Thr His Gly Ile Trp

1090 1095 1100

Lys Val Glu Lys Gln Asp Ile Val Glu Leu Leu Lys Lys Ile Phe Gly

1105 1110 1115 1120

Gln Gln Thr Ser Val Val Gly Asp Leu Lys Thr Lys Ile Thr Asn Asp

1125 1130 1135

Asn Val Asn Asp Leu Lys Tyr Thr Ile Asp Leu Ile Gln Gln Ile Arg

1140 1145 1150

Asn Thr Gly Phe Asn Glu Ile Asp Asn Asp Phe Ile Leu Ser Pro Val

1155 1160 1165

Arg Asp Glu Lys Gly Asn His Phe Asp Ser Arg Lys Asp Gly Ala Ile

1170 1175 1180

Leu Ser Asn Gly Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly

1185 1190 1195 1200

Val Leu Ala Phe Glu Arg Ile Asn Ala Lys Pro Glu Lys Pro Glu Leu

1205 1210 1215

Tyr Ile Ala Asp Val Glu Trp Asp Lys Trp Leu Gln Ser Lys

1220 1225 1230

<210> 6

<211> 1256

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC06的AA序列

<400> 6

Met Asp Ser Phe Thr Gln Phe Thr Gly Leu Tyr Ser Leu Ser Lys Thr

1 5 10 15

Leu Arg Phe Glu Leu Lys Pro Ile Gly Lys Thr Leu Ala Tyr Ile Glu

20 25 30

Asn Lys Gly Leu Leu Val Gln Asp Glu His Arg Ala Asp Ser Tyr Lys

35 40 45

Ile Val Lys Lys Ile Ile Asp Glu Tyr His Lys Ser Phe Ile Glu Lys

50 55 60

Ser Leu Asn Gly Leu Cys Leu Asp Gly Leu Glu Asp Tyr Tyr Phe Tyr

65 70 75 80

Tyr Gln Ile Pro Lys Lys Asp Asp Asn Gln Lys Lys Ile Val Glu Asp

85 90 95

Ile Leu Thr Lys Leu Arg Lys Gln Ile Ala Glu Arg Phe Ser Lys Gln

100 105 110

Asp Ile Tyr Lys Asn Leu Phe Ala Lys Glu Leu Ile Lys Asp Asp Leu

115 120 125

Asn Ser Phe Val Gln Glu Val Glu Gln Lys Asp Leu Ile Lys Glu Phe

130 135 140

Glu Asn Phe Thr Thr Tyr Phe Thr Gly Phe His Glu Asn Arg Lys Asn

145 150 155 160

Met Tyr Ser Ala Glu Asp Lys Ser Thr Ala Ile Ala Phe Arg Leu Ile

165 170 175

His Gln Asn Leu Pro Lys Phe Leu Asp Asn Met Arg Ala Phe Asn Lys

180 185 190

Ile Ser Val Ser Pro Leu Ala Glu Lys Phe Lys His Ile Leu Ser Asp

195 200 205

Ser Glu Leu Gly Pro Ile Val Gln Val Val Ala Met Glu Asp Val Phe

210 215 220

Asn Leu Ala Tyr Phe Asn Glu Thr Leu Thr Gln Ser Gly Ile Asp Ile

225 230 235 240

Tyr Asn His Leu Leu Gly Gly Tyr Thr Pro Glu Glu Gly Lys Glu Lys

245 250 255

Ile Lys Gly Leu Asn Glu Tyr Ile Asn Leu Tyr Asn Gln Thr Val Lys

260 265 270

Lys Glu Glu Arg Leu Pro Lys Leu Lys Pro Leu Phe Lys Gln Ile Leu

275 280 285

Ser Asp Arg Ser Thr Ala Ser Phe Ile Pro Glu Gln Tyr Lys Asn Asp

290 295 300

Asn Glu Val Leu Glu Ser Ile Glu Lys Leu Tyr Gln Glu Ile Lys Glu

305 310 315 320

His Val Phe His Ser Leu Lys Glu Leu Phe Val His Ile Asn Glu Tyr

325 330 335

Asp Leu His Lys Ile Tyr Leu Arg Asn Asp Val Ser Met Thr Asp Ile

340 345 350

Ser Gln Lys Met Phe Gly Asp Trp Gly Val Phe Thr Lys Ala Met Asn

355 360 365

Leu Tyr Phe Asp Lys Gln Tyr Lys Gly Lys Ala Lys Leu Gly Thr Glu

370 375 380

Lys Tyr Glu Asp Glu Gln Lys Lys Tyr Phe Ser Asn Gln Glu Ser Phe

385 390 395 400

Ser Ile Gly Tyr Ile Asn Glu Cys Leu Leu Leu Leu Gly Ser Asn Tyr

405 410 415

His Lys Lys Val Glu Asp Tyr Phe Lys Val Ala Gly Lys Thr Glu Glu

420 425 430

Gln Val Gln Met Leu Phe Glu Ile Ile Glu Thr Lys Tyr Gln Asn Ile

435 440 445

Gln Asp Leu Leu Asn Ser Pro Tyr Pro Thr Glu Lys Asn Leu Ala Gln

450 455 460

Asp Gln Val Gln Val Asp Lys Ile Lys Gly Leu Leu Asp Ser Ile Lys

465 470 475 480

Asn Leu Gln Trp Phe Ile Lys Pro Leu Leu Gly Lys Gly Asn Glu Ala

485 490 495

Glu Lys Asp Glu Arg Phe Tyr Gly Glu Phe Thr Ala Leu Trp Glu Thr

500 505 510

Leu Asp Gln Ile Thr Pro Leu Tyr Asn Lys Val Arg Asn Tyr Met Thr

515 520 525

Arg Lys Pro Tyr Ser Thr Glu Lys Met Lys Leu Asn Phe Asp Asn Ser

530 535 540

Thr Leu Leu Asp Gly Trp Asp Ile Asn Lys Glu Pro Asp Asn Thr Ser

545 550 555 560

Val Val Leu Arg Lys Asp Gly Leu Phe Tyr Leu Gly Ile Met Asp Lys

565 570 575

Lys Tyr Asn Lys Thr Phe Lys Gln Glu Phe Ile Glu Ser Asn Glu Pro

580 585 590

Cys Phe Glu Lys Met Glu Tyr Lys Leu Leu Pro Gly Ala Asn Lys Met

595 600 605

Leu Pro Lys Val Phe Phe Ser Asn Ser Arg Ile Glu Glu Phe Asn Pro

610 615 620

Thr Val Asp Leu Leu Glu Asn Tyr Lys Asn Gln Thr His Lys Lys Gly

625 630 635 640

Asp Lys Phe Asn Ile Ala His Cys Arg Asn Leu Ile Asp Phe Phe Lys

645 650 655

Gln Ser Ile Asn Lys His Asp Asp Trp Lys Gln Phe Gly Phe Ala Phe

660 665 670

Ser Asp Thr Lys Asn Tyr Asp Asp Leu Ser Gly Phe Tyr Arg Glu Val

675 680 685

Glu Gln Gln Gly Tyr Lys Ile Thr Phe Arg Asn Ile Pro Glu Lys Phe

690 695 700

Ile Asn Gln Met Val Glu Glu Ser Lys Leu Tyr Leu Phe Gln Ile Tyr

705 710 715 720

Asn Lys Asp Phe Ser Pro Tyr Ser Lys Gly Thr Pro Asn Met His Thr

725 730 735

Leu Tyr Trp Lys Met Leu Phe Asp Thr Glu Asn Leu Lys Asp Val Val

740 745 750

Tyr Lys Leu Asn Gly Gln Ala Glu Val Phe Tyr Arg Lys Ala Ser Ile

755 760 765

Asn Asp Glu Asn Ile Val Val His Lys Ala Asn Glu Val Ile Ile Asn

770 775 780

Lys Asn Thr Leu Asn Glu Lys Lys Gln Ser Arg Phe Asp Tyr Asp Ile

785 790 795 800

Ile Lys Asp Lys Arg Tyr Thr Ile Asp Lys Phe Gln Phe His Val Pro

805 810 815

Ile Thr Met Asn Phe Lys Ala Arg Gly Leu Asn Asn Ile Asn Leu Glu

820 825 830

Val Asn Gln Tyr Leu Gln Lys Glu Asn Asp Ile His Ile Ile Gly Ile

835 840 845

Asp Arg Gly Glu Arg His Leu Leu Tyr Leu Ser Leu Ile Asn Ser Lys

850 855 860

Gly Asn Ile Ile Glu Gln Tyr Ser Leu Asn Glu Ile Ile Asn Glu Tyr

865 870 875 880

Asn Gly Asn His Tyr His Thr Asn Tyr His Asp Leu Leu Asp Lys Arg

885 890 895

Glu Gly Asn Arg Thr Glu Glu Arg Gln Asn Trp Lys Thr Ile Glu Ser

900 905 910

Ile Lys Glu Leu Lys Glu Gly Tyr Leu Ser Gln Val Val His Lys Ile

915 920 925

Ser Glu Leu Met Val Glu Tyr Asn Ala Ile Val Val Leu Glu Asp Leu

930 935 940

Asn Met Gly Phe Ile Arg Gly Arg Gln Lys Val Glu Lys Ser Val Tyr

945 950 955 960

Gln Gln Phe Glu Lys Met Leu Ile Asp Lys Leu Asn Tyr Leu Val Asp

965 970 975

Lys Lys Lys Lys Ser Phe Glu Leu Gly Gly Thr Leu His Ala Tyr Gln

980 985 990

Leu Thr Asn Lys Phe Glu Ser Phe Gln Lys Met Gly Lys Gln Ser Gly

995 1000 1005

Phe Leu Phe Tyr Ile Pro Ala Trp Asn Thr Ser Lys Met Asp Pro Val

1010 1015 1020

Thr Gly Phe Val Asn Leu Phe Asp Thr Arg Tyr Glu Asn Val Val Lys

1025 1030 1035 1040

Ala Lys Ala Phe Phe Asn Lys Phe Glu Ser Ile Arg Tyr Asn Lys Asp

1045 1050 1055

Lys Asp Tyr Phe Glu Phe Glu Val Lys Lys Tyr Ser Asp Phe Asn Ala

1060 1065 1070

Lys Ala Glu Asp Thr Arg Gln Glu Trp Ile Ile Cys Thr His Gly Glu

1075 1080 1085

Arg Ile Ile Asn Tyr Arg Asn Pro Glu Lys Asn Asn Glu Trp Asp Asp

1090 1095 1100

Lys Thr Val His Pro Thr Thr Glu Leu Lys Ser Leu Phe Thr Ser Lys

1105 1110 1115 1120

Asn Ile Ile Phe Glu Asn Gly Ser Cys Leu Lys Glu Gln Ile Ala Leu

1125 1130 1135

Gln Lys Asp Thr Asp Lys Glu Phe Phe Glu Gly Leu Leu Lys Gln Phe

1140 1145 1150

Lys Asn Thr Leu Gln Met Arg Asn Ser Lys Thr Lys Ser Glu Ile Asp

1155 1160 1165

Tyr Leu Phe Ser Pro Val Ser Asn Glu Asn Gly Val Phe Phe Asp Ser

1170 1175 1180

Arg Asp Tyr Val Asp Ile Asp Asn Arg Asp Arg Lys Phe Cys Val Ser

1185 1190 1195 1200

Thr Gly Lys Pro Thr Leu Pro Val Asn Ala Asp Ala Asn Gly Ala Tyr

1205 1210 1215

Asn Ile Ala Arg Lys Gly Leu Trp Ile Val Glu Gln Ile Lys Asn Pro

1220 1225 1230

Asn Thr Asp Leu Lys Lys Leu Lys Leu Ala Met Thr Asn Lys Glu Trp

1235 1240 1245

Leu Gln Phe Val Gln Asn Lys Gly

1250 1255

<210> 7

<211> 1274

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC07的AA序列

<400> 7

Met Asp Asn Ala Phe Ser Asp Phe Thr Gln Lys Tyr Thr Leu Ser Lys

1 5 10 15

Thr Leu Arg Phe Glu Leu Arg Pro Val Gly Asn Thr Glu Lys Met Leu

20 25 30

Glu Asp Glu Lys Val Phe Glu Lys Asp Lys Leu Ile Gln Glu Lys Tyr

35 40 45

Ile Lys Thr Lys Pro Tyr Phe Asp Leu Leu His Arg Glu Phe Val Glu

50 55 60

Glu Ala Leu Lys Asp Val Asp Ile Ser Gly Leu His Asn Tyr Phe Glu

65 70 75 80

Thr Tyr Gln Lys Trp Ala Lys Asp Lys Lys Lys Tyr Gln Lys Glu Leu

85 90 95

Gln Asn Lys Glu Gln Ile Leu Arg Lys Glu Ile Leu Val Phe Leu Asp

100 105 110

Ser Thr Ala Lys Tyr Trp Ala Glu Lys Lys Tyr Ser Glu Leu Arg Ile

115 120 125

Lys Lys Lys Asp Ile Glu Ile Phe Phe Glu Glu Asp Val Phe Thr Ile

130 135 140

Leu Lys Lys Arg Tyr Gly Glu Asp Ser Glu Ala Gln Ile Ile Asp Glu

145 150 155 160

Val Ser Gly Glu Thr Val Ser Ile Phe Asp Ser Trp Lys Gly Phe Thr

165 170 175

Gly Tyr Phe Lys Lys Phe Gln Glu Thr Arg Lys Asn Leu Tyr Arg Asp

180 185 190

Asp Gly Thr Ala Thr Ala His Ala Thr Arg Ile Ile Asp Gln Asn Leu

195 200 205

Lys Arg Phe Cys Asp Asn Leu Glu Ile Ile Lys Arg Ile Ala Gly Ile

210 215 220

Ile Glu Phe Ser Glu Val Glu Gly Asn Phe Lys His Ser Met Gly Asp

225 230 235 240

Val Phe Ser Leu Ser Phe Tyr Asn Lys Cys Leu Leu Gln Asp Gly Ile

245 250 255

Asn Phe Tyr Asn Arg Ile Leu Gly Gly Glu Val Leu Gln Asp Gly Thr

260 265 270

Lys Leu Lys Gly Ile Asn Glu Leu Ile Asn Lys Tyr Arg Gln Asp Asn

275 280 285

Lys Gly Val Lys Ile Pro Phe Leu Lys Leu Leu Asp Lys Gln Ile Leu

290 295 300

Ser Glu Lys Glu Glu Phe Leu Asp Gly Ile Glu Asp Asp Lys Glu Leu

305 310 315 320

Leu Ala Val Leu Lys Lys Phe Tyr Glu Val Ala Glu Lys Lys Thr Ser

325 330 335

Ile Leu Lys Ser Leu Ile Gln Asp Phe Ala Gln Asn Asn Arg Gln Tyr

340 345 350

Asn Leu Glu Glu Val Tyr Ile Ser Lys Glu Ala Phe Asn Thr Ile Ser

355 360 365

Arg Lys Trp Thr His Glu Thr Ser Lys Phe Glu Glu Trp Leu Tyr Asn

370 375 380

Val Met Lys Pro Asn Lys Pro Thr Gly Leu Lys Tyr Asp Lys Lys Glu

385 390 395 400

Glu Ser Tyr Lys Phe Pro Asp Phe Ile Pro Leu Ser Tyr Ile Gln Thr

405 410 415

Ala Leu Glu Gln Ala Asp Ile Asp Gly Asp Phe Trp Lys Glu His Tyr

420 425 430

Ser Glu Asn Ser Lys Ala Asn Asp Gly Cys Leu Met Gly Asp Glu Ser

435 440 445

Ile Trp Glu Gln Phe Ile Lys Ile Phe Glu Tyr Glu Phe Gln Ser Leu

450 455 460

Phe Glu Lys Glu Ile Ile Asp Arg Glu Thr Gly Gln Pro Lys Lys Asn

465 470 475 480

Gly Tyr Asn Tyr Val Lys Asp Asp Phe Lys Gly Leu Leu Asn Gly Glu

485 490 495

Asn Phe Ser Val Glu Ile Ile Lys Asp Phe Ala Asp Thr Val Leu Ser

500 505 510

Ile Tyr Gln Met Ala Lys Tyr Phe Ala Ile Glu Lys Lys Arg Lys Trp

515 520 525

Leu Asp Glu Tyr Asp Thr Gly Asp Phe Tyr Glu Asn Pro Glu Phe Gly

530 535 540

Tyr Lys Leu Phe Tyr Asp Asp Ala Tyr Lys Glu Ile Val Gln Thr Tyr

545 550 555 560

Asn Asn Leu Arg Asn Tyr Leu Thr Lys Lys Ser Tyr Ser Glu Glu Lys

565 570 575

Trp Lys Leu Asn Phe Glu Asn Pro Thr Leu Ala Asp Gly Trp Asp Lys

580 585 590

Asn Lys Glu Pro Asp Asn Ser Ala Val Ile Leu Arg Lys Asp Gly Arg

595 600 605

Tyr Tyr Leu Gly Leu Met Lys Lys Gly Cys Asn Lys Ile Phe Asp Asp

610 615 620

Arg Asn Lys Val Glu Phe Ser Gly Gly Val Asp Lys Asp Lys Tyr Glu

625 630 635 640

Lys Ile Val Tyr Lys Phe Phe Pro Asp Gln Ala Lys Met Phe Pro Lys

645 650 655

Val Cys Phe Ser Ala Lys Gly Leu Asp Phe Phe Gln Pro Ser Glu Glu

660 665 670

Ile Leu Asn Ile Tyr Lys Asn Ser Glu Phe Lys Lys Gly Asp Thr Phe

675 680 685

Ser Val Gln Ser Met Gln Lys Leu Ile Asp Phe Tyr Lys Asp Cys Leu

690 695 700

Thr Lys Tyr Glu Gly Trp Ile Ala Tyr Glu Phe Lys His Leu Lys Ser

705 710 715 720

Thr Asp Leu Tyr Arg Asn Asn Ile Ser Glu Phe Phe Ser Asp Val Ala

725 730 735

Glu Asp Gly Tyr Lys Ile Thr Phe Gln Asp Ile Ser Asp Asn Tyr Ile

740 745 750

Asp Lys Lys Asn Gln Ser Glu Glu Leu Tyr Leu Phe Glu Ile His Asn

755 760 765

Lys Asp Trp Asn Leu Lys Asp Glu Val Lys Lys Thr Gly Ser Lys Asn

770 775 780

Leu His Thr Leu Tyr Phe Glu Ala Leu Phe Ser His Glu Asn Ile Gln

785 790 795 800

Asn Asn Phe Pro Ile Lys Leu Asn Gly Gln Ala Glu Val Phe Tyr Arg

805 810 815

Pro Lys Thr Asp Glu Glu Lys Leu Val Lys Lys Lys Asp Lys Lys Gly

820 825 830

Arg Glu Val Ile Asp His Lys Arg Tyr Ala Glu Asn Lys Ile Phe Phe

835 840 845

His Val Pro Leu Thr Leu Asn Arg Gly Lys Gly Asp Ala Tyr Gln Phe

850 855 860

Asn Ala Lys Ile Asn Asn Phe Leu Ala Asn Asn Ser Asp Ile Asn Val

865 870 875 880

Ile Gly Val Asp Arg Gly Glu Lys His Leu Ala Tyr Tyr Ser Val Ile

885 890 895

Asn Gln Lys Gly Glu Thr Leu Asp Ser Gly Ser Leu Asn Val Val Asn

900 905 910

Lys Ile Asn Tyr Gly Glu Lys Leu Gln Glu Lys Ala Ser Asn Arg Lys

915 920 925

Gln Ser Ile Arg Asp Trp Lys Ala Val Glu Gly Ile Lys Asn Leu Lys

930 935 940

Lys Gly Tyr Ile Ser Gln Val Val Arg Lys Leu Ala Asp Leu Ala Ile

945 950 955 960

Glu His Asn Ala Ile Ile Ile Phe Glu Asp Leu Asn Met Arg Phe Lys

965 970 975

Gln Ile Arg Gly Gly Ile Glu Lys Ser Val Tyr Gln Gln Leu Glu Gly

980 985 990

Ala Leu Ile Glu Lys Leu Ser Phe Leu Val Asn Lys Gly Glu Lys Asp

995 1000 1005

Pro Lys Gln Ala Gly Asn Leu Leu Lys Ala Tyr Gln Leu Ala Ala Pro

1010 1015 1020

Phe Thr Thr Phe Lys Asp Met Gly Lys Gln Thr Gly Ile Ile Phe Tyr

1025 1030 1035 1040

Thr Gln Ala Ser Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Trp Arg

1045 1050 1055

Pro Asn Leu Tyr Leu Lys Tyr Thr Asn Ala Glu Lys Thr Lys Glu Asp

1060 1065 1070

Ile Gly Asn Phe Ser Asn Ile Glu Phe Lys Asn Gly Ile Phe Glu Phe

1075 1080 1085

Thr Tyr Asp Leu Arg Asn Phe Gln Lys Gln Lys Glu Tyr Pro Lys Lys

1090 1095 1100

Thr Glu Trp Thr Leu Cys Ser Cys Val Glu Arg Phe Arg Trp Asn Arg

1105 1110 1115 1120

Val Leu Asn Gln Asn Lys Gly Gly Tyr Asp His Tyr Glu Asp Ile Thr

1125 1130 1135

His Asn Phe Arg Asp Leu Phe Glu Lys Tyr Asp Ile Asn Phe Met Ser

1140 1145 1150

Ala Asp Ile Lys Gly Gln Ile Asp Thr Leu Asp Ala Lys Gly Asn Glu

1155 1160 1165

Asn Phe Phe Lys Asp Phe Ile Phe Phe Phe Asn Leu Ile Cys Gln Ile

1170 1175 1180

Arg Asn Thr Gln Gln Asp Lys Asp Gly Asp Glu Asn Asp Phe Ile Leu

1185 1190 1195 1200

Ser Pro Ile Lys Pro Phe Phe Asp Ser Arg Asp Ser Lys Lys Phe Gly

1205 1210 1215

Glu Asn Leu Pro Asn Asn Gly Asp Asp Asn Gly Ala Tyr Asn Ile Ser

1220 1225 1230

Arg Lys Gly Ile Ile Ile Leu Asn Lys Ile Ser Glu Phe Phe Asp Glu

1235 1240 1245

Asn Gly Gly Cys Glu Lys Met Lys Trp Gly Asp Leu Tyr Ile Ser His

1250 1255 1260

Lys Asp Trp Asp Asp Phe Ala Arg Gln Ile

1265 1270

<210> 8

<211> 1335

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC08的AA序列

<400> 8

Met Met Gln Ile Met Lys Asn Phe Asp Lys Phe Thr Asn Leu Tyr Ser

1 5 10 15

Val Ser Lys Thr Leu Arg Phe Glu Leu Arg Pro Glu Pro Lys Thr Leu

20 25 30

Glu Tyr Met Arg Ser Asn Leu Arg Phe Asp Lys Asn Leu Gln Thr Phe

35 40 45

Leu Ala Asp Gln Glu Ile Glu Asp Ala Tyr Gln Ala Leu Lys Pro Ile

50 55 60

Phe Asp Ser Leu His Glu Arg Phe Ile Thr Glu Ser Leu Glu Ser Gly

65 70 75 80

Ser Ala Gln Lys Ile Asp Phe Ser Lys Tyr Leu Glu Lys Tyr Arg Asn

85 90 95

Lys Arg Asp Leu Gly Ile Lys Ala Leu Glu Gly Thr Glu Lys Leu Leu

100 105 110

Arg Asn Asn Phe Ala Glu Ile Tyr Lys Ala Thr Ala Lys Ser Trp Lys

115 120 125

Glu Asn Ala Gly Lys Asp Gly Lys Gly Lys Glu Val Phe Lys Lys Glu

130 135 140

Gly Phe Asn Ile Leu Thr Glu Lys Gly Ile Leu Glu Tyr Ile Glu Lys

145 150 155 160

Asn Ile Asp Ser Phe Ser Ala Ile Lys Ser Pro Glu Glu Ile Arg Gly

165 170 175

Ala Leu Gly Ala Phe Asp Gly Phe Phe Thr Tyr Phe Thr Gly Phe Asn

180 185 190

Gln Asn Arg Glu Asn Tyr Tyr Glu Thr Lys Lys Glu Ala Ser Thr Ala

195 200 205

Val Ala Thr Arg Ile Val His Glu Asn Leu Pro Lys Phe Cys Asp Asn

210 215 220

Ile Leu Ile Phe Asp Glu Arg Ala Glu Asp Tyr Ile Gly Ala Tyr Lys

225 230 235 240

Ala Leu Gln Lys Met Gly Arg Ala Leu Val Asn Lys Glu Gly Gly Glu

245 250 255

Leu Pro Ser Ile Ser Gly Asp Leu Phe Lys Ile Thr Phe Phe Asn Lys

260 265 270

Cys Phe Ser Gln Lys Gln Ile Glu Glu Tyr Asn Thr Ala Ile Gly Asn

275 280 285

Ala Asn Ser Leu Val Asn Leu Phe Asn Gln Ala Lys Arg Asp Glu Asp

290 295 300

Gly Tyr Lys Lys Leu Ala Leu Phe Lys Thr Leu Tyr Lys Gln Ile Gly

305 310 315 320

Cys Asp Lys Lys Asp Ser Leu Phe Phe Ala Val Thr His Asp Arg Arg

325 330 335

Ala Asp Ala Glu Lys Ala Arg Glu Asn Gly Gln Glu Ala Phe Ser Val

340 345 350

Glu Glu Val Leu Val Leu Ala Lys His Ala Gly Glu Lys Tyr Phe Asn

355 360 365

Lys Gly Asn Asp Asp Gly Glu Val Asn Thr Thr Gln Glu Phe Ile Ser

370 375 380

Tyr Ile Lys Asp Arg Ser Asp Tyr Gln Gly Ile Tyr Trp Ser Lys Ala

385 390 395 400

Ala Leu Asn Thr Ile Ser Asn Lys Tyr Phe Asp Asn Trp Tyr Glu Leu

405 410 415

Ile Asp Gln Leu Lys Glu Ala Lys Val Phe Thr Lys Thr Gly Ser Gly

420 425 430

Ser Glu Asp Asn Val Lys Ile Pro Asp Ala Ile Glu Leu Glu Gly Phe

435 440 445

Phe Gln Val Leu Asn Lys Ile Gln Asp Trp Lys Thr Val Phe Phe Lys

450 455 460

Lys Ser Ile Thr Ala Asp Pro Gln Lys Leu Gly Ile Ile Glu Ser Ser

465 470 475 480

Glu Thr Ala Ser Ala Ala Leu Leu Ser Leu Ile Phe Asp Asp Val Ala

485 490 495

Lys His Thr Lys Leu Phe Ile Asp Gln Ser Glu Asp Ile Leu Lys Val

500 505 510

Glu Asn Phe Val Lys Pro Glu Asn Lys Glu Asp Ile Lys Arg Trp Leu

515 520 525

Asp His Ser Leu Ala Ile Asn Gln Met Leu Lys Tyr Phe Leu Val Lys

530 535 540

Glu Ser Arg Thr Lys Gly Ala Pro Ile Asp Pro Thr Leu Thr Lys Ala

545 550 555 560

Leu Asp Thr Leu Leu Arg Ser Gln Asp Ala Glu Trp Phe Lys Trp Tyr

565 570 575

Asp Val Leu Arg Asn Tyr Leu Thr Lys Lys Pro Gln Asp Gly Thr Lys

580 585 590

Glu Asn Lys Leu Lys Leu Ser Phe Glu Asn Gly Thr Leu Ala Asn Gly

595 600 605

Trp Asp Val Asn Lys Glu Pro Asp Asn Phe Cys Val Ile Leu Gln Asn

610 615 620

Pro Glu Gly Lys Lys Phe Leu Ala Ile Ile Ala Arg Gln Glu Gly Gln

625 630 635 640

Lys Gly Phe Asn Gln Val Phe Ala Lys Lys His Asp Asn Pro Leu Tyr

645 650 655

Lys Val Asp Glu Gly Gly Val Phe Trp Ser Lys Met Glu Tyr Lys Leu

660 665 670

Leu Pro Gly Pro Asn Lys Met Leu Pro Lys Cys Leu Met Pro Lys Ser

675 680 685

Asn Arg Glu Lys Tyr Gly Ala Thr Glu Glu Val Leu Lys Ile Tyr Asn

690 695 700

Gln Gly Ser Phe Lys Lys Thr Glu Ser Asn Phe Ser Lys Lys Asp Leu

705 710 715 720

Ser Arg Leu Ile Asn Phe Tyr Lys Ser Ala Leu Gln Gln Tyr Glu Asp

725 730 735

Trp Arg Cys Phe Asn Phe Ser Phe Arg Ala Thr Asp Ser Tyr Glu Asp

740 745 750

Ile Gly Gln Phe Tyr Arg Asp Val Glu Ser Gln Gly Tyr Lys Leu Asp

755 760 765

Phe Gln Ser Ile Asn Thr Asp Val Leu Asp Glu Leu Val Glu Glu Gly

770 775 780

Lys Ile Tyr Leu Phe Glu Ile Lys Asn Gln Asp Ser Asn Gln Gly Lys

785 790 795 800

Ser Ser Ile His Arg Asp Asn Leu His Thr Met Tyr Trp Asn Ala Leu

805 810 815

Phe Gln Glu Val Leu Asn Arg Pro Lys Leu Asn Gly Gly Ala Glu Leu

820 825 830

Phe Tyr Arg Lys Ala Leu Ser Pro Glu Lys Ile Lys Glu Leu Gly Ser

835 840 845

Val Asp Lys Asn Gly Lys Arg Ile Ile Arg Asn Tyr Arg Phe Ser Lys

850 855 860

Glu Lys Phe Ile Phe His Ile Pro Ile Thr Leu Asn Phe Cys Leu Ser

865 870 875 880

Asp Thr Arg Val Asn Asp Thr Val Asn Gln Glu Leu Ser Arg Thr Ser

885 890 895

Ser Ser His Phe Leu Gly Ile Asp Arg Gly Glu Lys His Leu Ala Tyr

900 905 910

Tyr Phe Leu Val Asp Gln Asn Gly Lys Ile Val Leu Asp Glu Tyr Gly

915 920 925

Lys Ala Val Gln Gly Thr Leu Asn Ile Pro Phe Leu Asp Asn Asn Gly

930 935 940

Asn Val Arg Lys Ile Lys Ala Lys Arg Arg Ser Leu Asp Glu Asn Gly

945 950 955 960

Lys Glu Lys Ile Glu Glu Val Trp Cys Lys Asp Tyr Asn Glu Leu Leu

965 970 975

Glu Ala Arg Ala Gly Asp Arg Ala Tyr Ala Arg Lys Asn Trp Gln Thr

980 985 990

Ile Gly Asn Ile Lys Glu Leu Lys Glu Gly Tyr Ile Ser Gln Val Val

995 1000 1005

Arg Lys Ile Val Asp Leu Ala Ile Glu Tyr Glu Ala Phe Ile Val Leu

1010 1015 1020

Glu Asp Leu Asn Val Gly Phe Lys Arg Gly Arg Gln Lys Ile Glu Lys

1025 1030 1035 1040

Ser Val Tyr Gln Lys Leu Glu Leu Ala Leu Ala Lys Lys Leu Asn Phe

1045 1050 1055

Val Val Asp Lys Ser Ala Lys Ile Gly Gly Leu Lys Ser Val Thr Asn

1060 1065 1070

Ala Leu Gln Leu Ala Pro Pro Val Ser Asn Phe Gly Asp Ile Glu Gly

1075 1080 1085

Arg Lys Gln Phe Gly Ile Met Leu Tyr Thr Arg Ala Asn Tyr Thr Ser

1090 1095 1100

Gln Thr Asp Pro Ala Thr Gly Trp Arg Lys Ser Ile Tyr Leu Lys Arg

1105 1110 1115 1120

Gly Ser Glu Glu Ser Ile Arg Lys Gln Ile Ile Asp Ser Phe Glu Glu

1125 1130 1135

Ile Gly Phe Asp Gly Glu Asp Tyr Phe Phe Thr Tyr Thr Asp Ser Val

1140 1145 1150

Ala Gly Arg Thr Trp Ile Leu Tyr Ser Gly Lys Asn Gly Gly Ser Leu

1155 1160 1165

Asp Arg Phe Tyr Gly Lys Arg Asp Asn Asp Lys Asn Gln Trp Val Ser

1170 1175 1180

Met Arg Gln Asp Val Ser Lys Gln Leu Asp Gly Ile Leu Ala Asn Phe

1185 1190 1195 1200

Glu Lys Asp Arg Ser Ile Leu Ala Gln Ile Ile Asp Gly Glu Val Asp

1205 1210 1215

Leu Ile Lys Val Glu Gln Lys Tyr Thr Ala Trp Glu Ser Phe Arg Ser

1220 1225 1230

Thr Ile Asp Leu Ile Gln Gln Ile Arg Asn Thr Gly Thr Ser Glu Arg

1235 1240 1245

Asp Gly Asp Phe Ile Leu Ser Pro Val Arg Asp Glu Arg Gly Ile His

1250 1255 1260

Phe Asp Ser Arg Asp Thr Arg Glu Gly Met Pro Thr Ser Gly Asp Ala

1265 1270 1275 1280

Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Thr Ile Met Gly Glu His

1285 1290 1295

Ile Lys Arg Glu Tyr Ser Arg Met Phe Ile Ser Asp Glu Glu Trp Asp

1300 1305 1310

Ala Trp Leu Ala Gly Lys Gln Val Trp Glu Lys Trp Leu Lys Asp Asn

1315 1320 1325

Glu Lys Ile Leu Lys Lys Lys

1330 1335

<210> 9

<211> 1274

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC09的AA序列

<400> 9

Met Lys Asn Ser Leu Glu Asp Phe Thr Asn Leu Tyr Ser Leu Gln Lys

1 5 10 15

Thr Leu Arg Phe Glu Leu Lys Pro Ile Gly Asn Thr Gln Ser Met Leu

20 25 30

Glu Glu Asp Gly Val Phe Asp Thr Asp Glu Lys Arg Lys Ile Ala Tyr

35 40 45

Ser Lys Thr Lys Pro Tyr Ile Asp Arg Leu His Arg Glu Phe Ile Glu

50 55 60

Glu Ser Leu Ser Asp Ala Gln Ile Ser Lys Leu Asp Glu Tyr Phe Lys

65 70 75 80

Ala Tyr Val Asp Tyr Lys Lys Asp Lys Lys Asp Thr Lys Arg Phe Asn

85 90 95

Arg Ile Lys Gln Phe Lys Ser Val Leu Arg Lys Glu Val Val Asp His

100 105 110

Phe Asn Lys Gln Gly Lys Glu Trp Thr Thr Val Lys Phe Ala His Leu

115 120 125

Lys Ile Lys Lys Lys Asp Leu Glu Val Leu Phe Glu Lys Gln Leu Pro

130 135 140

Asn Ile Leu Lys Glu Glu Tyr Gly Thr Glu Lys Glu Thr Gln Ile Ile

145 150 155 160

Asp Glu Asp Ser Gly Glu Val Thr Ser Ile Phe Asp Met Trp Asn Gly

165 170 175

Phe Met Gly Tyr Phe Thr Lys Phe Phe Glu Thr Arg Lys Asn Phe Tyr

180 185 190

Lys Ser Asp Gly Thr Ser Thr Ala Ile Ala Thr Arg Ile Ile Asp Gln

195 200 205

Asn Leu Asp Arg Phe Ile Glu Asn Ile Leu Ile Tyr Asp Ser Ile Lys

210 215 220

Pro Lys Ile Asp Thr Ser Glu Val Arg Glu Phe Phe Asn Leu Glu Ser

225 230 235 240

Asp Thr Ile Phe Ser Met Glu Phe Tyr Asn Asn Cys Leu Leu Gln Ala

245 250 255

Gly Ile Asp Gln Tyr Asn Asn Phe Leu Gly Gly Lys Thr Leu Glu Asn

260 265 270

Gly Arg Lys Ile Arg Gly Ile Asn Glu Leu Ile Asn Lys Tyr Arg Gln

275 280 285

Glu Asn Pro Glu Asp Lys Ile Pro Phe Leu Lys Lys Leu Asp Lys Gln

290 295 300

Ile His Ser Glu Lys Glu Lys Phe Ile Gln Gln Ile Glu Thr Leu Glu

305 310 315 320

Asp Leu Lys Glu Glu Leu Gln Lys Phe Tyr Asn Ser Ser Asn Glu Lys

325 330 335

Ile Lys Ile Leu Asp Asn Leu Leu Ser Arg Ile Glu Glu Phe Lys Pro

340 345 350

Glu Gly Ile Phe Ile Ser Lys Gln Ala Phe Asn Thr Ile Ser Arg Arg

355 360 365

Trp Thr Asp Gln Ser Glu Ala Phe Glu Thr Ser Leu Phe Glu Ser Leu

370 375 380

Lys Glu Glu Lys Pro Ile Thr Gly Thr Ala Lys Lys Lys Asp Asp Gly

385 390 395 400

Tyr Asn Phe Pro Glu Phe Ile Ser Leu Gln Ser Ile Arg Asn Thr Leu

405 410 415

Lys Lys Val Gln Gly Glu Glu Arg Phe Trp Lys Glu Arg Tyr Tyr Arg

420 425 430

Asp Asn Ser Glu Ser Gly Ile Leu Ala Gly Asn Glu Glu Ile Trp Thr

435 440 445

Gln Phe Leu Met Ile Phe Lys Ser Glu Phe Asn Ser Lys Phe Glu Arg

450 455 460

Asn Asp Pro Glu Asp Asn Gly Thr Ile Gly Tyr Asn Leu Phe Lys Glu

465 470 475 480

Asp Leu Glu Lys Leu Leu Lys Asp Leu Lys Ile Thr Lys Asp Thr Lys

485 490 495

Ser Ile Ile Lys Arg Phe Ala Asp Glu Ala Leu His Ile Tyr Gln Val

500 505 510

Gly Lys Tyr Phe Ala Leu Glu Lys Asp Arg Val Trp Ile Ser Ser Tyr

515 520 525

Asp Asp Leu Leu Asp Thr Phe Tyr Thr Asp Pro Asn Thr Gly Tyr Leu

530 535 540

Ser Phe Tyr Glu Gly Ala Tyr Glu Gln Ile Val Gln Pro Tyr Asn Met

545 550 555 560

Ile Arg Asn Tyr Leu Thr Arg Lys Pro Tyr Ser Asp Glu Lys Trp Lys

565 570 575

Leu Asn Phe Glu Asn Pro Thr Leu Ala Asn Gly Trp Asp Lys Asn Lys

580 585 590

Glu Thr Asp Asn Ser Ser Ile Met Leu Arg Lys Glu Gly Ala Tyr Tyr

595 600 605

Leu Gly Ile Met Lys Lys Gly Lys Asn Lys Leu Phe Glu Glu Arg Asn

610 615 620

Arg Gln Leu Phe Glu Pro Lys Asn Gly Glu Asp Thr Tyr Glu Lys Leu

625 630 635 640

Ser Tyr Lys Leu Phe Pro Asp Pro Ala Lys Met Ile Pro Lys Val Cys

645 650 655

Phe Ser Asn Lys Asn Ile Gln Met Phe Ser Pro Ser Thr Glu Ile Met

660 665 670

Asn Ile Tyr Asn Gly Glu Thr Phe Lys Lys Asn Ser Asp Asp Phe Ser

675 680 685

Val Ser Ser Met Gln Lys Leu Ile Ala Phe Tyr Thr Lys Cys Leu Ser

690 695 700

Gln Tyr Glu Gly Trp Lys Tyr Tyr Asp Phe Lys Tyr Ile Lys Ser Pro

705 710 715 720

Asp Gln Tyr Lys Asp Asn Ile Gly Glu Phe Tyr Asn Asp Val Ala Lys

725 730 735

Ser Gly Tyr Arg Val Trp Phe Glu Asn Ile Ser Gln Ser Tyr Val Asp

740 745 750

Ser Lys Asn Thr Met Gly Glu Leu Tyr Leu Phe Lys Ile His Asn Lys

755 760 765

Asp Trp Asn Gln Lys Asp Lys Lys Thr Lys Val Gly Ser Lys Asn Leu

770 775 780

His Thr His Tyr Phe Glu Glu Leu Phe Ser Gln Asp Asn Ile Glu Asn

785 790 795 800

Asn Phe Pro Leu Lys Leu Asn Gly Glu Ala Glu Val Phe Tyr Arg Pro

805 810 815

Lys Thr Asn Pro Glu Lys Leu Gly Thr Lys Lys Asp Ser Lys Gly Arg

820 825 830

Glu Val Ile Asp Arg Lys Arg Tyr Ala Ser Asp Lys Val Leu Phe His

835 840 845

Val Pro Ile Thr Leu Asn Arg Thr Pro Val Thr Thr Thr Lys Leu Asn

850 855 860

Lys Glu Ile Asn Gly Phe Leu Ala Asn Asn Pro Ser Ile Asn Ile Ile

865 870 875 880

Gly Val Asp Arg Gly Glu Lys His Leu Val Tyr Tyr Ser Val Val Asn

885 890 895

Gln Arg Gly Lys Met Leu Glu Ser Gly Ser Phe Asn Thr Ile Asn Gly

900 905 910

Val Asp Tyr His Gly Lys Leu Glu Glu Arg Ala Asp Arg Arg Glu Gln

915 920 925

Ala Arg Arg Asp Trp Gln Asp Val Glu Gly Ile Lys Asn Leu Lys Lys

930 935 940

Gly Tyr Ile Ser Leu Val Val Arg Glu Leu Ala Asn Leu Ser Ile Lys

945 950 955 960

Tyr Asn Ala Ile Ile Val Met Glu Asp Leu Asn Met Arg Phe Lys Gln

965 970 975

Ile Arg Gly Gly Ile Glu Lys Ser Ala Tyr Gln Gln Leu Glu Lys Ala

980 985 990

Leu Ile Glu Lys Leu Asn Tyr Leu Val Asn Lys Thr Glu Thr Asp Pro

995 1000 1005

Gln Lys Thr Gly His Ile Leu Lys Ala Tyr Gln Leu Thr Ser Pro Ile

1010 1015 1020

Lys Ser Phe Lys Glu Met Gly Lys Gln Thr Gly Ile Ile Phe Tyr Thr

1025 1030 1035 1040

Gln Ala Ser Tyr Thr Ser Val Thr Asp Pro Ile Thr Gly Trp Arg Pro

1045 1050 1055

Asn Leu Tyr Leu Lys Tyr Ser Ser Ala Ser Lys Ala Lys Ser Asp Ile

1060 1065 1070

Leu Lys Phe Ser Lys Ile Ser Tyr Asn Thr Asn Asn Asn Arg Phe Glu

1075 1080 1085

Phe Thr Tyr Asp Leu Arg Asn Phe Val Asn Met Lys Ala Tyr Pro Gln

1090 1095 1100

Lys Thr Ala Trp Thr Ile Cys Ser Asn Val Glu Arg Phe Arg Trp Asp

1105 1110 1115 1120

Arg Lys Gly Asn Lys Asn Asn Gly Glu Tyr Ile Gln Tyr Lys Asp Leu

1125 1130 1135

Thr Glu Asn Phe Lys Thr Phe Phe Glu Glu Val Ser Ile Asn Tyr Lys

1140 1145 1150

Gly Asp Ile Leu Phe Gln Ile Lys Asn Leu Ser Glu Lys Gly Asn Glu

1155 1160 1165

Lys Phe Phe Arg Asp Leu Ile Phe Tyr Ile Ser Leu Ile Ser Gln Ile

1170 1175 1180

Arg Asn Thr Gln Lys Asp Lys Lys Gly Asp Glu Asn Asp Phe Ile Leu

1185 1190 1195 1200

Ser Pro Val Glu Pro Phe Phe Asp Ser Arg Lys Ser Ser Thr Phe Gly

1205 1210 1215

Glu Asn Leu Pro Leu Asn Gly Asp Ala Asn Gly Ala Tyr Asn Ile Ala

1220 1225 1230

Arg Lys Gly Ile Ile Met Leu Asn Lys Ile Ser Lys Gly Ser Lys Asn

1235 1240 1245

Lys Val Lys Glu Asp Ile Gly Trp Gly Asp Leu Tyr Ile Pro His Thr

1250 1255 1260

Glu Trp Asp Asp Phe Ala Thr Gly Ser Ile

1265 1270

<210> 10

<211> 1456

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC10的AA序列

<400> 10

Met Ser Thr Lys Arg Ser Phe Ser Asp Phe Thr Asn Leu Tyr Ser Val

1 5 10 15

Ser Lys Thr Leu Arg Phe Glu Leu Lys Pro Ile Gly Lys Thr Leu Glu

20 25 30

Asn Met Arg Glu Arg Ile Tyr Asn Asp Lys Lys Asp Tyr Asp Ser Ala

35 40 45

Leu Gln Thr Phe Leu His Asp Gln Ala Ile Glu Asp Ala Tyr Lys Thr

50 55 60

Leu Lys Pro Ile Leu Asp Ser Leu His Glu Glu Phe Ile Asn Thr Ser

65 70 75 80

Leu Asn Ser Ser Lys Ala Lys Asn Ile Asp Leu Ser Glu Tyr Leu Asn

85 90 95

Ala Tyr Arg Glu Arg Gly Asn Asp Thr Lys Thr Gly Glu Glu Ser Lys

100 105 110

Leu Ser Gly Ile Glu Lys Ser Leu Arg Lys Ala Ile Gly Glu Thr Tyr

115 120 125

Leu Thr Trp Ala Lys Ser Phe Thr Glu Gln Ala Lys Asn Leu Ile Gly

130 135 140

Ile Ile Glu Asp Ile Trp Asp Thr Glu Glu Glu Trp Asp Glu Glu Lys

145 150 155 160

Lys Thr Lys Trp Leu Phe Lys Lys Lys Asn Phe Glu Leu Leu Thr Glu

165 170 175

Ser Gly Ile Leu Val Phe Ile Glu Lys Lys Leu Asp Thr Met Asn Ile

180 185 190

Ser Glu Gln Glu Lys Thr Asp Ile Lys Lys Ala Leu Glu Glu Phe Lys

195 200 205

Gly Phe Phe Thr Tyr Phe Ser Gly Tyr Asn Gln Asn Arg Lys Asn Tyr

210 215 220

Tyr Glu Thr Lys Ala Glu Lys Lys Thr Ala Ile Ala Thr Arg Ile Val

225 230 235 240

His Glu Asn Leu Pro Lys Phe Cys Asp Asn Val Ile Leu Phe His Gly

245 250 255

Tyr Gln Lys Ile Leu Lys Asp Gly Ser Lys Arg Glu Tyr Lys Lys Lys

260 265 270

Glu Glu Tyr Leu Gly Met Tyr Ala Phe Leu Lys Leu Arg Asn Ile Glu

275 280 285

Thr Cys Ile Lys Asp Ala Glu Ser Gly Glu Met Ile Glu Leu Tyr Ala

290 295 300

Ile Thr Glu Asp Ile Phe Asp Ile Ser Phe Phe Ser Ser Cys Leu Ala

305 310 315 320

Gln Arg Glu Ile Asp Glu His Asn Arg Ile Ile Gly Gly Ile Asp Lys

325 330 335

Tyr Asn Arg Ile Ile Gly His Tyr Asn Ala Leu Ile Asn Leu Tyr Asn

340 345 350

Gln Ala Arg Lys Lys Asp Glu Lys Phe Thr Lys Leu Ser Pro Phe Lys

355 360 365

Glu Leu Tyr Lys Gln Ile Trp Cys Gly Asn Lys Lys Trp Ser Trp Ile

370 375 380

Lys Ala Ile Thr His Asp Thr Asp Glu Gln Ile Leu Ala Asp Thr Asn

385 390 395 400

His Thr Gly Glu Ala Ile Ser Val Glu Arg Ile Leu Ser Leu Ala Ser

405 410 415

Lys Ala Gly Lys Lys Tyr Phe Gln Pro Trp Lys Ser Thr Asp Asp Gly

420 425 430

Ile Lys Thr Val Pro Asp Phe Leu Asp Trp Leu Arg Gly Gln Thr Asp

435 440 445

Trp Asn Gly Ile Tyr Trp Ser Lys Ala Ala Ile Asn Ser Ile Ser Asn

450 455 460

Val Tyr Phe Pro Asn Trp Gly Ser Ile Lys Glu Thr Met Lys Gly Asp

465 470 475 480

Lys Thr Leu Val Ser Tyr Asp Lys Lys Arg Glu Glu Gln Ile Lys Ile

485 490 495

Asn Glu Ala Val Glu Leu Ser Gly Leu Phe Asp Ile Leu Asp Ser Thr

500 505 510

Asp Gly Asp Trp Lys Gln Glu Trp Val Leu Phe Lys Ala Ser Leu Thr

515 520 525

Lys Leu Leu Asp Ala Ser Ala Glu Asn Ala Glu Glu Asn Ser Lys Arg

530 535 540

Ala Arg Arg Lys Asp Ile Ile Asp Arg Ser Ser Ser Pro Ser Gln Ala

545 550 555 560

Leu Leu Ala Leu Ile Thr Asp Phe Ile Glu Glu Asn Met Lys His Phe

565 570 575

Leu Asp Gln Ser His Thr Ile Leu Arg Leu Thr Glu Tyr Ser Ser Pro

580 585 590

Lys Ser Lys Glu Ala Ile Lys Ser Trp Met Asp Leu Ala Leu Ser Val

595 600 605

Ser Gln Thr Ile Arg Tyr Phe Arg Val Lys Glu Ser Lys Thr Lys Gly

610 615 620

Asp Thr Leu Asn Ala Glu Leu Val Gly Ile Leu Thr Asn Leu Leu Asp

625 630 635 640

Ala Glu Asp Ala Thr Trp Phe Glu Trp Tyr Asp Leu Leu Arg Asn Tyr

645 650 655

Leu Thr Lys Lys Pro Gln Asp Asp Ala Lys Glu Asn Lys Leu Lys Leu

660 665 670

Asn Phe Ala Asn Ser Thr Leu Ala Ala Gly Trp Asp Val Asn Lys Glu

675 680 685

Thr Asp Asn Thr Cys Val Ile Leu Gln Asn Pro Glu Trp Lys Thr Tyr

690 695 700

Leu Ala Val Met Asn Lys Asn Lys Lys Asn Val Phe Gln Lys Glu Trp

705 710 715 720

Asn Glu Trp Arg Trp Lys Lys Lys Thr Thr Lys Leu Asn Pro Leu Tyr

725 730 735

Glu Ile Asp Trp Gly Glu Ser Trp Lys Lys Met Glu Tyr Asp Phe Trp

740 745 750

Ser Asp Val Ser Lys Met Ile Pro Lys Cys Ser Thr Gln Leu Lys Lys

755 760 765

Val Ile Lys His Phe Lys Glu Ser Asp Glu Asp Phe Ile Phe Pro Ser

770 775 780

Gly Tyr Lys Val Thr Ser Gly Glu Arg Phe Ile Glu Glu Cys Arg Ile

785 790 795 800

Thr Lys Glu Gln Phe Glu Leu Asn Asn Lys Val Tyr Lys Arg Asp Gly

805 810 815

Asp Arg Ile Ile Ser Ala Phe Arg Tyr Glu Leu Ser Glu Thr Glu Glu

820 825 830

Lys Thr Tyr Ile Lys Ser Phe Gln Lys Gly Tyr Leu Asp Met Leu Leu

835 840 845

Lys Ser Asn Asn Leu Pro Glu Thr Glu Gln Glu Ile Tyr Arg Lys Lys

850 855 860

Tyr Glu Asp Ser Leu Ser Lys Trp Ile Asn Phe Cys Lys Tyr Phe Ile

865 870 875 880

Trp Lys Tyr Pro Lys Thr Ser Leu Phe Glu Tyr Gln Phe Asp Glu Thr

885 890 895

Asp His Tyr Lys Ser Val Asp Lys Phe Asn Leu Asp Val Asp Ile Trp

900 905 910

Ser Tyr Lys Leu Lys Val Asp Thr Lys Ile Asn Lys Thr Ile Leu Asp

915 920 925

Thr Leu Val Glu Asn Gly Asp Ile Tyr Leu Phe Glu Ile Lys Asn Gln

930 935 940

Asp Ser Asn Ile Gly Lys Trp Glu Asn His Lys Asn Asn Leu His Thr

945 950 955 960

Thr Tyr Trp Lys Ser Ile Phe Glu Ser Val Gln Asn Arg Pro Lys Leu

965 970 975

Asn Gly Glu Ala Glu Ile Phe Tyr Met Lys Pro Leu Ser Pro Glu Lys

980 985 990

Leu Gln Lys Lys Ile Asp Lys Lys Gly Lys Glu Ile Ile Asp Gly Tyr

995 1000 1005

Arg Phe Ser Arg Glu Arg Phe Ile Phe His Cys Pro Ile Thr Leu Asn

1010 1015 1020

Phe Cys Leu Gly Asn Glu Lys Ile Asn Asn Ile Ile Asn Phe Glu Leu

1025 1030 1035 1040

Ser Pro Lys Ser Asp Ile Tyr Phe Leu Gly Leu Asp Arg Gly Glu Lys

1045 1050 1055

His Leu Val Tyr Tyr Ser Ile Val Asp Gln Asn Gly Lys Met Ile Asp

1060 1065 1070

Gln Trp Ser Phe Asn Glu Ile Lys Trp Lys Asp Tyr His Ala Leu Leu

1075 1080 1085

Thr Lys Arg Glu Trp Asp Arg Met Glu Ser Arg Lys Asn Trp Gln Thr

1090 1095 1100

Ile Ser Asn Ile Ala Lys Leu Lys Glu Trp Tyr Ile Ser Leu Val Ile

1105 1110 1115 1120

His Glu Ile Ile Glu Lys Leu Lys Leu Asn Pro Trp Phe Ile Val Leu

1125 1130 1135

Glu Asp Leu Asn Thr Gly Phe Lys Arg Gly Arg Gln Lys Ile Glu Lys

1140 1145 1150

Ser Ile Tyr Gln Lys Phe Glu Leu Ala Leu Ala Lys Lys Leu Asn Phe

1155 1160 1165

Val Val Asp Lys Ser Ala Lys Leu Gly Glu Val Gly Ser Val Thr Asn

1170 1175 1180

Ala Leu Gln Leu Thr Pro Pro Val Ser Asn Tyr Gly Asp Ile Glu Asn

1185 1190 1195 1200

Arg Lys Gln Val Gly Ile Met Leu Tyr Thr Arg Ala Asn Tyr Thr Ser

1205 1210 1215

Gln Thr Asp Pro Ala Thr Gly Trp Arg Lys Thr Ile Tyr Leu Lys Thr

1220 1225 1230

Gly Ser Glu Glu Asn Ile Lys Glu Gln Ile Val Thr Gln Phe Ser Asp

1235 1240 1245

Ile Gly Phe Asp Gly Lys Asp Tyr Tyr Phe Glu Tyr Thr Asp Lys Ile

1250 1255 1260

Gly Lys Thr Trp Ile Leu Tyr Ser Gly Lys Asn Gly Lys Ser Leu Thr

1265 1270 1275 1280

Arg Phe Arg Gly Val Arg Gly Lys Glu Lys Asn Glu Trp Asn Ile Lys

1285 1290 1295

Glu Ile Asn Val Arg Asn Met Leu Asp Gly Ile Phe Ala Asn Phe Asp

1300 1305 1310

Lys Asp Arg Ser Phe Leu Ser Gln Ile Leu Asp Glu Trp Val Glu Ile

1315 1320 1325

Lys Lys Ile Asp Glu His Thr Ala Trp Glu Ser Leu Arg Phe Ala Ile

1330 1335 1340

Asp Leu Ile Gln Gln Ile Arg Asn Ser Gly Asp Lys Thr Gln Trp Glu

1345 1350 1355 1360

Asp Asp Asn Phe Leu Phe Ser Pro Val Arg Asp Ala Gln Gly Asn His

1365 1370 1375

Phe Asp Thr Arg Glu Gln Lys Glu Gly Leu Pro Lys Asp Ala Asp Ala

1380 1385 1390

Asn Gly Ala Tyr Asn Ile Ala Arg Lys Trp Ile Ile Met Asn Glu His

1395 1400 1405

Ile Arg Ile Asn Glu Asp Thr Lys Asp Leu Asp Leu Phe Val Ser Asp

1410 1415 1420

Glu Glu Trp Asp Met Trp Leu Thr Asp Arg Glu Lys Trp Lys Glu Met

1425 1430 1435 1440

Leu Pro Ile Phe Ala Ser Arg Lys Ala Met Glu Lys Arg Arg Gly Lys

1445 1450 1455

<210> 11

<211> 1317

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC11的AA序列

<400> 11

Met Ser Gln Asn Asn Thr Phe Glu Lys Phe Thr Asn Gln Tyr Ser Leu

1 5 10 15

Ser Lys Thr Leu Arg Phe Glu Leu Arg Pro Val Gly Asn Thr Glu Gln

20 25 30

Met Leu Glu Asp Glu Asn Val Phe Lys Lys Asp Glu Ile Ile Arg Lys

35 40 45

Lys Tyr Glu Gln Thr Lys Pro Phe Ile Asp Lys Leu His Lys Glu Val

50 55 60

Ile Lys Asp Ser Leu His Gly Lys Lys Ile Glu Gly Leu Asp Asp Tyr

65 70 75 80

Phe Lys Lys Phe Glu Ile Tyr Ser Lys Asn Lys Lys Asp Ser Lys Ile

85 90 95

Lys Lys Glu Phe Thr Asp Lys Glu Ser Glu Leu Arg Lys Gln Leu Asn

100 105 110

Ser His Phe Lys Ala Glu Ser Leu Phe Ser Glu Lys Val Phe Ser Leu

115 120 125

Leu Lys Glu Lys Tyr Gly Thr Glu Asp Glu Ser Phe Val Lys Asp Glu

130 135 140

Asn Gly Asn Phe Val Leu Asp Thr Val Gly Glu Lys Ile Ser Ile Phe

145 150 155 160

Asp Glu Trp Lys Gly Phe Thr Gly Tyr Phe Thr Lys Phe Gln Lys Thr

165 170 175

Arg Glu Asn Phe Tyr Lys Asp Asp Gly Thr Ser Thr Ala Ile Val Thr

180 185 190

Arg Thr Ile Asp Glu Asn Leu Tyr Arg Phe Cys Glu Asn Ile Lys His

195 200 205

Phe Glu Ser Ile Lys Asn Arg Val Asn Phe Ser Glu Ile Glu Lys Asn

210 215 220

Phe Asn Phe Lys Leu Glu Asn Leu Phe Lys Ala Asp Phe Tyr Asn Ser

225 230 235 240

Cys Leu Leu Gln Asp Gly Ile Asp Lys Tyr Asn Asp Ile Leu Gly Gly

245 250 255

Lys Thr Leu Glu Ser Gly Glu Lys Leu Lys Gly Leu Asn Glu Ile Ile

260 265 270

Asn Lys Tyr Arg Gln Asp Asn Lys Val Glu Lys Ile Gly Phe Phe Lys

275 280 285

Met Leu Asp Lys Gln Ile Leu Gly Asp Lys Glu Lys Pro Ser Phe Ile

290 295 300

Glu Ser Ile Ala Asp Asp Asn Glu Leu Leu Leu Lys Leu Lys Glu Phe

305 310 315 320

Tyr Thr Asn Ala Glu Glu Lys Thr Glu Val Leu Lys Lys Leu Phe Ser

325 330 335

Asp Phe Ser Lys Asn Asn Asp Ser Tyr Asp Leu Ser Lys Ile Tyr Ile

340 345 350

Asn Lys Val Gly Ile Asn Thr Ile Leu Leu Lys Trp Phe Asp Val Ala

355 360 365

Gly Arg Ser Asp Phe Glu Lys Asn Ile Ser Thr Gln Thr Lys Lys Glu

370 375 380

Lys Ile Val Thr Phe Asp Lys Asp Ser Asn Ser Tyr Lys Phe Pro Glu

385 390 395 400

Phe Leu Ala Phe Ser His Ile Lys Glu Ala Leu Ser Asn Gly Thr Tyr

405 410 415

Glu Val Lys Glu Ile Trp Lys Glu Arg Tyr Tyr Gln Ser Glu Asn Lys

420 425 430

Glu Lys Ser Glu Lys Ala Pro Leu Lys Lys Asp Ser Ala Ile Ser His

435 440 445

Trp Glu Glu Phe Leu Gln Ile Phe Ser Tyr Glu Phe Asp Leu Leu Phe

450 455 460

Val Gly Ala Glu Ser Gln Ala Gly Tyr Asn Ser Asn Lys Asn Leu Phe

465 470 475 480

Glu Ser Leu Ile Lys Lys Asn Glu Lys Gly Phe Ser Ile Ser Pro Glu

485 490 495

Glu Lys Leu Val Ile Lys Asn Phe Val Asp Asn Thr Leu Trp Ile Tyr

500 505 510

Gln Met Ala Lys Tyr Phe Ala Ile Glu Lys Lys Arg Lys Trp Leu Glu

515 520 525

Ser Glu Tyr Pro Thr Asp Ser Ser Phe Tyr Asp Ser Glu Glu Phe Gly

530 535 540

Phe Lys Asn Lys Phe Tyr Asp Asp Ala Tyr Asp Lys Ile Val Lys Leu

545 550 555 560

Arg Met Leu Leu Gln Ser His Leu Thr Lys Lys Pro Phe Ser Thr Asp

565 570 575

Lys Trp Lys Leu Asn Phe Glu Asn Pro Thr Leu Ala Lys Gly Trp Asp

580 585 590

Lys Asn Lys Glu Ser Asp Asn Ser Ala Val Leu Leu Arg Lys Glu Gly

595 600 605

Arg Tyr Tyr Leu Ala Val Met Lys Lys Gly Asn Asn Lys Ile Phe Asp

610 615 620

Asp Lys Asn Lys Ser Asn Phe Leu Glu Asn Ile Glu Gly Gly Lys Tyr

625 630 635 640

Glu Lys Met Val Tyr Lys Gln Met Ser Asp Pro Ser Lys Asp Ile Gln

645 650 655

Asn Leu Met Val Ile Asp Asp Lys Thr Val Arg Lys Val Gly Lys Lys

660 665 670

Asp Pro Leu Asp Gly Val Asn Arg Arg Leu Glu Glu Leu Lys Lys Glu

675 680 685

Tyr Leu Pro Arg Asp Ile Asn Thr Ile Arg Glu Gln Lys Ala Tyr Leu

690 695 700

Lys Ser Ser Asp Asn Phe Asn Leu Gly Asp Ala Asn Leu Phe Ile Asn

705 710 715 720

Tyr Tyr Lys Asp Arg Leu Val Glu Tyr His Lys Asp Ile Phe Val Phe

725 730 735

Ser Phe Arg Asp Arg Tyr Ser Asp Phe His Asp Phe Ser Lys His Val

740 745 750

Ala Glu Gln Thr Tyr Ser Leu Ser Phe Glu Asp Ile Ser Glu Phe Tyr

755 760 765

Ile Gln Glu Lys Asn Asn Asn Gly Glu Leu Phe Leu Phe Glu Ile His

770 775 780

Asn Lys Asp Trp Asn Leu Glu Lys Lys Gly Gly Asp Arg Lys Ser Gly

785 790 795 800

Ala Lys Asn Leu His Thr Val Tyr Phe Glu Ser Leu Phe Ser Lys Glu

805 810 815

Asn Glu Asn Asn Asn Phe Ser Ile Lys Leu Asn Gly Glu Ala Glu Leu

820 825 830

Phe Tyr Arg Pro Lys Thr Asp Glu Gln Lys Leu Gly Asn Lys Asn Asp

835 840 845

Leu Lys Gly Lys Ile Val Leu Asn Lys Lys Arg Tyr Ala Glu Asn Lys

850 855 860

Thr Phe Ile His Ile Pro Ile Thr Leu Asn Arg Val Ala Ser Glu Ser

865 870 875 880

Lys Tyr Phe Asn Gln Lys Leu Asn Asp Phe Leu Val Gly Asn Pro Asp

885 890 895

Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Lys His Leu Ile Tyr Tyr

900 905 910

Ala Gly Ile Asn Gln Ala Gly Glu Phe Leu Lys Asp Glu Lys Gly Asn

915 920 925

Leu Val Leu Gly Ser Leu Asn Thr Ile Asn Asp Val Asn Tyr Ala Gln

930 935 940

Lys Leu Glu Glu Arg Ala Lys Gly Arg Val Lys Ala Lys Gln Asp Trp

945 950 955 960

Gln Glu Ile Glu Asn Ile Lys Asp Leu Lys Arg Gly Tyr Ile Ser Leu

965 970 975

Val Val Arg Glu Leu Ala Asp Leu Ile Ile Lys His Asn Ala Ile Ile

980 985 990

Val Phe Glu Asp Leu Asn Met Arg Phe Lys Gln Ile Arg Gly Gly Ile

995 1000 1005

Glu Lys Ser Val Tyr Gln Gln Leu Glu Lys Ala Leu Ile Asp Lys Leu

1010 1015 1020

Asn Phe Leu Val Asn Lys Gly Glu Lys Asp Pro Thr Lys Ala Gly His

1025 1030 1035 1040

Leu Leu Arg Ala Phe Gln Leu Thr Ala Pro Ile Ser Ala Tyr Lys Asp

1045 1050 1055

Met Gly Lys Gln Thr Gly Val Ile Phe Tyr Thr Gln Ala Ser Tyr Thr

1060 1065 1070

Ser Lys Thr Cys Pro Glu Cys Gly Phe Arg Pro Asn Val Arg Trp Glu

1075 1080 1085

Pro Lys Ser Ile Lys Asp Lys Ile Lys Glu Gly Lys Leu Glu Ile Thr

1090 1095 1100

Tyr Lys Glu Asp Gly Phe Glu Ile Ser Tyr Lys Leu Ser Asp Phe Ser

1105 1110 1115 1120

Lys Ser Gln Asn Gln Ser Lys Arg Arg Asn Ile Leu Tyr Thr Asn Val

1125 1130 1135

Ser Lys Gln Asp Lys Phe Asn Leu Asn Thr Lys Asp Ala Val Arg Cys

1140 1145 1150

Lys Trp Phe Arg Lys Thr Leu Ser Glu Asn Glu Leu Asn Lys Gly Glu

1155 1160 1165

Gln Lys Leu Asn Ile Gln Thr Glu Thr Gly Val Asn Ile Glu Tyr Lys

1170 1175 1180

Ile Ser Asp Cys Leu Ile Gly Leu Phe Glu Lys Tyr Gly Leu Asp Tyr

1185 1190 1195 1200

Gln Asn Asn Leu Gln Glu Glu Ile Lys Asn Ser Gly Asp Ser Leu Pro

1205 1210 1215

Val Lys Phe Tyr Asp Lys Leu Ser Phe Tyr Leu His Leu Leu Thr Asn

1220 1225 1230

Thr Arg Ser Ser Val Ser Gly Thr Asp Ile Asp His Ile Asn Cys Pro

1235 1240 1245

Asn Cys Gly Phe Cys Ser Lys Asn Gly Phe Lys Gly Gly Glu Phe Asn

1250 1255 1260

Gly Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Ile Ile Ile

1265 1270 1275 1280

Leu Asp Lys Leu Lys Asn Tyr Lys Thr Glu Asn Ser Asn Leu Glu Lys

1285 1290 1295

Met Thr Trp Gly Asp Leu Phe Ile Asp Ile Asp Glu Trp Asp Lys Phe

1300 1305 1310

Thr Gln Asn Lys Thr

1315

<210> 12

<211> 1385

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC12的AA序列

<400> 12

Met Glu Thr Lys Asn Lys Ser Ile Trp Gly Asp Phe Thr Asn Lys Tyr

1 5 10 15

Ser Leu Ser Lys Thr Leu Arg Phe Glu Leu Val Pro Val Gly Lys Thr

20 25 30

Arg Glu Asn Ile Gln Lys His Asn Pro Glu Phe Val Gln Asp Gln Lys

35 40 45

Ile Glu Glu Ala Tyr Gln Ile Leu Lys Ser Val Phe Asp Lys Ile His

50 55 60

Glu Asp Phe Ile Thr Lys Ser Leu Glu Ser Asp Glu Ala Lys Ser Ile

65 70 75 80

Asn Phe Ser Glu Tyr Phe Asp Leu Tyr Lys Lys Trp Asn Glu Leu Lys

85 90 95

Lys Lys Lys Thr Asn Glu Lys Asn Ile Glu Ile Lys Lys Glu Ile Gln

100 105 110

Asn Glu Ile Glu Lys Ile Tyr Lys Asp Asn Gly Gly Ser Lys Gly Glu

115 120 125

Ile Gln Lys Ile Glu Asp Glu Leu Arg Lys Arg Phe Glu Glu Ile Phe

130 135 140

Lys Ile Gln Gly Lys Ile Phe Lys Glu Lys Ala Cys Glu Leu Asn Ile

145 150 155 160

Lys Glu Gly Gln Glu Lys Asp Asp Asp Glu Glu Lys Asp Asp Asn Lys

165 170 175

Lys Gly Phe Arg Lys Leu Leu Lys Ala Lys Phe Leu Tyr Asp Tyr Leu

180 185 190

Cys Asn Leu Ile Glu Ser Lys Asn Ile Ile Tyr Lys Asp Phe Phe Glu

195 200 205

Asn Ile Lys Asn Lys Glu Gly Glu Ser Ile Ser Lys Glu Lys Thr Lys

210 215 220

Asp Ala Leu Ile Arg Phe Lys Gly Phe Thr Thr Tyr Phe Gly Gly Phe

225 230 235 240

Glu Leu Asn Arg Leu Asn Tyr Tyr Thr Thr Lys Glu Glu Lys Ser Thr

245 250 255

Ala Val Ala Thr Arg Ile Val Asn Gln Asn Leu Pro Lys Phe Cys Asp

260 265 270

Asn Val Ile Leu Phe Glu Ile Lys Lys Ser Glu Tyr Leu Lys Ile Asp

275 280 285

Glu Phe Leu Lys Asn Lys Asn Ile Ser Leu Ile Ser Lys Asn Gln Asn

290 295 300

Gly Gly Glu Val Glu Leu His Lys Ile Asn Lys Asn Phe Phe Glu Met

305 310 315 320

Met Phe Phe Ser Lys Cys Leu Ser Gln Lys Glu Ile Gln Lys Tyr Asn

325 330 335

Leu Glu Ile Gly Asn Ala Asn Asn Leu Ile Asn Arg Tyr Asn Gln Gln

340 345 350

Gln Ser Asp Lys Ser Gln Lys Leu Lys Leu Phe Lys Thr Leu His Lys

355 360 365

Gln Ile Gly Cys Gly Asp Arg Gly Gly Phe Ile Pro Ser Ile Lys Gly

370 375 380

Glu Glu Asp Leu Arg Glu Arg Leu Gln Glu Ile Lys Asn Asn Ser Ile

385 390 395 400

Glu Tyr Phe Glu Asn Ile Asn Asp Phe Ile Glu Tyr Leu Lys Asn His

405 410 415

Glu Asn Tyr Glu Asn Val Tyr Trp Ser Asp Lys Ala Ile Asn Thr Ile

420 425 430

Ser Ser Lys Tyr Phe Ser Asp Trp Leu Asn Leu Lys Lys Glu Ile Trp

435 440 445

Gly Lys Arg Asp Arg Lys Gly Asn Leu Lys Asp Glu Glu Thr Lys Ile

450 455 460

Pro Arg Ala Val Gln Leu Lys Asp Leu Leu Glu Asn Leu Asp Lys Ile

465 470 475 480

Thr Asp Trp Lys Leu Glu Gly Arg Leu Phe Lys Leu Ser Leu Phe Glu

485 490 495

Asn Gly Arg Lys Ala Lys Lys Leu Gln Gln Glu Asp Leu Asn Lys Phe

500 505 510

Asn Lys Asn Lys Ile Glu Asn Glu Leu Glu Ile Glu Lys Leu Gln Ile

515 520 525

Ile Glu Gln Asn Pro Ser Pro Phe Gln Ala Leu Leu Asn Met Ile Phe

530 535 540

Ala Asp Ile Lys Ser Lys Glu Ser Ala Phe Leu Glu Ser Arg Ile Phe

545 550 555 560

Glu Ile Ser Asp Phe Val His Asn Glu Asp Lys Gln Ile Ile Lys Gln

565 570 575

Trp Leu Asp Ser Ile Leu Ala Ile Asn Gln Ile Ile Lys Tyr Trp Arg

580 585 590

Val Lys Asp Thr Phe Gly Thr Glu Gly Thr Leu Asp Glu Lys Leu Lys

595 600 605

Asn Ile Ile Tyr Ser Glu Lys Asn Pro Thr Arg Phe Tyr Asp Ile Ile

610 615 620

Arg Asn Tyr Leu Thr Lys Lys Pro Gln Asp Glu Leu Asn Lys Leu Lys

625 630 635 640

Leu Asn Phe Glu Asn Ser Thr Leu Ala Gln Gly Leu Asp Val Asn Lys

645 650 655

Glu Lys Asp Asn Phe Cys Ile Ile Leu Arg Asp Asp Lys Gln Asn Gln

660 665 670

Tyr Leu Gly Ile Leu Asn Ser Lys Asn Lys Asn Ile Phe Glu Ile Asp

675 680 685

Gln Asn Glu Asp Ile Tyr Gln Asp Asp Gly Leu Gly Trp Ser Lys Met

690 695 700

Met Tyr Lys Leu Ile Pro Gly Ala Ser Lys Thr Leu Pro Lys Ile Phe

705 710 715 720

Phe Ser Lys Arg Trp Thr Glu Asn Asn Pro Thr Pro Asp Glu Ile Ser

725 730 735

Lys Ile Lys Lys Gly Glu Thr Phe Lys Lys Gly Asp Asn Phe Ile Lys

740 745 750

Arg Asp Leu His Glu Leu Ile Asn Phe Tyr Lys Ala Asn Leu Glu Lys

755 760 765

Tyr Pro Ser Val Asn Glu Ser Trp Ala Lys Leu Phe Ile Phe Asn Phe

770 775 780

Ser Asp Thr Lys Thr Tyr Glu Ser Ile Asp Gln Phe Tyr Asn Glu Val

785 790 795 800

Asp Lys Gln Gly Tyr Lys Val Ser Phe Ile Ser Ile Asn Lys Asn Thr

805 810 815

Leu Asp Asn Phe Ile Asp Lys Glu Lys Leu Tyr Leu Phe Gln Ile Lys

820 825 830

Asn Lys Asp Asn Asn Leu Asp Lys Gly Glu Lys Lys Gln Ser Asn Lys

835 840 845

Asn Leu His Ser Ile Tyr Trp Glu Ala Ile Phe Gly Lys Ala Leu Asn

850 855 860

Lys Pro Lys Leu Asn Gly Gly Ala Glu Ile Phe Tyr Arg Pro Ala Leu

865 870 875 880

Ser Glu Lys Lys Ile Ser Glu Leu Lys Ile Lys Asp Lys Asn Gly Lys

885 890 895

Asn Ile Ile Ile Ile Lys Asn Tyr Arg Tyr Ser Lys Asp Lys Phe Ile

900 905 910

Phe His Cys Pro Ile Thr Leu Asn Phe Ser Ala Lys Ser Ser Lys Leu

915 920 925

Asn Asp Glu Ile Asn Asp His Ile Lys Asn Lys Lys Glu Phe Cys Phe

930 935 940

Met Gly Ile Asp Arg Gly Glu Lys His Leu Ala Tyr Tyr Ser Leu Val

945 950 955 960

Asn Gln Asn Gly Lys Ile Leu Asp Lys Gly Gln Gly Thr Leu Asn Leu

965 970 975

Pro Phe Val Asp Lys Asp Gly Asn Lys Arg Cys Ile Lys Thr Glu Lys

980 985 990

Tyr Phe Glu Glu Asp Lys Lys Glu Asn Glu Lys Trp Lys Pro Arg Ile

995 1000 1005

Ile Asp Cys Pro Asp Tyr Asn Cys Leu Leu Asp Ala Arg Ala Ser Asn

1010 1015 1020

Arg Asp Leu Ala Arg Lys Asn Trp Gln Thr Ile Gly Thr Ile Lys Glu

1025 1030 1035 1040

Leu Lys Glu Gly Tyr Ile Ser Gln Val Val Arg Lys Ile Val Asp Leu

1045 1050 1055

Ala Ile Glu Asn Asn Ala Phe Ile Val Leu Glu Asn Leu Asn Ile Gly

1060 1065 1070

Phe Lys Arg Gly Arg Gln Lys Ile Glu Lys Gln Val Tyr Gln Lys Leu

1075 1080 1085

Glu Leu Ala Leu Ala Arg Lys Leu Asn Phe Leu Val Asp Lys Lys Ala

1090 1095 1100

Ile Ile Gly Glu Val Gly Ser Val Thr Lys Ala Leu Gln Leu Thr Pro

1105 1110 1115 1120

Pro Val Asn Asn Phe Gly Asp Ile Gly Gly Lys Ser Gln Phe Gly Ile

1125 1130 1135

Met Phe Tyr Thr Lys Ala Asp Tyr Thr Ser Gln Thr Asp Pro Val Thr

1140 1145 1150

Gly Trp Arg Lys Ser Ile Tyr Leu Lys Arg Gly Pro Glu Asp Tyr Ile

1155 1160 1165

Lys Asp Gln Ile Leu Gly Asn Lys Asn Lys Asn Ile Glu Pro Ala Phe

1170 1175 1180

Glu Asp Ile Cys Phe Asp Gly Gln Asp Tyr Cys Phe Thr Tyr Ile Asn

1185 1190 1195 1200

Lys Asn Thr Gly Lys Lys Trp Thr Leu Tyr Ser Ser Lys Asn Gly Lys

1205 1210 1215

Ser Leu Asp Arg Tyr His Arg Glu Leu Val Tyr Glu Asn Ser Asp Lys

1220 1225 1230

Lys Trp Leu Pro Lys Lys Gln Asp Val Leu Glu Met Leu Asn Asn Leu

1235 1240 1245

Phe Glu Gly Phe Asp Lys Lys Lys Ser Leu Leu Lys Gln Leu Glu Thr

1250 1255 1260

Lys Asn Pro Asn Lys Thr Gly Glu His Pro Ala Trp Glu Ser Leu Arg

1265 1270 1275 1280

Phe Thr Ile Asp Leu Ile Gln Gln Ile Arg Asn Thr Gly Ile Lys Glu

1285 1290 1295

Arg Asp Glu Asp Phe Ile Leu Ser Pro Val Arg Asp Lys Lys Gly Asp

1300 1305 1310

His Phe Asp Ser Arg Glu Ala Ser Pro Asp Leu Pro Asn Ser Gly Asp

1315 1320 1325

Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Ile Ile Met Ala Lys

1330 1335 1340

His Ile Glu Lys Gly Tyr Phe Leu Tyr Ile Ser Asp Glu Glu Trp Asp

1345 1350 1355 1360

Ala Trp Leu Ala Gly Glu Glu Cys Trp Asn Arg Trp Ala Glu Lys Asn

1365 1370 1375

Thr Lys Ser Leu Leu Lys Asn Asn Tyr

1380 1385

<210> 13

<211> 1385

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC13的AA序列

<400> 13

Met Ser Thr Lys Thr Ile Phe Ser Asp Phe Thr Asn Leu Tyr Glu Leu

1 5 10 15

Ser Lys Thr Leu Arg Phe Glu Leu Lys Pro Val Gly Glu Thr Glu Asn

20 25 30

Leu Leu Asn Glu Asn Gln Val Phe Leu Thr Asp Lys Ile Arg Gln Lys

35 40 45

Lys Tyr Glu Glu Ile Lys Pro Phe Leu Asp Glu Phe His Leu Asp Phe

50 55 60

Ile His Phe Cys Leu Ser Asp Leu His Leu Asp Tyr Thr Glu Tyr Lys

65 70 75 80

Lys Ser Leu Asp Asn Tyr Gln Lys Asp Lys Lys Asn Lys Asp Leu Glu

85 90 95

Lys Lys Lys Glu Asn Glu Glu Lys Lys Leu Arg Glu Gln Ile Val Gly

100 105 110

Lys Phe Asp Ser Lys Val Glu Asp Phe Leu Lys Thr Phe Gly Lys Val

115 120 125

Glu Lys Ile Lys Gly Lys Lys Asp Asn Glu Lys Phe Lys Val Ser Leu

130 135 140

Gly Lys Asp Trp Glu Ile Glu Phe Ser Lys Asn Asn Tyr Glu Phe Leu

145 150 155 160

Phe Glu Ile Gly Ile Phe Asp Leu Met Lys Lys Lys Phe Glu Gly Asn

165 170 175

Gly Asp Ile Tyr Val Ala Asp Lys Glu Thr Gly Glu Ile Tyr Gln Asp

180 185 190

Glu Lys Thr Gly Lys Asp Ile Thr Ile Phe Asp Asp Trp Asn Gly Trp

195 200 205

Leu Gly Tyr Leu Thr Lys Phe Phe Glu Thr Arg Lys Asn Leu Tyr Lys

210 215 220

Ser Asp Gly Thr Ser Thr Ala Ile Ala Thr Arg Ile Ile Asn Glu Asn

225 230 235 240

Leu Lys Lys Tyr Cys Glu Asn Leu Asp Ile Tyr Asn Lys Leu Ser Gln

245 250 255

Ile Glu Asn Leu Lys Asn Lys Phe Gln Asn Leu Glu Ala Asp Phe Gly

260 265 270

Ile Lys Leu Glu Lys Phe Phe Ser Leu Glu Asn Tyr Asn Ser Cys Ile

275 280 285

Leu Gln Asn Gly Ile Glu Asn Tyr Asn Asp Ile Arg Gly Gly Lys Leu

290 295 300

Glu Lys Asn Asn Asn Lys Ile Pro Gly Ile Asn Glu Tyr Ile Asn Lys

305 310 315 320

Tyr Arg Gln Asp Ser Gly Glu Lys Leu Pro Phe Leu Gln Lys Leu Asp

325 330 335

Lys Gln Ile Leu Ala Gly Gly Lys Glu Asn Phe Ile Glu Gln Ile Glu

340 345 350

Asn Glu Pro Ser Phe Glu Lys Cys Leu Lys Asn Phe Tyr Asn Asn Ser

355 360 365

Ile Lys Lys Val Asp Ile Leu Thr Gln Ile Phe Gln Asp Leu Ser Thr

370 375 380

Tyr Thr Asn Glu Asp Tyr Lys Thr Ile Tyr Phe Ser Lys Glu Ala Phe

385 390 395 400

Asn Thr Leu Ser His Lys Phe Thr Asp Gln Val Leu Asn Phe Glu Lys

405 410 415

Leu Val Phe Glu Glu Leu Leu Leu Asn Lys Leu Val Glu Lys Lys Asp

420 425 430

Phe Asp Lys Lys Glu Glu Lys Tyr Lys Phe Pro Asp Phe Ile Pro Leu

435 440 445

Phe Tyr Val Lys Lys Gly Leu Glu Asn Tyr His Thr Lys Asn Leu Phe

450 455 460

Tyr Lys Ser Arg Tyr Tyr Glu Asn Glu Ile Ile Glu Glu Asp Asn Asp

465 470 475 480

Asn Ile Trp Gln Lys Phe Cys Thr Ile Leu Asn Tyr Glu Phe Gln Ser

485 490 495

Leu Leu Ser Asn Thr Ile Ile Asn Gln Asn Gly Glu Glu Ile Glu Val

500 505 510

Gly Phe Thr Ile Ser Lys Asn Lys Leu Glu Lys Ile Leu Asp Asn Phe

515 520 525

Ser Leu Gly Glu Asn Asn Asn Gly Ile Ile Lys Asp Phe Ala Asp Ile

530 535 540

Ser Lys Thr Ile Tyr Gln Met Gly Lys Tyr Phe Ala Leu Glu Lys Lys

545 550 555 560

Arg Glu Trp Asn Asn Asn Phe Asp Leu Asn Asp Asp Phe Tyr Lys Thr

565 570 575

Glu Tyr Ser Gln Glu Asn Glu Lys Tyr Gly Tyr Leu Glu Phe Tyr Asn

580 585 590

Glu Ala Tyr Glu Gln Ile Ile Val Pro Tyr Asn Leu Met Arg Asn Phe

595 600 605

Ile Ala Lys Lys Pro Trp Glu Asp Asn Lys Lys Trp Lys Leu Asn Phe

610 615 620

Glu Asn Ser Ser Leu Leu Lys Gly Trp Asp Lys Glu Phe Glu Ser Tyr

625 630 635 640

Gly Ser Tyr Ile Phe Glu Lys Ala Gly Leu Tyr Tyr Leu Gly Ile Ile

645 650 655

Asn Gly Thr Lys Leu Asn Lys Asn Glu Ile Glu Lys Leu Tyr Asn Tyr

660 665 670

Asn Ala Asn Asn Gly Ala Lys Arg Phe Val Tyr Asp Phe Gln Lys Pro

675 680 685

Asp Asn Lys Asn Thr Pro Arg Leu Phe Ile Arg Ser Lys Gly Asp Asn

690 695 700

Phe Ala Pro Ser Val Lys Glu Leu Asn Leu Pro Ile Asn Asn Ile Ile

705 710 715 720

Glu Ile Tyr Asp Lys Glu Leu Tyr Lys Lys Asp Lys Glu Lys Pro Asn

725 730 735

Lys His Lys Glu Ser Leu Met Lys Leu Ile Asp Tyr Phe Lys Leu Gly

740 745 750

Phe Arg Lys His Ile Ser Tyr Lys His Phe Asn Phe Val Trp Lys Glu

755 760 765

Ser Asn Lys Tyr Asp Asn Ile Ala Asp Phe Tyr Arg Asp Val Glu Lys

770 775 780

Ser Cys Tyr Lys Pro Tyr Trp Glu Glu Asp Ile Asn Phe Asp Glu Leu

785 790 795 800

Lys Asn Leu Thr Lys Glu Lys Arg Met Tyr Leu Phe Gln Ile Tyr Asn

805 810 815

Lys Asn Phe Glu Leu Asp Glu Ser Ile Ser Thr Asp Asp Tyr Thr Phe

820 825 830

Lys Gly Asn Gly Lys Asp Ser Val His Thr Met Tyr Phe Lys Gly Leu

835 840 845

Phe Ser Lys Asp Asn Leu Glu Asn Lys Asn Gly Val Asn Leu Lys Leu

850 855 860

Ser Gly Gly Gly Glu Leu Phe Phe Arg Pro Lys Ser Ile Glu Lys Lys

865 870 875 880

Ile Asp Lys Asn Arg Lys Ser Lys Arg Glu Ile Ile Glu Asn Lys Arg

885 890 895

Tyr Ser Lys Asp Lys Ile Leu Leu His Phe Pro Ile Gln Val Asn Phe

900 905 910

Lys Glu Asn Lys Thr Ser Asn Phe Asn Asn Tyr Ile Asn Asn Phe Leu

915 920 925

Ala Asn Asn Pro Asp Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Lys

930 935 940

His Leu Ala Tyr Tyr Ser Val Ile Asn Gln Lys Gln Glu Ile Ile Glu

945 950 955 960

Ser Gly Ser Leu Asn Tyr Ile Tyr Gln Lys Asp Lys Asp Gly Lys Ile

965 970 975

Ile Gln Lys Ser Glu Lys Lys Ile Gln Glu Val Arg Asn Asp Glu Gly

980 985 990

Lys Ile Ile Asp Tyr Glu Leu Val Glu Thr Gly Lys Leu Val Asp Tyr

995 1000 1005

Glu Asp Tyr Gly Ile Leu Leu Asp Tyr Lys Glu Lys Lys Arg Arg Leu

1010 1015 1020

Gln Arg Gln Ser Trp Lys Glu Val Glu Gln Ile Lys Asp Leu Lys Lys

1025 1030 1035 1040

Gly Tyr Ile Ser Ala Val Val Arg Lys Ile Ala Asp Leu Ile Ile Glu

1045 1050 1055

His Asn Ala Ile Val Ile Phe Glu Asp Leu Asn Met Arg Phe Lys Gln

1060 1065 1070

Ile Arg Gly Gly Ile Glu Lys Ser Val Tyr Gln Gln Leu Glu Lys Ala

1075 1080 1085

Leu Ile Asp Lys Leu Asn Phe Leu Val Asn Lys Gly Glu Lys Asp Ser

1090 1095 1100

Glu Gln Ala Gly Asn Leu Leu Lys Ala Phe Gln Leu Thr Ala Pro Ile

1105 1110 1115 1120

Gly Thr Phe Lys Asp Met Gly Lys Gln Thr Gly Ile Ile Phe Tyr Thr

1125 1130 1135

Gln Ala Arg Tyr Thr Ser Lys Ile Asp Pro Leu Thr Gly Trp Arg Pro

1140 1145 1150

Asn Leu Tyr Ile Lys Lys Gln Ser Ala Glu Leu Asn Lys Glu Ser Ile

1155 1160 1165

Leu Lys Phe Asp Ser Ile Ile Trp Asn Lys Glu Lys Glu Tyr Phe Glu

1170 1175 1180

Ile Thr Tyr Asp Leu Glu Lys Phe Gln Ser Glu Ser Thr Lys Asn Leu

1185 1190 1195 1200

Lys Glu Lys Lys Glu Glu Lys Leu Glu Arg Thr Lys Trp Thr Leu Ser

1205 1210 1215

Thr Arg Val Glu Arg Phe Lys Trp Asn Lys Asn Leu Asn Asn Asn Lys

1220 1225 1230

Gly Gly Tyr Glu His Phe Glu Asn Leu Asn Ile His Phe Lys Glu Leu

1235 1240 1245

Phe Glu Lys Tyr Gly Leu Asp Ile Ser Gly Asp Ile Leu Lys Gln Ile

1250 1255 1260

His Asn Leu Glu Thr Lys Gly Asn Glu Ala Phe Phe Ser His Phe Leu

1265 1270 1275 1280

Asp Leu Phe Lys Leu Val Cys Gln Ile Arg Asn Thr Asn Gln Asp Lys

1285 1290 1295

Lys Gly Asn Glu Asn Asp Phe Ile Tyr Ser Pro Val Phe Pro Phe Phe

1300 1305 1310

Asp Ser Arg Lys Gln Asn Thr Val Gly Val Lys Asn Gly Asp Asp Asn

1315 1320 1325

Gly Ala Phe Asn Ile Ala Arg Lys Gly Ile Ile Ile Leu Glu Arg Ile

1330 1335 1340

Gly Lys Trp Lys Lys Glu Asn Asp Met Lys Ile Gln Lys Gly Glu Lys

1345 1350 1355 1360

Glu Met Tyr Pro Asp Leu Phe Ile Ser Asn Ile Gly Trp Asp Asn Phe

1365 1370 1375

Thr Gln Asn His Asn Ile Arg Asp Asn

1380 1385

<210> 14

<211> 1310

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC14的AA序列

<400> 14

Met Ser Gln Asn Asn Ile Lys Glu Lys Ser Ile Phe Asp Glu Phe Thr

1 5 10 15

Asn Lys Tyr Ser Leu Gln Lys Thr Leu Arg Phe Glu Leu Arg Pro Val

20 25 30

Leu Asn Thr Glu Gln Met Leu Thr Asp Ser Gly Ile Ile Lys Leu Asp

35 40 45

Glu Lys Arg Lys Leu Asn Tyr Glu Lys Thr Lys Pro Phe Leu Asn Arg

50 55 60

Leu His Gln Glu Phe Val Thr Glu Ser Leu Asn Gly Val Arg Leu Lys

65 70 75 80

Ser Leu Asp Gly Tyr Ala Val Leu Tyr Ala Asn Trp Lys Lys Ser Ile

85 90 95

Asp Lys Lys Glu Lys Asp Ala Ala Tyr Lys Val Leu Glu Lys Lys Glu

100 105 110

Leu Glu Ile Arg Gln Glu Ile Val Val Leu Phe Asp Glu Lys Ala Val

115 120 125

Glu Trp Ile Gly Lys Leu Pro Ala Asp Val Lys Lys Pro Lys Lys Pro

130 135 140

Asn Tyr Glu Phe Leu Phe Glu Pro Ala Ile Phe Ser Ile Leu Lys Lys

145 150 155 160

Lys Tyr Ser Asp Glu Val Gly Thr Thr Ile Asp Glu Glu Ser Ile Phe

165 170 175

Asp Ser Trp Asp Lys Trp Thr Ala Tyr Phe Gly Lys Phe Phe Glu Thr

180 185 190

Arg Lys Asn Phe Tyr Lys Ser Asp Gly Lys Ala Thr Ala Val Ala Thr

195 200 205

Arg Ile Val Asn Glu Asn Leu Arg Arg Phe Cys Asp Asp Val Ser Thr

210 215 220

Phe Glu Asn Ile Gln Ser Lys Ile Asp Leu Ser Pro Leu Glu Lys Glu

225 230 235 240

Phe Asp Val Ser Leu Lys Lys Val Phe Asp Ile Gln His Tyr Asn Gln

245 250 255

Cys Leu Asn Gln Ser Gly Ile Asp Ala Phe Asn Thr Leu Leu Gly Gly

260 265 270

Glu Val His Glu Asn Gly Glu Lys Ile Lys Gly Ile Asn Glu Tyr Ile

275 280 285

Asn Glu His Arg Gln Lys Thr Gly Glu Lys Leu Thr Arg Leu Lys Lys

290 295 300

Leu Asp Lys Gln Ile Gly Ser Asp Lys Glu Asn Phe Ile Asp Leu Ile

305 310 315 320

Glu Thr Asp Glu Gln Leu Lys Thr Thr Leu Val Thr Phe Ile Ala Asn

325 330 335

Ala Lys Glu Lys Val Asp Leu Leu Asp Lys Ser Val Ser Tyr Leu Thr

340 345 350

Lys Asp Thr Asp Val Lys Leu Ser Gly Ile Phe Phe Arg Lys Glu Ala

355 360 365

Ile Asn Thr Ile Thr Arg Arg Trp Phe Val Ser His Glu Lys Ile Ser

370 375 380

Asp Ala Leu Val Ser Ala Phe Ser Asp Lys Asn Val Lys Phe Asp Gln

385 390 395 400

Lys Arg Glu Glu Tyr Lys Phe Pro Asp Phe Ile Ser Trp His Val Ile

405 410 415

Gln Asn Ala Val Glu Lys Leu Ala Ser Asp Gly Glu Glu Ile Trp Lys

420 425 430

Lys Tyr Tyr Leu Glu Glu Glu Lys Leu Ser Leu Leu Asp Lys Thr Pro

435 440 445

Trp Gln Gln Phe Leu Thr Val Phe Glu Cys Glu Tyr Asn Asn Leu Lys

450 455 460

Ser Lys Gly His Glu Ser Glu Gly Arg Ser Phe Thr Glu Leu Val Gln

465 470 475 480

Asp Ile Glu Ser Leu Leu Lys Thr Asp Thr Leu Asp Arg Asn Asp His

485 490 495

Val Thr Glu Ile Ile Lys Ser Phe Ser Asp Arg Val Leu Asn Ile Tyr

500 505 510

Arg Phe Ala Lys Tyr Phe Ala Leu Asp Lys Ser Cys Gln Trp Asn Pro

515 520 525

Asp Gly Leu Asp Thr Asp Asp Phe Tyr Val Ala Tyr Glu Gln Phe Tyr

530 535 540

Ser Asp Gly Tyr Glu Lys Ile Val Lys Val Tyr Asp Lys Val Arg Asn

545 550 555 560

Tyr Met Thr Lys Lys Pro Phe Asn Gln Asp Lys Trp Lys Leu Asn Phe

565 570 575

Glu Asn Pro Thr Leu Ala Asn Gly Trp Asp Lys Asn Lys Glu Thr Asp

580 585 590

Asn Thr Ala Ile Ile Leu Arg Arg Ala Gly Arg Tyr Tyr Leu Ala Val

595 600 605

Met Glu Arg Gly His Asn Thr Leu Phe Lys Lys Ile Pro Met Ser Ser

610 615 620

Ser Gly Tyr Gln Lys Met Thr Tyr Lys Leu Phe Pro Asp Pro Ser Lys

625 630 635 640

Met Met Pro Lys Val Cys Phe Ser Lys Lys Gly Leu Glu Phe Phe Lys

645 650 655

Pro Ser Ala Glu Ile Met Arg Ile Tyr Lys Asn Gly Glu Phe Lys Lys

660 665 670

Gly Asp Thr Phe Ser Leu Ser Ser Met His Val Leu Ile Asp Phe Tyr

675 680 685

Lys Asn Ala Leu Lys Thr Tyr Asp Gly Trp Thr Met Tyr Asp Phe Ser

690 695 700

Asn Leu Lys Lys Thr Ser Glu Tyr Thr Glu Asn Ile Gly Glu Phe Tyr

705 710 715 720

Arg Asp Val Ala Glu Ser Gly Tyr Gln Ile Asn Phe Asp Tyr Ile Ala

725 730 735

Glu Gln Tyr Ile Glu Asp Ala Asn Lys Glu Gly Lys Leu Tyr Leu Phe

740 745 750

Glu Ile His Asn Lys Asp Trp Asn Leu Lys Asp Gly Ala Ile Lys Thr

755 760 765

Gly Ser Lys Asn Ala His Thr Leu Tyr Phe Glu Gln Val Phe Ser Asp

770 775 780

Glu Asn Ala Gln Asn Asn Phe Val Val Lys Leu Asn Gly Glu Ala Glu

785 790 795 800

Leu Phe Phe Arg Pro Ala Thr Ser Thr Glu Lys Leu Gly Asn His Tyr

805 810 815

Asp Ser Lys Gly Asn Val Val Thr Lys Asn Lys Arg Tyr Ala His Asp

820 825 830

Lys Met Phe Phe His Val Pro Val Thr Leu Asn Arg Thr Ala Pro Asp

835 840 845

Ala Arg Lys Phe Asn Gln Ser Val Asn Val Phe Leu Ala Asn Asn Pro

850 855 860

Asp Thr Asn Ile Ile Gly Ile Asp Arg Gly Glu Lys His Leu Ala Tyr

865 870 875 880

Leu Ser Val Ile Asn Gln Lys Gly Asp Ile Leu Lys Ile Lys Ser Leu

885 890 895

Asn Lys Ile Glu Val Lys Asp Lys Asp Gly Asn Val Ile Lys Glu Asp

900 905 910

Asp Tyr Ala Lys Leu Leu Glu Asp Arg Ala Lys Asn Arg Glu Ser Ala

915 920 925

Arg Arg Asp Trp Lys Ser Val Glu Gln Ile Lys Asp Leu Lys Lys Gly

930 935 940

Tyr Ile Ser Asn Val Val Arg Glu Ile Ala Asp Leu Val Ile Lys Tyr

945 950 955 960

Asn Ala Ile Val Val Phe Glu Asp Leu Asn Met Arg Phe Lys Gln Val

965 970 975

Arg Gly Gly Ile Glu Lys Ser Val Tyr Gln Gln Leu Glu Lys Ala Leu

980 985 990

Ile Asp Lys Leu Asn Phe Leu Val Asp Lys Asn Glu Leu Asp Pro Gln

995 1000 1005

Lys Ala Gly His Ile Leu His Ala Tyr Gln Leu Thr Ala Pro Phe Glu

1010 1015 1020

Thr Phe Lys Asp Met Gly Lys Gln Thr Gly Val Leu Phe Tyr Thr Gln

1025 1030 1035 1040

Ala Glu Tyr Thr Ser Gln Thr Asp Pro Val Thr Gly Phe Arg Lys Asn

1045 1050 1055

Val Tyr Leu Ser Asn Ser Ala Thr Val Glu Lys Ile Lys Ala Phe Val

1060 1065 1070

Glu Met Phe Asp Val Ile Gly Trp Asp Asp Lys Leu Lys Ser Tyr Tyr

1075 1080 1085

Phe Lys Tyr Asn Pro Val Asn Phe Val Glu Thr Lys Phe Lys Glu Asn

1090 1095 1100

Thr Phe Ser Lys Asp Trp Val Ile Tyr Ala Asn Val Pro Arg Ile Lys

1105 1110 1115 1120

Arg Glu Arg Lys Asn Gly Tyr Trp Glu Ala Thr Val Val Asn Pro Asn

1125 1130 1135

Glu Glu Phe Leu Lys Leu Phe Lys Glu Trp Asp Phe Asp Asn Ile Tyr

1140 1145 1150

Val Glu Asp Ile Lys Glu Gln Ile Phe Gln Met Phe Glu Glu Gly Arg

1155 1160 1165

Leu Asp Gly Thr Lys Glu Phe Asp Gly Lys Asn Arg Asn Phe Trp His

1170 1175 1180

Ser Phe Ile Phe Leu Phe Asn Leu Met Leu Gln Val Arg Asn Ser Thr

1185 1190 1195 1200

Ala Thr Gln Tyr Lys Lys Asp Glu Asp Gly Asn Ile Ile Glu Thr Val

1205 1210 1215

Glu Gly Val Asp Phe Ile Ala Ser Pro Val Phe Pro Phe Phe Thr Thr

1220 1225 1230

Asp Gly Gly Asp Phe Thr Glu Gly Cys Val Asn Leu Ala Lys Leu Glu

1235 1240 1245

Asp Lys Phe Val Gly Ser Asn Ala Asp Lys Glu Arg Phe Lys Lys Glu

1250 1255 1260

Phe Asn Gly Asp Ala Asn Gly Ala Tyr Asn Ile Ala Arg Lys Gly Ile

1265 1270 1275 1280

Ile Met Leu Asn Asn Ile Lys Asn Asn Pro Glu Lys Pro Asp Leu Phe

1285 1290 1295

Val Ser Lys Lys Asp Trp Asp Lys Phe Ala Gln Ala Asn Gln

1300 1305 1310

<210> 15

<211> 1275

<212> PRT

<213> 未知细菌物种

<220>

<223> BMC15的AA序列

<400> 15

Met Asn Pro Thr Gln Thr Asp Lys Thr Pro Ser Lys Pro Phe Glu Lys

1 5 10 15

Phe Thr Asn Leu Tyr Cys Leu Ser Lys Thr Leu Arg Phe Glu Leu Lys

20 25 30

Pro Ile Gly Lys Thr Gln Lys Ile Leu Glu Asp Asn Lys Val Phe Glu

35 40 45

Asn Asp Lys Lys Arg Ala Lys Ser Tyr Glu Glu Ala Lys Lys Tyr Phe

50 55 60

Asn Lys Leu His Arg Glu Phe Ile Asp Glu Ser Leu Lys Asn Ile Thr

65 70 75 80

Leu Ser Asn Asn Leu Ile Glu Lys Phe Glu Lys Lys Tyr Leu Thr Trp

85 90 95

Lys Asn Ser Lys Asn Lys Asp Asn Ser Thr Glu Leu Lys Lys Ser Ala

100 105 110

Lys Arg Leu Arg Ile Val Ile Leu Glu Ser Phe Asn Lys Lys Ala Asn

115 120 125

Glu Trp Asn Ser Glu Tyr Ser Asn Gln Val Lys Asn Glu Lys Lys Lys

130 135 140

Lys Lys Ile Gln Glu Ile Thr Gly Ile Asp Leu Phe Phe Lys Val Glu

145 150 155 160

Val Phe Asp Phe Leu Ile His Lys Tyr Pro Glu Val Gln Ile Asn Gly

165 170 175

Glu Ser Ile Phe Ser Pro Phe Asn Lys Phe Ser Gly Tyr Phe Lys Lys

180 185 190

Phe His Glu Thr Arg Lys Asn Phe Tyr Lys Asp Asp Gly Thr Ser Thr

195 200 205

Ala Ile Pro Thr Arg Ile Ile Asp Val Asn Leu Glu Lys Phe Leu Glu

210 215 220

Asn Lys Asp Ile Tyr Tyr Thr Lys Tyr Phe Gln Lys Tyr Asn Ser Ile

225 230 235 240

Phe Asn Lys Glu Glu Thr Asp Ile Phe Lys Leu Glu Ser Phe Lys Asn

245 250 255

Cys Leu Thr Gln Ser Gln Ile Asp Lys Tyr Asn Glu Ser Ile Ala Thr

260 265 270

Leu Lys Ser Lys Ile Asn Asn Leu Arg Gln Asn Asn Pro Glu Val Asn

275 280 285

Lys His Asp Leu Pro Phe Phe Lys Glu Leu Phe Arg Gln Ile Leu Gly

290 295 300

Gln Pro Ile Lys Lys Glu Thr Glu Gln Asp Asn Phe Ile Glu Ile Leu

305 310 315 320

Thr Asn Asp Glu Val Phe Pro Val Leu Gln Lys Asn Ile Asp Glu Asn

325 330 335

Glu Leu Tyr Ile Pro Lys Ala Asp Thr Leu Phe Lys Glu Phe Leu Lys

340 345 350

Ser Gln Ile Gln Glu Thr Asn Glu Tyr Asn Ile Asn Glu Ile Tyr Val

355 360 365

Ala Ser Arg Phe Ile Asn Ser Ile Ser Asn Asn Trp Phe Ala Glu Trp

370 375 380

Asp Thr Ile Ile Asn Leu Leu Arg Thr Glu Leu Lys Ile Lys Gln Asn

385 390 395 400

Gln Lys Lys Leu Pro Asp Phe Ile Ser Ile Ala Ser Leu Lys Arg Val

405 410 415

Leu Gln Lys Ser Gln Asp Glu Ile Asp Ala Lys Asp Leu Phe Arg Asn

420 425 430

Asn Tyr Glu Asn Leu Phe Glu Ser Thr Thr Asp Phe Tyr Lys Ile Phe

435 440 445

Leu Lys Ile Trp Glu Leu Glu Phe Asn Asp Asn Ile Lys Lys Tyr Asn

450 455 460

Leu Glu Thr Glu Asn Ile Arg Lys Ile Ile Ile Glu Asp Lys Lys Tyr

465 470 475 480

Leu Pro Asn Lys Lys Ser Ile Leu Lys Asn Gly Glu Thr Gly Ile Ile

485 490 495

His Asn Glu Lys Ile Leu Asp Tyr Ala Gln Ser Ala Leu Asn Ile Tyr

500 505 510

Gln Met Met Lys Tyr Phe Ser Leu Glu Lys Gly Lys Glu Arg Glu Trp

515 520 525

Asn Pro Asp Gly Leu Asn Glu Asp Thr Thr Gly Gly Phe Tyr Asp Asp

530 535 540

Phe Asn Lys Tyr Tyr Gln Asn Val Asn Thr Trp Lys Tyr Phe Asn Glu

545 550 555 560

Phe Arg Asn Tyr Leu Thr Lys Lys Pro Tyr Lys Thr Asp Lys Ile Lys

565 570 575

Leu Tyr Phe Gly His Lys Ser Leu Leu Gly Gly Phe Thr Glu Ser Lys

580 585 590

Thr Glu Lys Ser Asn Asn Gly Thr Gln Tyr Gly Ala Tyr Leu Leu Arg

595 600 605

Lys Lys His Gly Leu Gly Gly Phe Asp Tyr Tyr Leu Gly Ile Ser Thr

610 615 620

Asp Pro His Leu Met Ser Tyr Phe Asp Pro Ile Asp Asp Ser Gly Asp

625 630 635 640

Ser Glu Tyr Glu Arg Leu Asn Tyr Tyr Gln Val Leu Thr Arg Thr Ile

645 650 655

Tyr Gly Pro Ser Tyr Glu Gly Asp Tyr Glu Leu Asp Lys Lys Asn Leu

660 665 670

Ser Glu Ile Glu Ile Ile Lys Lys Ile Lys Arg Ser Leu Ser Tyr Tyr

675 680 685

Thr Ser Arg Val Lys Lys Ile Gln Asp Ile Ile Asn Asn Asn Tyr Glu

690 695 700

Ser Val Arg Asp Ile His Lys Asp Ile Thr Asp Val Leu Lys Glu Phe

705 710 715 720

Gly Thr Ile Phe Asp Tyr Lys Val Ile Thr Asn Ser Gln Ile Gln Lys

725 730 735

Ala Phe Asn Cys Asp Lys Gly Phe Tyr Leu Phe Glu Ile Tyr Ser Lys

740 745 750

Asp Phe Ser Lys Glu Lys Gly Asp Lys Ser Lys Asn Ser Lys Asp Asn

755 760 765

Leu His Thr Thr Tyr Phe Lys Ser Leu Met Asp Arg Lys Gln Ser Thr

770 775 780

Phe Asp Leu Gly Ser Gly Glu Ile Phe Phe Arg Glu Lys Ser Val Gln

785 790 795 800

Ser Glu Ile Asp Ser Met Arg Lys Thr Lys Asn Lys Ile Thr Arg Phe

805 810 815

Lys Arg Tyr Thr Lys Asn Leu Ile Gln Phe Asn Leu Ser Ile Thr Leu

820 825 830

Asn Asn Asn Cys Thr Glu Val Pro Gln Asn Lys Asn Ala Arg Lys Ala

835 840 845

Phe Ile Asn Asn Phe Asn Ile Glu Leu Ser Lys Lys Leu Leu Thr Asn

850 855 860

Asn Ser Asp Ile Asn Ile Ile Gly Ile Asp Arg Gly Glu Lys His Leu

865 870 875 880

Ala Tyr Tyr Ser Val Ile Asp Gln Gln Ser Asn Ile Leu Glu Thr Gly

885 890 895

Ser Phe Asn Lys Ile Gln Glu Arg Lys Asp Arg Glu Pro Thr Asp Tyr

900 905 910

Gln Gln Lys Leu Asp Lys Ile Gln Lys Asp Arg Asp Trp Gln Arg Lys

915 920 925

Ser Trp Gln Glu Ile Ser Asn Ile Lys Asp Leu Lys Lys Gly Tyr Ile

930 935 940

Ser Gln Val Val Tyr Glu Ile Ser Lys Leu Val Lys Lys Tyr Asn Ala

945 950 955 960

Ile Ile Val Phe Glu Asp Leu Asn Ile Gly Phe Lys Arg Gly Arg Phe

965 970 975

Ala Ile Glu Lys Gln Val Tyr Gln Asn Leu Glu Leu Ser Leu Ala Lys

980 985 990

Lys Leu Asn Tyr Leu Val Phe Lys Asp Ala Asn Glu Gly Glu Ser Gly

995 1000 1005

His Tyr Leu Lys Ala Tyr Gln Leu Thr Ser Pro Val Asn Asn Phe Gln

1010 1015 1020

Asp Ile Gly Lys Gln Cys Gly Ile Ile Phe Tyr Ile Pro Ala Ser Tyr

1025 1030 1035 1040

Thr Ser Ala Ile Cys Pro Ser Cys Gly Phe His Lys Asn Ile Pro Thr

1045 1050 1055

Ser Ile Lys Lys Leu Ala Lys Asn Lys Glu Phe Val Glu Lys Phe Val

1060 1065 1070

Ile Thr Tyr Glu Leu Lys Lys Asp Arg Phe Tyr Phe Gly Tyr Lys Ile

1075 1080 1085

Asn Asp Phe Tyr Asn Ser Asn Leu Gln Asp Asn Val Ile Phe Tyr Ser

1090 1095 1100

Asn Val Glu Arg Leu Arg Tyr Lys Arg Asn Lys Asp Asn Arg Ser Gly

1105 1110 1115 1120

Glu Val Gln Glu Arg Leu Pro Asn Glu Glu Leu Lys Lys Leu Phe Glu

1125 1130 1135

Gln Asn His Ile Asn Tyr Lys Asp Asn Pro Gln Ile Ser Gly Gln Ile

1140 1145 1150

Lys Asn Gln Lys Leu Asp Asn Glu Lys Phe Tyr Lys Pro Leu Ile Tyr

1155 1160 1165

Glu Ile Ser Leu Ile Leu Gln Leu Arg Asn Ser Lys Thr Val Lys Ser

1170 1175 1180

Glu Asp Gly Thr Ile Asn Thr Asn Ile Asn Arg Asp Phe Ile Ser Cys

1185 1190 1195 1200

Pro Ala Cys Tyr Phe His Ser Glu Asn Asn Leu Met Asn Leu Pro Asn

1205 1210 1215

Lys Tyr Lys Gly Gly Lys Lys Phe Glu Phe Asn Gly Asp Ala Asn Gly

1220 1225 1230

Ala Tyr Asn Ile Ala Arg Lys Gly Ile Leu Leu Leu Asn Lys Leu Asn

1235 1240 1245

Asn Ile Lys Asp Ile Glu Lys Ile Glu Tyr Asn Asp Leu Asn Ile Ser

1250 1255 1260

Gln Glu Asp Trp Asp Asn Phe Val Lys Asn Pro

1265 1270 1275

<210> 16

<211> 3888

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC01的NT序列

<400> 16

atgatattta ataacttcac ccagaaattt tcactttcca aaactctccg ctttgagctt 60

cgtccggttg atgccggagg aaacgtaatt acggatttaa ctatatttga agaaaccatt 120

aaaaacgacc agaaacgata tgaggcctat cttgcgataa agcctttggt tgatgaaacg 180

cataagcatt ttattcaaac ggttttatcg ggtttaaccg atttaattaa atccgatgaa 240

atgaagaatt acttagagca taaaaatcta attcgccaaa aggatgttga agaaaaagtt 300

aaaactaaat caatagatgt gatcaataaa attgaaaagg attggcgaaa gcgggtttct 360

gatagtttca ctaaacatcc gcaatacaag aaaatgtttg ataaaacctt atttgccgat 420

gaaagcccgc tttataaatt agccgaaaat gattttcaga gaagtcagat taaaatcttt 480

gaaaaattca ccggttattt taatggtttc catgaaaacc ggaaaaacct atatgttgcc 540

gaaaaacaag gcacggcaat agccaatcgc gtaattaatg aaaatttgcc gaagttcata 600

gaaaacgcca ataagctaaa acgggcattt gaaaagtatc cggaattttt atccaaaata 660

tctgaggata aatcttttca ggcattgcta attaaaaatc aattatccct tgaaaaactt 720

cttcaaccac taacctttaa tttattaatt tctcagaccg gtattgatag ttataacgag 780

gttttaggcg gttatacacc ggaaaattca gaaccaatta aaggtttaaa tcagttaatt 840

aacttatatc gccagaaaat taatttagcc cgcaatgact ttcctaacct tgccccgtta 900

tacaaacagc ttttatccga ccgcgaaacc aatagcgttg tctacaagcc attggaaaat 960

gttgcagatg tttatagcag tgtatttgag ctttgccaaa atctactttc aaagcaaagc 1020

gatataaata agtggattga ggatattaat attagttccg gacaaatctg gatttataaa 1080

agccatctat caggcttatc ggttatgctc tttggtgaaa gcggctgggg attaattcct 1140

cgcattctaa atatttctga agatgacgaa gaggaaatta ttaagtcaaa aagtaaaaaa 1200

tcacagcaag aatatttttc atttgctgaa attggcaatg ccattaataa ttattctttt 1260

gaggatgtaa acataaaagc cttagcaaaa caaggcttat gcttatggca aaagcaggga 1320

aatgaacgat tgattaaatt tggtaaattg tttagccaaa tgcaaaatga actgcaaagc 1380

ccaaaagaaa aatgggatag caccgaaaag gaaaaaataa aagaattatt agatacagga 1440

ttggagtttg tgcattggct gaaagtaatt agcaatcagc ccgaagacaa agatgaagtt 1500

ttttatgccg agtggcaggc acttactgat acttggcggg gactgcccaa actatatgac 1560

cgcgtgagaa attttgcgac taaaaaagat tacagccaga ataagttaaa aattaatttt 1620

gataaaggca ctttgttaaa cggctgggat accaacaagg aaaccgacaa tttaggaata 1680

ttattagaaa ataaagggca gtattatttg ggaataatga aagattcctc aatatttgat 1740

taccaatggg atattgataa ttttcaaaat cccaattcca aacaatcggt ggcgaagaaa 1800

aaccttcacg aggcaatagt atcggataac acacaagatt gctggagcaa gattgtctac 1860

aagcttttac ccgggccaaa taaaatgtta ccgaaggtat ttttttccga taaacggcaa 1920

aaatattttg gtgccgatga aaaagtaata gatattaatg aaaatggtcg gcataaaaaa 1980

ggcgataatt ttaatatttc ggattgccat tatcttattg acttttataa aaccgcgatt 2040

aataagcatc ctgaatggag tcaatttaat tttaagtttt ctgctaccaa aagctacgaa 2100

gatataagcc agttttatca tgaagtgcag aatcaaggct atcggattga gtttgaccat 2160

atccgcaaag attacattca aaagatggta agtgagggca aactattttt attcaaaatt 2220

cacagtaaag atttttcgtc ttatgccaaa ggtcgcccga atatgcatac catttattgg 2280

cgggcaattt tcaatccgga aaatttagca aatgtggtcg taaaattaaa tggcgaagcc 2340

gaatttttct atcggaaatc gtcgaaagat cggattatca gtcatcccca gggtttagag 2400

gtttctaata aaaatcccag caacccgaaa aagaccagta gatttgctta tgatttaatt 2460

aaagacaaaa gatttactca ggataaattt ttctttcatg tgcccatcac cctgaatttc 2520

cgcgaaggtg aaggctatcg ttttaatcaa tcggttattc gcgagcttaa aaagtattat 2580

caaaccgaca aagccaacct gcatataatc ggcattgacc gcggcgagcg gcatttgctt 2640

tattattgcg taattaatgt ggcgagcgga aaaatagttg agcagggaag ttttaatcag 2700

atttccacaa actatacgcc tgagcaaatt accgatgacg gtgaaataat taaaggtgaa 2760

accgtaaata aaactaccga ctatcacaat ctgctaaata ccaaagaagg cgaccgccag 2820

aaagcccgca aaaactggca gaccattgaa aatattaaag agttaaaagc cggatattta 2880

tcgaatgtaa ttcacaaaat atcgcaatta atggtgaaat ataatgcctt cgtggttttg 2940

gaagaattga aatatggttt taagcgcggg cgttttaagg ttgaaaaaca ggtttatcaa 3000

aaatttgaaa aggcattaat tgataaatta aattatctgg tttttaaaga ccgcgctccg 3060

gcagaggtgg gcggggtatt aaatgctctg caattagctc caccggtggc aagttatata 3120

gatattggta aacaggcggg atttttattt tatgtccccg cccatcacac ctccaaaatc 3180

tgcccgtgga cgggttttgt cgattggtta aaaccgcgct acgatggcat tgacaaggcg 3240

aaagcatttt ttacttgttt tgagagcatc cattttaata cgcagaaaaa ttattttgaa 3300

tttgcctttg actatgaaaa gttccgtggg aatattaatc atctgcctga aggcctaaag 3360

cgaaccagct ggacattatg ctcacataac agccttcgcg acattgctac caaagataaa 3420

aacggaaatt ggccatataa gcaaattaat ctcaccgccg aattgcttga aatattaaaa 3480

accctaaatc cccgcaatgg tgaaaacctt gtggagcgga ttattgaaat gaatgataaa 3540

aagttttttg aatcattaat gtgggctttg cgagtattgt tgcaacttcg ttatggatac 3600

attaaacgta ataatgaagg cataattatt gaagaagttg attatattct ttcgccggtg 3660

gccaacgaaa atggtgagtt ttttgattcg cggaattttg taaatattga aaaggcagat 3720

tttcccaaag atgccgatgc caatggcgcg tataacattg ctcgcaaagg cttattatta 3780

atcgcccaga atattaataa tgccaaaatt aatgataagg gcgaggttaa atgcgacctg 3840

cagattgata aaaccacttg gtttaattgg gtgcaaagta aatcataa 3888

<210> 17

<211> 3324

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC02的NT序列

<400> 17

atgcccaaag aagattttga taaagcatgc atatatttgt ctaactttga taagttttca 60

acttattttg ttggttttaa tcagaatcga gagaatctgt atacagatga agaacaggct 120

acggcaatac cttatagaat tattaatgat aatatggtaa gacattttga taactgccgg 180

aagtttgaaa agatagttaa aaaatatgga gatatatcta atgtattatc tacttataaa 240

gagtttttcg ctcctgactg ttttaaaaat aaattaaatc agtctcaaat tgatcattac 300

aataatacta taggacatac tgcagatgat atatatggag taggaattaa tcagatactc 360

agtaaatata agcaagataa caaacttaat tccagcgatc ttcctttaat atctaaactt 420

tataagcaaa tacttagtga tactgagagc tatgcaatag aaaattttgc tgatgataaa 480

atgatgttga atgccgttga taaagaatac tcaaggatta aagagaatga tgtttttatc 540

aatatcgaga cgtgtatgaa tgaatatctt acattagaaa attctcatat gatttatcta 600

aaaaatgatt cctctcttac tgatatctct aataaattat gggaagattg ggcctttgtt 660

aaaaatgcta tacagaaata ttctaaagag atattatgcc ttagtgacaa gaaaatagaa 720

gacatgctaa aaatgagtca ttactctatt tcttttgttc agaattctgt ttattactat 780

gtggataatt atatggagtc atgtgaagat aaaagaaaat caattataga ttatataaaa 840

acattttatt ctataaaata taataatgta ttttcttgtt ataaagaagc agaagctgtt 900

ttaaggcttg actcgattca caagaatagg agatctcctg ttgacaagaa cggcatagga 960

ggagaaggtt ttgctcaaat agaaaaaata aaaaatttct tagatagtat attagaagtc 1020

aagaatttct tgaatcctct ttatctgata aaatccggaa aaatggcaga aatagaagat 1080

aagagtgaag agttctataa ccgtttcaat gagttatata attctctttc tgatacaacc 1140

tatttatata ataaagttag gaattatctt acaaaaaaac cttataaaaa agaaaaattt 1200

aaaatgaatt ttgaaaattc cacgctatta agcggatggg atgttaataa ggaaaattgc 1260

agtaactcta ttatactcat ccgtaatggg aagtactatc tgggtatcat tgataagcaa 1320

tgcggcaata tgtttaattt taaaattgat gcagaagata atgaaaaaaa gagaaaagaa 1380

aaagaagatc tggcagagga catcctttca gatggttctg attcatatta tgaaaaaatg 1440

gtatataagc ttctccctga tccttctaag atgttaccga aagttttttt tagcaataag 1500

agtatagact tttatgcccc ttcagaggat attaaatata ttagagaaaa tggacttttc 1560

aaaaaagatg ctaaaaataa aaaagctctt tatatatgga tagagtttat gcagaattct 1620

cttaaaaaac atcctgaatg gagtaattat tttaatttta actttaaacc atcaacagaa 1680

tatgctgatg tgtcagaatt ttataagcaa gtttctgatc aagggtactc tttgtctttt 1740

gataaaataa aagatagtta tatagaaagt aaaataaaat caggagaact tttcttattt 1800

gaaatttata ataaagattt ttctccatat agcaaaggta atccaaattt acatactatc 1860

tattggaaat caatttttga taaagaaaat cttagcaatg ttgttataaa attgaatggt 1920

caggcagaaa tattctttag accagcttct ttaaaaagaa atgaagtagt tgttcacaga 1980

gcgaaagaga atatattaaa taagaatcct cttaacccta aaaaagagag tatgtttgaa 2040

tatgatattg taaaagataa aagatatact caagataaat tcttctttca ctgtcctata 2100

actttaaatt ttaagtcagg gaatgtagga aaattcaatg ataaggttaa tcaatttctt 2160

aaaaataatc ctgatgtaaa tgttatagga ttcgaccgag gagaaagaca tttactctat 2220

tgcaatgttc ttaatcaaaa aggagagata atagagcaaa agagttttaa tgtgatagaa 2280

aataaaaata atggaattac ccaaaaagta gattatcaca acttgttgga ccgcaaggaa 2340

aaagaaagag atgcttctcg caaatcatgg tcaaccattg aaaatatcaa agaattaaag 2400

gaaggatatc tgtctaatgt ggttcatgaa atttctgaac taataattaa atataatgct 2460

atcctagttc tagaagattt aaatttcgaa tttaaaaaag gtagatttaa aatagaaaaa 2520

caagtatatc aaaaatttga aaaagcactt atagacaaac tcagctacat ggtttttaaa 2580

aaggaagaat ctaataaacc ggggcattct cttatggcat atcaattagc ttctcctttt 2640

gaaagtttcc aaaaactagg gaaacaatgt ggatttattt tttatgtaaa tagtaattat 2700

acttctaaaa tagatccagt aacaggattc gtaaatcttc taaaaatcaa atacgaatct 2760

gttgataaaa gttgtaaatt tattaatgat aagtttgatg atatcagata caatgccgat 2820

agggaatatt ttgaatttac ttttgataat ggcaaatgga ctgcttgttc tcatggaaaa 2880

gaaagatatc gttataatag aaatgataag aaatataatt gttttgatgt tactgaagaa 2940

ttaaaatcac tgtttaataa atatgaaata gattttaagg caggaacaga tattaaaaaa 3000

agcatatgcc aggtgcaaga caaaaacttt catagcgaat tattgtttaa tttatctctg 3060

atagttcagt taagacatac ttataaaaat ggagatatag aaaaagattt tatcttatct 3120

cctattatgg ataaggaaac tggaaaattc tttgattcta gagagtatga aaatttagaa 3180

aattctttgt tacctactaa tgcggattct aatggagcat acaacatcgc cagaaaagga 3240

ttattaactt taaggcagat agacaaggac ggaaaaccat caaatatatc caacaaagaa 3300

tggtttgatt tcgtccaaaa gtaa 3324

<210> 18

<211> 3972

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC03的NT序列

<400> 18

atggaaaaaa atttaaatta tttagaaaga tttactaagc attacaacac taaaaaaaca 60

ttaaaaaata agttaatacc ttatggtaat actgctgaga acatgataaa aaataatatc 120

atatcaaatg aaaaacaaat aattttatca gcaaaaaaac aaaaacaaag tattgatttt 180

ttacaaaaag aatatattga aaataaatta tcagaaatta cattaccata tttaaatgat 240

tattataatg aatttattaa aaataaaaaa gaacgtgata ctgatgttat agacaatatt 300

gaaatagcta tgagaaagca tatttcaaaa tctttaacag aaaatggaaa ccataaaaaa 360

tatttaaata aagaagtctt tgatataata tcagaaaaaa aagaattata ttatgatgta 420

acatttaaaa gaaatgccac atatctatca gattattttc aaagcagagt aaatttatat 480

aaagattcaa ataaatcatc tacgattgca tcaagatgta ttaatattaa tttaccaata 540

tttgcaaaaa atatagtatt atttaatttt attaaaaata aagcaaatat tatttttgat 600

gatttaaaag aaattacaga tgatgaatac actttggatt caattttttc aatcgatttc 660

tttaatatgg tattatctca aaaaggtata gattattata atactattct tggtggcatg 720

acaaaagaag atggaaaaaa aataaagggt ataaatgaat atattaattt atataatcaa 780

aatgtaaaag atgaaaaaaa taaattacct tatccaaaga aattaaaaaa acaattatta 840

agtgatatta attcttattc agcaagattt gaaaaatttg atacagaaca agaaatggtt 900

aaaagtatta aatctttagt tgaaaatgac ttatttcaag gagaattatt tgataaaaaa 960

gttgatattc taaaggaaac agaaagactt ttagaaagaa tatctgaata tgattcaaat 1020

gctttattta ttactgaaaa aaatatttca tatatttcaa tagatatttt taatgataag 1080

ttttttataa aaacagctat tgaatatttt tatgaaaata acatttgtcc agattatagg 1140

aaaatatatg ataatgcttc taaaaataaa cgtaagcaat taggaaaaga aaagaataaa 1200

gttattaaac aaaaatcttt ttctatttct tttttacaag atgctataac cttttatatt 1260

aaagatagtg gtattaataa aatctctgag aattgtatca ttaattattt taaaaagcat 1320

actatcaaac taacagaatt atttggaaaa gtatatgaag attataatgt aattaaacct 1380

attttagaac aacatttagt agaatatgaa ggtaaatcta tttcaaaaga ttctatcaaa 1440

agaagtaaaa ttaaattgtt ttcagaaaat ttaaaaaata tattttattt tattcgacca 1500

ttaaatatta tagaagaagc attaaattat gatacatctt tttatacacc attcaatata 1560

ttatttgaag aaataaaaaa attcaataaa ttatatgata aaataagaaa ttttattaca 1620

aaaaaaccat tcaatgatga agaaattaat ttatatttcg gtattccaaa tttagggggt 1680

ggttttatag atagccaaac agataaaagt aataatggta cacaatattg tacttatctt 1740

tttaggaaaa aaaatcaatt attaaattgg gaatatttcg ttggaattag taaaaataaa 1800

catttattta gagaaaaaga aaatatagaa ttaaattctg atgaaacatc atttcaaaga 1860

tattcatttt acacccccaa agataaatct atttatggta gttcttattt ttcagctaat 1920

gaaaaaaatt ataaagatga taaacaagaa tttatcaata ttatcaataa tatagtaaat 1980

aatagtggta atgaattagc tattaaagaa cttaaaaaat atattaataa ttctactgaa 2040

aattctgaaa cacctaatgg ttgtcttagt gttttaaaaa ataaatgtaa tgagatatat 2100

aatcttgtta ttaaccatga tgattttaaa gaaaaaaatg aagacattat caataaactt 2160

aaaaacactc ttagtaaatt atctaaagta ccacaagcta aagaactaat aaataaaaaa 2220

tataatttgt tttctgaaat catatcagat attagtgaaa tttgtctaac ttcaacccaa 2280

agatattatc ctattgatga tgaagaacta aattctgctt tgaatgatga aaataaacca 2340

ttatatttct ttaaaataag caataaagat ttaagtgctg atgaaaatat tcttaatggt 2400

aaaagaaaaa gtaaaggaaa agataatatt catactatga tattaagagc catgatggat 2460

gataatgtga caaatattat acccacctct tgtaaaatta gtatgagaga agcatctata 2520

aaaaaagatg atttagttat tcataaagct aatgaaccaa ttaaacttaa aaattctctg 2580

gctaataaaa aagaaagtac tttttcatat gatataacta aagatagaag atatagtagg 2640

gatgaatttt tctttagtat aacagcatct attaatagtg attgtaagga aaatgattat 2700

tactttaatc aaaaagttaa tgaatattta aaaaacaatt ccaaaattaa tttattagct 2760

gttgatttag gagaaacaaa tattattact ataagtgtta ttgaccaaaa aggtaatata 2820

attttgcaaa aagatttaga taaatttata aataaagaaa aaaatattat tactgatttt 2880

aatctacttc tttctaatcg ttctaaagaa agagatatag ctaaaagaga ttggcaagaa 2940

caacaacaaa ttaaaaatct taaagaaggt atgatttcat gcattataca tgagatatgt 3000

aaacttatga tagaacataa tgctattctt attatggaag atttagatgc taattttaaa 3060

aataggaaaa aaagaataga aaaagctatt taccaaaaat ttgaaatagc aatacttgaa 3120

aaattaaaca atttagtttt taaagatata ccaattaatg aagttggtag tgtaacgaaa 3180

cctcttcaat taagcgataa gtttgaaaca tatgaaaaag ttggtaatca aagtggattt 3240

gtttttaaag tatccccttt ttatacaagt ataattgacc caacaacagg atttattaat 3300

ttatttaaga aaaattttga aagtgttaaa tattctattg agttcttttc aaagtttgaa 3360

agtattagat ataatacaaa agaaaaatac tttgaatttg cttttgatta taagaacttt 3420

aaagaaatta aatatactga aaatataaaa actgattggg tggcttgtac aacaaatata 3480

gatagatatg agtatgataa aaagaacaaa atctataaaa aatatgatgt tactacggat 3540

ttaaaaaatc tttttgaaaa tgaagaaatt tattatcaaa agggtgaaaa cattcttgat 3600

gtgattttaa aaaagaataa tagagagttc tttgaaaaac tcacaaatct attaaagata 3660

actatgctat ttagatatag aaattcacat ttaaaattag actatatatc atcacctgta 3720

aagaatagta atggagaatt ttttagtact gaaaatggat tagagaatta tccgatagat 3780

tctgatacaa atggtgcata tcatattgct cttaaaggaa aaatgattct tgaccgtatt 3840

aatagtaatt catcagaaaa attagatact tatattagta ttgaagattg gcttaaattt 3900

attcaaaaat ttagtgtgaa taaaataact gaaaccaaaa agaataaaaa aataaatatt 3960

aaatatgttt aa 3972

<210> 19

<211> 3975

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC04的NT序列

<400> 19

atgaagaatc taactgaatt tacaggattg tatcctgtct ccaaaacttt gcgttttgaa 60

ttaaaaccaa ctgatgattt caattgggag acctttttgg aatctaccat ttttaaacat 120

gaccaagaaa gagctgaagc atatccaatc gtaaaggtta tagtggatca atttcataaa 180

tggtttattg aagatgcttt aaataaatcg actatcaatt ggaattcact ttatgatgct 240

tatttcgcac ctaaaaacga gaatagtgtt gaaaacttga gaaaagaaca agataaaatc 300

cgcaaagaaa tagtagatac atactttaaa aaacatgact ggtggaagta cgtttcaaaa 360

gaccacagta aattattcaa aatagaacta ccagctttat tatccgatga tgcttttatt 420

tatgaaatta acgataaata tccaaattat acgcaagaaa tactgattga tgctttagca 480

aaatttcaaa atttctctgt ttattttgga ggttatttta agaacaggga taatatgtat 540

aaatcggatg ctcagagcac ttcaattgct aatagaattg taaatgagaa ttttacaaag 600

ttcgcagata acataaaaat ttacaataga ctaaaagaaa actgtttatc tgaattacaa 660

aaagtagagt tagattttac agacgaatta actgggttga cctttgatga cattttttca 720

ccttcttatt tcaataaatg cctgactcaa aaaggaattg aaaaattgaa tctttatatt 780

ggaggaaaaa caggtaaaaa taaggaagat aaagtttttg gaataaaccg agttggaaat 840

gaatttttac aatttaataa agaatcgaag ttaaaactca aagaccttaa aatggtcaaa 900

ctttacaagc aaatattaag cgatcgagaa caaccttcgt ttttacctga gcaatttaga 960

aatgaagatg aattaatcaa aagtattgaa gatttccata atttgataac agagcaaaaa 1020

ctgtttgaaa ggttattgaa attaatggga agattaaaaa acggagaatg tgaagatttg 1080

aataaaatac atgttgttgg cagttcattg acacaattgt caaaagtatt atatggaaat 1140

tgggaagtac ttggtactgc tttacgcaac aaatttcaaa cgaataaaac aaaaaaagac 1200

aaacttgaaa gtgaaaaaga tattcaggag tggatggaac gaaaatcttt ttcccttgct 1260

caaatcatcg aagttgaatc atcattacag gatgataaaa gtataaaagt tatcgattta 1320

tttacgacat tcaatgcgtg gcaaaaggtt aacgaaaaac cacagttagt tgatttaatc 1380

aagttatgta aagatgattt tcaaacccgc tttagagcag tgaaagattt gattgaaaaa 1440

ggagaacaaa tccaaggtaa cgaatcggcc aaagaagaaa taaaagctgt actcgataac 1500

tatcagaatt tactccacgt agttaaattg ttgaatctgg gcaaaaaaga aagttatctg 1560

gataaagatg aaacttttta taatgaatat aaggagattt tatcatcaac tgagtcggat 1620

aatgtttgtc ttgaagatat tataccgttg tacaataagg tgagaagttt tctgacacga 1680

aaattagggg atgaggggaa aatgttgttg aaatttgatt gtagtacatt ggctgatggt 1740

tgggatgtgg gaaaagaatc tgccaacaac tctacaattt taattgataa tagtaagtat 1800

tacctgataa ttacaaatcc cgaaaataaa ccagacttaa gtactgcaat tacctctaat 1860

acagataatg tttataaaaa gattgtctat cgacaaattg ctgatcccac aaaagactta 1920

cctaatttga tggttattga tggcaaaaca caacgaaaaa caggcaataa agatgatgac 1980

ggaataaaca gagtattgga tcaacttaaa gacaaatatt taccacagga ggttaatcga 2040

attcgtaagt taggctcata tctaaaaact tcagaacact tcaataaaaa agattcacaa 2100

gtatatttag catattatat gcaaagactt attgaataca aacagggaga aatggaattc 2160

tcatttaaga attccgaaga atatgactct tattcggatt ttttagatga tattactaaa 2220

caaaaatact ctctttcatt tgtaaatgtc tcgaaggaaa ttataacaca atggatttca 2280

gaaggaaaaa tatttctctt tcagatttac aataaagatt ttgaagaaaa ggcaacaggt 2340

acacccaatt tacatacact ttattggaaa gaattgttta gtgaagaaaa tctaaaagat 2400

atagtttata aattaaatgg tgaagcggag cttttttacc ggaagaaaat ggacggcaaa 2460

ccatttacac acaacaaagg ggctgtttta gtgaataaaa cctttgcgga tggtagccct 2520

gtggaacctg aacattacaa agaatatgtt gaatatatta ccggaaaagt gattgaaaaa 2580

cagctttcaa aagaggcaaa agataaactc catcttgtaa aaacaaacaa agcaaaactc 2640

gatataatta aggataaacg ttattttcag cacaagttac tattccatgt tcccataaca 2700

attaatttca agagtgaagg agtgccaaaa tttaatgatt atactttaaa ctatctgaga 2760

gaaaataaaa aagatattaa cataattgga attgaccgtg gtgaacgaaa tttaatttat 2820

gtttctgtaa tcaatcagaa aggcgaaaat ataataccgc caaaacattt caatattgta 2880

gaatcggaca tgtttgggat ggaagataaa cgaaagttca actatcttga aaaactaata 2940

caaaaggagg gaaaccgaga tgatgcacgt aagaactgga gtaaaattga aactattaaa 3000

gacttaaaga caggttattt atcattagtg gttcatgaaa tagctaaatt agtggtagag 3060

catcatgcaa tcgttgtctt agaggatttg aactatggtt ttaaaagagg tagatttaat 3120

gttgaaagac agatttacca aaattttgag aagatgctga ttgaaaaact gaatcttctt 3180

gtttttaaaa ataattctaa ttctccggat tatggtaata ttttaaatgg tttacaactt 3240

actgcaccat ttggcagttt caaagaactt ggcaaacaga gtggatggct gttttatgta 3300

aatgcatctt acacttcaaa aattgaccct caaactggtt ttgcaaacct atttaatatg 3360

aaagatgcaa aaaaagatac gaagtcattc tttgaaaaga ttacagaaat taaatatgat 3420

gatggaatgt ttaaatttac atttgattat cgcaatggtt tttctattgt acaaacagat 3480

tataaaaata tatggactgt atgtaccaac gacaaacgta ttttagttag taaagataac 3540

atcagtggaa agttcaaaca tgaatatgtt gatattacag aaagcataaa aaatcttttt 3600

ataaacaaca atattaatga ttaccactcg atttcaaaag aaactatctt atcaatcaaa 3660

gaaaagaaat tctttgatga tttgttcttt tattttaagc ttagtcttca aatgcgaaat 3720

tcaataccaa attcggacat tgattatttg atttcaccag ttcagataaa ggggaagcct 3780

ttctttgatt ctagaattcc aaataatatt aatattgttg atgccgatgc aaatggggct 3840

taccatatag cattaaaagg attatatctg gttataaatg attttccaac tgagaaaaaa 3900

ggaaaatctg aatacctaaa gaaaataacc aacgaagatt ggtttgaatt tgcacaaagg 3960

cgtagtttaa aataa 3975

<210> 20

<211> 3693

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC05的NT序列

<400> 20

atgagatggg atgaaaaact ccagactttt ttgaatgatc aggaaattga ggatgcttat 60

caggttttaa agcctgtatt tgataaatta catgagaatt ttattattgg cagtttagaa 120

aatacaaata ataaaaaact gttttctttt gataaatact tgaaactaaa aaatgatttg 180

ttgcacgtta ataaaaaaga acaagagtca gattacaaaa agaaagaaaa ggaatttgag 240

acagagggaa agttattgcg taatactttt gcaacggttt ggataaatga aggcaaaaat 300

tttaagaata cgattgttgg aggtgaaaat gacagagaaa ttttaaagga aggggggtat 360

aaaattttaa cggaggcggg tattttgaag tatattaaaa tgaatattga taagtttgtg 420

gaattaaaat taaagaccag agaggatatt ttatggaaaa aagagaatag aaacttggta 480

gagatggctg atttggaaaa gtctttggga acaattgaat catggggtgt ttttgaagga 540

ttctttactt atttttcagg ttttaatcaa aatcgagaaa attattattc aactgatgaa 600

aaggcgacgg ctgtcgcaag tagagttatt gatgagaact taccaaaatt ttctgataat 660

gttttggaat ttaataaaaa gaatgatgtt tatataggga ttttttcgtt cttgaaagga 720

aaaaacattg tattgaaggg caagtcggga aacggggaag aacaagattt attgccaatc 780

accgagaaaa tttttgagat tgaatatttt aagaattgtt tatcggaagg agaaatagaa 840

aggtataatt cggatattgg aaatgcaaac tttttaatta atttatacaa tcagcaacaa 900

gataaaaaag aaaataaatt gcgaattttc aagactttat ataaacaaat cgggtgtggt 960

attaaaggtg attttattca gttaattaaa acggatgatg aattaaaaaa gatttttgaa 1020

gacttaaaaa ttacgggcga taattttttt aagaacacgc aaaatttgaa agagataatt 1080

ttgagtttgg aaaattttag cggaatttat tggtcagaca aagcgttaaa tacagtctct 1140

ggtaaatact ttgctaattg ggcgagtctg aaggagcttt taaaaaacgc taaaattttc 1200

aaaaaggaaa aagatgaaat taaaatacct caaactatag agttgtcgga tttgtttggg 1260

gtgttggatt ctaatgaact aatttttaag gaaagtttta atgaaaacga tgaattgaaa 1320

caaataattt taaagagcta tgagaaaaat tcaattaagc ttttaaagat gatttttgtg 1380

gatgttgaag aaaatcagaa gatttttggg aatcttaaag atgggttgcc gataaatgat 1440

tttaagaaag atgagaacac tcaaattatt aagacctggt tggatggttt gttgaataca 1500

aatcaaattt tgaaatattt taaagttcgt gaaagcaaaa ttaagggggc gccattgaat 1560

ccggaggttt ctgaaagact taataaaatt ttgaatgttg aaaatccgac tgttatttat 1620

gatgttgttc gtaattattt aacaaaaaaa ccaacagaag gcttgaataa gttgaaatta 1680

aattttgata atgcggtttt ggcggccggt tgggatgtta acaaggagtc tgagcgtggc 1740

tgtttgattt tgaaggatgg tgataacaaa aaatatttgg caattttaac gaataaaacg 1800

caaaagtttt ttggtgaaaa ggtgaagtat aaagaatttg ttggtgatga aaattggcaa 1860

aaaatggatt acaaattgtt accaggacct aataagatgt tacctaaggt tttgttgcct 1920

aagagtgata gatataaatt tggagcaact gatgaaatct tgaaaattta taatgagggt 1980

ggttttaaga aaaatgaacc aacttttacg aaggcaaaac tggccaaaat cgttgatttt 2040

tttaaggatg gtttgaaaaa ttatccgtct gcgaaaagta gttggtataa tttgtttgct 2100

tttgattttt ctgatacaga aaaatatgaa agtatagatc gattttatac tgaggtggag 2160

aagcaggggt ataaattatc ttggagcgct attagcaaaa attttatttt tgaaaaggtg 2220

gatgcaggtg atatgtattt atttgaaatt agaaataaag ataataactt aaaaaatggc 2280

aaagcaaaaa caggagcaaa aaatttacat acaatttatt gggggactat ttttggggag 2340

tcagaaaata aaccaaagtt aaatggtgag gcagaaattt tctatcgtcc agttgttaag 2400

gatttaatta aagataagga caaaaacgga gatataatca aagcgagcga aaaacgattt 2460

gaacaagaaa aatttgtttt tcattgtccg ataactttaa atttttgttt aaaatcaaca 2520

aggttgaatg atgtaataaa tcaaataatg attgaaaaca agaaagatgt ttgttttatc 2580

ggcattgatc gtggcgaaaa acaccttgct tattattcgg ttgttaatca aaagggtgaa 2640

attttggaac aggggagttt taacgaaatt aacggacaga attatgcgaa aaagttggaa 2700

gaaaaggctg ggcatagaga tgaagctaga aaaaactgga aaacaattgg tacaattaaa 2760

gaattaaaaa acggttatat ttcgcaggtt gtgcgaagaa tcgttgattt agcagtaaaa 2820

tataatgctt atattgtttt ggaggattta aatagtggat ttaagcgtgg tcgtcaaaaa 2880

attgaaaaat cagtgtatca gaaattggaa ttggcattgg ctaaaaaatt gaattttttg 2940

gttgataaga gtaagaaaga tggtgaaatt ggtagcgttc agaaggcttt gcagttgacg 3000

ccccctgcca ctaattttgc tgacattgaa aaagctaaac aatttggcat tatgctttat 3060

gttcgtgcta attatacttc tcaaacggat cctgtcactg gttggcgaaa gactatttac 3120

tttaaatcca ctacccaaga aaatttaaaa aaggaaattt gtgaaaagtt tagcgaaata 3180

ggatttgatg gaaatgacta ttattttgag tataaggatg agaatgcaga aaaaaaatgg 3240

acaatgtatt caggtgttag tggtaaaagc ttggatagat ttcggggaaa gaaagatacc 3300

catggtattt ggaaagttga aaaacaggat attgttgaat tgttaaagaa aatttttggt 3360

caacaaacaa gcgttgtagg cgatttgaaa acaaaaatta ccaacgacaa tgtaaatgac 3420

ttaaaatata caattgattt aattcagcaa attagaaata ccggatttaa tgaaatagac 3480

aatgatttta ttttatcgcc ggtaagagat gaaaagggga atcattttga tagtcggaaa 3540

gatggtgcaa ttttgtctaa tggtgatgca aatggtgcat ataatatagc tcgtaaagga 3600

gtgttggctt ttgagaggat taatgcgaaa ccggaaaaac cggagctgta tattgcagat 3660

gtggaatggg ataaatggtt acaatctaaa taa 3693

<210> 21

<211> 3771

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC06的NT序列

<400> 21

atggattcat tcacacagtt tacagggctt tattctctat cgaaaacatt gcggtttgag 60

ttgaaaccga taggaaaaac cttagcctat atcgaaaata aagggttatt agtacaagac 120

gaacaccgtg cagatagtta caaaatcgtt aaaaaaatta ttgacgaata ccataaatca 180

tttattgaaa aatctttaaa tgggttgtgt ttggatggat tggaggatta ttatttttat 240

tatcaaattc caaaaaaaga tgataatcag aaaaaaatag ttgaagatat actgacaaag 300

ctacgtaaac aaatagccga acgattttca aaacaggata tatataaaaa tctttttgca 360

aaggaactga ttaaagacga tttaaattct tttgtacaag aggtagaaca aaaggattta 420

attaaagaat ttgaaaattt tactacctat tttacaggat ttcacgagaa ccgaaaaaat 480

atgtattcgg cagaagataa atcaacggca atagctttcc gtcttattca tcaaaactta 540

cctaagtttc ttgacaatat gcgagcattt aataaaataa gtgtttcacc gcttgccgaa 600

aaatttaagc atattctttc cgacagcgaa ttaggtccta ttgtacaggt agtagcgatg 660

gaagatgtat ttaacttggc atattttaac gaaacactaa cacaatcggg tattgatata 720

tataaccatt tacttggtgg atatactccc gaagaaggga aagaaaaaat taaaggacta 780

aacgaatata tcaatctata taatcaaacc gttaaaaagg aagaacgttt acctaaactt 840

aaaccattgt ttaaacagat tttaagcgac cgttctacag catcgtttat tcccgaacaa 900

tataaaaacg ataatgaggt attagaaagt attgaaaaat tataccaaga aataaaggaa 960

catgtttttc attcattgaa agagctattt gtacacataa atgagtatga tttgcataaa 1020

atctatttac ggaatgatgt aagtatgacc gatatttcgc aaaaaatgtt tggtgattgg 1080

ggggtgttta caaaagccat gaatctatac tttgataaac agtacaaggg gaaagcaaag 1140

cttggtacgg aaaaatacga agatgaacaa aagaagtatt ttagcaatca agagagtttt 1200

tcaataggct atattaatga atgcttgttg cttttaggta gtaattatca taaaaaagtg 1260

gaagactatt ttaaagtggc tggaaaaacg gaagaacaag ttcaaatgct ttttgaaata 1320

attgaaacaa aatatcaaaa catacaagat ttgcttaatt caccctatcc gacagaaaaa 1380

aacttggcac aagaccaagt tcaggttgat aaaataaaag gactgttaga cagcattaaa 1440

aacttgcaat ggtttataaa accattgctt ggaaaaggga atgaagccga aaaagacgaa 1500

cgcttttacg gtgaatttac tgcactttgg gaaacacttg accaaattac acctctttat 1560

aataaggtgc gtaactacat gactcgcaaa ccatattcta ctgagaaaat gaagttaaat 1620

ttcgacaatt caactttatt agatggttgg gatataaata aagagcctga taatacaagt 1680

gttgttttgc gaaaggatgg tttattttat ttgggtataa tggataaaaa atataataaa 1740

acgtttaaac aggaatttat tgaatcgaat gagccgtgtt tcgagaaaat ggaatacaaa 1800

ttattacccg gagccaataa aatgttgcct aaggttttct tctcaaattc cagaatagag 1860

gaatttaatc ctactgtcga tttacttgaa aattataaaa accaaaccca taaaaaaggt 1920

gataaattta atattgccca ttgtcgaaat ttgattgatt tttttaaaca atccattaac 1980

aaacatgatg attggaaaca attcggcttt gctttttcag atacaaaaaa ctatgacgat 2040

ttaagcgggt tttatcgaga ggttgaacaa caaggttata aaattacctt tagaaatata 2100

cctgaaaaat ttataaatca aatggtggag gaaagtaaac tttacctttt ccaaatttat 2160

aacaaagatt tttcacccta tagtaaaggt acaccaaaca tgcatacttt gtactggaaa 2220

atgttatttg ataccgaaaa tcttaaagat gtggtgtata agctaaacgg acaggcggaa 2280

gttttttacc gaaaagccag tattaatgac gaaaacatag tggttcataa agccaatgaa 2340

gtcataatca acaaaaatac acttaacgag aagaaacaaa gtagatttga ctatgacatt 2400

ataaaagaca aacgttacac gatagataaa tttcagtttc atgtacctat taccatgaac 2460

tttaaagctc gcgggttgaa taacattaat ttggaagtta atcaatacct gcaaaaagaa 2520

aatgatattc acattatcgg tattgatcgt ggcgaacgcc atttattgta tctttcatta 2580

attaacagta agggaaatat cattgaacaa tattctttaa atgaaattat taatgaatat 2640

aatggcaatc attaccacac aaattatcac gatttattgg ataagcgaga aggaaatcgc 2700

accgaagaac ggcaaaattg gaaaacaatt gaaagtatta aggaactaaa agaaggatat 2760

ttgagtcaag ttgtgcataa aatatccgaa ttaatggttg aatacaatgc tattgttgtg 2820

cttgaggatt taaatatggg atttattcgg ggacgacaaa aagtagaaaa atcggtttat 2880

caacaatttg aaaaaatgct tattgataaa cttaattatt tggtagataa aaagaaaaaa 2940

tcgtttgaat taggaggaac tttacatgcg tatcaattaa ccaataagtt tgaaagtttt 3000

caaaagatgg gcaagcaaag cggttttctg ttttatattc cggcatggaa taccagcaaa 3060

atggatcctg ttaccggctt tgtaaatctg tttgacacac gctatgaaaa tgtagtgaaa 3120

gcaaaagcat tttttaataa atttgaatcc attcgataca acaaagacaa ggactatttt 3180

gaatttgaag taaaaaaata ttcagacttt aatgccaaag ccgaagacac acgtcaagag 3240

tggattattt gtactcacgg cgaacgaatt atcaattatc gtaatcctga aaaaaacaat 3300

gaatgggacg acaaaacagt acatccgaca acggaattaa aatcactctt tacatcaaaa 3360

aatattattt ttgaaaacgg gtcttgttta aaagaacaga ttgccttgca aaaagatacc 3420

gataaagaat tttttgaagg gttactgaaa caatttaaaa atacgcttca aatgcgaaac 3480

agtaaaacaa aatcagaaat tgattatcta ttttctccgg tatcaaatga aaatggtgtt 3540

ttttttgatt ctcgtgatta tgtggatatt gataatagag atagaaaatt ttgcgtctct 3600

acgggaaaac caacattgcc tgtcaatgcc gatgctaacg gtgcctacaa cattgctcgc 3660

aaagggttgt ggattgttga acaaataaaa aatccaaata cagacctgaa aaaactgaaa 3720

ttggcaatga ccaataaaga atggctacaa ttcgtacaaa acaaggggta a 3771

<210> 22

<211> 3825

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC07的NT序列

<400> 22

atggataatg ctttttctga ttttacccag aagtatacgc tttcaaaaac attgaggttt 60

gagttgaggc ctgtggggaa taccgaaaag atgttggaag atgagaaagt atttgaaaag 120

gacaagctta ttcaagagaa atatataaaa acaaagcctt attttgatct cttgcatagg 180

gagtttgttg aagaagcgct gaaggacgta gatatatcgg gattacataa ttattttgaa 240

acttaccaaa aatgggcgaa ggacaagaaa aaataccaaa aggaactcca aaacaaagaa 300

cagatcctaa gaaaggagat attagttttt ttagacagta cggcgaaata ttgggctgag 360

aaaaaatact cggaacttag aataaagaaa aaagacatag agattttctt tgaagaggat 420

gtatttacta tactaaagaa gcgatatgga gaagattctg aagctcaaat aatagatgaa 480

gtatctggtg aaaccgtttc tatttttgat tcttggaaag gctttacggg ctactttaaa 540

aaatttcagg aaacacgaaa gaatctatat agggatgatg gcaccgctac tgctcatgca 600

acacgaatta ttgatcagaa cttaaagcgc ttttgtgata atttggaaat tataaaaaga 660

atagcgggga tcattgaatt ttcggaagta gaaggtaatt ttaaacactc gatgggtgac 720

gtattttcct tgagttttta taataagtgt ctactgcaag atggtatcaa tttctacaat 780

aggattttgg gcggagaagt tctgcaggat ggtacaaaac taaaaggcat caatgaactt 840

atcaataaat atagacagga taataaagga gtaaaaattc ccttcctgaa attactggat 900

aaacaaattc ttagcgaaaa agaggagttt ttagatggaa ttgaagatga taaagaactt 960

ttagccgtac ttaaaaagtt ttacgaagtg gctgagaaga agacatctat cctaaagtcg 1020

cttatccaag attttgctca aaataatagg caatacaatc ttgaggaggt ttatatatca 1080

aaagaggcat tcaatactat ttcgcgtaaa tggacgcatg aaacctcaaa atttgaagaa 1140

tggctttata atgtaatgaa gccaaataag ccgactggat taaaatatga caaaaaagaa 1200

gaaagttata aattcccgga ttttattccc ctgtcttata tccaaaccgc cttggagcaa 1260

gccgatattg atggggattt ttggaaggag cattattctg agaactcaaa agcaaatgat 1320

ggttgcctaa tgggagatga gtctatttgg gaacagttca taaagatttt cgaatatgaa 1380

tttcaatctc tttttgaaaa ggaaattatt gatagggaaa ccggtcaacc caaaaaaaat 1440

ggatataatt atgttaagga tgattttaaa ggattactca atggagagaa tttttctgta 1500

gaaataatca aggattttgc cgatactgta ttgagtattt atcaaatggc aaagtacttt 1560

gccattgaga aaaaaagaaa atggctagat gagtacgaca caggggattt ttatgaaaac 1620

cctgaatttg gctataagtt attttatgat gatgcctata aagagatagt tcaaacctac 1680

aataacctga gaaattattt aactaaaaag tcttatagcg aagaaaaatg gaagttaaat 1740

tttgaaaacc caacacttgc cgatggatgg gataagaata aggagcctga taattcagct 1800

gtgattttga ggaaagatgg aaggtattat ttagggttaa tgaaaaaagg gtgcaataaa 1860

atttttgatg acagaaataa ggtggagttt tctggaggag tagataagga taaatatgag 1920

aaaatagttt ataaattttt tccggatcag gcaaaaatgt ttccaaaagt ttgtttttct 1980

gctaagggat tggatttttt tcaaccttct gaagaaattt tgaatattta taaaaattca 2040

gaatttaaaa agggcgatac tttttcagtt caaagtatgc agaagcttat cgatttttat 2100

aaagattgtc ttacgaaata tgaaggatgg atcgcatatg aatttaaaca tttgaagtct 2160

acagatttgt accgaaacaa tattagtgaa ttttttagtg acgttgccga agatggttat 2220

aaaataactt ttcaagatat ttcggacaac tatattgata aaaaaaatca gagtgaggaa 2280

ctctatctct ttgaaattca caacaaagac tggaatctaa aagatgaggt taaaaaaaca 2340

ggatcaaaga atctgcatac tctttatttt gaggcgctct tttcccatga aaacattcag 2400

aataatttcc ctataaagct taatggacaa gctgaagttt tttatagacc taaaactgat 2460

gaagaaaagc ttgtgaagaa gaaagataaa aagggtagag aagtcattga tcataagaga 2520

tatgctgaaa ataagatatt ttttcatgtt cctttaacct taaatagggg caagggggat 2580

gcctatcaat ttaatgctaa aatcaataat ttccttgcca ataactccga tattaatgtt 2640

attggagtag ataggggaga aaagcattta gcttattatt cggttattaa tcaaaagggt 2700

gaaactttgg atagtggatc gcttaatgtc gtgaataaaa ttaactatgg cgaaaagctt 2760

caagaaaaag catccaatag aaaacaatct ataagggatt ggaaggctgt agaaggtatt 2820

aaaaatctta aaaagggtta tatttcacaa gtggtgcgta agttagcgga tttggctatt 2880

gaacataatg ccatcatcat ctttgaagat ctaaatatgc gctttaaaca aattcgagga 2940

ggtattgaaa aaagcgtgta tcagcaactg gaaggagcct tgatagaaaa attgagtttt 3000

cttgtaaata agggagaaaa agaccccaaa caggccggaa atttattgaa ggcttatcag 3060

cttgccgctc ctttcacaac ctttaaggat atgggcaagc agaccggaat cattttctat 3120

acgcaagctt cttatacttc aaagattgat ccattaacag gatggagacc caatctatat 3180

ctgaaatata ctaacgcaga aaaaacaaaa gaggatattg ggaatttctc aaatattgaa 3240

tttaaaaatg ggatatttga gtttacttat gatctaagaa acttccaaaa gcagaaggag 3300

tatcctaaga aaactgaatg gacgctctgc tcatgcgttg aaagatttcg atggaataga 3360

gttctaaatc aaaacaaagg cggatatgac cattatgaag atataactca taattttcga 3420

gatctttttg aaaaatatga cataaacttt atgagcgctg atatcaaagg tcaaatagat 3480

actcttgatg caaaaggcaa tgagaatttt ttcaaggatt ttattttttt ctttaactta 3540

atttgtcaaa tcagaaatac tcaacaagat aaagatggtg atgaaaatga ttttattctt 3600

tctcccataa agccattttt tgatagccgt gattctaaaa aatttggtga aaatttaccg 3660

aataacggag atgataatgg agcttataac atttcccgta aaggaattat tatcttaaat 3720

aagatttcag aattctttga tgaaaatggt ggatgtgaga aaatgaaatg gggagattta 3780

tatatatctc ataaggattg ggatgacttt gctcgtcaaa tataa 3825

<210> 23

<211> 4008

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC08的NT序列

<400> 23

atgatgcaaa ttatgaaaaa tttcgataaa tttacgaatc tatattctgt ttctaagacc 60

ctacgatttg agttgaggcc agaaccaaaa acattggagt atatgcgtag caatctacgt 120

tttgataaaa atttacagac ctttctggct gaccaagaga tcgaggatgc atatcaagct 180

ttgaagccga tctttgattc tttgcatgaa aggtttatta cagaaagtct tgagtctgga 240

agtgcgcaga agattgattt ttcgaaatat ttagaaaaat atcgaaacaa aagagattta 300

ggaataaaag cccttgaagg tacagagaaa cttttaagga ataattttgc agagatttat 360

aaagcaactg caaagagctg gaaagagaat gccggtaagg atggaaaagg taaagaggtt 420

tttaaaaagg aagggtttaa cattcttaca gaaaaaggaa ttctcgaata tatcgaaaaa 480

aatatagata gtttttcggc tataaaatca cccgaagaaa taagaggagc cctaggggca 540

tttgatggtt tttttacata ctttacgggt tttaatcaga atcgtgagaa ttattatgaa 600

acaaaaaaag aagcctctac tgctgttgcg acccgtatcg ttcatgaaaa cttaccgaag 660

ttttgtgata atattttaat tttcgacgag agagcggaag actatatagg agcgtataaa 720

gcactccaga agatggggag agctttggta aacaaagaag gaggggagtt gccttctatt 780

tcaggcgatc tttttaagat tacgtttttc aataaatgtt tttctcagaa gcaaattgaa 840

gaatataaca cagcaattgg aaatgcgaat tctctagtta acttattcaa ccaggcaaaa 900

agagatgaag atggatataa gaagctcgcg ctttttaaaa ctctctacaa gcaaattggt 960

tgtgacaaga aagattcact cttttttgct gtcacacacg atagaagggc ggacgcagaa 1020

aaggcaaggg aaaatgggca agaggcattt tcggtggaag aggtgttggt tttagcaaaa 1080

catgccggcg agaagtattt caacaagggg aatgatgatg gcgaggttaa taccacccaa 1140

gagtttatat cctatatcaa agatcgatcg gattatcagg gtatatattg gtcaaaagca 1200

gcactcaata caatttcaaa taagtacttc gataattggt atgagttaat agatcaatta 1260

aaagaggcaa aggttttcac gaaaacaggt agtggcagtg aagataatgt aaaaataccc 1320

gatgctatag agctggaagg attttttcag gtactaaata aaatccaaga ctggaagacg 1380

gtatttttta agaaatcaat cacagctgac ccacaaaagc tagggattat tgaaagttcc 1440

gaaactgcat cagcagcgct tctttcactt atttttgacg atgtcgctaa acatacaaaa 1500

ctttttattg atcaatccga agatatttta aaggtagaaa attttgtaaa accagaaaac 1560

aaggaagaca ttaagcgctg gttagatcat tcattggcaa tcaatcagat gttgaaatat 1620

tttcttgtaa aggaatctag gacaaaagga gcgcctatcg atcctactct tacaaaggct 1680

ttggacacct tgttgcgctc gcaggatgcg gagtggttta aatggtatga cgtgcttcga 1740

aactatctta ctaaaaagcc gcaggatggt acgaaagaga ataaactcaa gttaagtttt 1800

gagaacggta ccctagctaa tgggtgggat gttaataagg agccagataa tttttgtgtc 1860

attctacaaa acccagaggg taaaaaattt cttgctatta ttgctcgcca agaaggacaa 1920

aaaggtttta atcaggtttt tgcgaagaag catgataacc cactctacaa ggttgatgag 1980

gggggagttt tctggagcaa gatggaatat aaacttttgc ctggaccaaa taagatgctc 2040

ccaaaatgtt tgatgcccaa gtcaaataga gagaagtatg gagctacaga agaagttttg 2100

aaaatatata accagggtag ctttaagaaa acagaatcga atttctcgaa aaaagatcta 2160

tccaggttaa ttaattttta taaatctgct ctccagcaat acgaagactg gcggtgcttt 2220

aatttttctt tccgagcaac ggattcttat gaggacatag gtcaattcta ccgagatgtc 2280

gaaagccaag gatataagtt ggatttccaa agcatcaaca ccgacgttct agatgagttg 2340

gttgaagaag gcaagattta tttgtttgaa ataaaaaatc aagactcaaa tcaaggcaaa 2400

agtagtattc atagagataa tttacatacg atgtattgga atgctctttt ccaagaagta 2460

ttaaatcgtc ctaaacttaa cgggggcgcg gaactgtttt atagaaaggc gctctcacca 2520

gagaaaataa aagaactagg atcggtagat aaaaatggca agaggatcat aagaaattat 2580

cgtttctcaa aagaaaagtt tatttttcat attccaatta cgttgaattt ttgtttaagt 2640

gacacaaggg tcaatgatac cgtcaatcaa gagttgtcaa gaacttcgag tagtcacttt 2700

cttggtattg accggggaga gaagcatctt gcgtactatt ttctggttga tcagaatggg 2760

aaaattgtgt tggacgagta tggaaaagca gtgcaaggca ctttgaatat accttttctc 2820

gacaacaacg gtaatgtgcg aaagataaaa gctaaaaggc gaagtctaga tgagaatgga 2880

aaggaaaaaa tagaggaggt ttggtgcaaa gactataacg agcttttaga ggctcgagcg 2940

ggtgatagag cctatgcacg caaaaactgg caaacaatag gaaatataaa ggagttgaag 3000

gaggggtata tctcacaggt ggtgcgaaaa atagttgatc ttgctataga atacgaagcg 3060

tttattgtcc tcgaagatct caatgttggc tttaagcgtg ggcggcaaaa aatcgaaaaa 3120

tctgtatatc aaaaactgga acttgcgctg gcgaaaaaac ttaactttgt tgtagataaa 3180

tctgcaaaaa taggaggatt aaagtctgtc actaatgcct tgcagcttgc gccccctgta 3240

tctaatttcg gagatattga aggtaggaag cagttcggga ttatgctcta cacgagagct 3300

aattacactt cacaaacaga ccccgctaca gggtggagga aaagcatcta tcttaagagg 3360

ggatcggaag aaagcattag gaagcaaatt atcgattcct ttgaggagat cggctttgac 3420

ggagaagatt attttttcac ttatacagat tcggtagcgg gtagaacctg gattttatat 3480

tcgggtaaaa atggaggctc tttggaccgt ttttatggca agcgtgacaa cgacaagaat 3540

cagtgggtaa gtatgcggca agatgtctct aagcaactag atgggatttt agcaaatttc 3600

gaaaaggatc gctcaattct tgcacaaatt attgatgggg aggttgacct cattaaagta 3660

gagcaaaaat atactgcctg ggaatcattt cgatctacta tcgatttaat tcagcaaatt 3720

cggaatacag gtactagtga gagagatgga gatttcattc tttcgcctgt tcgagacgag 3780

agaggtatcc attttgactc acgggatact agggaaggga tgcctacgtc tggggatgca 3840

aatggagcat acaatattgc aaggaagggg acgattatgg gcgagcatat aaaacgagag 3900

tatagtagaa tgtttatttc tgacgaagaa tgggacgcgt ggcttgctgg gaagcaagtt 3960

tgggaaaaat ggttgaagga caacgagaag atattaaaga aaaagtag 4008

<210> 24

<211> 3825

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC09的NT序列

<400> 24

atgaaaaatt ccttggagga ctttacgaat ctatacagtc tccaaaagac acttagattt 60

gagcttaaac caataggcaa tacacaatct atgttggaag aagatggtgt atttgatacc 120

gatgagaaaa ggaaaatcgc atattctaaa acaaaaccgt atatagaccg tttacaccgt 180

gaattcatcg aagagtccct cagtgatgca caaataagta aattggatga atacttcaaa 240

gcatatgtcg actataaaaa ggacaagaag gatactaaaa gattcaatag gataaaacaa 300

tttaaatcag tacttaggaa ggaagttgtt gatcacttca ataagcaagg gaaggaatgg 360

actactgtta aatttgctca cctgaaaata aaaaagaagg atctagaagt tttattcgaa 420

aagcagttgc caaacattct aaaagaagaa tatggtacag aaaaagaaac tcaaataatt 480

gatgaggaca gtggtgaagt aacatcgata ttcgatatgt ggaatgggtt tatgggttac 540

tttacaaagt tttttgagac aagaaaaaat ttctacaaat cagacggtac ctcaacagct 600

atcgcaacta ggattattga tcaaaacctc gaccgattta ttgaaaatat actcatatat 660

gactcaatta aacctaaaat cgataccagc gaggttaggg aattcttcaa tttagagtcc 720

gatactattt tttccatgga attctataat aattgcttac ttcaagctgg tatagaccaa 780

tacaacaact ttttaggtgg aaaaaccctt gaaaatggga ggaagataag aggtataaat 840

gaacttatca ataaatacag acaggagaac ccagaagata agatcccttt tctgaaaaag 900

cttgacaagc aaatacacag tgagaaggag aagtttattc agcaaataga aactcttgag 960

gacctgaaag aagaattaca aaagttttat aactcatcta atgagaaaat caaaattctg 1020

gataacctcc tcagtagaat tgaggagttc aagccagaag gtatttttat ttcaaagcaa 1080

gccttcaata caatctctag aagatggaca gaccaatcag aggcctttga aaccagccta 1140

tttgagtcac taaaagaaga gaaaccaata acagggacag caaagaaaaa agacgatggt 1200

tacaatttcc ctgaattcat atcattacaa agcattagaa atactcttaa aaaagttcaa 1260

ggggaggaaa ggttttggaa agaaaggtac taccgagaca acagtgaatc cggtatcttg 1320

gctggaaatg aagaaatctg gacgcagttt ttgatgatat ttaaaagtga attcaattca 1380

aaattcgaaa gaaatgatcc ggaagacaac ggcaccattg gatataattt atttaaagaa 1440

gatctcgaga aactacttaa agaccttaaa ataacaaaag acacaaaatc tataattaag 1500

agatttgctg atgaagctct gcatatatat caagttggca agtatttcgc attagaaaaa 1560

gacagggtat ggatcagtag ttatgatgac ttactagaca ctttttatac agaccctaat 1620

actgggtatt tgagtttcta cgaaggggcg tatgaacaga ttgtacagcc atacaatatg 1680

ataagaaact atctaacaag aaagccatat agtgacgaaa agtggaagct gaattttgag 1740

aaccctactc tagcgaatgg gtgggataaa aataaagaaa cagacaattc ctcaataatg 1800

ctccgaaaag aaggggctta ctatcttgga attatgaaaa agggaaagaa caaattattt 1860

gaggaacgaa atagacaact gtttgagcca aagaatggtg aagatactta tgaaaaactt 1920

agttataaac ttttcccaga tcctgcaaaa atgattccta aggtatgttt ttcgaataaa 1980

aacatacaga tgttttctcc ttctacagaa attatgaata tttataacgg agaaacattt 2040

aagaaaaata gtgatgattt ttcagtatct agtatgcaaa agctcattgc tttctatact 2100

aaatgtttga gtcaatatga aggttggaaa tattatgact tcaaatatat aaagtctcct 2160

gatcaatata aggataatat aggagagttt tataatgatg tcgcaaagtc tgggtataga 2220

gtctggtttg aaaatatctc tcagagttac gttgactcaa agaataccat gggagagttg 2280

tatttattca aaatacacaa caaagactgg aatcaaaaag acaaaaagac caaagtggga 2340

agtaaaaacc tacacacaca ttattttgaa gagctttttt cacaagacaa tattgaaaat 2400

aactttcctc ttaaacttaa tggggaagca gaggtctttt atagacctaa aacaaatcca 2460

gagaagcttg ggacaaaaaa agatagtaaa gggagagagg tcattgatag aaagagatat 2520

gcatcagata aggtactatt ccatgtccca ataaccctaa acagaactcc tgttacaact 2580

acaaagctca ataaagaaat caacggtttc ctagcaaata acccaagtat caatataatc 2640

ggagtagata gaggtgaaaa acatttagtc tattactctg tagtaaatca aagaggaaag 2700

atgctggaaa gcggcagttt taatacaata aacggtgttg actatcacgg taagctcgaa 2760

gaaagggcag accggagaga gcaggcacgc agagattggc aggatgttga agggattaaa 2820

aaccttaaaa aaggttatat atctttggtt gtaagagagt tagcaaatct ttctataaaa 2880

tataacgcta ttatagtaat ggaggatctc aacatgagat ttaagcagat aaggggagga 2940

atagaaaaaa gtgcatacca acagctagaa aaggcattaa ttgaaaaact aaactatctt 3000

gtaaataaga cggaaacaga tccacaaaag acaggtcata tactaaaagc ataccagcta 3060

acatccccta ttaaatcttt taaggaaatg ggcaagcaaa ctgggatcat tttttacact 3120

caagcatcct atacttccgt aacagaccca ataacagggt ggagaccaaa cctatatctt 3180

aaatatagta gtgcaagcaa agcgaagagt gacatactaa agttttcaaa aatctcatac 3240

aacacaaata acaatcgatt tgagtttaca tacgacctaa gaaattttgt aaatatgaag 3300

gcttatcctc agaagactgc ttggacaatc tgttcaaatg tggagcgatt caggtgggat 3360

aggaaaggaa ataagaataa tggagagtac atacaataca aggatctaac agaaaacttt 3420

aaaacattct ttgaagaagt tagtataaac tacaaaggag atatactgtt tcagattaaa 3480

aatctaagtg aaaaaggcaa tgaaaaattc tttagagacc ttatcttcta cattagtcta 3540

atttcccaaa ttagaaatac acaaaaagac aagaaaggag atgaaaatga tttcattctg 3600

tccccagtag aaccattctt tgacagtaga aaatctagta cttttggaga aaacctacca 3660

ctaaatggag atgcaaacgg tgcctataat attgcaagaa aagggattat tatgttaaat 3720

aaaatctcaa aaggttcaaa aaataaagtc aaagaagata taggatgggg agatctatac 3780

attccacata ctgagtggga cgattttgcc acagggagca tttag 3825

<210> 25

<211> 4371

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC10的NT序列

<400> 25

atgagtacaa aaaggagttt ttcagatttt acaaatttgt atagtgtaag taagactctt 60

agatttgagt tgaaaccaat cggaaagacg ctcgaaaata tgcgagagcg tatctataat 120

gacaaaaaag attatgattc tgccctacaa acatttctcc atgatcaagc aatagaagat 180

gcatataaaa cactcaagcc cattctagat agcttacacg aggaatttat caataccagt 240

ctcaattctt ccaaggcaaa gaatatagat ctttcagagt atctcaatgc atatagagaa 300

cgaggaaatg atacaaagac tggtgaagaa tcaaaattat caggaattga aaagagtctt 360

cgaaaggcga taggagaaac atatcttaca tgagcgaaga gctttaccga acaagcaaaa 420

aatttgattg gaataataga ggatatatga gatacggaag aagagtgaga cgaggaaaag 480

aaaacaaaat gactctttaa gaaaaagaat tttgaacttt tgactgagtc ggggattttg 540

gtatttatcg aaaaaaaact ggataccatg aacattagtg agcaagaaaa gacggatata 600

aaaaaggctc tcgaagagtt taaaggattt tttacctatt tttcaggtta caatcagaat 660

agaaagaatt actatgaaac aaaagcggaa aagaaaacag caatcgcgac acgaatcgtt 720

catgaaaatc tcccaaaatt ttgtgacaat gtcatcttgt ttcacggata tcaaaaaatt 780

ctcaaggatg gatcaaaacg agaatacaag aaaaaggagg agtatctcgg gatgtatgca 840

tttctcaaat taagaaacat agaaacatgc atcaaagatg ctgagtcagg agagatgatt 900

gagctctatg ctattactga agatattttt gatatatctt tcttctcatc gtgccttgct 960

caacgagaaa ttgatgagca taatcggatc attgggggaa ttgataaata caatcgtatt 1020

atcggacact acaatgctct catcaatctc tacaatcagg cacgcaagaa agacgaaaaa 1080

ttcacaaaac tctcgccatt caaagagctc tacaagcaaa tctgatgcgg taataagaag 1140

tgaagttgga tcaaagccat tactcatgat actgacgagc agattcttgc agataccaat 1200

catacaggag aggcaatttc tgtagagcgt attctgagtc ttgcgagcaa agcagggaag 1260

aaatattttc agccatgaaa aagcacggat gatgggataa aaacagttcc tgattttctc 1320

gattggctca ggggacaaac agattggaat ggcatatact ggtcgaaagc agcgatcaac 1380

agtatctcaa atgtctattt tccaaattgg ggaagcatca aagaaaccat gaaaggcgac 1440

aaaacgctcg tttcttacga caagaaacga gaggagcaaa tcaaaatcaa tgaagcagtg 1500

gagctttctg gactttttga tatattggat agcactgatg gtgactggaa gcaagaatga 1560

gtactcttca aagcaagtct tacaaaatta ttggacgcat cagcagaaaa tgccgaagaa 1620

aatagtaaac gagcgagaag aaaagatatc atcgatcgat catcgtctcc aagtcaagca 1680

cttcttgctc tcattacaga ctttatagaa gaaaatatga agcattttct cgatcaatct 1740

catacgattt tgagactgac cgagtatagc tctcccaaaa gcaaagaagc cataaaatca 1800

tggatggatc tggcactctc agtgagtcaa acaattcgat attttcgagt caaagaatcc 1860

aagaccaaag gggatactct caatgctgag ttggtgggta ttctcaccaa tctgctggat 1920

gcagaagatg caacttggtt tgagtggtat gatctcctcc gaaactatct caccaaaaag 1980

ccacaggatg atgcaaaaga gaataagttg aagttgaatt ttgcaaacag tactctcgca 2040

gctggctggg atgtgaataa ggaaacggat aatacctgtg tcatcttgca aaatcctgaa 2100

tgaaaaacat atcttgcggt gatgaataaa aataaaaaaa acgtttttca gaaagaatgg 2160

aatgaatgaa gatgaaagaa gaaaaccaca aaattgaatc cattgtatga aattgattga 2220

ggggaaagct ggaaaaagat ggagtatgat ttttggtctg atgtctcaaa aatgatacca 2280

aaatgttcaa ctcaattaaa aaaggtcatt aaacatttca aagaatccga cgaagatttt 2340

atttttccat ccggatataa ggttacttct ggagaaagat ttatcgaaga atgtagaatt 2400

acaaaagaac aatttgagct caacaataaa gtttataaga gggatggtga tagaattata 2460

agtgcattta gatatgagtt gtctgagacg gaagaaaaaa cttatatcaa atcttttcag 2520

aaagggtatt tggatatgtt gttaaaaagc aataatctcc cagaaactga acaagaaata 2580

taccgaaaaa aatatgaaga ttcgctcagt aaatggatta acttctgtaa gtatttcatt 2640

tgaaaatatc caaaaacttc actttttgaa tatcaatttg atgagactga tcattataag 2700

tctgtagata agttcaatct tgatgtagat atatgatcat ataagcttaa ggtagataca 2760

aagataaaca aaaccattct cgatactctg gtagaaaatg gagatattta tctttttgag 2820

ataaagaatc aagattctaa tattggaaaa tgagagaacc ataaaaacaa tcttcataca 2880

acttattgga aatcaatttt tgaatcagtc caaaatcgtc caaaactcaa tggagaagcg 2940

gagatatttt atatgaagcc actttctcct gaaaaattgc aaaagaaaat cgataaaaaa 3000

ggaaaagaaa taattgatgg atatagattc tctcgggaaa gatttatatt tcattgtcct 3060

attactctca atttttgtct tggtaatgaa aagatcaaca atatcatcaa ttttgagctt 3120

tcaccaaaat cagacatcta ttttctcggt cttgatcgag gagaaaaaca tctcgtctac 3180

tactctatag tggatcaaaa cggaaaaatg atagatcagt gaagttttaa tgaaatcaag 3240

tgaaaagatt atcatgcact tctgacaaag cgagaatgag atcgtatgga atctcgcaag 3300

aattggcaga caataagcaa tatcgccaaa ctcaaggagt gatatatttc acttgtcatc 3360

catgagatca tcgagaaact gaaattgaat ccatgattta tcgtactgga agatcttaat 3420

actggattca agcgaggacg ccaaaagatc gagaaatcca tctaccagaa gtttgaactt 3480

gcccttgcga aaaaactcaa tttcgtcgtg gacaaatcag caaaactcgg ggaagtcgga 3540

tcagttacca atgctcttca gctgactccg ccagtatcga attatggcga tatcgagaat 3600

cggaagcagg ttgggatcat gctctatacc cgagcaaact atacgtctca gacagatcct 3660

gcgacaggat ggagaaagac gatctatctc aaaacaggga gcgaggaaaa tatcaaagaa 3720

cagattgtca ctcaattttc tgatattggt tttgacggca aagattatta ttttgaatac 3780

actgataaaa tagggaagac ttggattcta tactctggga aaaatgggaa aagtctcact 3840

cgatttcgcg gagttcgcgg aaaagaaaaa aatgaatgga acataaaaga gataaatgtc 3900

cgaaatatgc ttgatggaat atttgcgaat tttgataagg atcgctcttt cctctctcaa 3960

atactcgatg aatgagtgga aatcaaaaaa atagatgaac atactgcttg ggaatcactt 4020

cgatttgcta ttgatctgat ccagcagatt cgaaattcag gagacaaaac tcaatgagaa 4080

gatgataatt ttctcttttc tcctgttcga gatgcacagg ggaatcactt cgatacacga 4140

gaacaaaaag aggggttacc aaaagatgca gatgcaaacg gtgcctacaa tattgcccgc 4200

aagtgaatca tcatgaatga acatattcgt atcaatgaag atacgaaaga tcttgatctc 4260

tttgtctctg atgaagagtg ggatatgtgg cttacagaca gagaaaaatg gaaagaaatg 4320

cttccgatat ttgcatcgag aaaagctatg gaaaaacgga gagggaaata g 4371

<210> 26

<211> 3954

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC11的NT序列

<400> 26

atgtcgcaga acaacacttt tgaaaagttt actaaccagt attcactaag caagaccttg 60

cggtttgaat tgcgtcctgt aggaaataca gaacaaatgc ttgaagatga aaacgtcttc 120

aaaaaagatg aaataattcg taaaaaatac gaacagacca aaccttttat tgataaatta 180

cataaagagg ttattaaaga ttctttacat ggtaagaaga ttgaaggttt ggatgattac 240

tttaaaaagt tcgaaattta ttcaaaaaat aagaaagatt ctaaaataaa gaaagaattt 300

acagataaag aatcggaatt aaggaaacaa ttaaatagtc actttaaggc agaaagttta 360

ttctctgaga aggtattttc gttgttgaaa gaaaagtatg gtacggagga tgagtctttt 420

gttaaagatg aaaacggtaa ttttgttcta gatactgttg gtgaaaaaat ttctattttt 480

gatgagtgga aaggttttac tggttacttt acgaaatttc agaaaactag agagaacttt 540

tataaagatg atggaacttc tacagcaatt gtaactagaa ctatagatga gaacttatat 600

agattttgtg aaaatatcaa gcattttgaa agtataaaaa atagagtaaa tttttcagaa 660

attgagaaga attttaactt caagttagaa aatttattta aggcggattt ttataattct 720

tgcttactcc aagacggaat agataaatac aatgatattt taggcggtaa aacattagaa 780

agtggtgaaa aattaaaagg tttaaatgag attataaata aatatcgtca agacaataag 840

gttgaaaaaa ttggcttttt caaaatgctt gataagcaaa ttttgggtga taaggaaaaa 900

ccaagtttta ttgaatcaat tgcagatgat aatgaattac tattaaaatt aaaagaattc 960

tatacaaacg cagaagaaaa aactgaagta ttaaaaaagt tatttagtga tttttctaag 1020

aataatgata gctacgattt atcaaaaatc tatataaata aagtcggaat aaatactatt 1080

ctgttaaagt ggtttgatgt tgctggtaga agtgattttg agaaaaatat ttctacacaa 1140

actaaaaaag aaaaaattgt tacttttgac aaagatagta atagttacaa gtttccggaa 1200

tttttagctt ttagtcacat aaaagaagct ttaagtaacg gaacttatga agttaaagaa 1260

atttggaaag aaagatatta tcagtcagaa aataaagaaa aatcagaaaa agcgccactg 1320

aaaaaagatt cagctatttc acattgggaa gaattcttac aaatttttag ttatgaattt 1380

gatttgttgt ttgtaggagc tgaaagccaa gcaggataca attcaaataa gaatcttttt 1440

gaaagtttaa taaaaaagaa tgagaaaggt ttttctattt ctcctgaaga aaaattagtc 1500

attaaaaatt ttgtagataa tactttgtgg atttatcaaa tggcaaaata ctttgctatt 1560

gaaaaaaaga gaaagtggct agagtcagaa tatccaactg atagtagttt ttacgacagt 1620

gaagaatttg gttttaaaaa taaattttac gatgatgcgt atgacaaaat tgtaaaatta 1680

agaatgcttt tacaaagtca tctaactaaa aaacctttta gtactgataa gtggaagttg 1740

aattttgaaa acccaactct agctaaaggt tgggataaaa ataaagaaag tgataattct 1800

gctgttcttc tgagaaaaga aggcaggtat tatttggcgg taatgaaaaa agggaataat 1860

aagatttttg atgataaaaa taaatcaaat tttttagaaa atatagaagg tggaaaatat 1920

gaaaaaatgg tttataagca gatgtctgat ccgtcaaaag atatacagaa tttgatggtt 1980

attgacgaca agaccgttag aaaggttgga aaaaaagacc ccctcgatgg tgttaataga 2040

cgacttgaag aattaaagaa ggaatattta cctagagaca taaatactat tcgagaacaa 2100

aaagcttatt taaaaagtag cgataacttc aacttagggg acgctaactt atttataaac 2160

tactataaag ataggctggt agaataccat aaggacattt ttgtctttag ttttagagac 2220

aggtactctg attttcacga tttctctaaa catgtagcag aacaaactta cagcctaagc 2280

tttgaagata tttctgagtt ttatattcaa gaaaagaata ataatggaga attattttta 2340

tttgaaatcc ataataaaga ttggaattta gaaaagaagg gtggagatag aaagagtggt 2400

gctaaaaact tgcatacggt ttattttgaa agcttatttt caaaagaaaa cgaaaataat 2460

aatttctcga ttaaattaaa tggtgaagcc gaattgtttt atagaccaaa aactgatgag 2520

caaaaattag gaaataaaaa tgatttgaaa ggaaaaattg tcttgaacaa aaaaagatat 2580

gctgaaaata aaacttttat tcatattcct attacattaa acagagttgc ttctgagtct 2640

aaatacttca atcaaaaatt gaatgatttt ttggttggga atcccgatat aaatatcatt 2700

ggtattgatc gtggggaaaa acatttaatt tactacgctg gaatcaatca agcaggggaa 2760

tttttgaaag acgaaaaagg aaatctagtt ttaggaagcc taaacaccat aaacgatgta 2820

aactacgctc aaaaactaga agaacgtgcc aaagggaggg taaaagctaa gcaagactgg 2880

caagaaatag aaaatataaa agatttgaaa agaggatata tttctttggt ggtaagggaa 2940

ttggctgatt tgattattaa acataacgct atcattgttt ttgaggatct caatatgaga 3000

tttaagcaaa ttcgtggcgg tattgaaaag agtgtctatc agcaattaga aaaagctcta 3060

attgataagt taaacttttt agttaataaa ggtgaaaaag atccgacaaa ggccggacat 3120

ctcttacgag cctttcaatt aactgctcct atttcggctt acaaagacat gggtaaacaa 3180

acaggggtga ttttctatac tcaagctagt tatacttcaa aaacctgtcc cgagtgcggt 3240

tttcgaccaa acgtcaggtg ggaaccaaaa tcaattaaag ataaaataaa agaaggtaag 3300

ttagaaatta cttataaaga agatggtttt gaaatttcct ataaattatc tgattttagt 3360

aaatctcaaa atcaatcaaa aagaagaaat attctttata ctaatgtttc caaacaagat 3420

aagtttaatt taaacaccaa agatgctgtt aggtgtaaat ggtttagaaa aactttgtca 3480

gaaaatgaat taaataaagg agagcaaaaa ttaaacattc agactgagac aggggtaaat 3540

attgagtata aaatttctga ttgtttgatt ggtttatttg agaaatatgg actagattat 3600

caaaataatc tacaagaaga aattaaaaac tcaggagact ctttgccagt taagttttac 3660

gacaaattaa gtttttatct gcatctctta acaaatacca gaagtagtgt ttcgggaact 3720

gatattgatc acattaattg tccgaactgt ggattctgta gcaagaatgg atttaaagga 3780

ggggagttta atggtgatgc taacggtgct tataatattg cacgaaaagg aattattatt 3840

ttggataagt taaaaaatta taagacagaa aattctaact tggaaaaaat gacttggggc 3900

gatttattca ttgatattga tgagtgggat aaatttaccc aaaacaaaac ctaa 3954

<210> 27

<211> 4158

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC12的NT序列

<400> 27

atggaaacaa aaaataaaag tatttgggga gattttacta ataaatactc tttaagtaag 60

actttgcggt ttgagttggt gccagtggga aagactcgtg aaaatattca aaaacacaac 120

cctgagtttg ttcaagacca aaaaattgaa gaagcttatc aaatattaaa atcggttttt 180

gataaaattc atgaagattt tattacaaaa agcttagaga gtgatgaagc taaaagtata 240

aatttttctg aatattttga tttatataaa aaatggaatg agttaaaaaa gaagaaaacc 300

aatgaaaaaa acatagagat aaaaaaagag attcaaaatg aaatagaaaa aatttataaa 360

gataatggtg gaagtaaagg tgaaattcaa aagatagaag acgaattaag aaaaaggttt 420

gaagaaatat ttaaaattca aggaaaaatt tttaaggaaa aagcgtgtga gttaaatatt 480

aaagaaggcc aagaaaaaga cgatgatgaa gagaaagatg ataataaaaa aggctttaga 540

aaattattaa aagcaaaatt tttatatgat tatttgtgta atttgattga aagtaaaaat 600

ataatatata aagatttttt tgaaaacata aagaataaag aaggagagag tatttcaaaa 660

gaaaagacaa aagacgctct tataagattt aaaggtttta caacttattt tggcgggttt 720

gagttaaata gattaaatta ttatacaaca aaggaagaaa aatctactgc tgtagcaaca 780

agaattgtta accaaaattt accaaagttt tgtgacaatg ttattctttt tgaaattaaa 840

aaatcagaat acctcaaaat tgatgaattt ttgaaaaata aaaatatttc tttaatttct 900

aaaaatcaga atggaggtga ggttgaatta cataaaataa ataaaaattt ttttgaaatg 960

atgtttttta gcaaatgctt atctcaaaaa gaaattcaaa aatataattt agaaattgga 1020

aatgcaaata acttaataaa tagatacaac cagcaacaaa gtgacaaatc tcagaaatta 1080

aaattattta aaactttaca caaacaaatt ggatgcggag atagaggtgg atttattcca 1140

tcaattaaag gtgaggaaga cttaagggag agattgcaag aaattaagaa taacagtatt 1200

gagtattttg aaaatataaa tgactttatt gaatatttaa aaaatcatga aaattatgag 1260

aatgtttact ggtcagacaa ggcaataaat accatttctt caaaatattt ttctgattgg 1320

ttaaatctta aaaaagaaat ttggggcaaa agagacagaa agggtaactt aaaagatgaa 1380

gaaactaaaa ttccacgagc cgttcaatta aaggatctgc ttgaaaatct agacaaaatc 1440

acagactgga aattagaagg tagattattt aaactttctc tatttgaaaa tggtagaaaa 1500

gctaaaaaat tacagcaaga agatttaaat aaatttaata aaaataaaat agagaatgaa 1560

ctagagattg aaaaattgca gattatagaa caaaacccaa gtccttttca ggctttacta 1620

aatatgattt ttgctgatat caaaagcaag gaaagtgcct ttttagagag tcggatattt 1680

gagatttctg attttgttca caatgaagat aaacaaatta taaaacaatg gcttgattct 1740

attttagcta taaatcaaat tataaaatat tggagagtaa aagatacatt tggaaccgaa 1800

ggaactctcg atgaaaagtt aaagaatatt atttattcag aaaaaaatcc aacaagattt 1860

tatgatatta ttcgtaacta tctgacaaaa aaaccacaag acgagttaaa taaattgaag 1920

cttaattttg aaaattctac tcttgctcaa ggtttggacg taaataaaga aaaagacaat 1980

ttctgcataa ttttacgaga cgacaaacaa aatcaatatc taggtatttt gaatagtaaa 2040

aataaaaata tttttgagat agatcaaaat gaagatatat atcaggatga cggtctgggt 2100

tggagtaaaa tgatgtataa acttattcct ggagcgagca agacacttcc taaaatattt 2160

ttctcaaaga gatggacaga aaataaccca acaccagatg agattagtaa aataaaaaag 2220

ggtgagacct ttaaaaaagg ggataatttt attaagagag atcttcatga attaattaat 2280

ttttataagg ctaacttaga aaagtatcca tcggtaaatg agagttgggc taaactcttt 2340

atttttaatt ttagtgatac aaaaacatat gaaagtatcg atcaatttta caatgaggtt 2400

gataaacaag gatacaaggt ttcttttatc tcaataaata aaaacactct ggataatttt 2460

atagacaaag aaaaattata cctatttcag attaaaaata aagataataa tctagataaa 2520

ggcgagaaaa aacaaagcaa taaaaatctt catagtattt attgggaggc tatttttggt 2580

aaagccttaa ataaaccaaa attaaacggc ggggccgaaa ttttttatcg tccagctttg 2640

tcagagaaaa aaataagtga actaaaaatt aaagataaaa atggaaaaaa tataattata 2700

atcaaaaatt atagatattc aaaagataaa tttatttttc attgccctat aactctaaat 2760

tttagcgcaa agtcatctaa attaaatgat gaaataaatg atcacataaa aaacaaaaaa 2820

gaattttgtt ttatgggaat tgaccgtggc gaaaaacatc ttgcttatta ttctttggta 2880

aatcaaaatg gaaaaatttt agataaaggc caaggaactc taaatctccc atttgtagat 2940

aaagacggaa ataaaagatg cataaaaact gaaaaatatt ttgaggagga taaaaaagaa 3000

aacgagaaat ggaagccaag aattattgat tgtcctgatt ataattgtct tttggatgct 3060

cgtgcctcta atcgtgattt agctcgtaaa aactggcaga caattggaac tataaaagaa 3120

ttgaaagaag ggtatatttc ccaggttgtt agaaaaattg ttgatctggc aatagagaat 3180

aacgctttca tcgtattgga aaatctaaat attggtttta agaggggtcg tcaaaaaatt 3240

gaaaagcaag tatatcaaaa attagaactt gctttggcaa gaaaattaaa tttcttagtt 3300

gataaaaaag caataatcgg agaagttggt tcagtcacaa aagcgttaca acttactccg 3360

ccggtaaata acttcggtga cataggaggc aaaagtcaat ttggtataat gttttataca 3420

aaagctgatt atacttcaca aactgaccct gtcacgggtt ggcggaaaag tatttaccta 3480

aaaagggggc cggaagatta tatcaaagac caaatacttg gcaataaaaa caaaaatatt 3540

gaaccggcat tcgaagacat ttgttttgat ggacaagatt attgttttac ttatataaat 3600

aaaaatactg gtaaaaaatg gacactatac tcaagtaaaa acggtaaaag tttagataga 3660

tatcatcggg aattagtata tgaaaatagc gacaaaaaat ggttacccaa aaaacaagat 3720

gtcttagaga tgctcaataa tttatttgaa gggtttgata agaaaaaatc tttgcttaaa 3780

caattagaaa ctaaaaaccc aaataaaaca ggagaacacc ccgcttggga gtctctaaga 3840

tttacgattg atttaattca gcaaattaga aatacaggca taaaagaaag agatgaagac 3900

tttattcttt cgccggttag agataaaaaa ggtgatcatt ttgactcacg cgaggcttcc 3960

ccagatttac caaattcagg tgatgccaat ggcgcctata atattgctcg caaggggata 4020

attatggcga aacacattga aaaaggatac tttctttata ttagcgatga agaatgggac 4080

gcctggcttg ctggtgaaga atgctggaat cgttgggctg aaaaaaacac taaatctcta 4140

ctaaaaaaca attattga 4158

<210> 28

<211> 4158

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC13的NT序列

<400> 28

atgagtacaa aaacaatttt ttcagatttt accaatctct atgaattaag taaaactttg 60

aggtttgagt tgaagcctgt ttgagagact gaaaatttac taaatgaaaa tcaagtattt 120

ttaactgaca aaatcagaca aaaaaagtat gaagaaataa aaccattttt agatgaattt 180

catttggatt ttatacattt ttgtctttca gatttacatt tagattatac tgaatataaa 240

aaatctttgg ataattatca aaaagataaa aaaaataaag atttagaaaa gaaaaaagaa 300

aatgaagaaa aaaagctaag agaacaaatt gtttgaaaat ttgattcaaa agttgaagat 360

tttttaaaaa ctttttgaaa agttgaaaaa attaaatgaa aaaaagataa tgaaaaattt 420

aaagtaagtt tatgaaaaga ttgggaaata gaattttcta aaaataacta tgaatttctt 480

tttgaaatct gaatttttga tttgatgaaa aagaaatttg aatgaaactg agatatttat 540

gtcgcagata aagaaacttg agagatttat caggatgaaa aaacttgaaa agatatcact 600

atttttgatg attggaattg atggcttgga tatctaacaa agttttttga aacaagaaaa 660

aatttatata agtcagatgg aacttctact gcaattgcta caagaattat taatgaaaat 720

ttaaaaaaat attgtgaaaa tttagatatt tataataaac tttctcaaat tgagaattta 780

aaaaataagt ttcaaaactt agaagctgat ttttgaatta aattagaaaa gttttttagt 840

ttagagaatt ataactcttg tatattacaa aattgaatag aaaattataa tgatatcagg 900

tgatgaaaat tagaaaaaaa taacaataaa attccttgaa taaatgaata tataaataaa 960

tataggcaag atagttgaga aaagcttcct tttttacaaa aacttgataa acaaatttta 1020

gcctgatgaa aagaaaattt tatagaacaa atagaaaatg aaccatcttt tgaaaaatgt 1080

ttgaaaaatt tttataataa ttcaataaaa aaagtagaca ttttaactca aatttttcaa 1140

gatttaagta cctacactaa tgaagattat aaaactattt atttttcaaa agaagctttc 1200

aatacccttt ctcataaatt tacagaccaa gttttaaact ttgaaaaact tgtttttgag 1260

gaactcttac taaataaatt agttgagaaa aaggattttg ataaaaaaga agaaaaatat 1320

aaatttcctg attttatacc tttattttat gtgaagaaag gattagaaaa ttatcataca 1380

aaaaatttat tttataaatc aagatattat gaaaacgaaa ttatagaaga agataatgat 1440

aatatatggc agaaattttg tactattctc aattatgaat tccagtcttt actttcaaat 1500

acgataataa accaaaatgg agaagaaata gaagttgggt ttactatttc aaaaaataag 1560

ttagagaaaa ttttagataa cttctctctt tgagaaaata ataattgaat tattaaagat 1620

tttgcagata taagtaaaac tatctatcaa atgtgaaaat attttgcttt agagaaaaaa 1680

agagaatgga ataataattt tgatttaaat gatgattttt ataaaacaga atattctcaa 1740

gaaaatgaaa aatattgata tttagaattt tataatgaag cttatgaaca aattatagtt 1800

ccatataatt tgatgagaaa ttttatagca aaaaaacctt gggaagataa taaaaaatgg 1860

aaattaaatt ttgaaaattc tagtttattg aaatgatggg ataaagaatt tgaaagttat 1920

ggatcttata tttttgaaaa agcttgattg tattatttat gaataataaa ttgaacaaaa 1980

ttaaataaaa atgaaattga gaaactctat aattataatg caaataattg agcaaaaaga 2040

tttgtttatg attttcaaaa acctgataat aaaaataccc ctagattatt tattagatca 2100

aaatgagata attttgctcc atcagttaaa gaattaaatt taccaataaa taacattatt 2160

gaaatttatg ataaagaatt gtataaaaaa gacaaagaaa aacctaacaa acataaagaa 2220

agtttaatga aattgattga ttattttaag ctttgattta gaaaacatat atcttataag 2280

cattttaatt ttgtttggaa agagagtaat aaatatgata atattgctga tttttataga 2340

gatgtagaaa aatcatgtta taaaccatat tgggaagaag atataaattt tgatgaatta 2400

aaaaatctta caaaagaaaa aagaatgtat ttgtttcaaa tttataataa aaattttgaa 2460

ttagatgaaa gtatatctac agacgattat acttttaaat gaaattgaaa agatagtgtt 2520

catacaatgt attttaaatg attattttca aaagataatt tagaaaataa aaattgagta 2580

aatctaaaat taagctgatg atgagaattg ttttttagac caaaatctat agaaaaaaag 2640

atagataaaa atagaaaaag taaaagagaa attatagaaa ataaaagata ttctaaagat 2700

aaaatactat tacattttcc aattcaagta aattttaaag aaaataaaac ttcaaatttt 2760

aataattata taaacaattt tctcgcaaat aatccagata taaacattat ttgaatagat 2820

agatgagaaa aacatttagc ttactattca gtaataaacc aaaaacaaga aattattgaa 2880

agttgaagtc taaattatat ttatcaaaaa gataaagatg gaaaaattat tcaaaagtct 2940

gagaaaaaaa tacaagaagt gagaaatgat gaatgaaaaa tcattgatta tgaattagtg 3000

gaaacttgaa aattagtaga ttatgaagat tattgaatat tgcttgatta taaagagaaa 3060

aaaagaagat tgcaaagaca atcttggaag gaagtagaac aaataaaaga tttaaagaaa 3120

tgatatatct cagcagtggt aagaaaaatt gcagatttaa ttattgagca taatgcaata 3180

gttatatttg aagatttaaa tatgagattc aaacaaatta gatgatgaat agaaaaaagt 3240

gtgtatcaac aattagaaaa agctctcata gataaactca attttcttgt aaataaatga 3300

gaaaaagact cagaacaagc ttgaaattta ttaaaagctt ttcagttaac tgctccaatt 3360

tgaactttta aagatatgtg aaaacaaact ggaatcattt tttacactca agcccgttat 3420

acttcaaaaa ttgatccact cacttgatgg agaccaaatt tatatattaa aaaacaaagt 3480

gctgaactca ataaagaaag tattttaaaa tttgattcta ttatttggaa taaagagaaa 3540

gaatattttg aaataactta tgatttagag aaatttcaat cagaaagtac aaaaaattta 3600

aaagagaaaa aagaagaaaa attagaaaga acaaaatgga ctctatcaac aagagttgag 3660

agattcaaat ggaataaaaa tctcaataat aataaatgat gatatgaaca ctttgaaaat 3720

ttaaatatac atttcaaaga actttttgaa aaatattgat tagatatttc ttgagatatt 3780

ttaaagcaaa ttcataattt agaaacaaag tgaaatgaag cattttttag tcatttttta 3840

gatttattta aacttgtatg tcagattcga aacactaatc aagataaaaa atgaaacgaa 3900

aatgatttta tttattctcc agtttttcct ttttttgata gtagaaaaca aaatacagta 3960

tgagttaaaa atggagatga taattgagct tttaatatag caagaaaatg aattattatc 4020

ttggagagaa tttgaaaatg gaaaaaagaa aatgatatga aaatacaaaa atgagaaaag 4080

gaaatgtatc cagatttatt tatttcaaat ataggttggg ataattttac tcagaatcat 4140

aatattcgag ataattaa 4158

<210> 29

<211> 3933

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC14的NT序列

<400> 29

atgtcgcaga acaatataaa agagaaaagt atttttgatg aatttaccaa caagtattca 60

cttcaaaaga cattgcggtt tgaattacgt ccggtactaa atacggagca gatgcttacc 120

gacagtggaa taatcaaact ggatgaaaag cgaaagctga actatgaaaa aacgaaaccg 180

ttcctcaatc gactacatca agagtttgtc accgagtctc taaatggtgt gcgtttaaaa 240

tcactcgacg ggtacgcagt tttatatgca aattggaaga agagcataga taaaaaagaa 300

aaagatgcag cttataaagt tttagaaaaa aaggaactag aaatcagaca agagattgta 360

gttttgtttg atgagaaagc ggtagaatgg attggtaagt tacctgccga tgtaaaaaaa 420

ccgaaaaaac caaactatga gtttttgttt gaaccagcga ttttttcaat actcaagaaa 480

aaatatagtg atgaggtagg aactactatt gatgaggaat caatttttga tagttgggat 540

aaatggacgg cgtactttgg aaagtttttt gaaacacgca aaaattttta taaaagtgac 600

ggtaaggcta cagcagtagc gacaagaata gtgaatgaaa atttgagacg tttttgtgat 660

gatgtctcaa cttttgaaaa tatacaatca aaaatcgatt tgtcaccact cgaaaaggag 720

tttgacgtta gccttaaaaa ggtttttgat attcaacatt acaatcaatg tttgaaccag 780

tcaggtattg atgcgtttaa cacattactt ggaggagagg ttcacgagaa tggagaaaaa 840

attaagggaa ttaacgaata tataaacgag caccgacaaa aaactggcga aaaattaacg 900

cgcctaaaaa agttagataa acagattggc agtgataagg aaaattttat tgatctcatt 960

gagacagatg aacaattgaa gacaacgctt gtcacattta ttgcaaacgc taaggaaaaa 1020

gtagacttgt tagataagag cgtctcgtat ctcacgaaag atactgatgt taagttatct 1080

ggtatttttt tcagaaagga agcgattaac acaatcacca ggcgatggtt tgtgagtcat 1140

gaaaagatat cagatgcact cgtaagcgcc tttagtgata agaatgtaaa gttcgatcaa 1200

aagcgtgaag aatataagtt tccagatttt attagttggc acgtgataca aaatgcggtc 1260

gaaaaacttg ccagtgacgg agaagagatt tggaaaaagt attatcttga ggaagagaaa 1320

ttatcacttc ttgataaaac cccatggcag cagtttttga cagtttttga atgtgaatat 1380

aacaacctaa aatcgaaagg tcatgaaagc gaaggaagga gctttactga acttgtgcaa 1440

gatattgaat ctttgctgaa gacagacacc ctagatagaa acgaccatgt aacggagatt 1500

ataaaatcct tttctgatcg ggtgcttaat atctatcgat ttgcgaaata ttttgcactc 1560

gataaatctt gccaatggaa tcctgatggt ttggatactg acgattttta cgtggcctac 1620

gaacaattct atagtgatgg atacgagaag atcgttaagg tgtacgacaa ggttcgtaac 1680

tatatgacca agaagccgtt caatcaagac aaatggaaat tgaactttga gaacccaact 1740

ttggctaacg gctgggacaa gaacaaggag actgataaca ccgcaataat cttgcgacgg 1800

gcaggtcggt attatctagc ggtgatggaa aggggtcata ataccttgtt caagaaaata 1860

cctatgtcat cctctggcta tcaaaagatg acatataaat tgtttcctga tccatcaaag 1920

atgatgccga aggtttgttt ttcaaaaaaa ggattggaat tctttaaacc tagtgctgaa 1980

ataatgagaa tatacaaaaa cggcgagttt aaaaaaggtg acaccttctc actttcttca 2040

atgcacgttc tgattgattt ctataaaaat gcactcaaga catacgacgg ctggactatg 2100

tatgacttca gtaatttaaa gaaaacgagt gaatacacgg agaacattgg cgagttctat 2160

cgagatgtcg ctgaaagtgg gtatcaaatc aattttgact atattgcaga gcaatatata 2220

gaagatgcaa ataaagaagg aaaattgtat ctcttcgaaa tccataacaa ggattggaat 2280

ttgaaagatg gggcaataaa gaccggtagt aaaaatgcac acacgcttta ttttgagcag 2340

gttttttctg atgagaatgc ccaaaataat ttcgttgtta agctcaatgg agaagcagaa 2400

ttatttttcc gcccagcaac cagtactgaa aaacttggaa atcactacga tagtaaaggt 2460

aacgtagtta caaaaaacaa acgatatgca cacgataaaa tgtttttcca tgtgccagtg 2520

acgctcaacc gcactgctcc tgacgctcgt aaatttaatc aatcagtcaa tgtatttcta 2580

gcaaataatc ctgatacaaa catcatcggt attgatcggg gagagaagca tttggcctac 2640

ttgtccgtca ttaaccagaa aggagatata ttgaaaataa aatctttaaa caagattgag 2700

gttaaggata aggacggtaa tgtgataaag gaagatgatt atgcaaagct attggaagac 2760

cgtgctaaga atcgtgagag cgcacggcgc gactggaaga gtgtagaaca gattaaagac 2820

cttaagaaag gttatatctc aaatgttgta cgagagattg ccgacctcgt cattaaatac 2880

aatgcgattg tagtatttga agatttgaat atgcgcttca agcaagtgcg cggtggcatt 2940

gaaaagagcg tgtatcaaca acttgaaaag gccttgattg ataaacttaa cttcttggtt 3000

gataaaaatg aacttgatcc ccagaaggcg gggcacatat tgcatgcgta ccaactcact 3060

gcaccatttg agacgtttaa agatatggga aagcaaactg gcgtactctt ctacacccaa 3120

gctgaatata cgtcgcaaac cgatccggta acaggttttc gaaaaaatgt ctatctgagc 3180

aattcggcca ccgttgagaa gataaaagca tttgtggaaa tgtttgatgt aatcggctgg 3240

gacgataaac taaaaagtta ctacttcaaa tataacccgg ttaattttgt tgaaactaag 3300

tttaaggaaa atacgttctc aaaagattgg gtcatttatg ccaatgtgcc gcggattaag 3360

cgcgaacgca aaaatgggta ttgggaggca accgtggtca atccaaacga agaattcttg 3420

aaacttttta aggaatggga ttttgataac atctatgttg aagacataaa agaacagatc 3480

tttcagatgt ttgaagaggg aagattggac gggacaaaag aattcgatgg caaaaatcgt 3540

aatttttggc acagctttat cttcctattc aatcttatgt tgcaagtaag aaattctacc 3600

gcaacccaat acaagaagga tgaagatgga aatatcattg agactgttga gggtgttgat 3660

ttcattgctt caccagtttt tccattcttc accaccgatg gtggtgattt caccgaagga 3720

tgcgtgaatc tagcaaaact tgaagataaa tttgtcggta gtaatgctga caaagagcgg 3780

ttcaagaaag aatttaacgg agacgcaaac ggtgcataca acattgctcg aaagggaatt 3840

attatgttga acaatattaa aaataatccg gagaaaccag acctgttcgt gtctaaaaaa 3900

gactgggaca aatttgctca agcaaatcaa taa 3933

<210> 30

<211> 3828

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC15的NT序列

<400> 30

atgaatccaa cccaaacaga caaaaccccc tcaaagccat ttgaaaaatt taccaattta 60

tactgcttgt caaaaaccct gaggtttgag ctgaaaccaa tcggcaaaac gcaaaagata 120

cttgaagata ataaggtttt cgaaaatgat aaaaagagag ctaaaagtta tgaggaagca 180

aaaaaatatt ttaataaatt acatagagaa tttattgacg aatcactcaa aaatattaca 240

ttatctaata atctaattga aaaatttgaa aaaaaatatc ttacctggaa aaatagtaaa 300

aataaagata atagtactga actaaaaaag tctgctaaaa gattgagaat agtaattctt 360

gagagtttca ataaaaaagc taatgaatgg aactctgaat attcaaacca agttaagaac 420

gaaaagaaaa aaaagaaaat acaagaaata acaggaatag atttgttttt caaagttgaa 480

gtttttgatt ttcttataca taaatacccc gaggtgcaga taaatggtga gagtattttt 540

agtccattca acaaatttag tggttacttt aaaaaatttc acgaaacaag aaaaaacttt 600

tataaagacg atggaacctc cacagcaatt cccacaagaa taattgatgt taatttagaa 660

aaatttttag aaaataagga catctactat acaaaatact tccaaaaata caattctatt 720

tttaataagg aagaaacaga tattttcaaa ctggaatcgt ttaaaaattg tttaacacaa 780

tctcagattg ataaatataa tgaatcaata gccacattaa aatcaaaaat caacaactta 840

cgtcaaaata atcctgaggt taataaacat gatcttccat tttttaaaga attattcagg 900

caaattctag gtcaaccaat taaaaaggag acagaacagg ataacttcat agagatacta 960

acaaatgacg aggtttttcc tgttttacaa aaaaatattg atgaaaacga gttatatatt 1020

cctaaagctg atactctctt taaagaattt ttaaagagcc aaattcaaga gacaaatgaa 1080

tataatatta acgaaatata tgttgcaagt cgttttataa actcaatatc caacaactgg 1140

tttgctgaat gggataccat tattaattta ttacgtactg aactaaaaat taaacagaat 1200

cagaaaaaac ttccagattt catctcgatt gcttcattaa aaagagtgtt acaaaaatcc 1260

caagacgaga tagatgctaa agatttattt agaaacaact atgagaatct tttcgaatct 1320

acaactgact tctataaaat atttcttaaa atttgggaat tagaatttaa tgataatatt 1380

aaaaaatata atttggagac agagaatata agaaaaataa taatagaaga taaaaagtat 1440

cttccaaata aaaagagtat attaaagaat ggtgagacag gtattattca taatgaaaaa 1500

atattagatt atgcacaatc tgcattaaat atttatcaaa tgatgaaata tttttctttg 1560

gaaaaaggaa aagaaagaga gtggaatccg gacggtctga atgaagatac aacaggagga 1620

ttttatgatg atttcaataa atactaccaa aatgtaaaca cgtggaaata ttttaatgaa 1680

ttcagaaatt atttaacaaa aaaaccatat aagacagata agattaaatt atactttggt 1740

cacaagagtt tattaggtgg ttttactgaa agcaaaacag agaaaagcaa taacggcaca 1800

caatatgggg cttatttatt aagaaagaag catggacttg gaggctttga ttattatctt 1860

ggaattagta cagaccccca tttaatgagt tattttgatc caattgatga ttcaggagat 1920

agcgaatatg aaaggttaaa ttattatcaa gtattaacaa gaacaatcta cggtccttct 1980

tatgaaggcg attatgaatt ggacaaaaaa aatttatcag aaatagagat aataaaaaaa 2040

ataaaaagat cattatctta ttacacttca agggttaaaa aaatccagga cataatcaat 2100

aataattatg agtctgttag agatattcat aaagatataa ctgatgtact aaaagaattt 2160

ggaacaatct ttgattataa agtaataaca aacagtcaaa tacaaaaggc atttaattgc 2220

gataaaggat tttatctctt tgagatatat tcgaaggatt tttcaaaaga gaaaggtgat 2280

aaaagtaaga atagcaaaga taatttacat acaacgtatt ttaaatcatt aatggataga 2340

aagcagtcta catttgattt aggtagtgga gaaatatttt tcagagaaaa atctgttcag 2400

tctgaaattg attctatgag aaaaactaag aataaaataa ctaggttcaa acgatacaca 2460

aaaaatttaa ttcagttcaa tttatcaatt acccttaata ataactgtac agaagttcct 2520

cagaataaaa atgcaagaaa agcatttatt aataatttta atattgaact aagtaagaaa 2580

ctgttaacaa ataattcaga cataaatatt attggtattg accgtggaga aaaacatttg 2640

gcctattact ctgttataga ccagcaaagc aacattcttg aaacaggctc ttttaataaa 2700

attcaagaga gaaaagacag agaacctacc gattaccaac aaaaattaga taaaattcaa 2760

aaggacagag actggcaaag aaaatcatgg caggaaatat caaatatcaa agatttaaaa 2820

aaaggctata tttctcaagt tgtctatgaa ataagtaagt tagttaaaaa atataatgca 2880

atcatagtat ttgaagacct gaatatagga tttaagcgtg gccgttttgc aatagaaaaa 2940

caagtatacc aaaatttgga gctttcatta gcgaaaaaat taaattattt agtttttaaa 3000

gatgcaaatg aaggagaatc aggacattat ctaaaagcat atcaacttac atcccctgtc 3060

aacaattttc aagatattgg taaacaatgt ggtataatat tttatattcc tgccagctac 3120

acatcagcaa tttgtccttc atgtggattc cataaaaaca taccaacatc aattaaaaaa 3180

cttgcaaaga ataaagaatt tgtagaaaaa tttgttataa cttatgaact taaaaaagat 3240

cgtttttatt ttggttacaa aataaatgat ttttacaatt ctaatttgca agataatgtc 3300

attttctact caaatgtaga aagattaaga tataaaagaa ataaggataa ccgaagtggc 3360

gaagtacaag aacgattgcc taatgaagaa ttaaaaaaac tctttgagca aaatcacatt 3420

aattataaag ataatcctca aatatctggt caaattaaaa atcaaaaact agataatgaa 3480

aaattttata aacctcttat atatgaaatc tctttaattt tacaattaag gaatagtaaa 3540

acagtaaaaa gtgaagatgg aacaattaac acaaatataa atcgggattt catttcttgt 3600

ccagcttgtt attttcattc agagaataat ttaatgaatc ttcctaataa gtataaagga 3660

ggaaaaaaat ttgaatttaa cggagatgca aatggagcat ataatattgc acgaaaaggc 3720

attttgcttt taaacaaatt aaataatatc aaagatatag aaaaaattga atataatgac 3780

ctcaatatat cacaagaaga ctgggataac tttgtcaaaa acccctaa 3828

<210> 31

<211> 19

<212> DNA

<213> 人工序列

<220>

<223> 茎环共有序列

<220>

<221> 变体

<222> 1,11

<223> /替代="a,c,g,t"

<400> 31

natttctact nttgtagat 19

<210> 32

<211> 19

<212> DNA

<213> 未知细菌物种

<220>

<223> BMC01-15茎环序列

<400> 32

gatttctact attgtagat 19

<210> 33

<211> 3888

<212> DNA

<213> 人工序列

<220>

<223> BMC01的核苷酸序列 (大肠杆菌密码子优化)

<400> 33

atgatcttca acaacttcac ccagaaattc agcctgagca aaaccctgcg ttttgaactg 60

cgtccggttg atgccggtgg taatgttatt accgatctga ccattttcga agaaaccatc 120

aaaaacgatc agaaacgcta tgaagcatac ctggcaatta aaccgctggt tgatgaaacc 180

cataaacact ttattcagac cgttctgagt ggtctgacag atctgattaa atccgatgag 240

atgaagaact acctggaaca caaaaatctg atccgtcaga aagatgtgga agagaaagtt 300

aaaaccaaaa gcatcgacgt gatcaacaaa atcgaaaaag attggcgtaa acgtgtgagc 360

gatagcttta ccaaacatcc gcagtacaaa aagatgttcg ataaaaccct gtttgccgat 420

gaaagtccgc tgtataaact ggccgaaaat gattttcagc gtagccagat taaaatcttc 480

gagaaattta ccggctactt caacggcttt catgagaatc gtaaaaatct gtatgtggcg 540

gaaaaacagg gcaccgcaat tgcaaatcgt gtgattaatg aaaacctgcc gaaatttatc 600

gagaacgcca ataaactgaa acgtgccttt gaaaaatatc ccgaatttct gtccaaaatc 660

agcgaggata aaagctttca ggcactgctg attaaaaacc agctgagcct ggaaaaactg 720

ctgcagccgc tgacctttaa tctgctgatt tctcagaccg gtatcgatag ctataatgaa 780

gtgttaggtg gttacacacc ggaaaatagc gaaccgatta aaggcctgaa tcaactgatt 840

aatctgtacc gccagaaaat taacctggca cgtaacgatt ttccgaatct ggcaccgctg 900

tacaaacagc tgctgagcga tcgtgaaacc aatagcgttg tgtataaacc tctggaaaat 960

gtggcagatg tttatagcag tgtgtttgaa ctgtgtcaga acctgctgag caaacagagc 1020

gatattaata aatggatcga ggatatcaac atcagcagcg gtcagatttg gatctataaa 1080

agccatctga gcggtctgtc agttatgctg tttggtgaaa gtggttgggg tctgattccg 1140

cgtattctga atattagcga agatgatgaa gaagagatca tcaaatccaa aagcaaaaag 1200

agccagcaag agtatttcag ctttgcggaa attggtaacg ccatcaacaa ctatagcttt 1260

gaggatgtga acattaaagc actggcaaaa cagggtctgt gtctgtggca gaaacaaggt 1320

aatgaacgtc tgatcaaatt cggcaaactg tttagccaga tgcagaatga actgcagagc 1380

ccgaaagaaa aatgggatag caccgaaaaa gagaaaatca aagaactgct ggatacaggc 1440

ctggaatttg ttcattggct gaaagttatt agcaaccagc cggaagataa agatgaagtc 1500

ttttatgccg aatggcaggc actgaccgat acctggcgtg gtctgccgaa actgtatgat 1560

cgtgttcgta attttgccac caaaaaagat tacagccaga acaagctgaa gatcaacttt 1620

gataaaggca ccctgctgaa tggttgggat accaataaag aaaccgataa tctgggtatc 1680

ctgctggaaa ataaaggcca gtattatctg ggcatcatga aagatagcag catctttgat 1740

tatcagtggg acattgacaa ttttcagaac ccgaatagca aacagtcagt ggccaaaaaa 1800

aacctgcatg aagcaattgt tagcgataac acccaggatt gttggagcaa aattgtgtac 1860

aaactgttac cgggtccgaa taaaatgctg ccgaaagtgt tttttagtga caagcgccag 1920

aaatactttg gtgcggatga aaaagtgatc gacattaacg aaaatggccg tcataaaaaa 1980

ggcgacaact ttaacattag cgattgccac tatctgatcg acttctataa aaccgccatc 2040

aataaacatc cggaatggtc ccagttcaac ttcaaattta gcgcgaccaa aagctacgaa 2100

gatatcagcc agttttatca cgaagttcag aatcagggtt atcgcatcga gtttgatcat 2160

atccgcaaag actatatcca gaaaatggtg agcgaaggta aactgttcct gtttaaaatc 2220

cacagcaaag attttagcag ctatgcaaaa ggtcgtccga atatgcatac catctattgg 2280

cgtgccattt ttaacccgga aaatctggcc aatgttgtgg ttaaactgaa tggtgaagcc 2340

gaattctttt atcgcaaaag cagtaaagat cgcattatca gccatccgca aggtctggaa 2400

gttagcaata aaaacccgag caatccgaaa aaaaccagtc gttttgcata cgacctgatc 2460

aaagataaac gtttcacgca ggacaagttt tttttccatg ttccgattac gctgaatttt 2520

cgtgaaggtg aaggttatcg ctttaaccag agcgttattc gtgagctgaa aaaatactat 2580

cagaccgata aagccaacct gcatatcatt ggtattgatc gtggtgaacg tcatctgctg 2640

tattattgcg ttattaatgt tgccagcggc aaaatcgttg aacagggtag cttcaatcag 2700

atcagcacca attatacacc ggaacaaatt accgatgatg gcgaaatcat taaaggcgaa 2760

accgtgaata aaaccaccga ttatcataac ctgcttaaca ccaaagaagg tgatcgccag 2820

aaagcacgta aaaattggca gaccattgaa aacatcaaag agctgaaagc cggttatctg 2880

agcaatgtga ttcataaaat cagccagctg atggtgaaat ataacgcctt tgttgttctg 2940

gaagaactga aatatggttt taaacgcggt cgcttcaagg ttgagaaaca ggtttatcag 3000

aaatttgaga aagccctgat cgacaagctt aactatctgg tgtttaaaga tcgtgcccct 3060

gccgaagttg gtggtgttct gaatgcactg cagctggcac ctccggttgc aagctatatt 3120

gatattggta aacaggcagg cttcctgttt tatgttccgg cacatcatac cagcaaaatt 3180

tgtccgtgga ccggttttgt ggactggctg aaaccgcgtt atgatggcat tgataaagcg 3240

aaagcattct ttacctgttt cgaaagcatc catttcaaca cgcagaaaaa ctattttgag 3300

ttcgccttcg actatgagaa atttcgcggt aacattaatc atctgccgga aggtctgaaa 3360

cgtaccagct ggaccctgtg tagccataat agcctgcgtg atattgccac gaaagataaa 3420

aatggtaact ggccgtataa gcagatcaat ctgacagcag aactgcttga aattctgaaa 3480

accctgaatc cgcgtaatgg cgaaaacctg gttgaacgta ttatcgagat gaacgacaaa 3540

aagttcttcg agagcctgat gtgggcactg cgtgttctgc tgcaactgcg ttatggttat 3600

atcaaacgta ataacgaggg catcatcatc gaagaggttg attatattct gagtccggtg 3660

gcaaatgaaa acggcgaatt ttttgatagc cgcaactttg tgaatatcga gaaagccgat 3720

tttcccaaag atgcagatgc aaatggtgca tataacattg cacgtaaagg tctgctgctg 3780

attgcacaga atattaacaa cgccaaaatc aacgataaag gcgaggttaa atgtgacctg 3840

cagattgata aaacaacctg gtttaactgg gttcagagca aaagctaa 3888

<210> 34

<211> 1191

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<220>

<223> Nucleotide sequence of the araC-ParaBAD inducible promotor system

<400> 34

tatggagaaa cagtagagag ttgcgataaa aagcgtcagg taggatccgc taatcttatg 60

gataaaaatg ctatggcata gcaaagtgtg acgccgtgca aataatcaat gtggactttt 120

ctgccgtgat tatagacact tttgttacgc gtttttgtca tggctttggt cccgctttgt 180

tacagaatgc ttttaataag cggggttacc ggtttggtta gcgagaagag ccagtaaaag 240

acgcagtgac ggcaatgtct gatgcaatat ggacaattgg tttcttctct gaatggcggg 300

agtatgaaaa gtatggctga agcgcaaaat gatcccctgc tgccgggata ctcgtttaat 360

gcccatctgg tggcgggttt aacgccgatt gaggccaacg gttatctcga tttttttatc 420

gaccgaccgc tgggaatgaa aggttatatt ctcaatctca ccattcgcgg tcagggggtg 480

gtgaaaaatc agggacgaga atttgtttgc cgaccgggtg atattttgct gttcccgcca 540

ggagagattc atcactacgg tcgtcatccg gaggctcgcg aatggtatca ccagtgggtt 600

tactttcgtc cgcgcgccta ctggcatgaa tggcttaact ggccgtcaat atttgccaat 660

acggggttct ttcgcccgga tgaagcgcac cagccgcatt tcagcgacct gtttgggcaa 720

atcattaacg ccgggcaagg ggaagggcgc tattcggagc tgctggcgat aaatctgctt 780

gagcaattgt tactgcggcg catggaagcg attaacgagt cgctccatcc accgatggat 840

aatcgggtac gcgaggcttg tcagtacatc agcgatcacc tggcagacag caattttgat 900

atcgccagcg tcgcacagca tgtttgcttg tcgccgtcgc gtctgtcaca tcttttccgc 960

cagcagttag ggattagcgt cttaagctgg cgcgaggacc aacgtatcag ccaggcgaag 1020

ctgcttttga gcaccacccg gatgcctatc gccaccgtcg gtcgcaatgt tggttttgac 1080

gatcaactct atttctcgcg ggtatttaaa aaatgcaccg gggccagccc gagcgagttc 1140

cgtgccggtt gtgaagaaaa agtgaatgat gtagccgtca agttgtcata a 1191

<210> 35

<211> 177

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<220>

<223> Nucleotide sequence of the fdT expression terminator

<400> 35

aattttctgt atgaggtttt gctaaacaac tttcaacagt ttcagcggag tgagaataga 60

aaggaacaac taaaggaatt gcgaataata attttttcac gttgaaaatc tccaaaaaaa 120

aaggctccaa aaggagcctt taattgtatc ggtttatcag cttgctttcg aggtgaa 177

<210> 36

<211> 10216

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC01-Ec载体系统

<400> 36

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgatcttc aacaacttca cccagaaatt cagcctgagc 480

aaaaccctgc gttttgaact gcgtccggtt gatgccggtg gtaatgttat taccgatctg 540

accattttcg aagaaaccat caaaaacgat cagaaacgct atgaagcata cctggcaatt 600

aaaccgctgg ttgatgaaac ccataaacac tttattcaga ccgttctgag tggtctgaca 660

gatctgatta aatccgatga gatgaagaac tacctggaac acaaaaatct gatccgtcag 720

aaagatgtgg aagagaaagt taaaaccaaa agcatcgacg tgatcaacaa aatcgaaaaa 780

gattggcgta aacgtgtgag cgatagcttt accaaacatc cgcagtacaa aaagatgttc 840

gataaaaccc tgtttgccga tgaaagtccg ctgtataaac tggccgaaaa tgattttcag 900

cgtagccaga ttaaaatctt cgagaaattt accggctact tcaacggctt tcatgagaat 960

cgtaaaaatc tgtatgtggc ggaaaaacag ggcaccgcaa ttgcaaatcg tgtgattaat 1020

gaaaacctgc cgaaatttat cgagaacgcc aataaactga aacgtgcctt tgaaaaatat 1080

cccgaatttc tgtccaaaat cagcgaggat aaaagctttc aggcactgct gattaaaaac 1140

cagctgagcc tggaaaaact gctgcagccg ctgaccttta atctgctgat ttctcagacc 1200

ggtatcgata gctataatga agtgttaggt ggttacacac cggaaaatag cgaaccgatt 1260

aaaggcctga atcaactgat taatctgtac cgccagaaaa ttaacctggc acgtaacgat 1320

tttccgaatc tggcaccgct gtacaaacag ctgctgagcg atcgtgaaac caatagcgtt 1380

gtgtataaac ctctggaaaa tgtggcagat gtttatagca gtgtgtttga actgtgtcag 1440

aacctgctga gcaaacagag cgatattaat aaatggatcg aggatatcaa catcagcagc 1500

ggtcagattt ggatctataa aagccatctg agcggtctgt cagttatgct gtttggtgaa 1560

agtggttggg gtctgattcc gcgtattctg aatattagcg aagatgatga agaagagatc 1620

atcaaatcca aaagcaaaaa gagccagcaa gagtatttca gctttgcgga aattggtaac 1680

gccatcaaca actatagctt tgaggatgtg aacattaaag cactggcaaa acagggtctg 1740

tgtctgtggc agaaacaagg taatgaacgt ctgatcaaat tcggcaaact gtttagccag 1800

atgcagaatg aactgcagag cccgaaagaa aaatgggata gcaccgaaaa agagaaaatc 1860

aaagaactgc tggatacagg cctggaattt gttcattggc tgaaagttat tagcaaccag 1920

ccggaagata aagatgaagt cttttatgcc gaatggcagg cactgaccga tacctggcgt 1980

ggtctgccga aactgtatga tcgtgttcgt aattttgcca ccaaaaaaga ttacagccag 2040

aacaagctga agatcaactt tgataaaggc accctgctga atggttggga taccaataaa 2100

gaaaccgata atctgggtat cctgctggaa aataaaggcc agtattatct gggcatcatg 2160

aaagatagca gcatctttga ttatcagtgg gacattgaca attttcagaa cccgaatagc 2220

aaacagtcag tggccaaaaa aaacctgcat gaagcaattg ttagcgataa cacccaggat 2280

tgttggagca aaattgtgta caaactgtta ccgggtccga ataaaatgct gccgaaagtg 2340

ttttttagtg acaagcgcca gaaatacttt ggtgcggatg aaaaagtgat cgacattaac 2400

gaaaatggcc gtcataaaaa aggcgacaac tttaacatta gcgattgcca ctatctgatc 2460

gacttctata aaaccgccat caataaacat ccggaatggt cccagttcaa cttcaaattt 2520

agcgcgacca aaagctacga agatatcagc cagttttatc acgaagttca gaatcagggt 2580

tatcgcatcg agtttgatca tatccgcaaa gactatatcc agaaaatggt gagcgaaggt 2640

aaactgttcc tgtttaaaat ccacagcaaa gattttagca gctatgcaaa aggtcgtccg 2700

aatatgcata ccatctattg gcgtgccatt tttaacccgg aaaatctggc caatgttgtg 2760

gttaaactga atggtgaagc cgaattcttt tatcgcaaaa gcagtaaaga tcgcattatc 2820

agccatccgc aaggtctgga agttagcaat aaaaacccga gcaatccgaa aaaaaccagt 2880

cgttttgcat acgacctgat caaagataaa cgtttcacgc aggacaagtt ttttttccat 2940

gttccgatta cgctgaattt tcgtgaaggt gaaggttatc gctttaacca gagcgttatt 3000

cgtgagctga aaaaatacta tcagaccgat aaagccaacc tgcatatcat tggtattgat 3060

cgtggtgaac gtcatctgct gtattattgc gttattaatg ttgccagcgg caaaatcgtt 3120

gaacagggta gcttcaatca gatcagcacc aattatacac cggaacaaat taccgatgat 3180

ggcgaaatca ttaaaggcga aaccgtgaat aaaaccaccg attatcataa cctgcttaac 3240

accaaagaag gtgatcgcca gaaagcacgt aaaaattggc agaccattga aaacatcaaa 3300

gagctgaaag ccggttatct gagcaatgtg attcataaaa tcagccagct gatggtgaaa 3360

tataacgcct ttgttgttct ggaagaactg aaatatggtt ttaaacgcgg tcgcttcaag 3420

gttgagaaac aggtttatca gaaatttgag aaagccctga tcgacaagct taactatctg 3480

gtgtttaaag atcgtgcccc tgccgaagtt ggtggtgttc tgaatgcact gcagctggca 3540

cctccggttg caagctatat tgatattggt aaacaggcag gcttcctgtt ttatgttccg 3600

gcacatcata ccagcaaaat ttgtccgtgg accggttttg tggactggct gaaaccgcgt 3660

tatgatggca ttgataaagc gaaagcattc tttacctgtt tcgaaagcat ccatttcaac 3720

acgcagaaaa actattttga gttcgccttc gactatgaga aatttcgcgg taacattaat 3780

catctgccgg aaggtctgaa acgtaccagc tggaccctgt gtagccataa tagcctgcgt 3840

gatattgcca cgaaagataa aaatggtaac tggccgtata agcagatcaa tctgacagca 3900

gaactgcttg aaattctgaa aaccctgaat ccgcgtaatg gcgaaaacct ggttgaacgt 3960

attatcgaga tgaacgacaa aaagttcttc gagagcctga tgtgggcact gcgtgttctg 4020

ctgcaactgc gttatggtta tatcaaacgt aataacgagg gcatcatcat cgaagaggtt 4080

gattatattc tgagtccggt ggcaaatgaa aacggcgaat tttttgatag ccgcaacttt 4140

gtgaatatcg agaaagccga ttttcccaaa gatgcagatg caaatggtgc atataacatt 4200

gcacgtaaag gtctgctgct gattgcacag aatattaaca acgccaaaat caacgataaa 4260

ggcgaggtta aatgtgacct gcagattgat aaaacaacct ggtttaactg ggttcagagc 4320

aaaagctaat ggtctagagg tcgaaattca aattgtgagc ggataacaat ttgaattttc 4380

tgtatgaggt tttgctaaac aactttcaac agtttcagtg gagtgagaat agaaaggaac 4440

aactaaagga attgcgaata ataatttttt cacgttgaaa atctccaaaa aaaaaggctc 4500

caaaaggagc ctttaattgt atcggtttat cagcttgctt tcgaggtgaa ttttgaccct 4560

ctagcgaaaa tgcaagagca aagacgaaaa catgccacac atgaggaata ccgattctct 4620

cattaacata ttcaggccag ttatctgggc ttaaaagcag aagtccaacc cagataacga 4680

tcatatacat ggttctctcc agaggttcat tactgaacac tcgtccgaga ataacgagtg 4740

gatcccctcc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt 4800

acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 4860

ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 4920

gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta 4980

aatttttgtt aaatcagctc attttttaac caataggccg actgcgatga gtggcagggc 5040

ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggtgc tacgcctgaa 5100

taagtgataa taagcggatg aatggcagaa attcgaaagc aaattcgacc cggtcgtcgg 5160

ttcagggcag ggtcgttaaa tagccgctta tgtctattgc tggtttaccg gtttattgac 5220

taccggaagc agtgtgaccg tgtgcttctc aaatgcctga ggccagtttg ctcaggctct 5280

ccccgtggag gtaataattg acgatatgat catttattct gcctcccaga gcctgataaa 5340

aacggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc 5400

catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgag gcggctacag 5460

ccgatagtct ggaacagcgc acttacgggt tgctgcgcaa cccaagtgct accggcgcgg 5520

cagcgtgacc cgtgtcggcg gctccaacgg ctcgccatcg tccagaaaac acggctcatc 5580

gggcatcggc aggcgctgct gcccgcgccg ttcccattcc tccgtttcgg tcaaggctgg 5640

caggtctggt tccatgcccg gaatgccggg ctggctgggc ggctcctcgc cggggccggt 5700

cggtagttgc tgctcgcccg gatacagggt cgggatgcgg cgcaggtcgc catgccccaa 5760

cagcgattcg tcctggtcgt cgtgatcaac caccacggcg gcactgaaca ccgacaggcg 5820

caactggtcg cggggctggc cccacgccac gcggtcattg accacgtagg ccgacacggt 5880

gccggggccg ttgagcttca cgacggagat ccagcgctcg gccaccaagt ccttgactgc 5940

gtattggacc gtccgcaaag aacgtccgat gagcttggaa agtgtctttt ggctgaccac 6000

cacggcgttc tggtggccca tctgcgccac gaggtgatgc agcagcattg ccgccgtggg 6060

tttcctcgca ataagcccgg cccacgcctc atgcgctttg cgttccgttt gcacccagtg 6120

accgggcttg ttcttggctt gaatgccgat ttctctggac tgcgtggcca tgcttatctc 6180

catgcggtag ggtgccgcac ggttgcggca ccatgcgcaa tcagctgcaa cttttcggca 6240

gcgcgacaac aattatgcgt tgcgtaaaag tggcagtcaa ttacagattt tctttaacct 6300

acgcaatgag ctattgcggg gggtgccgca atgagctgtt gcgtaccccc cttttttaag 6360

ttgttgattt ttaagtcttt cgcatttcgc cctatatcta gttctttggt gcccaaagaa 6420

gggcacccct gcggggttcc cccacgcctt cggcgcggct ccccctccgg caaaaagtgg 6480

cccctccggg gcttgttgat cgactgcgcg gccttcggcc ttgcccaagg tggcgctgcc 6540

cccttggaac ccccgcactc gccgccgtga ggctcggggg gcaggcgggc gggcttcgcc 6600

ttcgactgcc cccactcgca taggcttggg tcgttccagg cgcgtcaagg ccaagccgct 6660

gcgcggtcgc tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg gcaagcgaag 6720

cgcgcaggcc gcaggccgga ggcttttccc cagagaaaat taaaaaaatt gatggggcaa 6780

ggccgcaggc cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg tagccggtgg 6840

gcaatccctg tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg 6900

gttgtccacg ggccgagcga agcgagccag ccggtggccg ctcgcggcca tcgtccacat 6960

atccacgggc tggcaaggga gcgcagcgac cgcgcagggc gaagcccgga gagcaagccc 7020

gtagggcgcc gcagccgccg taggcggtca cgactttgcg aagcaaagtc tagtgagtat 7080

actcaagcat tgagtggccc gccggaggca ccgccttgcg ctgcccccgt cgagccggtt 7140

ggacaccaaa agggaggggc aggcatggcg gcatacgcga tcatgcgatg caagaagctg 7200

gcgaaaatgg gcaacgtggc ggccagtctc aagcacgcct accgcgagcg cgagacgccc 7260

aacgctgacg ccagcaggac gccagagaac gagcactggg cggccagcag caccgatgaa 7320

gcgatgggcc gactgcgcga gttgctgcca gagaagcggc gcaaggacgc tgtgttggcg 7380

gtcgagtacg tcatgacggc cagcccggaa tggtggaagt cggccagcca agaacagcag 7440

gcggcgttct tcgagaaggc gcacaagtgg ctggcggaca agtacggggc ggatcgcatc 7500

gtgacggcca gcatccaccg tgacgaaacc agcccgcaca tgaccgcgtt cgtggtgccg 7560

ctgacgcagg acggcaggct gtcggccaag gagttcatcg gcaacaaagc gcagatgacc 7620

cgcgaccaga ccacgtttgc ggccgctgtg gccgatctag ggctgcaacg gggcatcgag 7680

ggcagcaagg cacgtcacac gcgcattcag gcgttctacg aggccctgga gcggccacca 7740

gtgggccacg tcaccatcag cccgcaagcg gtcgagccac gcgcctatgc accgcaggga 7800

ttggccgaaa agctgggaat ctcaaagcgc gttgagacgc cggaagccgt ggccgaccgg 7860

ctgacaaaag cggttcggca ggggtatgag cctgccctac aggccgccgc aggagcgcgt 7920

gagatgcgca agaaggccga tcaagcccaa gagacggccc gagaccttcg ggagcgcctg 7980

aagcccgttc tggacgccct ggggccgttg aatcgggata tgcaggccaa ggccgccgcg 8040

atcatcaagg ccgtgggcga aaagctgctg acggaacagc gggaagtcca gcgccagaaa 8100

caggcccagc gccagcagga acgcgggcgc gcacatttcc ccgaaaagtg ccacctgaac 8160

cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat 8220

cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt 8280

cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc 8340

cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat 8400

cgccatgggt cacgacgaga tcctcgccgt cgggcatccg cgccttgagc ctggcgaaca 8460

gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 8520

cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 8580

tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 8640

caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 8700

cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca 8760

gccacgatag ccgcgctgcc tcgtcttgga gttcattcag ggcaccggac aggtcggtct 8820

tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 8880

cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 8940

ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag 9000

atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc 9060

agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 9120

ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 9180

ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc 9240

accgtttctg cggactggct ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg 9300

agtgcttgcg gcagcgtgaa gctagctgca taatgtgcct gtcaaatgga cgaagcaggg 9360

attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt taccaattat 9420

gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg 9480

ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca 9540

ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg 9600

atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt 9660

gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg 9720

tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga 9780

tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc 9840

gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg 9900

tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt 9960

gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga 10020

taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc 10080

aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg 10140

cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg 10200

agataaccgt tggcct 10216

<210> 37

<211> 10300

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC03-Ec载体系统

<400> 37

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatggaaaag aacctgaatt acctggaacg cttcaccaaa 480

cattacaaca ccaaaaagac cctgaagaac aaactgattc cgtatggtaa taccgcagag 540

aacatgatca aaaacaacat catcagcaac gagaagcaga ttattctgag cgcaaagaaa 600

cagaaacaga gcattgattt tctgcaaaaa gagtacatcg agaacaagct gagcgaaatt 660

accctgccgt atctgaatga ttattacaac gagttcatta aaaacaagaa agagcgcgat 720

accgacgtca ttgataacat tgaaattgcc atgcgcaagc acattagcaa aagcctgacc 780

gaaaatggca accacaagaa atatctgaac aaagaggtgt ttgatatcat cagcgagaag 840

aaagaactgt attatgacgt gacctttaaa cgcaatgcaa cctatctgag cgattatttt 900

cagagccgtg tgaacctgta taaagacagc aataaaagca gcaccattgc cagccgttgc 960

attaacatta atctgccgat tttcgccaaa aacatcgtgc tgtttaactt catcaagaac 1020

aaggcgaaca tcatcttcga tgacctgaaa gaaattaccg atgatgagta taccctggat 1080

agcattttta gcatcgattt ctttaacatg gtgctgagcc agaaaggcat cgattactat 1140

aataccattt taggtggcat gaccaaagag gatggcaaaa agattaaagg catcaacgag 1200

tacatcaacc tgtacaatca gaacgtgaaa gacgagaaaa acaaactgcc ttatccgaag 1260

aaactgaaga aacagctgct gagcgatatc aatagctata gcgcacgctt tgaaaaattc 1320

gataccgaac aagaaatggt gaaaagcatt aaaagcctgg tggaaaatga cctgtttcag 1380

ggtgaactgt ttgataaaaa ggtggatatc ctgaaagaga cagaacgtct gctggaacgt 1440

attagcgaat atgatagcaa tgccctgttt atcaccgaaa agaacattag ctatatcagc 1500

atcgacatct tcaacgacaa attctttatc aaaaccgcca tcgagtactt ctacgagaat 1560

aacatttgtc cggattaccg caaaatctat gataacgcca gcaaaaacaa gcgtaaacag 1620

ctgggtaaag aaaagaataa ggtgatcaag cagaaaagct ttagcatcag ctttctgcag 1680

gatgccatta ccttctatat caaagatagc ggcatcaaca agatcagcga aaactgcatt 1740

atcaactact tcaagaagca caccatcaaa ctgaccgagc tgtttggtaa agtgtatgag 1800

gattacaacg tgatcaaacc gattctggaa cagcatctgg ttgaatatga aggtaaaagc 1860

atcagcaagg atagcatcaa acgcagcaaa atcaaactgt ttagcgaaaa cctgaaaaac 1920

atcttctatt tcattcgtcc gctgaacatt attgaagagg ccctgaatta tgataccagc 1980

ttttataccc cgttcaacat cctgtttgaa gagatcaaaa agtttaacaa actgtacgat 2040

aaaatccgca actttatcac caagaaaccg ttcaacgatg aggaaattaa cctgtatttt 2100

ggcattccga atttaggcgg tggttttatt gatagccaga ccgataaaag caataacggc 2160

acccagtatt gtacctacct gtttcgtaaa aagaaccaac tgctgaactg ggaatatttt 2220

gtgggcatta gcaagaataa acacctgttc cgcgaaaaag aaaacatcga actgaatagt 2280

gatgaaacga gctttcagcg ttatagtttc tataccccga aagacaaaag catttatggc 2340

agcagctatt ttagcgccaa tgagaaaaac tacaaggacg acaaacaaga gttcatcaac 2400

atcattaaca acatcgtgaa taacagcggt aacgaactgg caattaaaga gctgaagaaa 2460

tatatcaaca acagcaccga aaatagcgaa accccgaatg gttgtctgag cgttctgaaa 2520

aataagtgca acgagattta caacctggtg atcaaccacg atgacttcaa agagaaaaac 2580

gaggacatca tcaataaact taaaaacacc ctgtcgaagc tgagcaaagt tccgcaggca 2640

aaagaactga tcaacaaaaa gtataacctg ttcagcgaga tcattagcga tattagtgaa 2700

atttgtctga ccagcacgca gcgttattat ccgatcgatg atgaagaact gaacagcgca 2760

ctgaacgatg aaaataaacc gctgtacttc tttaaaatca gcaacaaaga tctgagtgcc 2820

gatgaaaaca ttctgaatgg taaacgcaaa agcaaaggca aagataacat ccataccatg 2880

attctgcgtg ccatgatgga tgataatgtg accaacatta ttccgaccag ctgcaaaatt 2940

agcatgcgtg aagcaagcat caaaaaggat gatctggtta tccataaagc caacgaaccg 3000

attaagctga aaaactcact ggccaataag aaagaaagca ccttcagcta tgatatcacc 3060

aaagatcgtc gttatagccg tgatgaattc tttttcagca ttaccgccag cattaacagc 3120

gattgcaaag agaacgatta ttactttaac cagaaagtga acgagtacct gaagaataat 3180

agcaagatta acctgctggc agttgatctg ggtgaaacca atattatcac cattagcgtg 3240

attgatcaga aaggtaacat catactgcag aaggatctgg acaagtttat caacaaagag 3300

aagaatatca ttaccgattt caacctgctg ctgagtaatc gtagcaaaga acgtgatatt 3360

gcaaaacgcg attggcaaga acagcagcag attaagaatc tgaaagaagg tatgattagc 3420

tgcatcatcc acgaaatttg caaactgatg attgaacata acgccatcct gatcatggaa 3480

gatctggatg ccaatttcaa aaatcgcaaa aagcgcatcg agaaagccat ctatcagaaa 3540

tttgaaatcg cgatcctgga aaagctgaat aacctggtgt ttaaagatat cccgattaat 3600

gaagttggca gcgttaccaa accgctgcag ctgagtgata aattcgaaac ctatgaaaaa 3660

gtgggtaacc agagcggctt tgtctttaaa gttagcccgt tttataccag cattattgat 3720

ccgaccaccg gctttataaa cctgtttaag aaaaacttcg aaagcgtgaa atacagcatt 3780

gagttcttca gcaaatttga gagcattcgc tataacacca aagaaaaata cttcgagttc 3840

gccttcgact ataagaattt caaagaaatc aaatacaccg agaacattaa aaccgattgg 3900

gttgcatgta ccaccaacat tgatcgctat gagtatgaca agaagaacaa gatctacaag 3960

aagtacgacg ttaccaccga tctgaaaaac ctgttcgaaa acgaagagat ctattatcag 4020

aagggcgaaa atatcctgga tgtgattctg aagaagaata accgcgaatt ctttgagaaa 4080

ctgacgaacc tgctgaaaat caccatgctg tttcgttatc gtaatagcca tctgaaactg 4140

gactatatta gcagtccggt gaaaaatagc aatggcgagt tctttagcac agaaaatggc 4200

ctggaaaact atccgattga tagcgatacc aatggtgcgt atcatattgc actgaaaggc 4260

aaaatgattc tggatcgcat taatagcaat tcgagcgaaa aactggatac ctacatcagc 4320

attgaagatt ggctgaaatt catccagaag tttagcgtca acaaaatcac ggaaaccaaa 4380

aagaacaaaa agatcaacat caaatatgtg taatggtcta gaggtcgaaa ttcaaattgt 4440

gagcggataa caatttgaat tttctgtatg aggttttgct aaacaacttt caacagtttc 4500

agtggagtga gaatagaaag gaacaactaa aggaattgcg aataataatt ttttcacgtt 4560

gaaaatctcc aaaaaaaaag gctccaaaag gagcctttaa ttgtatcggt ttatcagctt 4620

gctttcgagg tgaattttga ccctctagcg aaaatgcaag agcaaagacg aaaacatgcc 4680

acacatgagg aataccgatt ctctcattaa catattcagg ccagttatct gggcttaaaa 4740

gcagaagtcc aacccagata acgatcatat acatggttct ctccagaggt tcattactga 4800

acactcgtcc gagaataacg agtggatccc ctccaattcg ccctatagtg agtcgtatta 4860

cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca 4920

acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg aagaggcccg 4980

caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggaaat tgtaagcgtt 5040

aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag 5100

gccgactgcg atgagtggca gggcggggcg taattttttt aaggcagtta ttggtgccct 5160

taaacgcctg gtgctacgcc tgaataagtg ataataagcg gatgaatggc agaaattcga 5220

aagcaaattc gacccggtcg tcggttcagg gcagggtcgt taaatagccg cttatgtcta 5280

ttgctggttt accggtttat tgactaccgg aagcagtgtg accgtgtgct tctcaaatgc 5340

ctgaggccag tttgctcagg ctctccccgt ggaggtaata attgacgata tgatcattta 5400

ttctgcctcc cagagcctga taaaaacggt gaatccgtta gcgaggtgcc gccggcttcc 5460

attcaggtcg aggtggcccg gctccatgca ccgcgacgca acgcggggag gcagacaagg 5520

tatagggcgg cgaggcggct acagccgata gtctggaaca gcgcacttac gggttgctgc 5580

gcaacccaag tgctaccggc gcggcagcgt gacccgtgtc ggcggctcca acggctcgcc 5640

atcgtccaga aaacacggct catcgggcat cggcaggcgc tgctgcccgc gccgttccca 5700

ttcctccgtt tcggtcaagg ctggcaggtc tggttccatg cccggaatgc cgggctggct 5760

gggcggctcc tcgccggggc cggtcggtag ttgctgctcg cccggataca gggtcgggat 5820

gcggcgcagg tcgccatgcc ccaacagcga ttcgtcctgg tcgtcgtgat caaccaccac 5880

ggcggcactg aacaccgaca ggcgcaactg gtcgcggggc tggccccacg ccacgcggtc 5940

attgaccacg taggccgaca cggtgccggg gccgttgagc ttcacgacgg agatccagcg 6000

ctcggccacc aagtccttga ctgcgtattg gaccgtccgc aaagaacgtc cgatgagctt 6060

ggaaagtgtc ttttggctga ccaccacggc gttctggtgg cccatctgcg ccacgaggtg 6120

atgcagcagc attgccgccg tgggtttcct cgcaataagc ccggcccacg cctcatgcgc 6180

tttgcgttcc gtttgcaccc agtgaccggg cttgttcttg gcttgaatgc cgatttctct 6240

ggactgcgtg gccatgctta tctccatgcg gtagggtgcc gcacggttgc ggcaccatgc 6300

gcaatcagct gcaacttttc ggcagcgcga caacaattat gcgttgcgta aaagtggcag 6360

tcaattacag attttcttta acctacgcaa tgagctattg cggggggtgc cgcaatgagc 6420

tgttgcgtac cccccttttt taagttgttg atttttaagt ctttcgcatt tcgccctata 6480

tctagttctt tggtgcccaa agaagggcac ccctgcgggg ttcccccacg ccttcggcgc 6540

ggctccccct ccggcaaaaa gtggcccctc cggggcttgt tgatcgactg cgcggccttc 6600

ggccttgccc aaggtggcgc tgcccccttg gaacccccgc actcgccgcc gtgaggctcg 6660

gggggcaggc gggcgggctt cgccttcgac tgcccccact cgcataggct tgggtcgttc 6720

caggcgcgtc aaggccaagc cgctgcgcgg tcgctgcgcg agccttgacc cgccttccac 6780

ttggtgtcca accggcaagc gaagcgcgca ggccgcaggc cggaggcttt tccccagaga 6840

aaattaaaaa aattgatggg gcaaggccgc aggccgcgca gttggagccg gtgggtatgt 6900

ggtcgaaggc tgggtagccg gtgggcaatc cctgtggtca agctcgtggg caggcgcagc 6960

ctgtccatca gcttgtccag cagggttgtc cacgggccga gcgaagcgag ccagccggtg 7020

gccgctcgcg gccatcgtcc acatatccac gggctggcaa gggagcgcag cgaccgcgca 7080

gggcgaagcc cggagagcaa gcccgtaggg cgccgcagcc gccgtaggcg gtcacgactt 7140

tgcgaagcaa agtctagtga gtatactcaa gcattgagtg gcccgccgga ggcaccgcct 7200

tgcgctgccc ccgtcgagcc ggttggacac caaaagggag gggcaggcat ggcggcatac 7260

gcgatcatgc gatgcaagaa gctggcgaaa atgggcaacg tggcggccag tctcaagcac 7320

gcctaccgcg agcgcgagac gcccaacgct gacgccagca ggacgccaga gaacgagcac 7380

tgggcggcca gcagcaccga tgaagcgatg ggccgactgc gcgagttgct gccagagaag 7440

cggcgcaagg acgctgtgtt ggcggtcgag tacgtcatga cggccagccc ggaatggtgg 7500

aagtcggcca gccaagaaca gcaggcggcg ttcttcgaga aggcgcacaa gtggctggcg 7560

gacaagtacg gggcggatcg catcgtgacg gccagcatcc accgtgacga aaccagcccg 7620

cacatgaccg cgttcgtggt gccgctgacg caggacggca ggctgtcggc caaggagttc 7680

atcggcaaca aagcgcagat gacccgcgac cagaccacgt ttgcggccgc tgtggccgat 7740

ctagggctgc aacggggcat cgagggcagc aaggcacgtc acacgcgcat tcaggcgttc 7800

tacgaggccc tggagcggcc accagtgggc cacgtcacca tcagcccgca agcggtcgag 7860

ccacgcgcct atgcaccgca gggattggcc gaaaagctgg gaatctcaaa gcgcgttgag 7920

acgccggaag ccgtggccga ccggctgaca aaagcggttc ggcaggggta tgagcctgcc 7980

ctacaggccg ccgcaggagc gcgtgagatg cgcaagaagg ccgatcaagc ccaagagacg 8040

gcccgagacc ttcgggagcg cctgaagccc gttctggacg ccctggggcc gttgaatcgg 8100

gatatgcagg ccaaggccgc cgcgatcatc aaggccgtgg gcgaaaagct gctgacggaa 8160

cagcgggaag tccagcgcca gaaacaggcc cagcgccagc aggaacgcgg gcgcgcacat 8220

ttccccgaaa agtgccacct gaaccccaga gtcccgctca gaagaactcg tcaagaaggc 8280

gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg aggaagcggt 8340

cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct atgtcctgat 8400

agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg ccattttcca 8460

ccatgatatt cggcaagcag gcatcgccat gggtcacgac gagatcctcg ccgtcgggca 8520

tccgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc tcttcgtcca 8580

gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg atgcgatgtt 8640

tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc cgcattgcat 8700

cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga tcctgccccg 8760

gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg agcacagctg 8820

cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct tggagttcat 8880

tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc gctgacagcc 8940

ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag ccgaatagcc 9000

tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc atgcgaaacg 9060

atcctcatcc tgtctcttga tcagatcttg atcccctgcg ccatcagatc cttggcggca 9120

agaaagccat ccagtttact ttgcagggct tcccaacctt accagagggc gccccagctg 9180

gcaattccgg ttcgcttgct gtccataaaa ccgcccagtc tagctatcgc catgtaagcc 9240

cactgcaagc tacctgcttt ctctttgcgc ttgcgttttc ccttgtccag atagcccagt 9300

agctgacatt catccggggt cagcaccgtt tctgcggact ggctttctac gtgttccgct 9360

tcctttagca gcccttgcgc cctgagtgct tgcggcagcg tgaagctagc tgcataatgt 9420

gcctgtcaaa tggacgaagc agggattctg caaaccctat gctactccgt caagccgtca 9480

attgtctgat tcgttaccaa ttatgacaac ttgacggcta catcattcac tttttcttca 9540

caaccggcac ggaactcgct cgggctggcc ccggtgcatt ttttaaatac ccgcgagaaa 9600

tagagttgat cgtcaaaacc aacattgcga ccgacggtgg cgataggcat ccgggtggtg 9660

ctcaaaagca gcttcgcctg gctgatacgt tggtcctcgc gccagcttaa gacgctaatc 9720

cctaactgct ggcggaaaag atgtgacaga cgcgacggcg acaagcaaac atgctgtgcg 9780

acgctggcga tatcaaaatt gctgtctgcc aggtgatcgc tgatgtactg acaagcctcg 9840

cgtacccgat tatccatcgg tggatggagc gactcgttaa tcgcttccat gcgccgcagt 9900

aacaattgct caagcagatt tatcgccagc agctccgaat agcgcccttc cccttgcccg 9960

gcgttaatga tttgcccaaa caggtcgctg aaatgcggct ggtgcgcttc atccgggcga 10020

aagaaccccg tattggcaaa tattgacggc cagttaagcc attcatgcca gtaggcgcgc 10080

ggacgaaagt aaacccactg gtgataccat tcgcgagcct ccggatgacg accgtagtga 10140

tgaatctctc ctggcgggaa cagcaaaata tcacccggtc ggcaaacaaa ttctcgtccc 10200

tgatttttca ccaccccctg accgcgaatg gtgagattga gaatataacc tttcattccc 10260

agcggtcggt cgataaaaaa atcgagataa ccgttggcct 10300

<210> 38

<211> 10303

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC04-Ec载体系统

<400> 38

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgaaaaac ctgaccgaat tcaccggtct gtatccggtt 480

agcaaaaccc tgcgttttga actgaaaccg accgatgatt ttaactggga aacctttctg 540

gaaagcacca tctttaaaca tgatcaagaa cgtgcagaag cctatccgat tgttaaagtt 600

attgtggacc agttccacaa gtggtttatt gaagatgcac tgaacaaaag cacgatcaat 660

tggaatagcc tgtatgatgc atatttcgca ccgaaaaatg aaaacagcgt tgaaaatctg 720

cgcaaagagc aggataaaat ccgcaaagaa attgtggata cctacttcaa gaaacacgac 780

tggtggaaat atgtgagcaa agatcatagc aagctgttca aaattgaact gcctgcactg 840

ctgagtgatg atgcatttat ctatgagatc aacgacaagt atccgaacta tacccaagaa 900

attctgattg atgcactggc caaatttcag aactttagcg tttatttcgg tggctatttc 960

aaaaaccgcg acaacatgta taaaagtgat gcacagagca ccagcattgc caatcgtatt 1020

gttaatgaga acttcaccaa attcgccgac aacatcaaaa tctataaccg cctgaaagaa 1080

aactgtctga gcgaactgca gaaagtcgaa ctggatttta ccgatgaact gaccggtctg 1140

acctttgatg atatctttag cccgagctac ttcaacaaat gtctgaccca gaaaggcatc 1200

gaaaaactga atctgtatat tggtggtaag accggcaaaa acaaagagga taaagttttt 1260

ggcattaacc gcgtgggtaa tgaattcctg cagtttaaca aagagagcaa gctgaaactg 1320

aaagacctga aaatggtgaa actgtacaag cagattctga gcgatcgtga acagccgagc 1380

tttctgccgg aacagtttcg taatgaagat gagctgatta aaagcatcga ggattttcat 1440

aacctgatca ccgaacagaa actgtttgaa cgtctgctga aattgatggg tcgtctgaaa 1500

aatggcgaat gtgaagatct gaataaaatc catgttgtgg gtagctcact gacccagctg 1560

agcaaagttc tgtatggtaa ttgggaagtt ctgggcaccg cactgcgcaa taaattccag 1620

accaataaaa ccaaaaagga caagctggaa agcgagaaag atatccaaga atggatggaa 1680

cgtaaaagct ttagcctggc acagattatt gaagttgaaa gcagcctgca ggatgacaaa 1740

agcattaaag tgattgacct gttcaccacc tttaatgcat ggcagaaagt taacgaaaaa 1800

ccgcagctgg ttgatctgat taaactgtgc aaagatgatt ttcagacccg ttttcgtgca 1860

gtgaaagatc tgattgaaaa aggtgagcag attcagggca atgaaagcgc aaaagaagaa 1920

attaaagccg tcctggataa ctatcagaac ctgctgcatg ttgtgaaact gctgaatctg 1980

ggtaagaaag aaagctatct ggataaggat gaaaccttct ataacgagta caaagaaatc 2040

ctgagcagca ccgaaagcga taatgtttgt ctggaagata ttatcccgct gtataacaaa 2100

gttcgtagct ttctgacccg taaactgggt gatgaaggta aaatgctgct gaagtttgat 2160

tgtagcaccc tggcagatgg ttgggatgtt ggtaaagaaa gcgccaataa tagcaccatt 2220

ctgatcgata acagcaaata ctatctgatc atcacgaacc cggaaaataa accggatctg 2280

agcaccgcaa ttaccagcaa taccgataat gtgtacaaaa agattgtgta tcgccagatt 2340

gcagatccga ccaaagatct gccgaatctg atggttattg atggtaaaac ccagcgcaaa 2400

accggcaata aagatgatga tggtattaat cgtgttctgg accagctgaa agataaatat 2460

ctgccgcaag aagtgaatcg cattcgcaaa ctgggtagct atctgaaaac cagcgaacat 2520

ttcaacaaaa aggatagcca ggtttatctg gcctattata tgcagcgcct gatcgaatat 2580

aaacagggcg aaatggaatt cagctttaag aacagcgaag agtatgatag ctatagcgat 2640

ttcctggatg atatcaccaa gcagaaatat tcactgagct ttgtgaacgt gtcgaaagaa 2700

attatcacac agtggattag cgagggcaaa attttcctgt ttcagatcta caacaaggac 2760

ttcgaagaaa aagcaaccgg tacaccgaat ctgcataccc tgtattggaa agaactgttt 2820

agcgaagaga acctgaaaga catcgtgtat aaactgaatg gtgaagcgga actgttctac 2880

cgtaaaaaga tggatggtaa accgttcacg cataacaaag gtgcagttct ggtgaataaa 2940

acctttgcag atggtagtcc ggtggaaccg gaacattata aagaatatgt ggaatacatc 3000

accggcaagg tgattgagaa acagctgagt aaagaagcca aagacaaact gcatctggtg 3060

aaaaccaata aagcgaaact ggacatcatc aaggacaaac gctattttca gcacaaactg 3120

ctgtttcatg ttccgatcac catcaacttt aaaagcgaag gtgttccgaa attcaacgat 3180

tacaccctga attatctgcg cgagaacaag aaagacatta acatcattgg tatcgatcgc 3240

ggtgaacgca atctgattta tgttagcgtt attaatcaga aaggcgagaa cattatccct 3300

ccgaaacatt tcaatatcgt ggaaagtgat atgttcggca tggaagataa acgcaaattc 3360

aactacctgg aaaagctgat tcagaaagaa ggtaatcgcg acgacgcacg taaaaattgg 3420

agtaaaattg aaacgatcaa agaccttaaa accggctatc tgagcctggt tgttcatgaa 3480

attgcaaaac tggttgttga acatcatgcc attgtggtgc tggaagatct taactatggt 3540

tttaaacgcg gtcgctttaa tgtggaacgt cagatttacc agaattttga gaaaatgctg 3600

atcgagaagc tgaacctgct tgtgtttaaa aacaatagca acagtccgga ttatggcaat 3660

attctgaatg gtctgcagct gaccgcaccg tttggtagct ttaaagaact gggtaaacag 3720

agcggttggc tgttttatgt taatgcaagc tataccagca aaatcgatcc gcagaccggc 3780

tttgcaaacc tgtttaatat gaaagacgcc aagaaggata ccaagagctt tttcgagaaa 3840

atcaccgaga tcaaatatga cgacggcatg ttcaaattca ccttcgatta tcgtaacggc 3900

tttagcattg ttcagaccga ctataaaaac atttggaccg tttgcaccaa cgacaaacgt 3960

attctggttt ccaaagataa catctcgggc aaatttaagc acgagtatgt ggatattacc 4020

gaaagcatca aaaacctgtt catcaacaac aacattaacg actatcatag catcagcaaa 4080

gaaaccatcc tgtccatcaa agaaaagaag ttctttgacg acctgttctt ttacttcaaa 4140

ctgagcctgc agatgcgtaa tagcattccg aatagcgata tcgattatct gatttcaccg 4200

gtgcagatta aaggcaaacc gttctttgat agccgtattc cgaacaatat taacatcgtt 4260

gatgcagatg caaatggtgc ctatcatatt gcactgaaag gcctgtatct ggtgattaat 4320

gattttccca cagagaaaaa gggcaaaagc gagtacctga aaaagatcac caatgaagat 4380

tggtttgaat tcgcacagcg tcgtagcctg aaataatggt ctagaggtcg aaattcaaat 4440

tgtgagcgga taacaatttg aattttctgt atgaggtttt gctaaacaac tttcaacagt 4500

ttcagtggag tgagaataga aaggaacaac taaaggaatt gcgaataata attttttcac 4560

gttgaaaatc tccaaaaaaa aaggctccaa aaggagcctt taattgtatc ggtttatcag 4620

cttgctttcg aggtgaattt tgaccctcta gcgaaaatgc aagagcaaag acgaaaacat 4680

gccacacatg aggaataccg attctctcat taacatattc aggccagtta tctgggctta 4740

aaagcagaag tccaacccag ataacgatca tatacatggt tctctccaga ggttcattac 4800

tgaacactcg tccgagaata acgagtggat cccctccaat tcgccctata gtgagtcgta 4860

ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 4920

ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 4980

ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga aattgtaagc 5040

gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa 5100

taggccgact gcgatgagtg gcagggcggg gcgtaatttt tttaaggcag ttattggtgc 5160

ccttaaacgc ctggtgctac gcctgaataa gtgataataa gcggatgaat ggcagaaatt 5220

cgaaagcaaa ttcgacccgg tcgtcggttc agggcagggt cgttaaatag ccgcttatgt 5280

ctattgctgg tttaccggtt tattgactac cggaagcagt gtgaccgtgt gcttctcaaa 5340

tgcctgaggc cagtttgctc aggctctccc cgtggaggta ataattgacg atatgatcat 5400

ttattctgcc tcccagagcc tgataaaaac ggtgaatccg ttagcgaggt gccgccggct 5460

tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg gaggcagaca 5520

aggtataggg cggcgaggcg gctacagccg atagtctgga acagcgcact tacgggttgc 5580

tgcgcaaccc aagtgctacc ggcgcggcag cgtgacccgt gtcggcggct ccaacggctc 5640

gccatcgtcc agaaaacacg gctcatcggg catcggcagg cgctgctgcc cgcgccgttc 5700

ccattcctcc gtttcggtca aggctggcag gtctggttcc atgcccggaa tgccgggctg 5760

gctgggcggc tcctcgccgg ggccggtcgg tagttgctgc tcgcccggat acagggtcgg 5820

gatgcggcgc aggtcgccat gccccaacag cgattcgtcc tggtcgtcgt gatcaaccac 5880

cacggcggca ctgaacaccg acaggcgcaa ctggtcgcgg ggctggcccc acgccacgcg 5940

gtcattgacc acgtaggccg acacggtgcc ggggccgttg agcttcacga cggagatcca 6000

gcgctcggcc accaagtcct tgactgcgta ttggaccgtc cgcaaagaac gtccgatgag 6060

cttggaaagt gtcttttggc tgaccaccac ggcgttctgg tggcccatct gcgccacgag 6120

gtgatgcagc agcattgccg ccgtgggttt cctcgcaata agcccggccc acgcctcatg 6180

cgctttgcgt tccgtttgca cccagtgacc gggcttgttc ttggcttgaa tgccgatttc 6240

tctggactgc gtggccatgc ttatctccat gcggtagggt gccgcacggt tgcggcacca 6300

tgcgcaatca gctgcaactt ttcggcagcg cgacaacaat tatgcgttgc gtaaaagtgg 6360

cagtcaatta cagattttct ttaacctacg caatgagcta ttgcgggggg tgccgcaatg 6420

agctgttgcg tacccccctt ttttaagttg ttgattttta agtctttcgc atttcgccct 6480

atatctagtt ctttggtgcc caaagaaggg cacccctgcg gggttccccc acgccttcgg 6540

cgcggctccc cctccggcaa aaagtggccc ctccggggct tgttgatcga ctgcgcggcc 6600

ttcggccttg cccaaggtgg cgctgccccc ttggaacccc cgcactcgcc gccgtgaggc 6660

tcggggggca ggcgggcggg cttcgccttc gactgccccc actcgcatag gcttgggtcg 6720

ttccaggcgc gtcaaggcca agccgctgcg cggtcgctgc gcgagccttg acccgccttc 6780

cacttggtgt ccaaccggca agcgaagcgc gcaggccgca ggccggaggc ttttccccag 6840

agaaaattaa aaaaattgat ggggcaaggc cgcaggccgc gcagttggag ccggtgggta 6900

tgtggtcgaa ggctgggtag ccggtgggca atccctgtgg tcaagctcgt gggcaggcgc 6960

agcctgtcca tcagcttgtc cagcagggtt gtccacgggc cgagcgaagc gagccagccg 7020

gtggccgctc gcggccatcg tccacatatc cacgggctgg caagggagcg cagcgaccgc 7080

gcagggcgaa gcccggagag caagcccgta gggcgccgca gccgccgtag gcggtcacga 7140

ctttgcgaag caaagtctag tgagtatact caagcattga gtggcccgcc ggaggcaccg 7200

ccttgcgctg cccccgtcga gccggttgga caccaaaagg gaggggcagg catggcggca 7260

tacgcgatca tgcgatgcaa gaagctggcg aaaatgggca acgtggcggc cagtctcaag 7320

cacgcctacc gcgagcgcga gacgcccaac gctgacgcca gcaggacgcc agagaacgag 7380

cactgggcgg ccagcagcac cgatgaagcg atgggccgac tgcgcgagtt gctgccagag 7440

aagcggcgca aggacgctgt gttggcggtc gagtacgtca tgacggccag cccggaatgg 7500

tggaagtcgg ccagccaaga acagcaggcg gcgttcttcg agaaggcgca caagtggctg 7560

gcggacaagt acggggcgga tcgcatcgtg acggccagca tccaccgtga cgaaaccagc 7620

ccgcacatga ccgcgttcgt ggtgccgctg acgcaggacg gcaggctgtc ggccaaggag 7680

ttcatcggca acaaagcgca gatgacccgc gaccagacca cgtttgcggc cgctgtggcc 7740

gatctagggc tgcaacgggg catcgagggc agcaaggcac gtcacacgcg cattcaggcg 7800

ttctacgagg ccctggagcg gccaccagtg ggccacgtca ccatcagccc gcaagcggtc 7860

gagccacgcg cctatgcacc gcagggattg gccgaaaagc tgggaatctc aaagcgcgtt 7920

gagacgccgg aagccgtggc cgaccggctg acaaaagcgg ttcggcaggg gtatgagcct 7980

gccctacagg ccgccgcagg agcgcgtgag atgcgcaaga aggccgatca agcccaagag 8040

acggcccgag accttcggga gcgcctgaag cccgttctgg acgccctggg gccgttgaat 8100

cgggatatgc aggccaaggc cgccgcgatc atcaaggccg tgggcgaaaa gctgctgacg 8160

gaacagcggg aagtccagcg ccagaaacag gcccagcgcc agcaggaacg cgggcgcgca 8220

catttccccg aaaagtgcca cctgaacccc agagtcccgc tcagaagaac tcgtcaagaa 8280

ggcgatagaa ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc 8340

ggtcagccca ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct 8400

gatagcggtc cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt 8460

ccaccatgat attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg 8520

gcatccgcgc cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt 8580

ccagatcatc ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat 8640

gtttcgcttg gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg 8700

catcagccat gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc 8760

ccggcacttc gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag 8820

ctgcgcaagg aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttggagtt 8880

cattcagggc accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca 8940

gccggaacac ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata 9000

gcctctccac ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa 9060

acgatcctca tcctgtctct tgatcagatc ttgatcccct gcgccatcag atccttggcg 9120

gcaagaaagc catccagttt actttgcagg gcttcccaac cttaccagag ggcgccccag 9180

ctggcaattc cggttcgctt gctgtccata aaaccgccca gtctagctat cgccatgtaa 9240

gcccactgca agctacctgc tttctctttg cgcttgcgtt ttcccttgtc cagatagccc 9300

agtagctgac attcatccgg ggtcagcacc gtttctgcgg actggctttc tacgtgttcc 9360

gcttccttta gcagcccttg cgccctgagt gcttgcggca gcgtgaagct agctgcataa 9420

tgtgcctgtc aaatggacga agcagggatt ctgcaaaccc tatgctactc cgtcaagccg 9480

tcaattgtct gattcgttac caattatgac aacttgacgg ctacatcatt cactttttct 9540

tcacaaccgg cacggaactc gctcgggctg gccccggtgc attttttaaa tacccgcgag 9600

aaatagagtt gatcgtcaaa accaacattg cgaccgacgg tggcgatagg catccgggtg 9660

gtgctcaaaa gcagcttcgc ctggctgata cgttggtcct cgcgccagct taagacgcta 9720

atccctaact gctggcggaa aagatgtgac agacgcgacg gcgacaagca aacatgctgt 9780

gcgacgctgg cgatatcaaa attgctgtct gccaggtgat cgctgatgta ctgacaagcc 9840

tcgcgtaccc gattatccat cggtggatgg agcgactcgt taatcgcttc catgcgccgc 9900

agtaacaatt gctcaagcag atttatcgcc agcagctccg aatagcgccc ttccccttgc 9960

ccggcgttaa tgatttgccc aaacaggtcg ctgaaatgcg gctggtgcgc ttcatccggg 10020

cgaaagaacc ccgtattggc aaatattgac ggccagttaa gccattcatg ccagtaggcg 10080

cgcggacgaa agtaaaccca ctggtgatac cattcgcgag cctccggatg acgaccgtag 10140

tgatgaatct ctcctggcgg gaacagcaaa atatcacccg gtcggcaaac aaattctcgt 10200

ccctgatttt tcaccacccc ctgaccgcga atggtgagat tgagaatata acctttcatt 10260

cccagcggtc ggtcgataaa aaaatcgaga taaccgttgg cct 10303

<210> 39

<211> 10153

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC09-Ec载体系统

<400> 39

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgaagaac agcctggaag atttcaccaa tctgtatagc 480

ctgcagaaaa ccctgcgttt tgaactgaaa ccgattggta atacccagag catgctggaa 540

gaagatggcg tttttgatac cgatgaaaaa cgcaaaatcg cctacagcaa aaccaaaccg 600

tatattgatc gtctgcaccg cgaatttatt gaagaaagtc tgtcagatgc ccagatcagc 660

aaactggatg aatatttcaa agcctacgtg gactacaaaa aggacaagaa agataccaaa 720

cgcttcaacc gcatcaaaca gtttaaaagc gttctgcgta aagaagtggt ggatcacttt 780

aacaaacagg gtaaagaatg gaccaccgtg aaatttgcac atctgaaaat caaaaagaaa 840

gatctggaag tgctgttcga aaaacagctg ccgaatattc tgaaagaaga atacggcacc 900

gaaaaagaaa cccagattat tgatgaagat agcggtgaag tgaccagcat ttttgatatg 960

tggaatggct ttatgggcta tttcacgaaa tttttcgaaa cccgcaagaa cttctataaa 1020

agtgatggca ccagcaccgc aattgcaacc cgcattattg atcagaatct ggatcgcttt 1080

atcgagaaca tcctgatcta tgatagcatc aaaccgaaaa tcgataccag cgaagttcgc 1140

gaatttttca atctggaaag cgataccatc tttagcatgg aattctataa taactgtctg 1200

ctgcaggcag gcattgatca gtataacaat tttcttggtg gcaaaaccct ggaaaacggt 1260

cgtaaaattc gtggcattaa cgagctgatt aacaaatatc gtcaagagaa cccggaagat 1320

aaaatcccgt ttctgaaaaa gctggataag cagatccata gcgagaaaga gaaatttatc 1380

cagcagatcg aaacactgga agatctgaaa gaggaactgc agaaatttta caatagcagc 1440

aacgagaaga tcaaaattct ggataatctg ctgagccgca tcgaagaatt taaaccggaa 1500

ggtatcttca ttagcaaaca ggcctttaat accattagcc gtcgttggac cgatcagagc 1560

gaagcatttg aaaccagcct gtttgaaagc cttaaagaag aaaaaccgat taccggcacc 1620

gcaaagaaaa aggatgatgg ttataacttc ccggaattca ttagtctgca gagcattcgt 1680

aataccctga aaaaggttca gggtgaagaa cgtttttgga aagaacgtta ttatcgcgac 1740

aatagcgaaa gcggtattct ggcaggtaat gaagaaattt ggacccagtt tctgatgatc 1800

ttcaaaagcg aattcaacag caaattcgaa cgcaatgatc ctgaagataa tggcaccatt 1860

ggctacaacc tgtttaaaga ggatctggaa aaactgctga aagacctgaa aattaccaaa 1920

gacaccaaga gcatcattaa acgctttgca gatgaagccc tgcatattta tcaggtgggt 1980

aaatactttg cgctggaaaa agatcgtgtt tggattagca gctatgatga tctgcttgat 2040

accttttata ccgatccgaa taccggttat ctgagctttt atgaaggtgc ctatgagcag 2100

attgttcagc cgtataatat gatccgtaat tatctgaccc gtaaaccgta cagtgatgag 2160

aaatggaaac tgaactttga aaatccgaca ctggcaaatg gttgggacaa aaacaaagaa 2220

accgataaca gcagtatcat gctgcgcaaa gaaggtgcat attatctggg tatcatgaag 2280

aaaggcaaga acaaactgtt cgaagaacgt aatcgtcagc tgtttgaacc gaaaaatggt 2340

gaggatacgt atgagaaact gagctataaa ctgttcccgg atcctgcaaa aatgattccg 2400

aaagtttgct tcagcaacaa aaacatccag atgtttagcc cgagcaccga aatcatgaat 2460

atctataatg gcgaaacctt caagaaaaac tccgatgatt ttagcgttag cagcatgcag 2520

aaactgattg ccttctatac caaatgtctg agccagtatg aaggctggaa atactatgac 2580

ttcaaatata tcaaaagtcc ggatcagtac aaggataaca tcggcgagtt ttataacgat 2640

gttgccaaaa gcggttatcg tgtgtggttt gaaaacatta gccagagcta tgtggatagc 2700

aaaaatacca tgggtgaact gtacctgttc aagatccata acaaagactg gaaccagaaa 2760

gataaaaaga ccaaagtggg cagcaaaaac ctgcataccc attattttga agaactgttc 2820

agccaggaca acatcgaaaa taactttccg ctgaaactga atggtgaagc cgaagtgttt 2880

tatcgtccga aaaccaatcc tgaaaaactg ggcaccaaaa aggatagtaa aggtcgtgaa 2940

gtgattgatc gtaaacgtta tgcaagcgat aaggtgctgt ttcatgttcc gattacactg 3000

aatcgtacac cggttaccac caccaaactg aataaagaaa ttaatggctt cctggccaac 3060

aatccgagca ttaacattat tggtgttgat cgcggtgaaa aacacctggt gtattattca 3120

gtggtgaatc agcgtggtaa gatgctggaa agtggtagct ttaacacaat taacggcgtg 3180

gattatcacg gcaaattgga agaacgtgca gatcgtcgtg aacaggcacg tcgtgattgg 3240

caggatgttg aaggcattaa aaacctgaag aagggctata ttagcctggt tgttcgtgaa 3300

ctggccaatc tgagcatcaa atataacgcc attatcgtga tggaagatct taacatgcgc 3360

tttaagcaga ttcgtggtgg tatcgaaaaa tcagcatatc agcagttaga aaaagccctg 3420

atcgaaaagc tgaattacct ggtgaataaa accgaaaccg atccgcagaa aacaggtcat 3480

atcctgaaag cctatcagct gaccagtccg attaaaagct ttaaagagat gggtaaacag 3540

accggcatca ttttctatac ccaggcaagc tataccagcg ttacagatcc gatcaccggt 3600

tggcgtccga atctgtatct gaaatatagc agcgcaagca aagcgaaaag cgacattctg 3660

aaattcagca agattagcta caacaccaac aacaaccgct ttgagtttac ctatgatctg 3720

cgcaattttg tgaacatgaa ggcctatcct cagaaaaccg catggaccat ttgtagcaat 3780

gttgaacgtt ttcgttggga tcgcaaaggc aataagaata acggtgagta catccagtat 3840

aaggatctga ccgaaaactt taagaccttc ttcgaggaag tcagcattaa ctataaaggc 3900

gatattctgt tccagatcaa gaacctgagc gaaaaaggta acgaaaaatt ctttcgcgac 3960

ctgatctttt acattagcct gattagtcag attcgcaaca cccagaaaga caagaagggt 4020

gacgaaaacg attttattct gagtccggtt gaaccgttct ttgatagccg taaaagcagc 4080

acctttggtg aaaatctgcc gctgaacggt gatgcaaatg gtgcatacaa tattgcacgc 4140

aagggcatta ttatgctgaa caaaatttca aaaggcagca agaataaagt caaagaggat 4200

attggttggg gagatctgta tattccgcat accgaatggg atgattttgc aaccggtagc 4260

atttaatggt ctagaggtcg aaattcaaat tgtgagcgga taacaatttg aattttctgt 4320

atgaggtttt gctaaacaac tttcaacagt ttcagtggag tgagaataga aaggaacaac 4380

taaaggaatt gcgaataata attttttcac gttgaaaatc tccaaaaaaa aaggctccaa 4440

aaggagcctt taattgtatc ggtttatcag cttgctttcg aggtgaattt tgaccctcta 4500

gcgaaaatgc aagagcaaag acgaaaacat gccacacatg aggaataccg attctctcat 4560

taacatattc aggccagtta tctgggctta aaagcagaag tccaacccag ataacgatca 4620

tatacatggt tctctccaga ggttcattac tgaacactcg tccgagaata acgagtggat 4680

cccctccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca 4740

acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc 4800

tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg 4860

cagcctgaat ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat 4920

ttttgttaaa tcagctcatt ttttaaccaa taggccgact gcgatgagtg gcagggcggg 4980

gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggtgctac gcctgaataa 5040

gtgataataa gcggatgaat ggcagaaatt cgaaagcaaa ttcgacccgg tcgtcggttc 5100

agggcagggt cgttaaatag ccgcttatgt ctattgctgg tttaccggtt tattgactac 5160

cggaagcagt gtgaccgtgt gcttctcaaa tgcctgaggc cagtttgctc aggctctccc 5220

cgtggaggta ataattgacg atatgatcat ttattctgcc tcccagagcc tgataaaaac 5280

ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat 5340

gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgaggcg gctacagccg 5400

atagtctgga acagcgcact tacgggttgc tgcgcaaccc aagtgctacc ggcgcggcag 5460

cgtgacccgt gtcggcggct ccaacggctc gccatcgtcc agaaaacacg gctcatcggg 5520

catcggcagg cgctgctgcc cgcgccgttc ccattcctcc gtttcggtca aggctggcag 5580

gtctggttcc atgcccggaa tgccgggctg gctgggcggc tcctcgccgg ggccggtcgg 5640

tagttgctgc tcgcccggat acagggtcgg gatgcggcgc aggtcgccat gccccaacag 5700

cgattcgtcc tggtcgtcgt gatcaaccac cacggcggca ctgaacaccg acaggcgcaa 5760

ctggtcgcgg ggctggcccc acgccacgcg gtcattgacc acgtaggccg acacggtgcc 5820

ggggccgttg agcttcacga cggagatcca gcgctcggcc accaagtcct tgactgcgta 5880

ttggaccgtc cgcaaagaac gtccgatgag cttggaaagt gtcttttggc tgaccaccac 5940

ggcgttctgg tggcccatct gcgccacgag gtgatgcagc agcattgccg ccgtgggttt 6000

cctcgcaata agcccggccc acgcctcatg cgctttgcgt tccgtttgca cccagtgacc 6060

gggcttgttc ttggcttgaa tgccgatttc tctggactgc gtggccatgc ttatctccat 6120

gcggtagggt gccgcacggt tgcggcacca tgcgcaatca gctgcaactt ttcggcagcg 6180

cgacaacaat tatgcgttgc gtaaaagtgg cagtcaatta cagattttct ttaacctacg 6240

caatgagcta ttgcgggggg tgccgcaatg agctgttgcg tacccccctt ttttaagttg 6300

ttgattttta agtctttcgc atttcgccct atatctagtt ctttggtgcc caaagaaggg 6360

cacccctgcg gggttccccc acgccttcgg cgcggctccc cctccggcaa aaagtggccc 6420

ctccggggct tgttgatcga ctgcgcggcc ttcggccttg cccaaggtgg cgctgccccc 6480

ttggaacccc cgcactcgcc gccgtgaggc tcggggggca ggcgggcggg cttcgccttc 6540

gactgccccc actcgcatag gcttgggtcg ttccaggcgc gtcaaggcca agccgctgcg 6600

cggtcgctgc gcgagccttg acccgccttc cacttggtgt ccaaccggca agcgaagcgc 6660

gcaggccgca ggccggaggc ttttccccag agaaaattaa aaaaattgat ggggcaaggc 6720

cgcaggccgc gcagttggag ccggtgggta tgtggtcgaa ggctgggtag ccggtgggca 6780

atccctgtgg tcaagctcgt gggcaggcgc agcctgtcca tcagcttgtc cagcagggtt 6840

gtccacgggc cgagcgaagc gagccagccg gtggccgctc gcggccatcg tccacatatc 6900

cacgggctgg caagggagcg cagcgaccgc gcagggcgaa gcccggagag caagcccgta 6960

gggcgccgca gccgccgtag gcggtcacga ctttgcgaag caaagtctag tgagtatact 7020

caagcattga gtggcccgcc ggaggcaccg ccttgcgctg cccccgtcga gccggttgga 7080

caccaaaagg gaggggcagg catggcggca tacgcgatca tgcgatgcaa gaagctggcg 7140

aaaatgggca acgtggcggc cagtctcaag cacgcctacc gcgagcgcga gacgcccaac 7200

gctgacgcca gcaggacgcc agagaacgag cactgggcgg ccagcagcac cgatgaagcg 7260

atgggccgac tgcgcgagtt gctgccagag aagcggcgca aggacgctgt gttggcggtc 7320

gagtacgtca tgacggccag cccggaatgg tggaagtcgg ccagccaaga acagcaggcg 7380

gcgttcttcg agaaggcgca caagtggctg gcggacaagt acggggcgga tcgcatcgtg 7440

acggccagca tccaccgtga cgaaaccagc ccgcacatga ccgcgttcgt ggtgccgctg 7500

acgcaggacg gcaggctgtc ggccaaggag ttcatcggca acaaagcgca gatgacccgc 7560

gaccagacca cgtttgcggc cgctgtggcc gatctagggc tgcaacgggg catcgagggc 7620

agcaaggcac gtcacacgcg cattcaggcg ttctacgagg ccctggagcg gccaccagtg 7680

ggccacgtca ccatcagccc gcaagcggtc gagccacgcg cctatgcacc gcagggattg 7740

gccgaaaagc tgggaatctc aaagcgcgtt gagacgccgg aagccgtggc cgaccggctg 7800

acaaaagcgg ttcggcaggg gtatgagcct gccctacagg ccgccgcagg agcgcgtgag 7860

atgcgcaaga aggccgatca agcccaagag acggcccgag accttcggga gcgcctgaag 7920

cccgttctgg acgccctggg gccgttgaat cgggatatgc aggccaaggc cgccgcgatc 7980

atcaaggccg tgggcgaaaa gctgctgacg gaacagcggg aagtccagcg ccagaaacag 8040

gcccagcgcc agcaggaacg cgggcgcgca catttccccg aaaagtgcca cctgaacccc 8100

agagtcccgc tcagaagaac tcgtcaagaa ggcgatagaa ggcgatgcgc tgcgaatcgg 8160

gagcggcgat accgtaaagc acgaggaagc ggtcagccca ttcgccgcca agctcttcag 8220

caatatcacg ggtagccaac gctatgtcct gatagcggtc cgccacaccc agccggccac 8280

agtcgatgaa tccagaaaag cggccatttt ccaccatgat attcggcaag caggcatcgc 8340

catgggtcac gacgagatcc tcgccgtcgg gcatccgcgc cttgagcctg gcgaacagtt 8400

cggctggcgc gagcccctga tgctcttcgt ccagatcatc ctgatcgaca agaccggctt 8460

ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg gtggtcgaat gggcaggtag 8520

ccggatcaag cgtatgcagc cgccgcattg catcagccat gatggatact ttctcggcag 8580

gagcaaggtg agatgacagg agatcctgcc ccggcacttc gcccaatagc agccagtccc 8640

ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg aacgcccgtc gtggccagcc 8700

acgatagccg cgctgcctcg tcttggagtt cattcagggc accggacagg tcggtcttga 8760

caaaaagaac cgggcgcccc tgcgctgaca gccggaacac ggcggcatca gagcagccga 8820

ttgtctgttg tgcccagtca tagccgaata gcctctccac ccaagcggcc ggagaacctg 8880

cgtgcaatcc atcttgttca atcatgcgaa acgatcctca tcctgtctct tgatcagatc 8940

ttgatcccct gcgccatcag atccttggcg gcaagaaagc catccagttt actttgcagg 9000

gcttcccaac cttaccagag ggcgccccag ctggcaattc cggttcgctt gctgtccata 9060

aaaccgccca gtctagctat cgccatgtaa gcccactgca agctacctgc tttctctttg 9120

cgcttgcgtt ttcccttgtc cagatagccc agtagctgac attcatccgg ggtcagcacc 9180

gtttctgcgg actggctttc tacgtgttcc gcttccttta gcagcccttg cgccctgagt 9240

gcttgcggca gcgtgaagct agctgcataa tgtgcctgtc aaatggacga agcagggatt 9300

ctgcaaaccc tatgctactc cgtcaagccg tcaattgtct gattcgttac caattatgac 9360

aacttgacgg ctacatcatt cactttttct tcacaaccgg cacggaactc gctcgggctg 9420

gccccggtgc attttttaaa tacccgcgag aaatagagtt gatcgtcaaa accaacattg 9480

cgaccgacgg tggcgatagg catccgggtg gtgctcaaaa gcagcttcgc ctggctgata 9540

cgttggtcct cgcgccagct taagacgcta atccctaact gctggcggaa aagatgtgac 9600

agacgcgacg gcgacaagca aacatgctgt gcgacgctgg cgatatcaaa attgctgtct 9660

gccaggtgat cgctgatgta ctgacaagcc tcgcgtaccc gattatccat cggtggatgg 9720

agcgactcgt taatcgcttc catgcgccgc agtaacaatt gctcaagcag atttatcgcc 9780

agcagctccg aatagcgccc ttccccttgc ccggcgttaa tgatttgccc aaacaggtcg 9840

ctgaaatgcg gctggtgcgc ttcatccggg cgaaagaacc ccgtattggc aaatattgac 9900

ggccagttaa gccattcatg ccagtaggcg cgcggacgaa agtaaaccca ctggtgatac 9960

cattcgcgag cctccggatg acgaccgtag tgatgaatct ctcctggcgg gaacagcaaa 10020

atatcacccg gtcggcaaac aaattctcgt ccctgatttt tcaccacccc ctgaccgcga 10080

atggtgagat tgagaatata acctttcatt cccagcggtc ggtcgataaa aaaatcgaga 10140

taaccgttgg cct 10153

<210> 40

<211> 10486

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC13-Ec载体系统

<400> 40

ccatgcttat ctccatgcgg tagggtgccg cacggttgcg gcaccatgcg caatcagctg 60

caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga 120

ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc 180

cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt 240

ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc 300

cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca 360

aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg 420

ggcgggcttc gccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca 480

aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa 540

ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa 600

attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct 660

gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag 720

cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg 780

ccatcgtcca catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc 840

ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa 900

gtctagtgag tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc 960

cgtcgagccg gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg 1020

atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga 1080

gcgcgagacg cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag 1140

cagcaccgat gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga 1200

cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag 1260

ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg 1320

ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc 1380

gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa 1440

agcgcagatg acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca 1500

acggggcatc gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct 1560

ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta 1620

tgcaccgcag ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc 1680

cgtggccgac cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc 1740

cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct 1800

tcgggagcgc ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc 1860

caaggccgcc gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt 1920

ccagcgccag aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa 1980

gtgccacctg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg 2040

atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg 2100

ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc 2160

acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc 2220

ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg 2280

agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga 2340

tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg 2400

tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg 2460

gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc 2520

aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg 2580

cccgtcgtgg ccagccacga tagccgcgct gcctcgtctt ggagttcatt cagggcaccg 2640

gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg 2700

gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa 2760

gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct 2820

gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc 2880

cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt 2940

tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct 3000

acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc 3060

atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag 3120

cccttgcgcc ctgagtgctt gcggcagcgt gaagctagct gcataatgtg cctgtcaaat 3180

ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt 3240

cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg 3300

gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc 3360

gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag 3420

cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg 3480

gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat 3540

atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt 3600

atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc 3660

aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat 3720

ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt 3780

attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta 3840

aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc 3900

tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac 3960

caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc 4020

gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc 4080

attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact 4140

cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt 4200

cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta 4260

acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag 4320

aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc 4380

cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata 4440

cccgtttttt ggtaccgggc cccccctcga gtttatttta ggaggcaaaa atgagcacca 4500

aaaccatctt tagcgatttc accaatctgt acgaactgag caaaaccctg cgttttgaac 4560

tgaaaccggt tggtgaaacc gaaaatctgc tgaatgaaaa tcaggtgttc ctgaccgata 4620

aaatccgcca gaaaaaatac gaagagatca aaccgtttct ggatgaattt cacctggact 4680

ttattcactt ttgtctgagc gatctgcatc tggattatac cgaatacaaa aaaagcctgg 4740

acaactacca gaaggacaaa aaaaacaaag atctggaaaa aaagaaagag aacgaagaga 4800

aaaaactgcg cgagcagatt gtgggtaaat ttgatagcaa agtggaagat tttctgaaaa 4860

cctttggcaa ggtggaaaaa atcaaaggca aaaaggacaa cgagaagttc aaagttagcc 4920

tgggtaaaga ttgggagatc gaatttagca aaaacaacta cgaattcctg ttcgagatcg 4980

gcattttcga tctgatgaag aaaaagtttg aaggcaacgg cgatatttat gtggcagata 5040

aagaaaccgg tgagatctat caggatgaaa aaaccggtaa agacatcacc atcttcgatg 5100

attggaatgg ttggctgggt tatctgacca aatttttcga aacccgcaaa aacctgtata 5160

aaagtgatgg caccagcacc gcaattgcaa cccgtattat caatgaaaac ctgaagaaat 5220

actgcgagaa cctggacatc tataacaaac tgagccagat tgagaacctg aaaaacaaat 5280

tccaaaacct ggaagccgat ttcggcatta aactggaaaa attctttagc ctggaaaatt 5340

acaacagctg catcctgcag aatggcatcg aaaactataa tgatattcgc ggtggtaagc 5400

tggaaaagaa caataacaaa attccgggta tcaacgagta catcaataaa taccgtcagg 5460

atagcggtga aaaactgccg tttctgcaaa aactggataa gcagattctg gcaggcggta 5520

aagaaaactt tattgagcag atagaaaacg agccgagctt tgaaaaatgc ctgaagaact 5580

tttacaacaa cagcatcaag aaggtggata ttctgaccca gatttttcag gatctgagca 5640

cctataccaa cgaggactat aaaaccattt atttcagcaa agaggccttc aataccctga 5700

gccataaatt caccgatcag gtgctgaatt ttgagaaact ggtttttgaa gaactgctgc 5760

tgaataagct ggtcgagaaa aaagacttcg acaaaaaaga agaaaaatac aagttcccgg 5820

atttcatccc gctgttctat gttaaaaaag gccttgaaaa ctatcacacc aagaacctgt 5880

tctataaaag ccgctattat gagaacgaaa ttatcgaaga ggataacgac aacatctggc 5940

agaaattttg caccatcctg aactatgaat tccagagcct gctgagcaat accattatta 6000

accagaatgg cgaagaaatc gaagtgggtt ttaccatcag taagaacaag cttgagaaga 6060

tcctggacaa ttttagtctg ggcgaaaata acaacggcat catcaaagat tttgccgaca 6120

tcagcaaaac gatttatcag atgggtaaat acttcgccct tgaaaaaaaa cgcgaatgga 6180

acaacaactt cgacctgaac gatgattttt acaaaaccga gtatagccaa gaaaacgaaa 6240

aatacggtta tctggaattt tacaacgagg cctatgagca gattattgtg ccgtataatc 6300

tgatgcgcaa ctttatcgca aaaaagccgt gggaagataa caagaaatgg aaactgaact 6360

tcgaaaatag cagtctgctg aaaggttggg ataaagaatt cgaaagctat ggcagctaca 6420

tctttgagaa agcaggtctg tattatctgg gcatcattaa tggcaccaaa ctgaacaaaa 6480

acgagatcga gaaactgtac aattataacg ccaataatgg tgccaaacgc ttcgtgtatg 6540

atttccagaa accggacaac aaaaatacac cgcgtctgtt tattcgtagc aaaggcgata 6600

actttgcacc gagcgttaaa gaactgaatc tgccgattaa taacatcatc gaaatctatg 6660

ataaagagct gtacaagaaa gataaagaga agccgaacaa gcataaagaa agcctgatga 6720

aactgatcga ctatttcaaa ctgggctttc gcaaacacat cagctataaa cactttaact 6780

tcgtgtggaa agaaagcaac aaatatgaca acattgccga tttttatcgc gacgttgaaa 6840

aaagctgcta taaaccgtat tgggaagagg acatcaactt cgatgaactg aagaatctga 6900

cgaaagaaaa acgcatgtac ctgtttcaga tctacaataa gaacttcgag ctggatgaaa 6960

gcattagcac cgatgattac acgtttaaag gcaatggtaa agatagcgtg cacaccatgt 7020

atttcaaagg cctgtttagc aaagataacc ttgagaataa aaacggcgtc aacctgaaac 7080

tgagcggtgg tggtgaactg ttttttcgtc cgaaaagcat tgagaaaaaa atcgacaaga 7140

accgcaaaag caaacgcgaa atcattgaga acaaacgcta cagcaaagac aaaatcctgc 7200

tgcattttcc gattcaggtg aacttcaaag aaaacaagac cagcaacttc aacaactaca 7260

tcaacaattt tctggccaac aatcccgaca ttaacattat tggtattgac cgtggcgaaa 7320

aacacctggc atattatagc gttatcaacc agaaacaaga gattattgaa agcggcagcc 7380

tgaactacat ctatcagaaa gacaaagatg gcaaaatcat tcagaaaagc gagaaaaaga 7440

ttcaagaggt gcgtaatgat gaaggcaaga tcattgatta tgaactggtt gaaaccggca 7500

aactggtgga ttatgaagat tatggtatcc tgctggacta caaagaaaaa aagcgtcgtc 7560

tgcagcgtca gagctggaaa gaagttgaac aaatcaagga tctgaagaag ggctatatta 7620

gcgcagttgt tcgtaaaatt gcggatctga ttatcgaaca taacgccatc gtgatctttg 7680

aggacctgaa tatgcgcttt aaacaaatcc gtggtggtat tgaaaaatcc gtgtatcagc 7740

agttagaaaa agccctgatt gataagctga acttcctggt taacaaaggc gagaaagata 7800

gtgaacaggc aggtaattta ctgaaagcgt ttcagctgac cgcaccgatt ggcaccttta 7860

aagatatggg taaacagacc ggcattatct tttataccca ggcacgttat accagcaaaa 7920

ttgatccgct gaccggttgg cgtccgaatc tgtatatcaa aaaacagagt gccgagctga 7980

acaaagagag cattctgaaa ttcgatagca tcatctggaa taaagaaaaa gagtacttcg 8040

aaatcaccta tgacctggaa aagtttcaga gcgaaagtac caaaaatctg aaagagaaga 8100

aagaagagaa gctggaacgt accaaatgga ccctgagcac ccgtgttgaa cgttttaaat 8160

ggaataagaa ccttaataac aacaaaggtg gctacgaaca cttcgagaat ctgaacatcc 8220

atttcaaaga actgtttgag aaatatggcc tggatattag cggtgatatc ctgaagcaaa 8280

tccataatct ggaaaccaaa ggtaacgaag cctttttcag ccattttctg gacctgttta 8340

aactggtgtg tcagattcgt aataccaacc aggataaaaa gggtaacgag aacgatttca 8400

tttacagtcc ggtgtttccg tttttcgata gccgtaaaca gaataccgtt ggcgttaaaa 8460

atggtgatga taacggtgca ttcaacattg cacgtaaagg cattattatc ctggaacgca 8520

ttggcaagtg gaagaaagaa aacgatatga agattcagaa aggtgaaaaa gagatgtatc 8580

cggacctgtt cattagcaat attggctggg ataatttcac gcagaatcat aacattcgcg 8640

ataattaatg gtctagaggt cgaaattcaa attgtgagcg gataacaatt tgaattttct 8700

gtatgaggtt ttgctaaaca actttcaaca gtttcagtgg agtgagaata gaaaggaaca 8760

actaaaggaa ttgcgaataa taattttttc acgttgaaaa tctccaaaaa aaaaggctcc 8820

aaaaggagcc tttaattgta tcggtttatc agcttgcttt cgaggtgaat tttgaccctc 8880

tagcgaaaat gcaagagcaa agacgaaaac atgccacaca tgaggaatac cgattctctc 8940

attaacatat tcaggccagt tatctgggct taaaagcaga agtccaaccc agataacgat 9000

catatacatg gttctctcca gaggttcatt actgaacact cgtccgagaa taacgagtgg 9060

atcccctcca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta 9120

caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc 9180

cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg 9240

cgcagcctga atggcgaatg gaaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa 9300

atttttgtta aatcagctca ttttttaacc aataggccga ctgcgatgag tggcagggcg 9360

gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggtgct acgcctgaat 9420

aagtgataat aagcggatga atggcagaaa ttcgaaagca aattcgaccc ggtcgtcggt 9480

tcagggcagg gtcgttaaat agccgcttat gtctattgct ggtttaccgg tttattgact 9540

accggaagca gtgtgaccgt gtgcttctca aatgcctgag gccagtttgc tcaggctctc 9600

cccgtggagg taataattga cgatatgatc atttattctg cctcccagag cctgataaaa 9660

acggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg gcccggctcc 9720

atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgagg cggctacagc 9780

cgatagtctg gaacagcgca cttacgggtt gctgcgcaac ccaagtgcta ccggcgcggc 9840

agcgtgaccc gtgtcggcgg ctccaacggc tcgccatcgt ccagaaaaca cggctcatcg 9900

ggcatcggca ggcgctgctg cccgcgccgt tcccattcct ccgtttcggt caaggctggc 9960

aggtctggtt ccatgcccgg aatgccgggc tggctgggcg gctcctcgcc ggggccggtc 10020

ggtagttgct gctcgcccgg atacagggtc gggatgcggc gcaggtcgcc atgccccaac 10080

agcgattcgt cctggtcgtc gtgatcaacc accacggcgg cactgaacac cgacaggcgc 10140

aactggtcgc ggggctggcc ccacgccacg cggtcattga ccacgtaggc cgacacggtg 10200

ccggggccgt tgagcttcac gacggagatc cagcgctcgg ccaccaagtc cttgactgcg 10260

tattggaccg tccgcaaaga acgtccgatg agcttggaaa gtgtcttttg gctgaccacc 10320

acggcgttct ggtggcccat ctgcgccacg aggtgatgca gcagcattgc cgccgtgggt 10380

ttcctcgcaa taagcccggc ccacgcctca tgcgctttgc gttccgtttg cacccagtga 10440

ccgggcttgt tcttggcttg aatgccgatt tctctggact gcgtgg 10486

<210> 41

<211> 165

<212> DNA

<213> 巨大芽孢杆菌(Bacillus megaterium)

<220>

<223> 来自巨大芽孢杆菌(Bacillus megaterium)的

PsacB RNA聚合酶II启动子的核苷酸序列

<400> 41

gcccatgcaa cagaaactat aaaaaataca gagaatgaaa agaaacagat agatttttta 60

gttctttagg cccgtagtct gcaaatcctt ttatgatttt ctatcaaaca aaagaggaaa 120

atagaccagt tgcaatccaa acgagagtct aatagaatga ggtcg 165

<210> 42

<211> 169

<212> DNA

<213> 大肠杆菌(Escherichia coli)

<220>

<223> 大肠杆菌rrnB基因的转录T1和T2终止区

的核苷酸序列

<400> 42

tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa 60

acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc 120

atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttat 169

<210> 43

<211> 24

<212> DNA

<213> 人工序列

<220>

<223> malQ靶特异性24 bp间隔子序列

<400> 43

gtggcggtac gttgatgcat cgcg 24

<210> 44

<211> 5039

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/gRNA-Ec载体系统的核苷酸序列

<400> 44

gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt 60

cttagacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt 120

tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa atgcttcaat 180

aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt 240

ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg 300

ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga 360

tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc 420

tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac 480

actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg 540

gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca 600

acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg 660

gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg 720

acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg 780

gcgaactact tactctagct tcccggcaac aattaataga ctggatggag gcggataaag 840

ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg 900

gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct 960

cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac 1020

agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac caagtttact 1080

catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga 1140

tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt 1200

cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct 1260

gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc 1320

taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc 1380

ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc 1440

tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg 1500

ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt 1560

cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg 1620

agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 1680

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 1740

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 1800

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1860

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1920

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1980

cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc 2040

cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca 2100

acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc 2160

cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg 2220

accatgatta cgccaagctc ggaattaacc ctcactaaag ggaacaaaag ctggagctct 2280

gcagcccatg caacagaaac tataaaaaat acagagaatg aaaagaaaca gatagatttt 2340

ttagttcttt aggcccgtag tctgcaaatc cttttatgat tttctatcaa acaaaagagg 2400

aaaatagacc agttgcaatc caaacgagag tctaatagaa tgaggtcgga tttctactat 2460

tgtagatgtg gcggtacgtt gatgcatcgc gggccggcat ggtcccagcc tcctcgctgg 2520

cgccggctgg gcaacatgct tcggcatggc gaatgggact ttttttttgc ctggcggcag 2580

tagcgcggtg gtcccacctg accccatgcc gaactcagaa gtgaaacgcc gtagcgccga 2640

tggtagtgtg gggtctcccc atgcgagagt agggaactgc caggcatcaa ataaaacgaa 2700

aggctcagtc gaaagactgg gcctttcgtt ttatggtacc caattcgccc tatagtgagt 2760

cgtattacaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 2820

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 2880

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa aggcaggccg ggccgtggtg 2940

gccacggcct ctaggccaga tccagcggca tctgggttag tcgagcgcgg gccgcttccc 3000

atgtctcacc agggcgagcc tgtttcgcga tctcagcatc tgaaatcttc ccggccttgc 3060

gcttcgctgg ggccttaccc accgccttgg cgggcttctt cggtccaaaa ctgaacaaca 3120

gatgtgtgac cttgcgcccg gtctttcgct gcgcccactc cacctgtagc gggctgtgct 3180

cgttgatctg cgtcacggct ggatcaagca ctcgcaactt gaagtccttg atcgagggat 3240

accggccttc cagttgaaac cactttcgca gctggtcaat ttctatttcg cgctggccga 3300

tgctgtccca ttgcatgagc agctcgtaaa gcctgatcgc gtgggtgctg tccatcttgg 3360

ccacgtcagc caaggcgtat ttggtgaact gtttggtgag ttccgtcagg tacggcagca 3420

tgtctttggt gaacctgagt tctacacggc cctcaccctc ccggtagatg attgtttgca 3480

cccagccggt aatcatcaca ctcggtcttt tccccttgcc attgggctct tgggttaacc 3540

ggacttcccg ccgtttcagg cgcagggccg cttctttgag ctggttgtag gaagattcga 3600

tagggacacc cgccatcgtc gctatgtcct ccgccgtcac tgaatacatc acttcatcgg 3660

tgacaggctc gctcctcttc acctggctaa tacaggccag aacgatccgc tgttcctgaa 3720

cactgaggcg atacgcggcc tcgaccaggg cattgctttt gtaaaccatt gggggtgagg 3780

ccacgttcga cattccttgt gtataagggg acactgtatc tgcgtcccac aatacaacaa 3840

atccgtccct ttacaacaac aaatccgtcc cttcttaaca acaaatccgt cccttaatgg 3900

caacaaatcc gtcccttttt aaactctaca ggccacggat tacgtggcct gtagacgtcc 3960

taaaaggttt aaaagggaaa aggaagaaaa gggtggaaac gcaaaaaacg caccactacg 4020

tggccccgtt ggggccgcat ttgtgcccct gaaggggcgg gggaggcgtc tgggcaatcc 4080

ccgttttacc agtcccctat cgccgcctga gagggcgcag gaagcgagta atcagggtat 4140

cgaggcggat tcacccttgg cgtccaacca gcggcaccag cggcgcctga gaggcctaca 4200

gagcggttgg acaccaaggg gaggggctaa gaccggttta tcagtcccct ttccctcgtt 4260

tctttccaac gcgatagccc agcaaggccg ccaccgttgc caccgtcacc ccagcaagca 4320

cagccagtgg cgtgtaattg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga 4380

ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa 4440

gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa 4500

tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc 4560

ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga 4620

taccgcgaga tccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 4680

gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt 4740

gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg 4800

ccgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg 4860

cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac acccgccaac 4920

acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt 4980

gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcga 5039

<210> 45

<211> 674

<212> DNA

<213>大肠杆菌(Escherichia coli)

<220>

<223> 用于在大肠杆菌中有附加型繁殖

源自pMB1的高拷贝复制(pUC)的核苷酸序列

<400> 45

atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 60

ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 120

cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 180

tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 240

gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 300

aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 360

tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 420

aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 480

aactacggct acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc 540

ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 600

ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 660

atcttttcta cggg 674

<210> 46

<211> 1884

<212> DNA

<213> 假单胞菌属(Pseudomonas)

<220>

<223> 用于在假单胞菌属中附加型载体繁殖

源自pRO1614的复制子区的核苷酸序列

<400> 46

gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 60

caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 120

cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg atctcgcggt 180

atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 240

gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 300

attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 360

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 420

atcccttaac gtgagttttc gttccactga gcgtcagacc ccaattacac gccactggct 480

gtgcttgctg gggtgacggt ggcaacggtg gcggccttgc tgggctatcg cgttggaaag 540

aaacgaggga aaggggactg ataaaccggt cttagcccct ccccttggtg tccaaccgct 600

ctgtaggcct ctcaggcgcc gctggtgccg ctggttggac gccaagggtg aatccgcctc 660

gataccctga ttactcgctt cctgcgccct ctcaggcggc gataggggac tggtaaaacg 720

gggattgccc agacgcctcc cccgcccctt caggggcaca aatgcggccc caacggggcc 780

acgtagtggt gcgttttttg cgtttccacc cttttcttcc ttttcccttt taaacctttt 840

aggacgtcta caggccacgt aatccgtggc ctgtagagtt taaaaaggga cggatttgtt 900

gccattaagg gacggatttg ttgttaagaa gggacggatt tgttgttgta aagggacgga 960

tttgttgtat tgtgggacgc agatacagtg tccccttata cacaaggaat gtcgaacgtg 1020

gcctcacccc caatggttta caaaagcaat gccctggtcg aggccgcgta tcgcctcagt 1080

gttcaggaac agcggatcgt tctggcctgt attagccagg tgaagaggag cgagcctgtc 1140

accgatgaag tgatgtattc agtgacggcg gaggacatag cgacgatggc gggtgtccct 1200

atcgaatctt cctacaacca gctcaaagaa gcggccctgc gcctgaaacg gcgggaagtc 1260

cggttaaccc aagagcccaa tggcaagggg aaaagaccga gtgtgatgat taccggctgg 1320

gtgcaaacaa tcatctaccg ggagggtgag ggccgtgtag aactcaggtt caccaaagac 1380

atgctgccgt acctgacgga actcaccaaa cagttcacca aatacgcctt ggctgacgtg 1440

gccaagatgg acagcaccca cgcgatcagg ctttacgagc tgctcatgca atgggacagc 1500

atcggccagc gcgaaataga aattgaccag ctgcgaaagt ggtttcaact ggaaggccgg 1560

tatccctcga tcaaggactt caagttgcga gtgcttgatc cagccgtgac gcagatcaac 1620

gagcacagcc cgctacaggt ggagtgggcg cagcgaaaga ccgggcgcaa ggtcacacat 1680

ctgttgttca gttttggacc gaagaagccc gccaaggcgg tgggtaaggc cccagcgaag 1740

cgcaaggccg ggaagatttc agatgctgag atcgcgaaac aggctcgccc tggtgagaca 1800

tgggaagcgg cccgcgctcg actaacccag atgccgctgg atctggccta gaggccgtgg 1860

ccaccacggc ccggcctgcc tttc 1884

<210> 47

<211> 570

<212> DNA

<213> 诺尔斯氏链霉菌(Streptomyces noursei)

<220>

<223> 诺尔丝菌素乙酰转移酶基因(nat1)的核苷酸序列

<400> 47

atgaccactc ttgacgacac ggcttaccgg taccgcacca gtgtcccggg ggacgccgag 60

gccatcgagg cactggatgg gtccttcacc accgacaccg tcttccgcgt caccgccacc 120

ggggacggct tcaccctgcg ggaggtgccg gtggacccgc ccctgaccaa ggtgttcccc 180

gacgacgaat cggacgacga atcggacgcc ggggaggacg gcgacccgga ctcccggacg 240

ttcgtcgcgt acggggacga cggcgacctg gcgggcttcg tggtcgtctc gtactccggc 300

tggaaccgcc ggctgaccgt cgaggacatc gaggtcgccc cggagcaccg ggggcacggg 360

gtcgggcgcg cgttgatggg gctcgcgacg gagttcgccc gcgagcgggg cgccgggcac 420

ctctggctgg aggtcaccaa cgtcaacgca ccggcgatcc acgcgtaccg gcggatgggg 480

ttcaccctct gcggcctgga caccgccctg tacgacggca ccgcctcgga cggcgagcag 540

gcgctctaca tgagcatgcc ctgcccctga 570

<210> 48

<211> 90

<212> DNA

<213> 枯草芽孢杆菌(Bacillus subtilis)

<220>

<223> 来自枯草芽孢杆菌的Veg蛋白基因的N末端90bp

核苷酸编码序列片段

<400> 48

atggcgaaga cgttgtccga tattaaaaga tcgcttgatg ggaatttagg taaaaggctg 60

acgttaaaag caaacggtgg ccggatccat 90

<210> 49

<211> 660

<212> DNA

<213> 人工序列

<220>

<223> veg-nat1基因融合的核苷酸序列

<400> 49

atggcgaaga cgttgtccga tattaaaaga tcgcttgatg ggaatttagg taaaaggctg 60

acgttaaaag caaacggtgg ccggatccat atgaccactc ttgacgacac ggcttaccgg 120

taccgcacca gtgtcccggg ggacgccgag gccatcgagg cactggatgg gtccttcacc 180

accgacaccg tcttccgcgt caccgccacc ggggacggct tcaccctgcg ggaggtgccg 240

gtggacccgc ccctgaccaa ggtgttcccc gacgacgaat cggacgacga atcggacgcc 300

ggggaggacg gcgacccgga ctcccggacg ttcgtcgcgt acggggacga cggcgacctg 360

gcgggcttcg tggtcgtctc gtactccggc tggaaccgcc ggctgaccgt cgaggacatc 420

gaggtcgccc cggagcaccg ggggcacggg gtcgggcgcg cgttgatggg gctcgcgacg 480

gagttcgccc gcgagcgggg cgccgggcac ctctggctgg aggtcaccaa cgtcaacgca 540

ccggcgatcc acgcgtaccg gcggatgggg ttcaccctct gcggcctgga caccgccctg 600

tacgacggca ccgcctcgga cggcgagcag gcgctctaca tgagcatgcc ctgcccctga 660

<210> 50

<211> 822

<212> DNA

<213>枯草芽孢杆菌(Bacillus subtilis)

<220>

<223> veg-nat1基因融合上游5’-UTR区域的核苷酸序列，

其含有来自枯草芽孢杆菌veg RNA聚合酶II启动子

<400> 50

atgcgagcca tttggacggg ttcgatcgcc ttcgggctgg tgaacgtgcc ggtcaaggtg 60

tacagcgcta ccgcagacca cgacatcagg ttccaccagg tgcacgccaa ggacaacgga 120

cgcatccggt acaagcgcgt ctgcgaggcg tgtggcgagg tggtcgacta ccgcgatctt 180

gcccgggcct acgagtccgg cgacggccaa atggtggcga tcaccgacga cgacatcgcc 240

agcttgcctg aagaacgcag ccgggagatc gaggtgttgg agttcgtccc cgccgccgac 300

gtggacccga tgatgttcga ccgcagctac tttttggagc ctgattcgaa gtcgtcgaaa 360

tcgtatgtgc tgctggctaa gacactcgcc gagaccgacc ggatggcgat cgtgcatttc 420

acgctgcgca acaagaccag gctggcggcg ttgcgcgtca aggatttcgg caagcgagag 480

gtgatgatgg tgcacacgtt gctgtggccc gatgagatcc gcgaccccga cttcccggtg 540

ctggaccaga aggtggagat caaacccgcg gaactcaaga tggccggcca ggtggtggac 600

tcgatggccg acgacttcaa tccggaccgc taccacgaca cctaccagga gcagttacag 660

gagctgatcg acaccaaact cgaaggtggg caggcattta ccgccgagga ccaaccgagg 720

ttgctggacg agcccgaaga cgtctccgac ctgctcgcca agctggaggc cagcgtgaag 780

gcgcgctcga aggccaactc aaacgtccca acgcctccgt ga 822

<210> 51

<211> 822

<212> DNA

<213> 结核分枝杆菌(Mycobacterium tuberculosis)

<220>

<223> 来自结核分枝杆菌的ku基因的核苷酸序列

<400> 51

atgcgagcca tttggacggg ttcgatcgcc ttcgggctgg tgaacgtgcc ggtcaaggtg 60

tacagcgcta ccgcagacca cgacatcagg ttccaccagg tgcacgccaa ggacaacgga 120

cgcatccggt acaagcgcgt ctgcgaggcg tgtggcgagg tggtcgacta ccgcgatctt 180

gcccgggcct acgagtccgg cgacggccaa atggtggcga tcaccgacga cgacatcgcc 240

agcttgcctg aagaacgcag ccgggagatc gaggtgttgg agttcgtccc cgccgccgac 300

gtggacccga tgatgttcga ccgcagctac tttttggagc ctgattcgaa gtcgtcgaaa 360

tcgtatgtgc tgctggctaa gacactcgcc gagaccgacc ggatggcgat cgtgcatttc 420

acgctgcgca acaagaccag gctggcggcg ttgcgcgtca aggatttcgg caagcgagag 480

gtgatgatgg tgcacacgtt gctgtggccc gatgagatcc gcgaccccga cttcccggtg 540

ctggaccaga aggtggagat caaacccgcg gaactcaaga tggccggcca ggtggtggac 600

tcgatggccg acgacttcaa tccggaccgc taccacgaca cctaccagga gcagttacag 660

gagctgatcg acaccaaact cgaaggtggg caggcattta ccgccgagga ccaaccgagg 720

ttgctggacg agcccgaaga cgtctccgac ctgctcgcca agctggaggc cagcgtgaag 780

gcgcgctcga aggccaactc aaacgtccca acgcctccgt ga 822

<210> 52

<211> 2280

<212> DNA

<213> 结核分枝杆菌(Mycobacterium tuberculosis)

<220>

<223> 来自结核分枝杆菌的ligD基因的核苷酸序列

<400> 52

atgggttcgg cgtcggagca acgggtgacg ctgaccaacg ccgacaaggt gctctatccc 60

gccaccggga ccacaaagtc cgatatcttc gactactacg ccggtgttgc cgaagtcatg 120

ctcggccaca tcgcgggacg gccggcgacg cgcaagcgct ggcctaacgg cgtcgaccaa 180

cccgcgttct tcgaaaagca gttggcgttg tcggcgccgc cttggctgtc acgtgcaacg 240

gtggcgcacc ggtccgggac gacgacctat ccgatcatcg atagcgcaac cgggctggcc 300

tggatcgccc aacaggcggc gctggaggtg cacgtgccgc agtggcggtt tgtcgccgag 360

cccggatcag gtgagttaaa tccgggcccg gcaacgcgtt tggtgttcga cctggacccg 420

ggcgaaggcg tgatgatggc ccagctggcc gaggtggcgc gcgcggttcg tgatcttctc 480

gccgatatcg ggttggtcac cttcccggtc accagcggca gcaagggatt gcatctgtac 540

acaccgctgg atgagccggt gagcagcagg ggagccacgg tgttggccaa gcgcgtcgcg 600

cagcgattgg agcaggcgat gcccgcgttg gtcacctcga ccatgaccaa aagcctgcgg 660

gccgggaagg tgtttgtgga ctggagccag aacagcggct cgaagaccac catcgcgccg 720

tactcactac gtggccggac gcatccgacc gtcgcggcgc cacgcacctg ggcggagctc 780

gacgaccccg cactgcgtca gctctcctac gacgaggtgc tgacccggat tgcccgcgac 840

ggcgatctgc tcgagcggct ggatgccgac gctccggtag cggaccggtt gacccgatac 900

cgccgcatgc gcgacgcatc gaaaactccc gagccgattc ccacggcgaa acccgttacc 960

ggagacggca atacgttcgt catccaggag catcacgcgc gtcggccgca ctacgatttc 1020

cggctggaat gcgacggcgt gctggtctcg tgggcggtac cgaaaaacct gcccgacaac 1080

acatcggtta accatctagc gatacacacc gaggaccacc cgctggaata cgccacgttc 1140

gagggcgcga ttcccagcgg ggagtacggc gccggcaagg tgatcatctg ggactccggc 1200

acttacgaca ccgagaagtt ccacgatgac ccgcacacgg gggaggtcat cgtgaatctg 1260

cacggcggcc ggatctctgg gcgttatgcg ctgattcgga ccaacggcga tcggtggctg 1320

gcgcaccgcc taaagaatca gaaagaccag aaggtgttcg agttcgacaa tctggcccca 1380

atgcttgcca cgcacggcac ggtggccggt ctaaaggcca gccagtgggc gttcgaaggc 1440

aagtgggacg gctaccggtt gctggttgag gctgaccacg gcgccgtgcg gctgcggtcc 1500

cgcagcgggc gcgatgtcac cgccgagtat ccgcaattgc gggcattggc ggaggatctc 1560

gccgatcacc acgtggtgct ggacggcgag gccgtcgtac ttgactcctc tggtgtgccc 1620

agcttcagcc agatgcagaa tcggggccgc gacacccgtg tcgagttctg ggcgttcgac 1680

ctgctctacc tcgacggccg cgcgctgcta ggcacccgct accaagaccg gcgtaagctg 1740

ctcgaaaccc tagctaacgc aaccagtctc accgttcccg agctgctgcc cggtgacggc 1800

gcccaagcgt ttgcgtgctc gcgcaagcac ggctgggagg gcgtgatcgc caagaggcgt 1860

gactcgcgct atcagccggg ccggcgctgc gcgtcgtggg tcaaggacaa gcactggaac 1920

acccaggaag tcgtcattgg tggctggcgc gccggggaag gcgggcgcag cagtggcgtc 1980

gggtcgctgc tcatgggcat ccccggtcca ggtgggctgc agttcgccgg gcgggtcggt 2040

accggcctca gcgaacgcga actggccaac ctcaaggaga tgctggcgcc gctgcatacc 2100

gacgagtccc ccttcgacgt accactgccc gcgcgtgacg ccaagggcat cacatatgtc 2160

aagccggcgc tggttgcaga ggtgcgctac agcgagtgga ctccggaggg ccggctgcgt 2220

caatcaagct ggcgtgggct gcggccggac aagaaaccca gtgaggtggt gcgcgaatga 2280

<210> 53

<211> 176

<212> DNA

<213> 枯草芽孢杆菌(Bacillus subtilis)

<220>

<223> ligD编码序列上游5’-UTR区域的核苷酸序列，

其含有来自枯草芽孢杆菌veg RNA聚合酶II启动子

<400> 53

ctcatgtttg acagcttatc atcgaattat aggaatagag caaacaagca aaggaaattt 60

tgtcaaaata attttattga caacgtctta ttaacgttga tataatttaa attttatttg 120

acaaaaatgg gctcgtgttg tacaataaat gtaactagtt aaggaggtaa taatat 176

<210> 54

<211> 47

<212> DNA

<213> T7噬菌体

<220>

<223> T7转录终止子的核苷酸序列

<400> 54

tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttg 47

<210> 55

<211> 362

<212> DNA

<213> 巨大芽孢杆菌(Bacillus megaterium)

<220>

<223> ku基因上游5’-UTR区域的核苷酸序列，

其含有来自巨大芽孢杆菌SacB RNA聚合酶II启动子

<400> 55

cccatgcaac agaaactata aaaaatacag agaatgaaaa gaaacagata gattttttag 60

ttctttaggc ccgtagtctg caaatccttt tatgattttc tatcaaacaa aagaggaaaa 120

tagaccagtt gcaatccaaa cgagagtcta atagaatgag gtcgaaaagt aaatcgcgcg 180

ggtttgttac tgataaagca ggcaagacct aaaatgtgta aagggcaaag tgtatacttt 240

ggcgtcaccc cttacatatt ttaggtcttt ttttattgtg cgtaactaac ttgccatctt 300

caaacaggag ggctggaaga agcagaccgc taacacagta cataaaaaag gagacatgaa 360

cg 362

<210> 56

<211> 8883

<212> DNA

<213> 人工序列

<220>

<223> pUCP-SK-Pveg-LigD_PsacB-Ku NHEJ载体系统的核苷酸序列

<400> 56

gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg 60

caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc 120

cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg atctcgcggt 180

atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg 240

gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg 300

attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa 360

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 420

atcccttaac gtgagttttc gttccactga gcgtcagacc ccaattacac gccactggct 480

gtgcttgctg gggtgacggt ggcaacggtg gcggccttgc tgggctatcg cgttggaaag 540

aaacgaggga aaggggactg ataaaccggt cttagcccct ccccttggtg tccaaccgct 600

ctgtaggcct ctcaggcgcc gctggtgccg ctggttggac gccaagggtg aatccgcctc 660

gataccctga ttactcgctt cctgcgccct ctcaggcggc gataggggac tggtaaaacg 720

gggattgccc agacgcctcc cccgcccctt caggggcaca aatgcggccc caacggggcc 780

acgtagtggt gcgttttttg cgtttccacc cttttcttcc ttttcccttt taaacctttt 840

aggacgtcta caggccacgt aatccgtggc ctgtagagtt taaaaaggga cggatttgtt 900

gccattaagg gacggatttg ttgttaagaa gggacggatt tgttgttgta aagggacgga 960

tttgttgtat tgtgggacgc agatacagtg tccccttata cacaaggaat gtcgaacgtg 1020

gcctcacccc caatggttta caaaagcaat gccctggtcg aggccgcgta tcgcctcagt 1080

gttcaggaac agcggatcgt tctggcctgt attagccagg tgaagaggag cgagcctgtc 1140

accgatgaag tgatgtattc agtgacggcg gaggacatag cgacgatggc gggtgtccct 1200

atcgaatctt cctacaacca gctcaaagaa gcggccctgc gcctgaaacg gcgggaagtc 1260

cggttaaccc aagagcccaa tggcaagggg aaaagaccga gtgtgatgat taccggctgg 1320

gtgcaaacaa tcatctaccg ggagggtgag ggccgtgtag aactcaggtt caccaaagac 1380

atgctgccgt acctgacgga actcaccaaa cagttcacca aatacgcctt ggctgacgtg 1440

gccaagatgg acagcaccca cgcgatcagg ctttacgagc tgctcatgca atgggacagc 1500

atcggccagc gcgaaataga aattgaccag ctgcgaaagt ggtttcaact ggaaggccgg 1560

tatccctcga tcaaggactt caagttgcga gtgcttgatc cagccgtgac gcagatcaac 1620

gagcacagcc cgctacaggt ggagtgggcg cagcgaaaga ccgggcgcaa ggtcacacat 1680

ctgttgttca gttttggacc gaagaagccc gccaaggcgg tgggtaaggc cccagcgaag 1740

cgcaaggccg ggaagatttc agatgctgag atcgcgaaac aggctcgccc tggtgagaca 1800

tgggaagcgg cccgcgctcg actaacccag atgccgctgg atctggccta gaggccgtgg 1860

ccaccacggc ccggcctgcc tttcaggctg cgcaactgtt gggaagggcg atcggtgcgg 1920

gcctcttcgc tattacgcca gctggcgaaa gggggatgtg ctgcaaggcg attaagttgg 1980

gtaacgccag ggttttccca gtcacgacgt tgtaaaacga cggccagtga attgtaatac 2040

gactcactat agggcgaatt gggtaccggg ccccccctcg aggtcgacgg tatcgataag 2100

cttgatatcg aattctcatg tttgacagct tatcatcgaa ttataggaat agagcaaaca 2160

agcaaaggaa attttgtcaa aataatttta ttgacaacgt cttattaacg ttgatataat 2220

ttaaatttta tttgacaaaa atgggctcgt gttgtacaat aaatgtaact agttaaggag 2280

gtaataatat atgggttcgg cgtcggagca acgggtgacg ctgaccaacg ccgacaaggt 2340

gctctatccc gccaccggga ccacaaagtc cgatatcttc gactactacg ccggtgttgc 2400

cgaagtcatg ctcggccaca tcgcgggacg gccggcgacg cgcaagcgct ggcctaacgg 2460

cgtcgaccaa cccgcgttct tcgaaaagca gttggcgttg tcggcgccgc cttggctgtc 2520

acgtgcaacg gtggcgcacc ggtccgggac gacgacctat ccgatcatcg atagcgcaac 2580

cgggctggcc tggatcgccc aacaggcggc gctggaggtg cacgtgccgc agtggcggtt 2640

tgtcgccgag cccggatcag gtgagttaaa tccgggcccg gcaacgcgtt tggtgttcga 2700

cctggacccg ggcgaaggcg tgatgatggc ccagctggcc gaggtggcgc gcgcggttcg 2760

tgatcttctc gccgatatcg ggttggtcac cttcccggtc accagcggca gcaagggatt 2820

gcatctgtac acaccgctgg atgagccggt gagcagcagg ggagccacgg tgttggccaa 2880

gcgcgtcgcg cagcgattgg agcaggcgat gcccgcgttg gtcacctcga ccatgaccaa 2940

aagcctgcgg gccgggaagg tgtttgtgga ctggagccag aacagcggct cgaagaccac 3000

catcgcgccg tactcactac gtggccggac gcatccgacc gtcgcggcgc cacgcacctg 3060

ggcggagctc gacgaccccg cactgcgtca gctctcctac gacgaggtgc tgacccggat 3120

tgcccgcgac ggcgatctgc tcgagcggct ggatgccgac gctccggtag cggaccggtt 3180

gacccgatac cgccgcatgc gcgacgcatc gaaaactccc gagccgattc ccacggcgaa 3240

acccgttacc ggagacggca atacgttcgt catccaggag catcacgcgc gtcggccgca 3300

ctacgatttc cggctggaat gcgacggcgt gctggtctcg tgggcggtac cgaaaaacct 3360

gcccgacaac acatcggtta accatctagc gatacacacc gaggaccacc cgctggaata 3420

cgccacgttc gagggcgcga ttcccagcgg ggagtacggc gccggcaagg tgatcatctg 3480

ggactccggc acttacgaca ccgagaagtt ccacgatgac ccgcacacgg gggaggtcat 3540

cgtgaatctg cacggcggcc ggatctctgg gcgttatgcg ctgattcgga ccaacggcga 3600

tcggtggctg gcgcaccgcc taaagaatca gaaagaccag aaggtgttcg agttcgacaa 3660

tctggcccca atgcttgcca cgcacggcac ggtggccggt ctaaaggcca gccagtgggc 3720

gttcgaaggc aagtgggacg gctaccggtt gctggttgag gctgaccacg gcgccgtgcg 3780

gctgcggtcc cgcagcgggc gcgatgtcac cgccgagtat ccgcaattgc gggcattggc 3840

ggaggatctc gccgatcacc acgtggtgct ggacggcgag gccgtcgtac ttgactcctc 3900

tggtgtgccc agcttcagcc agatgcagaa tcggggccgc gacacccgtg tcgagttctg 3960

ggcgttcgac ctgctctacc tcgacggccg cgcgctgcta ggcacccgct accaagaccg 4020

gcgtaagctg ctcgaaaccc tagctaacgc aaccagtctc accgttcccg agctgctgcc 4080

cggtgacggc gcccaagcgt ttgcgtgctc gcgcaagcac ggctgggagg gcgtgatcgc 4140

caagaggcgt gactcgcgct atcagccggg ccggcgctgc gcgtcgtggg tcaaggacaa 4200

gcactggaac acccaggaag tcgtcattgg tggctggcgc gccggggaag gcgggcgcag 4260

cagtggcgtc gggtcgctgc tcatgggcat ccccggtcca ggtgggctgc agttcgccgg 4320

gcgggtcggt accggcctca gcgaacgcga actggccaac ctcaaggaga tgctggcgcc 4380

gctgcatacc gacgagtccc ccttcgacgt accactgccc gcgcgtgacg ccaagggcat 4440

cacatatgtc aagccggcgc tggttgcaga ggtgcgctac agcgagtgga ctccggaggg 4500

ccggctgcgt caatcaagct ggcgtgggct gcggccggac aagaaaccca gtgaggtggt 4560

gcgcgaatga taagttggct gctgccaccg ctgagcaata actagcataa ccccttgggg 4620

cctctaaacg ggtcttgagg ggttttttgc ccatgcaaca gaaactataa aaaatacaga 4680

gaatgaaaag aaacagatag attttttagt tctttaggcc cgtagtctgc aaatcctttt 4740

atgattttct atcaaacaaa agaggaaaat agaccagttg caatccaaac gagagtctaa 4800

tagaatgagg tcgaaaagta aatcgcgcgg gtttgttact gataaagcag gcaagaccta 4860

aaatgtgtaa agggcaaagt gtatactttg gcgtcacccc ttacatattt taggtctttt 4920

tttattgtgc gtaactaact tgccatcttc aaacaggagg gctggaagaa gcagaccgct 4980

aacacagtac ataaaaaagg agacatgaac gatgcgagcc atttggacgg gttcgatcgc 5040

cttcgggctg gtgaacgtgc cggtcaaggt gtacagcgct accgcagacc acgacatcag 5100

gttccaccag gtgcacgcca aggacaacgg acgcatccgg tacaagcgcg tctgcgaggc 5160

gtgtggcgag gtggtcgact accgcgatct tgcccgggcc tacgagtccg gcgacggcca 5220

aatggtggcg atcaccgacg acgacatcgc cagcttgcct gaagaacgca gccgggagat 5280

cgaggtgttg gagttcgtcc ccgccgccga cgtggacccg atgatgttcg accgcagcta 5340

ctttttggag cctgattcga agtcgtcgaa atcgtatgtg ctgctggcta agacactcgc 5400

cgagaccgac cggatggcga tcgtgcattt cacgctgcgc aacaagacca ggctggcggc 5460

gttgcgcgtc aaggatttcg gcaagcgaga ggtgatgatg gtgcacacgt tgctgtggcc 5520

cgatgagatc cgcgaccccg acttcccggt gctggaccag aaggtggaga tcaaacccgc 5580

ggaactcaag atggccggcc aggtggtgga ctcgatggcc gacgacttca atccggaccg 5640

ctaccacgac acctaccagg agcagttaca ggagctgatc gacaccaaac tcgaaggtgg 5700

gcaggcattt accgccgagg accaaccgag gttgctggac gagcccgaag acgtctccga 5760

cctgctcgcc aagctggagg ccagcgtgaa ggcgcgctcg aaggccaact caaacgtccc 5820

aacgcctccg tgacgaaatt caaattgtga gcggcgaaat tcaaattgtg agcggataac 5880

aatttgaatt ttctgtatga ggttttgcta aacaactttc aacagtttca gtggagtgag 5940

aatagaaagg aacaactaaa ggaattgcga ataataattt tttcacgttg aaaatctcca 6000

aaaaaaaagg ctccaaaagg agcctttaat tgtatcggtt tatcagcttg ctttcgaggt 6060

gaattttgac cctctagcga aaatgcaaga gcaaagacga aaacatgcca cacatgagga 6120

ataccgattc tctcattaac atattcaggc cagttatctg ggcttaaaag cagaagtcca 6180

acccagataa cgatcatata catggttctc tccagaggtt cattactgaa cactcgtccg 6240

agaataacga gtggatccac tagttctaga gcggccgcca ccgcggtgga gctccagctt 6300

ttgttccctt tagtgagggt taattccgag cttggcgtaa tcatggtcat agctgtttcc 6360

tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 6420

taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 6480

cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 6540

gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 6600

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 6660

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 6720

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 6780

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 6840

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 6900

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 6960

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 7020

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 7080

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 7140

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga cagtatttgg 7200

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 7260

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 7320

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 7380

cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 7440

ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 7500

tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 7560

atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 7620

tggccccagt gctgcaatga taccgccggt catttcgaac cccagagtcc cgcccatggt 7680

caggggcagg gcatgctcat gtagagcgcc tgctcgccgt ccgaggcggt gccgtcgtac 7740

agggcggtgt ccaggccgca gagggtgaac cccatccgcc ggtacgcgtg gatcgccggt 7800

gcgttgacgt tggtgacctc cagccagagg tgcccggcgc cccgctcgcg ggcgaactcc 7860

gtcgcgagcc ccatcaacgc gcgcccgacc ccgtgccccc ggtgctccgg ggcgacctcg 7920

atgtcctcga cggtcagccg gcggttccag ccggagtacg agacgaccac gaagcccgcc 7980

aggtcgccgt cgtccccgta cgcgacgaac gtccgggagt ccgggtcgcc gtcctccccg 8040

gcgtccgatt cgtcgtccga ttcgtcgtcg gggaacacct tggtcagggg cgggtccacc 8100

ggcacctccc gcagggtgaa gccgtccccg gtggcggtga cgcggaagac ggtgtcggtg 8160

gtgaaggacc catccagtgc ctcgatggcc tcggcgtccc ccgggacact ggtgcggtac 8220

cggtaagccg tgtcgtcaag agtggtcata tggatccggc caccgtttgc ttttaacgtc 8280

agccttttac ctaaattccc atcaagcgat cttttaatat cggacaacgt cttcgccatt 8340

gcatccacct cactacattt attgtacaac acgagcccat ttttgtcaaa taaaatttaa 8400

attatatcaa cgttaataag acgttgtcaa taaaattatt ttgacaaaat ttcctttgct 8460

tgtttgctct attcctataa ttcgatgata agctgtcaaa catgagagat cttgatcccc 8520

tgcgccatca gatccttggc ggcaagaaag ccatccagtt tactttgcag ggcttcccaa 8580

ccttaccaga gggcgcccca gctggcaatt ccggttcgct tgctgtccat aaaaccgccc 8640

agtctagcta tcgccatgta agcccactgc aagctacctg ctttctcttt gcgcttgcgt 8700

tttcccttgt ccagatagcc cagtagctga cattcatccg gggtcagcac cgtttctgcg 8760

gactggcttt ctacgtgttc cgcttccttt agcagccctt gcgccctgag tgcttgcggc 8820

agcgtgaagt atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag 8880

gcg 8883

<210> 57

<211> 9652

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC02-Ec载体系统

<400> 57

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgccgaaa gaggattttg ataaagcctg catttatctg 480

agcaacttcg ataaattcag cacctatttt gtgggcttta atcagaatcg cgaaaacctg 540

tataccgatg aagaacaggc aaccgcaatt ccgtatcgta ttatcaatga taacatggtg 600

cgccactttg ataactgccg taaatttgag aagatcgtga aaaagtatgg cgacattagc 660

aatgttctga gcacctataa agaatttttc gcaccggatt gcttcaaaaa caaactgaat 720

cagagccaga tcgaccacta taataacacc attggtcata ccgcagatga tatttatggt 780

gtgggtatta accagatcct gagcaaatac aaacaggaca ataaactgaa tagcagcgat 840

ctgccgctga ttagcaaact gtataaacaa attctgagcg acaccgaaag ctatgccatt 900

gaaaattttg ccgacgacaa aatgatgctg aacgccgttg ataaagaata cagccgcatt 960

aaagaaaacg acgtgttcat taacatcgaa acctgcatga atgaatacct gacactggaa 1020

aatagccaca tgatctatct gaaaaatgat agcagcctga ccgacatcag taataaactg 1080

tgggaagatt gggcctttgt gaaaaatgcc attcagaagt acagcaaaga aattctgtgt 1140

ctgtccgaca aaaagatcga ggatatgctg aaaatgagcc actatagcat tagctttgtt 1200

cagaacagcg tgtattatta cgtggataac tatatggaaa gctgcgagga taaacgcaaa 1260

agcatcatcg attacatcaa gaccttctat agcatcaaat acaacaacgt gtttagctgc 1320

tataaagagg cagaagcagt tctgcgtctg gatagcattc ataaaaatcg tcgtagtccg 1380

gtggataaaa atggtattgg tggtgaaggt tttgcccaga ttgagaaaat caaaaacttt 1440

ctggacagca tcctggaagt gaagaatttt ctgaatccgc tgtatctgat caaaagcggt 1500

aaaatggcag aaatcgaaga taagagcgaa gagttttata atcgcttcaa cgagctgtat 1560

aatagcctga gcgataccac ctatctgtat aacaaagtgc gtaactacct gaccaaaaag 1620

ccgtacaaga aagaaaaatt caaaatgaac tttgaaaaca gcaccctgct gagcggttgg 1680

gatgttaata aagaaaattg ctccaacagc atcatcctga ttcgcaatgg taaatactat 1740

ctggggatca tcgataaaca gtgcggcaat atgttcaact tcaaaatcga tgccgaagat 1800

aacgagaaaa agcgcaaaga aaaagaagat ctggcggaag atattctgag tgatggtagc 1860

gatagctact atgagaaaat ggtgtataaa ctgctgccgg atccgagcaa aatgctgccg 1920

aaagtgtttt tcagcaacaa gagcattgat ttctatgcac cgagcgagga cattaaatac 1980

attcgtgaaa atggcctgtt caagaaagac gccaaaaata agaaagccct gtatatctgg 2040

atcgaattta tgcagaacag cctgaaaaag catccggaat ggtccaacta ttttaacttt 2100

aacttcaaac cgagcaccga gtatgcagat gttagcgaat tctataaaca ggttagcgat 2160

cagggttatt ccctgagctt tgataagatc aaagatagct atatcgagag caagatcaaa 2220

agtggtgaac tgtttctgtt cgagatctac aacaaagatt tcagcccgta tagcaaaggt 2280

aatccgaacc tgcataccat ctattggaaa tccatctttg acaaagagaa cctgagcaac 2340

gttgtgatta aactgaacgg tcaggccgaa atctttttcc gtccggcaag cctgaaacgt 2400

aatgaagttg ttgttcatcg tgcgaaagaa aacatcctga ataaaaaccc gctgaacccg 2460

aagaaagaat ccatgtttga atacgacatc gtgaaagata aacgctacac ccaggacaaa 2520

ttctttttcc attgtccgat tacgctgaac tttaaaagcg gcaatgttgg caaattcaac 2580

gacaaagtta accagtttct gaagaataac ccggatgtga atgtgattgg ttttgatcgt 2640

ggtgaacgtc atctgctgta ttgtaatgtg ctgaaccaga aaggcgaaat cattgaacag 2700

aaaagcttta acgtgatcga gaacaaaaac aacggcatta cccagaaagt ggattatcat 2760

aatctgctgg atcgtaaaga gaaagaacgc gacgcaagcc gtaaaagctg gtcaacaatt 2820

gaaaacatca aagagctgaa agagggctat ctgtcaaacg ttgttcatga aattagcgag 2880

ctgatcatca agtataatgc gattctggtt cttgaggatc tgaacttcga atttaagaaa 2940

ggtcgcttca agatcgagaa acaggtgtat cagaaattcg aaaaagccct gattgacaag 3000

ctgagctata tggtgtttaa gaaagaagag agcaacaaac cgggtcatag cctgatggca 3060

tatcagctgg caagcccgtt tgaaagcttc caaaaactgg gtaaacagtg tggctttatc 3120

ttctatgtga acagcaacta caccagcaaa attgatccgg ttaccggttt tgtgaatctg 3180

ctgaagatca aatatgagag cgtggacaaa agctgcaaat tcatcaacga taagttcgat 3240

gatatccgct ataatgccga tcgcgaatat tttgagttta ccttcgataa tggcaaatgg 3300

accgcatgta gtcatggtaa agaacgttat cgctataatc gcaacgacaa gaaatacaac 3360

tgtttcgacg ttaccgaaga actgaaaagc ctgtttaaca aatacgagat cgatttcaaa 3420

gcaggcacgg atattaagaa aagcatttgt caggtgcagg acaagaactt tcatagcgaa 3480

ctgctgttta atctgagcct gattgttcag ctgcgtcata cctataaaaa cggcgatatc 3540

gagaaagact ttattctgtc accgattatg gataaagaaa ccggcaaatt tttcgacagc 3600

cgtgaatatg aaaatctgga aaacagcctg ctgccgacca atgcagatag caatggtgca 3660

tataacattg cacgtaaagg tctgctgacc ctgcgtcaga tcgataaaga tggtaaaccg 3720

tccaacatct ccaataaaga atggtttgat tttgtgcaga aataatggtc tagaggtcga 3780

aattcaaatt gtgagcggat aacaatttga attttctgta tgaggttttg ctaaacaact 3840

ttcaacagtt tcagtggagt gagaatagaa aggaacaact aaaggaattg cgaataataa 3900

ttttttcacg ttgaaaatct ccaaaaaaaa aggctccaaa aggagccttt aattgtatcg 3960

gtttatcagc ttgctttcga ggtgaatttt gaccctctag cgaaaatgca agagcaaaga 4020

cgaaaacatg ccacacatga ggaataccga ttctctcatt aacatattca ggccagttat 4080

ctgggcttaa aagcagaagt ccaacccaga taacgatcat atacatggtt ctctccagag 4140

gttcattact gaacactcgt ccgagaataa cgagtggatc ccctccaatt cgccctatag 4200

tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc 4260

tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag 4320

cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa 4380

attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt 4440

tttaaccaat aggccgactg cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt 4500

tattggtgcc cttaaacgcc tggtgctacg cctgaataag tgataataag cggatgaatg 4560

gcagaaattc gaaagcaaat tcgacccggt cgtcggttca gggcagggtc gttaaatagc 4620

cgcttatgtc tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg 4680

cttctcaaat gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga 4740

tatgatcatt tattctgcct cccagagcct gataaaaacg gtgaatccgt tagcgaggtg 4800

ccgccggctt ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg 4860

aggcagacaa ggtatagggc ggcgaggcgg ctacagccga tagtctggaa cagcgcactt 4920

acgggttgct gcgcaaccca agtgctaccg gcgcggcagc gtgacccgtg tcggcggctc 4980

caacggctcg ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc 5040

gcgccgttcc cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat 5100

gccgggctgg ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata 5160

cagggtcggg atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg 5220

atcaaccacc acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca 5280

cgccacgcgg tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac 5340

ggagatccag cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg 5400

tccgatgagc ttggaaagtg tcttttggct gaccaccacg gcgttctggt ggcccatctg 5460

cgccacgagg tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca 5520

cgcctcatgc gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat 5580

gccgatttct ctggactgcg tggccatgct tatctccatg cggtagggtg ccgcacggtt 5640

gcggcaccat gcgcaatcag ctgcaacttt tcggcagcgc gacaacaatt atgcgttgcg 5700

taaaagtggc agtcaattac agattttctt taacctacgc aatgagctat tgcggggggt 5760

gccgcaatga gctgttgcgt accccccttt tttaagttgt tgatttttaa gtctttcgca 5820

tttcgcccta tatctagttc tttggtgccc aaagaagggc acccctgcgg ggttccccca 5880

cgccttcggc gcggctcccc ctccggcaaa aagtggcccc tccggggctt gttgatcgac 5940

tgcgcggcct tcggccttgc ccaaggtggc gctgccccct tggaaccccc gcactcgccg 6000

ccgtgaggct cggggggcag gcgggcgggc ttcgccttcg actgccccca ctcgcatagg 6060

cttgggtcgt tccaggcgcg tcaaggccaa gccgctgcgc ggtcgctgcg cgagccttga 6120

cccgccttcc acttggtgtc caaccggcaa gcgaagcgcg caggccgcag gccggaggct 6180

tttccccaga gaaaattaaa aaaattgatg gggcaaggcc gcaggccgcg cagttggagc 6240

cggtgggtat gtggtcgaag gctgggtagc cggtgggcaa tccctgtggt caagctcgtg 6300

ggcaggcgca gcctgtccat cagcttgtcc agcagggttg tccacgggcc gagcgaagcg 6360

agccagccgg tggccgctcg cggccatcgt ccacatatcc acgggctggc aagggagcgc 6420

agcgaccgcg cagggcgaag cccggagagc aagcccgtag ggcgccgcag ccgccgtagg 6480

cggtcacgac tttgcgaagc aaagtctagt gagtatactc aagcattgag tggcccgccg 6540

gaggcaccgc cttgcgctgc ccccgtcgag ccggttggac accaaaaggg aggggcaggc 6600

atggcggcat acgcgatcat gcgatgcaag aagctggcga aaatgggcaa cgtggcggcc 6660

agtctcaagc acgcctaccg cgagcgcgag acgcccaacg ctgacgccag caggacgcca 6720

gagaacgagc actgggcggc cagcagcacc gatgaagcga tgggccgact gcgcgagttg 6780

ctgccagaga agcggcgcaa ggacgctgtg ttggcggtcg agtacgtcat gacggccagc 6840

ccggaatggt ggaagtcggc cagccaagaa cagcaggcgg cgttcttcga gaaggcgcac 6900

aagtggctgg cggacaagta cggggcggat cgcatcgtga cggccagcat ccaccgtgac 6960

gaaaccagcc cgcacatgac cgcgttcgtg gtgccgctga cgcaggacgg caggctgtcg 7020

gccaaggagt tcatcggcaa caaagcgcag atgacccgcg accagaccac gtttgcggcc 7080

gctgtggccg atctagggct gcaacggggc atcgagggca gcaaggcacg tcacacgcgc 7140

attcaggcgt tctacgaggc cctggagcgg ccaccagtgg gccacgtcac catcagcccg 7200

caagcggtcg agccacgcgc ctatgcaccg cagggattgg ccgaaaagct gggaatctca 7260

aagcgcgttg agacgccgga agccgtggcc gaccggctga caaaagcggt tcggcagggg 7320

tatgagcctg ccctacaggc cgccgcagga gcgcgtgaga tgcgcaagaa ggccgatcaa 7380

gcccaagaga cggcccgaga ccttcgggag cgcctgaagc ccgttctgga cgccctgggg 7440

ccgttgaatc gggatatgca ggccaaggcc gccgcgatca tcaaggccgt gggcgaaaag 7500

ctgctgacgg aacagcggga agtccagcgc cagaaacagg cccagcgcca gcaggaacgc 7560

gggcgcgcac atttccccga aaagtgccac ctgaacccca gagtcccgct cagaagaact 7620

cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 7680

cgaggaagcg gtcagcccat tcgccgccaa gctcttcagc aatatcacgg gtagccaacg 7740

ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 7800

ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct 7860

cgccgtcggg catccgcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 7920

gctcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 7980

cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 8040

gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 8100

gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 8160

cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 8220

cttggagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 8280

gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 8340

agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 8400

tcatgcgaaa cgatcctcat cctgtctctt gatcagatct tgatcccctg cgccatcaga 8460

tccttggcgg caagaaagcc atccagttta ctttgcaggg cttcccaacc ttaccagagg 8520

gcgccccagc tggcaattcc ggttcgcttg ctgtccataa aaccgcccag tctagctatc 8580

gccatgtaag cccactgcaa gctacctgct ttctctttgc gcttgcgttt tcccttgtcc 8640

agatagccca gtagctgaca ttcatccggg gtcagcaccg tttctgcgga ctggctttct 8700

acgtgttccg cttcctttag cagcccttgc gccctgagtg cttgcggcag cgtgaagcta 8760

gctgcataat gtgcctgtca aatggacgaa gcagggattc tgcaaaccct atgctactcc 8820

gtcaagccgt caattgtctg attcgttacc aattatgaca acttgacggc tacatcattc 8880

actttttctt cacaaccggc acggaactcg ctcgggctgg ccccggtgca ttttttaaat 8940

acccgcgaga aatagagttg atcgtcaaaa ccaacattgc gaccgacggt ggcgataggc 9000

atccgggtgg tgctcaaaag cagcttcgcc tggctgatac gttggtcctc gcgccagctt 9060

aagacgctaa tccctaactg ctggcggaaa agatgtgaca gacgcgacgg cgacaagcaa 9120

acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg ccaggtgatc gctgatgtac 9180

tgacaagcct cgcgtacccg attatccatc ggtggatgga gcgactcgtt aatcgcttcc 9240

atgcgccgca gtaacaattg ctcaagcaga tttatcgcca gcagctccga atagcgccct 9300

tccccttgcc cggcgttaat gatttgccca aacaggtcgc tgaaatgcgg ctggtgcgct 9360

tcatccgggc gaaagaaccc cgtattggca aatattgacg gccagttaag ccattcatgc 9420

cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc attcgcgagc ctccggatga 9480

cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa tatcacccgg tcggcaaaca 9540

aattctcgtc cctgattttt caccaccccc tgaccgcgaa tggtgagatt gagaatataa 9600

cctttcattc ccagcggtcg gtcgataaaa aaatcgagat aaccgttggc ct 9652

<210> 58

<211> 10021

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC05-Ec载体系统

<400> 58

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgcgctgg gatgaaaaac tgcagacctt tctgaatgat 480

caagaaatcg aagatgccta ccaggttctg aaaccggtgt ttgataaact gcacgaaaac 540

tttattatcg gcagcctgga aaataccaac aacaaaaagc tgttcagctt cgacaaatac 600

ctgaaactga aaaacgatct gctgcacgtg aataagaaag aacaagagag cgactacaaa 660

aagaaagaga aagaatttga aaccgagggt aaactgctgc gtaatacctt tgcaaccgtt 720

tggattaatg agggcaaaaa cttcaagaac accattgttg gtggtgaaaa cgatcgcgaa 780

attctgaaag aaggtggcta taaaatcctg accgaagcag gtattctgaa atatatcaag 840

atgaacatcg ataaatttgt ggaactgaag ctgaaaaccc gtgaagatat tctgtggaag 900

aaagaaaatc gcaacctggt tgaaatggca gatctggaaa aatcactggg caccattgaa 960

agctggggtg tttttgaagg tttcttcacc tatttcagcg gctttaatca gaaccgcgaa 1020

aactattata gcaccgatga aaaagcaacc gcagttgcaa gccgtgttat tgatgaaaat 1080

ctgccgaaat ttagcgacaa cgtgctggaa ttcaataaaa agaacgatgt gtacatcggc 1140

atctttagct ttctgaaagg caaaaacatt gtgctgaaag gtaaaagcgg taatggtgaa 1200

gaacaggacc tgctgccgat taccgaaaag atttttgaaa tcgagtactt taaaaactgc 1260

ctgagcgaag gtgaaatcga acgttataat agcgatattg gcaacgccaa ctttctgatc 1320

aatctgtata accagcagca ggacaagaaa gaaaacaaac tgcgtatctt caagaccctg 1380

tataaacaaa ttggctgcgg tatcaaaggc gattttattc agctgatcaa aaccgatgat 1440

gagctgaaga agatctttga ggatctgaaa attaccggtg acaacttctt taagaatacc 1500

cagaacctga aagagattat cctgtcgctg gaaaacttca gcggtattta ttggagcgat 1560

aaagcactga ataccgtgag cggtaaatac tttgcaaatt gggcaagcct gaaagaactg 1620

ctgaaaaatg ccaaaatctt taagaaagaa aaagacgaga tcaaaatccc gcagaccatt 1680

gaactgagcg acctgtttgg tgttctggat agcaatgaac tgatcttcaa agaaagcttc 1740

aacgagaacg acgagctgaa acaaatcatc ctgaaaagct atgagaaaaa cagcatcaag 1800

ctgctgaaga tgattttcgt ggatgttgaa gagaaccaga aaatctttgg caatctgaaa 1860

gatggtctgc cgatcaacga tttcaagaaa gatgaaaaca cccagatcat caaaacctgg 1920

ctggatggcc tgctgaatac caatcagatc ctgaaatact tcaaagtgcg cgagagcaaa 1980

atcaaaggtg caccgctgaa tccggaagtt agcgaacgtc tgaataagat tctgaatgtt 2040

gaaaatccga ccgtgatcta tgatgtggtt cgtaattatc tgaccaaaaa gccgaccgaa 2100

ggcctgaata aactgaaact taattttgat aatgccgtgc tggcagccgg ttgggatgtt 2160

aataaagaaa gcgaacgtgg ttgcctgatt ctgaaggatg gtgataataa gaaatatctg 2220

gccatcctga ccaacaagac ccagaaattt ttcggtgaga aggtgaagta caaagaattc 2280

gtgggtgatg aaaactggca gaaaatggat tataaactgt taccgggtcc gaataagatg 2340

ctgccgaaag ttctgctgcc taaaagcgat cgttacaaat ttggtgccac agacgaaatc 2400

ctgaagattt ataacgaagg cggtttcaag aagaacgaac cgacctttac caaagcaaaa 2460

ctggccaaaa ttgtggactt tttcaaagac ggcctgaaaa attacccgag cgcaaaaagc 2520

agctggtata acctgtttgc atttgatttt agcgataccg agaaatacga aagcatcgat 2580

cgtttctata ccgaggttga aaaacagggc tataaactga gctggtcagc cattagcaaa 2640

aatttcatct tcgaaaaagt ggacgcaggc gatatgtacc tgtttgaaat tcgcaacaaa 2700

gataacaacc tgaagaacgg taaagcaaaa accggtgcca aaaatctgca taccatttat 2760

tggggtacga tctttggtga aagcgagaac aaaccgaaac tgaatggcga agcagaaatc 2820

ttttatcgtc cggttgttaa agacctgatc aaagacaagg ataaaaacgg cgatattatc 2880

aaagccagcg aaaaacgctt tgagcaagaa aaatttgtgt ttcattgccc gatcacgctg 2940

aacttttgtc tgaaaagcac acgcctgaat gatgtgatta accagattat gatcgagaat 3000

aaaaaggatg tgtgctttat tggcattgac cgtggcgaaa aacatctggc atattatagc 3060

gttgtgaatc agaaagggga aattctggaa cagggtagct ttaatgaaat taacggtcag 3120

aactacgcca agaaactgga agagaaagca ggtcatcgtg atgaagcacg taaaaactgg 3180

aaaaccattg gcacgatcaa agaactgaag aatggttata ttagccaggt ggttcgtcgt 3240

attgttgatc tggcagtgaa atataacgcc tacattgttc tggaagatct gaatagcggt 3300

tttaaacgtg gtcgtcagaa aattgagaaa agcgtgtatc agaaattaga actggcactg 3360

gctaaaaagc tgaatttcct ggttgacaaa agcaagaaag acggtgaaat tggtagcgtg 3420

cagaaagcac tgcagctgac ccctccggca accaattttg cagatattga aaaagcgaaa 3480

cagtttggca tcatgctgta tgttcgtgcg aattacacca gccagaccga tccggttacc 3540

ggttggcgta aaaccattta ctttaaatca accacgcaag agaacttaaa gaaagaaatc 3600

tgcgagaaat tctccgagat cggctttgat ggtaacgatt attacttcga gtataaggat 3660

gaaaacgccg agaagaaatg gaccatgtat agcggtgtta gcggtaaaag cctggatcgc 3720

tttcgtggta agaaagatac ccatggtatt tggaaagtgg aaaagcagga tattgtcgag 3780

ctgttaaaga agattttcgg tcagcagacc agcgttgttg gtgacctgaa aaccaaaatt 3840

accaacgata atgtgaacga tctgaaatac accatcgatc tgattcagca gattcgcaat 3900

acgggtttta acgagatcga taacgacttt attctgagtc cggttcgtga tgagaaaggc 3960

aatcattttg atagccgtaa agatggtgca attctgagca atggtgatgc aaatggtgca 4020

tataacattg cccgtaaagg tgtgctggca tttgaacgta ttaatgcaaa accggaaaag 4080

cccgaactgt atattgccga tgttgaatgg gataaatggc tgcagagcaa ataatggtct 4140

agaggtcgaa attcaaattg tgagcggata acaatttgaa ttttctgtat gaggttttgc 4200

taaacaactt tcaacagttt cagtggagtg agaatagaaa ggaacaacta aaggaattgc 4260

gaataataat tttttcacgt tgaaaatctc caaaaaaaaa ggctccaaaa ggagccttta 4320

attgtatcgg tttatcagct tgctttcgag gtgaattttg accctctagc gaaaatgcaa 4380

gagcaaagac gaaaacatgc cacacatgag gaataccgat tctctcatta acatattcag 4440

gccagttatc tgggcttaaa agcagaagtc caacccagat aacgatcata tacatggttc 4500

tctccagagg ttcattactg aacactcgtc cgagaataac gagtggatcc cctccaattc 4560

gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg 4620

ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 4680

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 4740

cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 4800

agctcatttt ttaaccaata ggccgactgc gatgagtggc agggcggggc gtaatttttt 4860

taaggcagtt attggtgccc ttaaacgcct ggtgctacgc ctgaataagt gataataagc 4920

ggatgaatgg cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg 4980

ttaaatagcc gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt 5040

gaccgtgtgc ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat 5100

aattgacgat atgatcattt attctgcctc ccagagcctg ataaaaacgg tgaatccgtt 5160

agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc 5220

aacgcgggga ggcagacaag gtatagggcg gcgaggcggc tacagccgat agtctggaac 5280

agcgcactta cgggttgctg cgcaacccaa gtgctaccgg cgcggcagcg tgacccgtgt 5340

cggcggctcc aacggctcgc catcgtccag aaaacacggc tcatcgggca tcggcaggcg 5400

ctgctgcccg cgccgttccc attcctccgt ttcggtcaag gctggcaggt ctggttccat 5460

gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta gttgctgctc 5520

gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg attcgtcctg 5580

gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact ggtcgcgggg 5640

ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg ggccgttgag 5700

cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt ggaccgtccg 5760

caaagaacgt ccgatgagct tggaaagtgt cttttggctg accaccacgg cgttctggtg 5820

gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc tcgcaataag 5880

cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg gcttgttctt 5940

ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc ggtagggtgc 6000

cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta 6060

tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca atgagctatt 6120

gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag 6180

tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg 6240

gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg 6300

ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg 6360

cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgccttcga ctgcccccac 6420

tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc 6480

gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg 6540

ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc 6600

agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc 6660

aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt ccacgggccg 6720

agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca cgggctggca 6780

agggagcgca gcgaccgcgc agggcgaagc ccggagagca agcccgtagg gcgccgcagc 6840

cgccgtaggc ggtcacgact ttgcgaagca aagtctagtg agtatactca agcattgagt 6900

ggcccgccgg aggcaccgcc ttgcgctgcc cccgtcgagc cggttggaca ccaaaaggga 6960

ggggcaggca tggcggcata cgcgatcatg cgatgcaaga agctggcgaa aatgggcaac 7020

gtggcggcca gtctcaagca cgcctaccgc gagcgcgaga cgcccaacgc tgacgccagc 7080

aggacgccag agaacgagca ctgggcggcc agcagcaccg atgaagcgat gggccgactg 7140

cgcgagttgc tgccagagaa gcggcgcaag gacgctgtgt tggcggtcga gtacgtcatg 7200

acggccagcc cggaatggtg gaagtcggcc agccaagaac agcaggcggc gttcttcgag 7260

aaggcgcaca agtggctggc ggacaagtac ggggcggatc gcatcgtgac ggccagcatc 7320

caccgtgacg aaaccagccc gcacatgacc gcgttcgtgg tgccgctgac gcaggacggc 7380

aggctgtcgg ccaaggagtt catcggcaac aaagcgcaga tgacccgcga ccagaccacg 7440

tttgcggccg ctgtggccga tctagggctg caacggggca tcgagggcag caaggcacgt 7500

cacacgcgca ttcaggcgtt ctacgaggcc ctggagcggc caccagtggg ccacgtcacc 7560

atcagcccgc aagcggtcga gccacgcgcc tatgcaccgc agggattggc cgaaaagctg 7620

ggaatctcaa agcgcgttga gacgccggaa gccgtggccg accggctgac aaaagcggtt 7680

cggcaggggt atgagcctgc cctacaggcc gccgcaggag cgcgtgagat gcgcaagaag 7740

gccgatcaag cccaagagac ggcccgagac cttcgggagc gcctgaagcc cgttctggac 7800

gccctggggc cgttgaatcg ggatatgcag gccaaggccg ccgcgatcat caaggccgtg 7860

ggcgaaaagc tgctgacgga acagcgggaa gtccagcgcc agaaacaggc ccagcgccag 7920

caggaacgcg ggcgcgcaca tttccccgaa aagtgccacc tgaaccccag agtcccgctc 7980

agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac 8040

cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg 8100

tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc 8160

cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga 8220

cgagatcctc gccgtcgggc atccgcgcct tgagcctggc gaacagttcg gctggcgcga 8280

gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac 8340

gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg 8400

tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag 8460

atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag 8520

tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg 8580

ctgcctcgtc ttggagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg 8640

ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg 8700

cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat 8760

cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatctt gatcccctgc 8820

gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct 8880

taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa accgcccagt 8940

ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt 9000

cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac 9060

tggctttcta cgtgttccgc ttcctttagc agcccttgcg ccctgagtgc ttgcggcagc 9120

gtgaagctag ctgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta 9180

tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct 9240

acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat 9300

tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg 9360

gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg 9420

cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc 9480

gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg 9540

ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta 9600

atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa 9660

tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc 9720

tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc 9780

cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc 9840

tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt 9900

cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg 9960

agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc 10020

t 10021

<210> 59

<211> 10153

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC07-Ec载体系统

<400> 59

ccatgcttat ctccatgcgg tagggtgccg cacggttgcg gcaccatgcg caatcagctg 60

caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga 120

ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc 180

cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt 240

ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc 300

cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca 360

aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg 420

ggcgggcttc gccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca 480

aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa 540

ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa 600

attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct 660

gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag 720

cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg 780

ccatcgtcca catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc 840

ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa 900

gtctagtgag tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc 960

cgtcgagccg gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg 1020

atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga 1080

gcgcgagacg cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag 1140

cagcaccgat gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga 1200

cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag 1260

ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg 1320

ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc 1380

gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa 1440

agcgcagatg acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca 1500

acggggcatc gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct 1560

ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta 1620

tgcaccgcag ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc 1680

cgtggccgac cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc 1740

cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct 1800

tcgggagcgc ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc 1860

caaggccgcc gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt 1920

ccagcgccag aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa 1980

gtgccacctg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg 2040

atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg 2100

ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc 2160

acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc 2220

ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg 2280

agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga 2340

tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg 2400

tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg 2460

gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc 2520

aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg 2580

cccgtcgtgg ccagccacga tagccgcgct gcctcgtctt ggagttcatt cagggcaccg 2640

gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg 2700

gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa 2760

gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct 2820

gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc 2880

cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt 2940

tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct 3000

acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc 3060

atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag 3120

cccttgcgcc ctgagtgctt gcggcagcgt gaagctagct gcataatgtg cctgtcaaat 3180

ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt 3240

cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg 3300

gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc 3360

gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag 3420

cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg 3480

gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat 3540

atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt 3600

atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc 3660

aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat 3720

ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt 3780

attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta 3840

aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc 3900

tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac 3960

caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc 4020

gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc 4080

attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact 4140

cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt 4200

cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta 4260

acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag 4320

aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc 4380

cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata 4440

cccgtttttt ggtaccgggc cccccctcga gtttatttta ggaggcaaaa atggataacg 4500

cctttagcga tttcacccag aaatataccc tgagcaaaac cctgcgtttt gaactgcgtc 4560

cggttggtaa taccgaaaaa atgctggaag atgagaaagt gttcgagaaa gacaaactga 4620

tccaagagaa atatatcaaa accaaaccgt acttcgatct gctgcatcgt gaatttgttg 4680

aagaggcact gaaagatgtt gatattagcg gtctgcacaa ctactttgaa acctatcaga 4740

aatgggccaa agacaagaag aaataccaga aagaactgca gaacaaagag cagattctgc 4800

gcaaagaaat tctggttttt ctggatagca ccgccaaata ttgggcagaa aaaaagtata 4860

gcgaactgcg catcaaaaag aaggatatcg aaatcttttt cgaagaggac gtgttcacca 4920

ttctgaaaaa acgttatggt gaagatagcg aagcccagat tattgatgaa gttagcggtg 4980

aaaccgtgag catttttgat agctggaaag gttttaccgg ctacttcaaa aaattccaag 5040

aaacccgcaa aaacctgtat cgtgatgatg gcaccgcaac cgcacatgca acccgtatta 5100

ttgatcagaa tctgaaacgc ttttgcgata acctggaaat cattaaacgt atcgcaggca 5160

tcatcgaatt tagcgaagtt gaaggcaact tcaaacatag catgggtgat gtttttagcc 5220

tgagcttcta taacaaatgt ctgctgcagg atggcatcaa cttttataac cgtattttag 5280

gtggtgaggt tctgcaggac ggcaccaaac tgaaaggtat taatgaactg atcaacaagt 5340

atcgccagga taacaaaggt gtgaaaatcc cgtttctgaa actgctggat aaacaaatcc 5400

tgagcgaaaa agaagaattt ctggacggca tcgaagatga taaagagctg ctggcagtac 5460

tgaaaaaatt ctatgaagtg gccgagaaaa aaaccagcat cctgaaaagc ctgattcagg 5520

attttgcaca gaataaccgt cagtacaatc tggaagaagt gtacattagc aaagaagcct 5580

ttaataccat tagccgcaaa tggacccatg aaaccagcaa atttgaagag tggctgtaca 5640

atgtgatgaa accgaataaa ccgaccggtc tgaaatacga caaaaaagag gaaagctata 5700

agttcccgga ttttattccg ctgagctata ttcagaccgc actggaacag gcagatattg 5760

atggtgattt ttggaaagaa cactacagcg aaaatagcaa agccaatgat ggttgtctga 5820

tgggagatga aagcatttgg gaacagttta tcaagatctt cgagtatgaa ttccagagcc 5880

tgtttgaaaa agaaatcatt gatcgtgaaa ccggtcagcc gaagaaaaat ggttataact 5940

atgtgaagga cgacttcaaa ggtctgctga atggcgaaaa ctttagcgtc gaaatcatca 6000

aagattttgc cgataccgtg ctgagcattt atcagatggc caaatacttt gccatcgaga 6060

aaaagcgtaa atggctggat gaatatgata ccggtgattt ctatgaaaac ccggaatttg 6120

gctacaaact gttctatgat gatgcgtata aagaaatcgt gcagacctat aataacctgc 6180

gcaattacct gaccaaaaaa tcctatagcg aagagaaatg gaagctgaat tttgaaaatc 6240

cgacactggc agatggttgg gataaaaaca aagaaccgga taattcagcc gtgatcctgc 6300

gtaaagatgg tcgttattat ctgggcctga tgaaaaaagg ctgcaacaaa attttcgacg 6360

accgcaacaa agtggaattt agtggtggtg tggataaaga caaatacgag aaaatcgtgt 6420

acaagttctt tccggatcag gcaaaaatgt ttccgaaagt ttgttttagc gcgaaaggcc 6480

tggatttttt tcagccgagc gaggaaattc tgaacatcta taaaaacagc gagttcaaaa 6540

aaggcgatac ctttagcgtt cagagcatgc agaaactgat cgatttctat aaagattgcc 6600

tgaccaagta tgaaggctgg attgcctatg aattcaaaca tctgaaaagc accgatctgt 6660

atcgcaataa catcagcgaa tttttcagtg atgttgccga agatggctat aaaatcacct 6720

ttcaggatat cagcgataac tacattgata aaaagaacca gtccgaggaa ctgtacctgt 6780

tcgaaattca taacaaagac tggaacctga aagacgaggt gaaaaaaaca ggtagcaaaa 6840

atctgcacac cctgtatttt gaagcactgt ttagccatga aaacatccag aacaactttc 6900

cgattaaact gaatggtcag gccgaagttt tttatcgtcc gaaaaccgat gaagagaaac 6960

tggtgaagaa aaaggataaa aaaggccgtg aagtgatcga ccataaacgc tatgcagaaa 7020

acaaaatctt ctttcatgtt ccgctgacac tgaatcgtgg taaaggtgat gcatatcagt 7080

ttaacgccaa gatcaataac tttctggcca acaacagcga tattaatgtt attggtgttg 7140

accgtggcga aaaacatctg gcatattata gcgtgattaa ccagaaaggc gaaaccctgg 7200

atagcggtag cctgaatgtt gtgaacaaaa ttaactatgg cgagaaactg caagagaaag 7260

ccagcaatcg taaacagagc attcgtgatt ggaaagcagt tgagggcatt aaaaacctga 7320

aaaagggcta tattagccag gttgttcgta aactggccga tctggcaatt gaacataatg 7380

cgatcatcat ctttgaggat ctgaacatgc gttttaagca gattcgtggt ggtattgaaa 7440

aaagcgtgta tcagcagctg gaaggtgcac tgattgaaaa actgagcttt ctggtgaata 7500

aaggcgagaa agatccgaaa caggcaggta atctgctgaa agcatatcag ctggcagcac 7560

cgtttaccac ctttaaagat atgggtaaac agaccggcat catcttctat acccaggcaa 7620

gctataccag taaaattgat ccgctgaccg gctggcgtcc gaatctgtat ctgaaatata 7680

caaatgccga aaaaaccaaa gaggacatcg gcaactttag caacatcgaa ttcaaaaacg 7740

gcatctttga atttacctac gatctgcgca acttccagaa acagaaagag tatccgaaaa 7800

aaacggaatg gaccctgtgt agctgtgttg aacgttttcg ttggaatcgt gttctgaacc 7860

agaataaagg tggctatgat cattatgagg atatcaccca taatttccgt gacctgtttg 7920

agaagtatga catcaatttc atgagcgcag atatcaaagg tcagattgat accctggatg 7980

ccaaaggcaa tgaaaacttc ttcaaagact ttatcttctt cttcaacctg atctgccaga 8040

ttcgtaatac ccagcaggac aaagatggtg atgaaaacga ttttatcctg agtccgatca 8100

aaccgttttt tgattcacgt gacagcaaga aatttggcga aaatctgccg aataatggcg 8160

acgataatgg tgcatacaat attagccgta agggcattat catcctgaac aagattagcg 8220

aattcttcga tgaaaatggt ggctgcgaga aaatgaaatg gggtgatctg tatatcagcc 8280

acaaagattg ggatgatttt gcccgtcaga tctaatggtc tagaggtcga aattcaaatt 8340

gtgagcggat aacaatttga attttctgta tgaggttttg ctaaacaact ttcaacagtt 8400

tcagtggagt gagaatagaa aggaacaact aaaggaattg cgaataataa ttttttcacg 8460

ttgaaaatct ccaaaaaaaa aggctccaaa aggagccttt aattgtatcg gtttatcagc 8520

ttgctttcga ggtgaatttt gaccctctag cgaaaatgca agagcaaaga cgaaaacatg 8580

ccacacatga ggaataccga ttctctcatt aacatattca ggccagttat ctgggcttaa 8640

aagcagaagt ccaacccaga taacgatcat atacatggtt ctctccagag gttcattact 8700

gaacactcgt ccgagaataa cgagtggatc ccctccaatt cgccctatag tgagtcgtat 8760

tacgcgcgct cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc 8820

caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc 8880

cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggaa attgtaagcg 8940

ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt tttaaccaat 9000

aggccgactg cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt tattggtgcc 9060

cttaaacgcc tggtgctacg cctgaataag tgataataag cggatgaatg gcagaaattc 9120

gaaagcaaat tcgacccggt cgtcggttca gggcagggtc gttaaatagc cgcttatgtc 9180

tattgctggt ttaccggttt attgactacc ggaagcagtg tgaccgtgtg cttctcaaat 9240

gcctgaggcc agtttgctca ggctctcccc gtggaggtaa taattgacga tatgatcatt 9300

tattctgcct cccagagcct gataaaaacg gtgaatccgt tagcgaggtg ccgccggctt 9360

ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa 9420

ggtatagggc ggcgaggcgg ctacagccga tagtctggaa cagcgcactt acgggttgct 9480

gcgcaaccca agtgctaccg gcgcggcagc gtgacccgtg tcggcggctc caacggctcg 9540

ccatcgtcca gaaaacacgg ctcatcgggc atcggcaggc gctgctgccc gcgccgttcc 9600

cattcctccg tttcggtcaa ggctggcagg tctggttcca tgcccggaat gccgggctgg 9660

ctgggcggct cctcgccggg gccggtcggt agttgctgct cgcccggata cagggtcggg 9720

atgcggcgca ggtcgccatg ccccaacagc gattcgtcct ggtcgtcgtg atcaaccacc 9780

acggcggcac tgaacaccga caggcgcaac tggtcgcggg gctggcccca cgccacgcgg 9840

tcattgacca cgtaggccga cacggtgccg gggccgttga gcttcacgac ggagatccag 9900

cgctcggcca ccaagtcctt gactgcgtat tggaccgtcc gcaaagaacg tccgatgagc 9960

ttggaaagtg tcttttggct gaccaccacg gcgttctggt ggcccatctg cgccacgagg 10020

tgatgcagca gcattgccgc cgtgggtttc ctcgcaataa gcccggccca cgcctcatgc 10080

gctttgcgtt ccgtttgcac ccagtgaccg ggcttgttct tggcttgaat gccgatttct 10140

ctggactgcg tgg 10153

<210> 60

<211> 10336

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC08-Ec载体系统

<400> 60

ccatgcttat ctccatgcgg tagggtgccg cacggttgcg gcaccatgcg caatcagctg 60

caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga 120

ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc 180

cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt 240

ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc 300

cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca 360

aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg 420

ggcgggcttc gccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca 480

aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa 540

ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa 600

attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct 660

gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag 720

cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg 780

ccatcgtcca catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc 840

ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa 900

gtctagtgag tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc 960

cgtcgagccg gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg 1020

atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga 1080

gcgcgagacg cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag 1140

cagcaccgat gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga 1200

cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag 1260

ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg 1320

ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc 1380

gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa 1440

agcgcagatg acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca 1500

acggggcatc gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct 1560

ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta 1620

tgcaccgcag ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc 1680

cgtggccgac cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc 1740

cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct 1800

tcgggagcgc ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc 1860

caaggccgcc gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt 1920

ccagcgccag aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa 1980

gtgccacctg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg 2040

atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg 2100

ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc 2160

acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc 2220

ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg 2280

agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga 2340

tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg 2400

tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg 2460

gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc 2520

aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg 2580

cccgtcgtgg ccagccacga tagccgcgct gcctcgtctt ggagttcatt cagggcaccg 2640

gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg 2700

gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa 2760

gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct 2820

gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc 2880

cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt 2940

tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct 3000

acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc 3060

atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag 3120

cccttgcgcc ctgagtgctt gcggcagcgt gaagctagct gcataatgtg cctgtcaaat 3180

ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt 3240

cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg 3300

gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc 3360

gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag 3420

cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg 3480

gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat 3540

atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt 3600

atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc 3660

aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat 3720

ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt 3780

attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta 3840

aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc 3900

tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac 3960

caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc 4020

gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc 4080

attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact 4140

cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt 4200

cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta 4260

acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag 4320

aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc 4380

cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata 4440

cccgtttttt ggtaccgggc cccccctcga gtttatttta ggaggcaaaa atgatgcaga 4500

tcatgaagaa cttcgacaaa ttcaccaatc tgtacagcgt tagcaaaacc ctgcgttttg 4560

aactgcgtcc ggaaccgaaa acactggaat atatgcgtag caatctgcgc tttgataaaa 4620

acctgcagac ctttctggca gatcaagaaa ttgaagatgc atatcaggca ctgaaaccga 4680

tttttgatag cctgcatgaa cgctttatta ccgaaagcct ggaaagcggt agcgcacaga 4740

aaattgattt tagcaaatat ctggaaaaat atcgcaacaa acgcgacctg ggtattaaag 4800

cactggaagg caccgaaaaa ctgctgcgta ataactttgc cgaaatctat aaagcaaccg 4860

ccaaaagctg gaaagaaaat gcaggtaaag atggcaaagg caaagaggtg ttcaaaaaag 4920

aaggctttaa catcctgacc gaaaaaggca ttctggaata catcgagaaa aacatcgata 4980

gctttagcgc cattaaaagt ccggaagaaa ttcgtggtgc actgggtgca tttgatggtt 5040

tttttaccta tttcaccggc ttcaatcaga accgcgaaaa ctattacgaa accaaaaaag 5100

aggcaagcac cgcagttgca acccgtattg ttcatgaaaa tctgccgaaa ttctgcgata 5160

acattctgat ctttgatgaa cgtgccgagg attatattgg tgcatataaa gccctgcaga 5220

aaatgggtcg tgcactggtt aataaagaag gtggcgaact gccgagcatt agcggtgacc 5280

tgtttaaaat caccttcttc aacaaatgct tcagccagaa gcagatcgaa gaatataaca 5340

ccgcaattgg taatgccaat agcctggtta acctgttcaa tcaggcaaaa cgtgatgaag 5400

atggctacaa aaaactggca ctgtttaaga ccctgtataa gcagattggc tgcgataaaa 5460

aagatagcct gttttttgca gtgacccatg atcgtcgtgc agatgcagaa aaagcacgtg 5520

aaaatggtca agaagcattt agcgtagaag aagttctggt tctggcaaaa catgcgggtg 5580

aaaagtattt caataaaggc aatgatgatg gcgaagtgaa taccacgcaa gaatttatca 5640

gctatattaa ggatcgcagc gattaccagg gtatctattg gagcaaagca gcactgaata 5700

ccatcagcaa caaatatttc gataactggt atgagctgat tgatcagctg aaagaagcca 5760

aagtttttac caaaaccggt agcggtagtg aggataatgt taaaattccg gatgccattg 5820

aactggaagg tttttttcag gtgctgaaca aaatccagga ttggaaaacc gtgttcttca 5880

aaaaaagcat taccgcagat ccgcagaaac tgggcattat tgaaagcagc gaaaccgcaa 5940

gcgcagcact gctgagcctg attttcgatg atgttgcaaa acacaccaaa ctgtttatcg 6000

atcagagcga ggatattctg aaagtggaaa attttgtgaa accggaaaac aaagaggaca 6060

ttaaacgttg gctggatcat agcctggcaa ttaatcagat gctgaagtat tttctggtga 6120

aagaaagccg taccaaaggt gcaccgattg atccgacact gaccaaagcg ctggataccc 6180

tgctgcgttc acaggatgca gaatggttta aatggtatga tgtgctgcgc aactacctga 6240

ccaaaaaacc gcaggatggc accaaagaaa ataaactgaa actgagcttt gaaaatggca 6300

ccctggcaaa tggttgggat gtgaataaag aaccggataa cttttgcgtg atcctgcaga 6360

atccggaagg caaaaaattc ctggccatta ttgcacgtca agaaggtcag aaaggtttta 6420

atcaggtgtt cgccaaaaaa catgataacc cgctgtataa agttgatgaa ggtggtgtct 6480

tttggtccaa gatggaatat aaactgttac cgggtccgaa taagatgctg cctaaatgtc 6540

tgatgccgaa aagcaatcgt gaaaaatatg gtgcaaccga ggaagtgctg aaaatttaca 6600

atcagggcag ctttaaaaag accgaaagca acttcagcaa aaaagatctg agtcgcctga 6660

tcaacttcta taaaagcgca ctgcagcagt atgaagattg gcgttgtttt aactttagct 6720

ttcgtgccac cgatagctat gaagatattg gtcagtttta tcgtgatgtg gaaagccagg 6780

gttataaact ggattttcag agcattaata ccgatgtgct ggatgaactg gtggaagaag 6840

gtaaaatcta cctgttcgaa atcaaaaacc aggatagcaa tcagggtaaa agcagcattc 6900

atcgtgataa tctgcatacc atgtattgga atgccctgtt tcaagaagta ctgaatcgtc 6960

cgaaactgaa tggtggtgca gaactgttct atcgtaaagc gctgtcaccg gaaaaaatca 7020

aagaactggg tagcgtggat aaaaacggca aacgtattat tcgcaactat cggttcagca 7080

aagagaagtt cattttccat attccgatca cgctgaactt ttgtctgagc gatacccgtg 7140

ttaatgatac cgttaatcaa gaactgagcc gtacctcaag cagtcatttt ctgggtattg 7200

atcgtggtga aaaacacctg gcatattact ttctggttga tcagaacggc aaaattgtgc 7260

tggacgaata tggtaaagca gttcagggca ccctgaatat tccgtttctg gataataatg 7320

gtaacgtgcg caaaattaag gccaaacgtc gtagcctgga tgagaatggc aaagaaaaaa 7380

tagaagaggt gtggtgcaaa gactataacg aactgctgga agcacgtgcc ggtgatcgtg 7440

catatgcccg taaaaattgg cagaccattg gcaacattaa agaactgaaa gagggctata 7500

ttagccaggt ggttcgtaaa attgttgatc tggccattga atatgaagcc tttatcgttc 7560

tggaagatct gaacgttggt tttaaacgtg gtcgtcagaa aatcgaaaaa agcgtgtatc 7620

agaaattaga actggccctg gcgaaaaaac tgaattttgt tgttgacaaa agcgccaaaa 7680

ttggtggtct gaaaagcgtt accaatgcgc tgcagctggc accgcctgtt agcaattttg 7740

gtgatattga aggtcgtaaa cagttcggca ttatgctgta tacccgtgca aattatacca 7800

gccagaccga tccggcaacc ggctggcgta aaagcattta tctgaaacgt ggtagcgaag 7860

aaagcattcg caagcagatt attgatagct tcgaagaaat tggctttgat ggcgaggatt 7920

actttttcac ctataccgat agcgttgcag gtcgcacctg gattctgtat agcggtaaaa 7980

atggtggtag tctggatcgc ttttatggca aacgtgataa cgataaaaat cagtgggtta 8040

gcatgcgcca ggatgtgagc aaacagctgg atggtatcct ggccaatttt gaaaaagatc 8100

gtagcattct ggcccagatt atcgatggtg aagtggatct gatcaaagtc gaacagaaat 8160

ataccgcatg ggaaagtttt cgtagcacca ttgatctgat tcagcagatt cgtaataccg 8220

gcaccagcga acgtgatggc gatttcattc tgagtccggt tcgtgatgaa cgcggtattc 8280

attttgatag tcgtgataca cgtgaaggta tgccgaccag cggtgatgca aatggtgcct 8340

ataacattgc acgtaaaggt acgattatgg gcgaacatat taaacgtgaa tacagccgca 8400

tgttcatttc cgatgaagaa tgggatgcat ggctggcagg taaacaggtt tgggaaaaat 8460

ggctgaaaga caacgagaaa atcctgaaaa agaaataatg gtctagaggt cgaaattcaa 8520

attgtgagcg gataacaatt tgaattttct gtatgaggtt ttgctaaaca actttcaaca 8580

gtttcagtgg agtgagaata gaaaggaaca actaaaggaa ttgcgaataa taattttttc 8640

acgttgaaaa tctccaaaaa aaaaggctcc aaaaggagcc tttaattgta tcggtttatc 8700

agcttgcttt cgaggtgaat tttgaccctc tagcgaaaat gcaagagcaa agacgaaaac 8760

atgccacaca tgaggaatac cgattctctc attaacatat tcaggccagt tatctgggct 8820

taaaagcaga agtccaaccc agataacgat catatacatg gttctctcca gaggttcatt 8880

actgaacact cgtccgagaa taacgagtgg atcccctcca attcgcccta tagtgagtcg 8940

tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 9000

acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 9060

gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa 9120

gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 9180

aataggccga ctgcgatgag tggcagggcg gggcgtaatt tttttaaggc agttattggt 9240

gcccttaaac gcctggtgct acgcctgaat aagtgataat aagcggatga atggcagaaa 9300

ttcgaaagca aattcgaccc ggtcgtcggt tcagggcagg gtcgttaaat agccgcttat 9360

gtctattgct ggtttaccgg tttattgact accggaagca gtgtgaccgt gtgcttctca 9420

aatgcctgag gccagtttgc tcaggctctc cccgtggagg taataattga cgatatgatc 9480

atttattctg cctcccagag cctgataaaa acggtgaatc cgttagcgag gtgccgccgg 9540

cttccattca ggtcgaggtg gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga 9600

caaggtatag ggcggcgagg cggctacagc cgatagtctg gaacagcgca cttacgggtt 9660

gctgcgcaac ccaagtgcta ccggcgcggc agcgtgaccc gtgtcggcgg ctccaacggc 9720

tcgccatcgt ccagaaaaca cggctcatcg ggcatcggca ggcgctgctg cccgcgccgt 9780

tcccattcct ccgtttcggt caaggctggc aggtctggtt ccatgcccgg aatgccgggc 9840

tggctgggcg gctcctcgcc ggggccggtc ggtagttgct gctcgcccgg atacagggtc 9900

gggatgcggc gcaggtcgcc atgccccaac agcgattcgt cctggtcgtc gtgatcaacc 9960

accacggcgg cactgaacac cgacaggcgc aactggtcgc ggggctggcc ccacgccacg 10020

cggtcattga ccacgtaggc cgacacggtg ccggggccgt tgagcttcac gacggagatc 10080

cagcgctcgg ccaccaagtc cttgactgcg tattggaccg tccgcaaaga acgtccgatg 10140

agcttggaaa gtgtcttttg gctgaccacc acggcgttct ggtggcccat ctgcgccacg 10200

aggtgatgca gcagcattgc cgccgtgggt ttcctcgcaa taagcccggc ccacgcctca 10260

tgcgctttgc gttccgtttg cacccagtga ccgggcttgt tcttggcttg aatgccgatt 10320

tctctggact gcgtgg 10336

<210> 61

<211> 10699

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC10-Ec载体系统

<400> 61

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgagcacc aaacgtagct ttagcgattt caccaatctg 480

tatagcgtta gcaaaaccct gcgttttgaa ctgaaaccga ttggtaaaac cctggaaaat 540

atgcgtgaac gcatctacaa cgataaaaag gattatgata gcgctctgca gacctttctg 600

catgatcagg caattgaaga tgcctataaa acactgaaac ccattctgga tagcctgcac 660

gaagaattta tcaataccag cctgaatagc agcaaagcca aaaacattga tctgagcgaa 720

tatctgaatg cctatcgtga acgtggtaat gataccaaaa ccggtgaaga aagcaaactg 780

agcggtattg aaaaatcact gcgtaaagca attggcgaaa cctatctgac ctgggcaaaa 840

agctttaccg aacaggccaa aaatctgatt ggcatcattg aagatatctg ggataccgaa 900

gaggaatggg atgaagaaaa gaaaaccaaa tggctgttca aaaagaaaaa cttcgaactg 960

ctgaccgaaa gcggtattct ggtttttatt gagaaaaagc tggataccat gaacatcagc 1020

gaacaagaga aaaccgatat taagaaagcc ctggaagaat tcaaaggctt ctttacctat 1080

ttcagcggct ataatcagaa ccgcaaaaac tactatgaaa ccaaggcaga gaaaaagacc 1140

gcaattgcaa cccgtattgt tcatgaaaat ctgccgaaat tttgcgacaa cgtgattctg 1200

tttcatggct atcagaaaat cctgaaagat ggtagcaaac gcgagtacaa gaagaaagag 1260

gaatatctgg gtatgtacgc ctttctgaaa ctgcgtaata ttgaaacctg catcaaagat 1320

gcggaaagcg gtgaaatgat tgaactgtat gcaatcaccg aggatatctt cgatattagc 1380

tttttcagca gctgtctggc acagcgtgaa attgatgaac ataatcgtat tatcggtggc 1440

atcgataaat acaaccgcat tattggtcat tataatgccc tgatcaacct gtataatcag 1500

gcacgtaaga aagacgagaa atttaccaaa ctgtccccgt tcaaagaact gtataaacaa 1560

atttggtgcg gcaacaaaaa gtggtcatgg attaaagcga ttacccatga taccgatgag 1620

cagattctgg cagataccaa tcatacaggt gaagcaatta gcgttgaacg tattctgagc 1680

ctggcaagca aagcaggtaa aaagtatttt cagccgtgga aaagcaccga tgatggtatt 1740

aaaaccgttc cggatttcct ggattggctg cgtggtcaga ccgattggaa tggtatttat 1800

tggagcaaag ccgcaattaa tagcatcagc aatgtgtatt ttccgaactg gggtagcatt 1860

aaagaaacca tgaaaggtga taaaacgctg gtgagctatg ataaaaagcg cgaagaacaa 1920

atcaaaatca acgaagcagt tgaactgagt ggcctgtttg atattctgga ttcaaccgat 1980

ggtgattgga aacaagaatg ggtgctgttt aaagcaagcc tgaccaaact gctggatgca 2040

agcgcagaaa atgccgaaga aaattcaaaa cgtgcccgtc gcaaagatat tattgatcgt 2100

agcagcagcc cgagccaggc actgctggca ctgattaccg attttattga agagaacatg 2160

aaacattttc tggaccagag ccataccatt ctgcgtctga ccgaatatag cagtccgaaa 2220

agcaaagaag ccatcaaaag ttggatggat ctggccctga gcgttagcca gaccattcgt 2280

tattttcgtg tgaaagaatc caaaaccaaa ggcgataccc tgaatgcaga actggttggt 2340

attctgacca atttactgga tgcagaagat gcaacctggt ttgagtggta tgatctgctg 2400

cgtaattatc tgaccaaaaa gccgcaggat gatgcgaaag aaaataaact gaaactgaac 2460

tttgccaaca gtaccctggc agcaggttgg gatgttaata aagaaacgga taatacctgc 2520

gtgatcctgc agaatccgga atggaaaaca tatctggccg tgatgaacaa aaacaaaaag 2580

aacgtgttcc agaaagaatg gaatgaatgg cgctggaaga agaaaacaac caaactgaat 2640

ccgctgtatg aaatcgattg gggtgaaagc tggaaaaaga tggaatatga tttttggagc 2700

gacgtgagca aaatgattcc gaaatgtagc acccagctga agaaagtgat caagcacttt 2760

aaagaaagcg acgaggattt tatctttccg agcggttata aagttaccag cggtgaacgt 2820

tttatcgaag aatgtcgtat taccaaagaa cagttcgagc tgaacaacaa agtgtataaa 2880

cgtgatggcg atcgcattat tagcgccttt cgttatgaac tgagcgaaac cgaggaaaag 2940

acctatatca aaagcttcca aaaaggctat ctggatatgc tgctgaagag taataatctg 3000

ccggaaaccg aacaagaaat ctaccgcaag aaatatgaag atagcctgag caaatggatc 3060

aacttctgca aatacttcat ctggaaatac ccgaaaacca gcctgttcga atatcagttt 3120

gatgaaaccg accactataa aagcgtggac aaatttaacc tggatgtgga tatttggagc 3180

tacaaactga aagtggacac caagattaac aaaaccatcc tggataccct ggtggaaaat 3240

ggtgatattt acctgttcga gatcaaaaac caggatagca atattggcaa atgggagaac 3300

cataaaaaca acctgcatac cacctactgg aaatccattt ttgaaagcgt tcagaatcgt 3360

ccgaaactga atggtgaagc cgaaatcttt tatatgaaac cgctgagtcc ggaaaaactg 3420

cagaaaaaga ttgacaagaa gggcaaagaa atcatcgatg gttatcgttt tagccgtgaa 3480

cgcttcattt ttcattgtcc gattacgctg aatttttgcc tgggcaatga gaagatcaac 3540

aacatcatta atttcgaact gagcccgaaa agcgatatct attttctggg tttagatcgc 3600

ggtgaaaaac acctggtgta ttattcaatt gtggaccaga acggtaaaat gatcgaccag 3660

tggtctttca acgagatcaa gtggaaagat tatcatgcgc tgctgacaaa acgcgaatgg 3720

gatcgtatgg aaagccgtaa aaattggcag accattagca acattgccaa gcttaaagag 3780

tggtatatta gcctggtgat ccatgagatt atcgagaagc tgaagctgaa tccgtggttt 3840

attgttctgg aagatctgaa caccggcttt aaacgtggtc gtcagaaaat tgaaaagagc 3900

atctaccaga aatttgaact ggcactggct aaaaagctga atttcgttgt tgataaaagc 3960

gccaaactgg gtgaagttgg tagcgttacc aatgcactgc agctgacccc tccggttagc 4020

aattatggcg atattgaaaa tcgtaaacag gtgggcatta tgctgtatac ccgtgcaaat 4080

tataccagcc agaccgatcc ggcaaccggc tggcgtaaaa ccatttatct gaaaaccggt 4140

agcgaggaaa acatcaaaga gcagattgtt acccagttta gcgatattgg ctttgatggc 4200

aaagattatt attttgaata taccgacaaa atcggcaaaa cctggattct gtatagtggc 4260

aaaaatggca aaagtctgac ccgttttcgt ggtgttcgtg gtaaagaaaa gaatgaatgg 4320

aacattaaag agatcaacgt ccgcaatatg ctggatggta tttttgcgaa ctttgataaa 4380

gatcgcagct ttctgagcca gatcctggat gaatgggttg aaattaagaa aatcgatgaa 4440

cacaccgcat gggaaagcct gcgctttgca atcgatctga ttcagcagat tcgtaacagt 4500

ggcgataaaa cccagtggga agatgataac tttctgttta gtccggttcg tgatgcacag 4560

ggtaaccatt ttgatacccg tgaacagaaa gaaggcctgc cgaaagacgc agatgcaaat 4620

ggtgcatata atattgcccg taagtggatc atcatgaacg aacatattcg cattaacgag 4680

gataccaaag atctggacct gtttgttagt gatgaagaat gggatatgtg gctgaccgat 4740

cgtgaaaaat ggaaagaaat gctgcctatt tttgcaagcc gcaaagcaat ggaaaaacgt 4800

cgcggtaaat aatggtctag aggtcgaaat tcaaattgtg agcggataac aatttgaatt 4860

ttctgtatga ggttttgcta aacaactttc aacagtttca gtggagtgag aatagaaagg 4920

aacaactaaa ggaattgcga ataataattt tttcacgttg aaaatctcca aaaaaaaagg 4980

ctccaaaagg agcctttaat tgtatcggtt tatcagcttg ctttcgaggt gaattttgac 5040

cctctagcga aaatgcaaga gcaaagacga aaacatgcca cacatgagga ataccgattc 5100

tctcattaac atattcaggc cagttatctg ggcttaaaag cagaagtcca acccagataa 5160

cgatcatata catggttctc tccagaggtt cattactgaa cactcgtccg agaataacga 5220

gtggatcccc tccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt 5280

tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca 5340

tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca 5400

gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atattttgtt aaaattcgcg 5460

ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgactgcga tgagtggcag 5520

ggcggggcgt aattttttta aggcagttat tggtgccctt aaacgcctgg tgctacgcct 5580

gaataagtga taataagcgg atgaatggca gaaattcgaa agcaaattcg acccggtcgt 5640

cggttcaggg cagggtcgtt aaatagccgc ttatgtctat tgctggttta ccggtttatt 5700

gactaccgga agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt ttgctcaggc 5760

tctccccgtg gaggtaataa ttgacgatat gatcatttat tctgcctccc agagcctgat 5820

aaaaacggtg aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg 5880

ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gaggcggcta 5940

cagccgatag tctggaacag cgcacttacg ggttgctgcg caacccaagt gctaccggcg 6000

cggcagcgtg acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa aacacggctc 6060

atcgggcatc ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc 6120

tggcaggtct ggttccatgc ccggaatgcc gggctggctg ggcggctcct cgccggggcc 6180

ggtcggtagt tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt cgccatgccc 6240

caacagcgat tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga acaccgacag 6300

gcgcaactgg tcgcggggct ggccccacgc cacgcggtca ttgaccacgt aggccgacac 6360

ggtgccgggg ccgttgagct tcacgacgga gatccagcgc tcggccacca agtccttgac 6420

tgcgtattgg accgtccgca aagaacgtcc gatgagcttg gaaagtgtct tttggctgac 6480

caccacggcg ttctggtggc ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt 6540

gggtttcctc gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca 6600

gtgaccgggc ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg ccatgcttat 6660

ctccatgcgg tagggtgccg cacggttgcg gcaccatgcg caatcagctg caacttttcg 6720

gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga ttttctttaa 6780

cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc cccctttttt 6840

aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt ggtgcccaaa 6900

gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc cggcaaaaag 6960

tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca aggtggcgct 7020

gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg ggcgggcttc 7080

gccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca aggccaagcc 7140

gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa ccggcaagcg 7200

aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa attgatgggg 7260

caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct gggtagccgg 7320

tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag cttgtccagc 7380

agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg ccatcgtcca 7440

catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc ggagagcaag 7500

cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa gtctagtgag 7560

tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc cgtcgagccg 7620

gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg atgcaagaag 7680

ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga gcgcgagacg 7740

cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag cagcaccgat 7800

gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga cgctgtgttg 7860

gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag ccaagaacag 7920

caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg ggcggatcgc 7980

atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc gttcgtggtg 8040

ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa agcgcagatg 8100

acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca acggggcatc 8160

gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct ggagcggcca 8220

ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta tgcaccgcag 8280

ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc cgtggccgac 8340

cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc cgcaggagcg 8400

cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct tcgggagcgc 8460

ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc caaggccgcc 8520

gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt ccagcgccag 8580

aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa gtgccacctg 8640

aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg atgcgctgcg 8700

aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg ccgccaagct 8760

cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc acacccagcc 8820

ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc ggcaagcagg 8880

catcgccatg ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg agcctggcga 8940

acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga tcgacaagac 9000

cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg tcgaatgggc 9060

aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg gatactttct 9120

cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc aatagcagcc 9180

agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg cccgtcgtgg 9240

ccagccacga tagccgcgct gcctcgtctt ggagttcatt cagggcaccg gacaggtcgg 9300

tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg gcatcagagc 9360

agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa gcggccggag 9420

aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct gtctcttgat 9480

cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt 9540

tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg 9600

tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc 9660

tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc 9720

agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag cccttgcgcc 9780

ctgagtgctt gcggcagcgt gaagctagct gcataatgtg cctgtcaaat ggacgaagca 9840

gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt cgttaccaat 9900

tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg gaactcgctc 9960

gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc gtcaaaacca 10020

acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag cttcgcctgg 10080

ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg gcggaaaaga 10140

tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat atcaaaattg 10200

ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt atccatcggt 10260

ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc aagcagattt 10320

atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat ttgcccaaac 10380

aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt attggcaaat 10440

attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta aacccactgg 10500

tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc tggcgggaac 10560

agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac caccccctga 10620

ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc gataaaaaaa 10680

tcgagataac cgttggcct 10699

<210> 62

<211> 10282

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC11-Ec载体系统

<400> 62

ccatgcttat ctccatgcgg tagggtgccg cacggttgcg gcaccatgcg caatcagctg 60

caacttttcg gcagcgcgac aacaattatg cgttgcgtaa aagtggcagt caattacaga 120

ttttctttaa cctacgcaat gagctattgc ggggggtgcc gcaatgagct gttgcgtacc 180

cccctttttt aagttgttga tttttaagtc tttcgcattt cgccctatat ctagttcttt 240

ggtgcccaaa gaagggcacc cctgcggggt tcccccacgc cttcggcgcg gctccccctc 300

cggcaaaaag tggcccctcc ggggcttgtt gatcgactgc gcggccttcg gccttgccca 360

aggtggcgct gcccccttgg aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg 420

ggcgggcttc gccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca 480

aggccaagcc gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa 540

ccggcaagcg aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa 600

attgatgggg caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct 660

gggtagccgg tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag 720

cttgtccagc agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg 780

ccatcgtcca catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc 840

ggagagcaag cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa 900

gtctagtgag tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc 960

cgtcgagccg gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg 1020

atgcaagaag ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga 1080

gcgcgagacg cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag 1140

cagcaccgat gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga 1200

cgctgtgttg gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag 1260

ccaagaacag caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg 1320

ggcggatcgc atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc 1380

gttcgtggtg ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa 1440

agcgcagatg acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca 1500

acggggcatc gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct 1560

ggagcggcca ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta 1620

tgcaccgcag ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc 1680

cgtggccgac cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc 1740

cgcaggagcg cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct 1800

tcgggagcgc ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc 1860

caaggccgcc gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt 1920

ccagcgccag aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa 1980

gtgccacctg aaccccagag tcccgctcag aagaactcgt caagaaggcg atagaaggcg 2040

atgcgctgcg aatcgggagc ggcgataccg taaagcacga ggaagcggtc agcccattcg 2100

ccgccaagct cttcagcaat atcacgggta gccaacgcta tgtcctgata gcggtccgcc 2160

acacccagcc ggccacagtc gatgaatcca gaaaagcggc cattttccac catgatattc 2220

ggcaagcagg catcgccatg ggtcacgacg agatcctcgc cgtcgggcat ccgcgccttg 2280

agcctggcga acagttcggc tggcgcgagc ccctgatgct cttcgtccag atcatcctga 2340

tcgacaagac cggcttccat ccgagtacgt gctcgctcga tgcgatgttt cgcttggtgg 2400

tcgaatgggc aggtagccgg atcaagcgta tgcagccgcc gcattgcatc agccatgatg 2460

gatactttct cggcaggagc aaggtgagat gacaggagat cctgccccgg cacttcgccc 2520

aatagcagcc agtcccttcc cgcttcagtg acaacgtcga gcacagctgc gcaaggaacg 2580

cccgtcgtgg ccagccacga tagccgcgct gcctcgtctt ggagttcatt cagggcaccg 2640

gacaggtcgg tcttgacaaa aagaaccggg cgcccctgcg ctgacagccg gaacacggcg 2700

gcatcagagc agccgattgt ctgttgtgcc cagtcatagc cgaatagcct ctccacccaa 2760

gcggccggag aacctgcgtg caatccatct tgttcaatca tgcgaaacga tcctcatcct 2820

gtctcttgat cagatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc 2880

cagtttactt tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt 2940

tcgcttgctg tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct 3000

acctgctttc tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc 3060

atccggggtc agcaccgttt ctgcggactg gctttctacg tgttccgctt cctttagcag 3120

cccttgcgcc ctgagtgctt gcggcagcgt gaagctagct gcataatgtg cctgtcaaat 3180

ggacgaagca gggattctgc aaaccctatg ctactccgtc aagccgtcaa ttgtctgatt 3240

cgttaccaat tatgacaact tgacggctac atcattcact ttttcttcac aaccggcacg 3300

gaactcgctc gggctggccc cggtgcattt tttaaatacc cgcgagaaat agagttgatc 3360

gtcaaaacca acattgcgac cgacggtggc gataggcatc cgggtggtgc tcaaaagcag 3420

cttcgcctgg ctgatacgtt ggtcctcgcg ccagcttaag acgctaatcc ctaactgctg 3480

gcggaaaaga tgtgacagac gcgacggcga caagcaaaca tgctgtgcga cgctggcgat 3540

atcaaaattg ctgtctgcca ggtgatcgct gatgtactga caagcctcgc gtacccgatt 3600

atccatcggt ggatggagcg actcgttaat cgcttccatg cgccgcagta acaattgctc 3660

aagcagattt atcgccagca gctccgaata gcgcccttcc ccttgcccgg cgttaatgat 3720

ttgcccaaac aggtcgctga aatgcggctg gtgcgcttca tccgggcgaa agaaccccgt 3780

attggcaaat attgacggcc agttaagcca ttcatgccag taggcgcgcg gacgaaagta 3840

aacccactgg tgataccatt cgcgagcctc cggatgacga ccgtagtgat gaatctctcc 3900

tggcgggaac agcaaaatat cacccggtcg gcaaacaaat tctcgtccct gatttttcac 3960

caccccctga ccgcgaatgg tgagattgag aatataacct ttcattccca gcggtcggtc 4020

gataaaaaaa tcgagataac cgttggcctc aatcggcgtt aaacccgcca ccagatgggc 4080

attaaacgag tatcccggca gcaggggatc attttgcgct tcagccatac ttttcatact 4140

cccgccattc agagaagaaa ccaattgtcc atattgcatc agacattgcc gtcactgcgt 4200

cttttactgg ctcttctcgc taaccaaacc ggtaaccccg cttattaaaa gcattctgta 4260

acaaagcggg accaaagcca tgacaaaaac gcgtaacaaa agtgtctata atcacggcag 4320

aaaagtccac attgattatt tgcacggcgt cacactttgc tatgccatag catttttatc 4380

cataagatta gcggatccta cctgacgctt tttatcgcaa ctctctactg tttctccata 4440

cccgtttttt ggtaccgggc cccccctcga gtttatttta ggaggcaaaa atgagccaga 4500

ataacacctt cgagaaattc accaatcagt acagcctgag caaaaccctg cgttttgaac 4560

tgcgtccggt tggtaatacc gagcagatgc tggaagatga aaacgtgttc aaaaaagatg 4620

agatcatccg caaaaaatac gagcagacca aaccgttcat tgacaaactg cataaagagg 4680

tgattaaaga tagcctgcac ggcaaaaaaa tcgaaggtct ggatgattac ttcaaaaaat 4740

tcgaaatcta tagcaaaaac aaaaaagaca gcaaaatcaa gaaagaattt accgataaag 4800

aaagcgagct gcgcaaacag ctgaatagcc attttaaagc agaaagcctg tttagcgaaa 4860

aagtgtttag cctgctgaaa gaaaaatatg gcaccgaaga tgagagcttc gtgaaagacg 4920

aaaatggtaa ttttgttctg gataccgtgg gcgaaaaaat cagcattttt gatgaatgga 4980

aaggcttcac cggctacttt accaaatttc agaaaacccg tgagaacttc tataaagatg 5040

atggcaccag caccgcaatt gttacccgta ccattgatga aaatctgtat cgcttttgcg 5100

agaacatcaa acactttgag agcattaaaa accgcgtgaa ctttagcgag atcgagaaaa 5160

actttaactt taaactggaa aacctgttta aagccgactt ctacaatagc tgtctgctgc 5220

aggatggtat cgataaatac aatgatatcc ttggcggtaa aaccctggaa agcggtgaaa 5280

aactgaaagg cctgaatgaa atcatcaaca aatatcgcca ggacaacaag gtggaaaaaa 5340

tcggtttctt caaaatgctg gataagcaga ttctgggcga taaagagaaa ccgagcttta 5400

ttgaaagcat tgccgatgat aatgagctgc tgctgaaact taaagagttt tataccaacg 5460

ccgaggaaaa aaccgaggtt ctgaaaaaac tgttcagcga cttcagcaaa aataacgata 5520

gctacgatct gagcaagatc tatattaaca aagtgggcat taacaccatt ctgctgaaat 5580

ggtttgatgt ggcaggtcgt agcgattttg aaaaaaacat tagcacccag accaaaaaag 5640

aaaaaattgt gaccttcgac aaggatagca acagctataa gtttccggaa tttctggcct 5700

tcagccatat taaagaagca ctgagcaacg gcacctatga agttaaagaa atctggaaag 5760

agcgctatta tcagagcgag aacaaagaga aatcagaaaa agcaccgctg aaaaaagata 5820

gcgcaattag ccattgggaa gagtttctgc agatctttag ctatgaattc gatctgctgt 5880

ttgttggtgc cgaaagccag gcaggttata atagcaataa gaacctgttt gagagcctga 5940

tcaaaaaaaa cgagaaaggg tttagcatca gtccggaaga aaaactggtg atcaagaact 6000

ttgttgataa caccctgtgg atttatcaga tggccaaata tttcgcgatc gaaaaaaagc 6060

gtaaatggct ggaaagtgaa tatccgaccg atagcagctt ttatgatagc gaagaattcg 6120

gcttcaaaaa caagttctat gatgacgcct atgataaaat cgtgaaactg cgtatgctgc 6180

tgcagagcca tctgacaaaa aaaccgttta gcaccgataa atggaagctg aactttgaaa 6240

atccgacact ggcaaaaggt tgggacaaga ataaagaatc agataattca gcagtgctgc 6300

tgcgtaaaga aggtcgttat tatctggccg ttatgaaaaa aggcaacaac aaaatcttcg 6360

atgacaagaa caaaagcaac tttctggaaa acatcgaagg cggtaaatac gagaaaatgg 6420

tttacaagca gatgagcgat ccgagcaaag atattcagaa tctgatggtg atcgacgata 6480

aaaccgttcg taaagtgggt aaaaaagatc cgctggatgg tgtgaatcgt cgtctggaag 6540

aactgaagaa agagtatctg cctcgcgata tcaataccat tcgtgaacag aaagcatacc 6600

tgaaaagcag cgataatttc aatctgggtg atgccaacct gttcatcaac tattacaaag 6660

atcgtctggt ggaataccac aaagacattt ttgtgtttag cttccgtgat cgctacagcg 6720

attttcatga ttttagcaaa catgttgcgg agcagaccta tagcctgagc tttgaagata 6780

ttagcgagtt ctacatccaa gagaaaaaca ataacggcga actgtttctg ttcgagatcc 6840

ataataaaga ctggaacctg gaaaaaaaag gtggcgatcg taaaagcggt gccaaaaatc 6900

tgcataccgt ttattttgaa tccctgttca gcaaagagaa cgagaacaac aacttcagca 6960

ttaaactgaa tggtgaagcc gagctgttct atcgtccgaa aaccgatgag cagaaactgg 7020

gtaataagaa tgatctgaaa ggcaagatcg tgctgaacaa aaaacgttat gccgaaaaca 7080

aaacctttat tcacattccg attacgctga atcgtgttgc aagcgaaagc aaatacttta 7140

accagaaact gaacgatttt ctggtgggca atccggatat taacattatt ggcattgatc 7200

gcggtgagaa acacctgatc tattatgcag gtattaatca ggcaggcgag tttctgaagg 7260

atgaaaaagg taatctggtg ctgggtagcc tgaataccat taatgatgtt aactacgccc 7320

aaaagttaga ggaacgtgca aaaggtcgtg ttaaagcaaa acaggattgg caagaaatcg 7380

aaaacatcaa agatctgaaa cgcggttata ttagcctggt tgttcgtgaa ctggcagatc 7440

tgattatcaa acataacgcc atcatcgtgt ttgaggatct gaatatgcgt tttaaacaaa 7500

ttcgcggtgg cattgagaaa agcgtttatc agcagctgga aaaagccctg attgataaac 7560

tgaacttcct ggtgaacaaa ggtgagaaag atccgaccaa agcaggtcat ctgctgcgtg 7620

catttcagct gaccgcaccg attagcgcat ataaagacat gggtaaacag accggtgtga 7680

ttttctatac ccaggcaagc tataccagca aaacctgtcc ggaatgtggt tttcgtccga 7740

atgttcgttg ggaaccgaaa agtatcaaag ataaaatcaa agaaggcaag ctggaaatca 7800

cctacaaaga agatggtttc gagatcagct ataaactgtc cgatttttcc aaaagccaga 7860

atcagagcaa acgtcgcaat attctgtata cgaatgtgag caaacaggat aaattcaacc 7920

tgaacaccaa agatgccgtt cgttgtaaat ggtttcgtaa aacactgagc gaaaacgaac 7980

tgaataaggg cgaacagaag ctgaatatcc agacagaaac cggtgttaac atcgagtata 8040

aaatcagcga ttgtctgatc ggcctgtttg aaaaatacgg tctggattat cagaacaacc 8100

tgcaagaaga gatcaaaaat tcaggtgata gtctgccggt gaaattctat gataagctga 8160

gcttttatct gcatctgctg accaatacac gtagcagcgt tagcggcacc gatattgatc 8220

atatcaattg tccgaattgc ggcttctgta gcaaaaatgg ctttaaaggc ggtgaattta 8280

atggtgatgc aaacggtgca tacaacattg cacgtaaagg tattatcatc ctggacaagc 8340

tgaagaacta taaaaccgaa aatagcaacc ttgaaaaaat gacctggggt gacctgttta 8400

ttgatattga tgagtgggac aaatttaccc agaacaagac ctaatggtct agaggtcgaa 8460

attcaaattg tgagcggata acaatttgaa ttttctgtat gaggttttgc taaacaactt 8520

tcaacagttt cagtggagtg agaatagaaa ggaacaacta aaggaattgc gaataataat 8580

tttttcacgt tgaaaatctc caaaaaaaaa ggctccaaaa ggagccttta attgtatcgg 8640

tttatcagct tgctttcgag gtgaattttg accctctagc gaaaatgcaa gagcaaagac 8700

gaaaacatgc cacacatgag gaataccgat tctctcatta acatattcag gccagttatc 8760

tgggcttaaa agcagaagtc caacccagat aacgatcata tacatggttc tctccagagg 8820

ttcattactg aacactcgtc cgagaataac gagtggatcc cctccaattc gccctatagt 8880

gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct 8940

ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc 9000

gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatggaaa 9060

ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt 9120

ttaaccaata ggccgactgc gatgagtggc agggcggggc gtaatttttt taaggcagtt 9180

attggtgccc ttaaacgcct ggtgctacgc ctgaataagt gataataagc ggatgaatgg 9240

cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg ttaaatagcc 9300

gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt gaccgtgtgc 9360

ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat aattgacgat 9420

atgatcattt attctgcctc ccagagcctg ataaaaacgg tgaatccgtt agcgaggtgc 9480

cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc aacgcgggga 9540

ggcagacaag gtatagggcg gcgaggcggc tacagccgat agtctggaac agcgcactta 9600

cgggttgctg cgcaacccaa gtgctaccgg cgcggcagcg tgacccgtgt cggcggctcc 9660

aacggctcgc catcgtccag aaaacacggc tcatcgggca tcggcaggcg ctgctgcccg 9720

cgccgttccc attcctccgt ttcggtcaag gctggcaggt ctggttccat gcccggaatg 9780

ccgggctggc tgggcggctc ctcgccgggg ccggtcggta gttgctgctc gcccggatac 9840

agggtcggga tgcggcgcag gtcgccatgc cccaacagcg attcgtcctg gtcgtcgtga 9900

tcaaccacca cggcggcact gaacaccgac aggcgcaact ggtcgcgggg ctggccccac 9960

gccacgcggt cattgaccac gtaggccgac acggtgccgg ggccgttgag cttcacgacg 10020

gagatccagc gctcggccac caagtccttg actgcgtatt ggaccgtccg caaagaacgt 10080

ccgatgagct tggaaagtgt cttttggctg accaccacgg cgttctggtg gcccatctgc 10140

gccacgaggt gatgcagcag cattgccgcc gtgggtttcc tcgcaataag cccggcccac 10200

gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg gcttgttctt ggcttgaatg 10260

ccgatttctc tggactgcgt gg 10282

<210> 63

<211> 10486

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC12-Ec载体系统

<400> 63

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatggaaacc aagaacaaaa gcatctgggg tgatttcacc 480

aacaaatata gcctgagcaa aaccctgcgt tttgaactgg ttccggttgg taaaacccgt 540

gaaaatattc agaaacacaa cccggaattt gtgcaggatc agaaaattga agaagcctac 600

cagattctga aaagcgtgtt tgataaaatc cacgaggact tcattaccaa gagcctggaa 660

agtgatgaag ccaaaagcat taacttcagc gagtatttcg acctgtacaa aaagtggaac 720

gagctgaaaa agaaaaagac caacgagaag aacatcgaga tcaagaaaga aatccagaac 780

gagatcgaga agatctacaa agataatggt ggtagcaaag gcgagatcca gaaaatcgag 840

gatgaactgc gtaaacgctt tgaagaaatc tttaaaatcc agggcaaaat cttcaaagag 900

aaagcctgcg aactgaacat taaagaaggt caagagaaag acgacgacga ggaaaaagac 960

gataataaga aaggttttcg caaactgctg aaagccaaat tcctgtatga ttatctgtgc 1020

aacctgatcg agagcaaaaa catcatctac aaggatttct tcgagaacat caaaaacaaa 1080

gaaggcgaga gcatcagcaa agaaaagaca aaagatgccc tgattcgctt taaaggcttt 1140

accacctatt ttggtggctt tgaactgaat cgcctgaact attataccac caaagaagag 1200

aaaagcaccg cagttgcaac ccgtattgtt aatcagaacc tgccgaaatt ttgcgataac 1260

gtgattctgt ttgaaatcaa aaagagcgaa tatctgaaaa tcgatgaatt cctgaaaaac 1320

aagaacatca gcctgatcag caaaaatcag aatggtggtg aagtggaact gcacaaaatc 1380

aacaaaaact ttttcgagat gatgttcttt agcaagtgcc tgagccagaa agagattcag 1440

aaatataacc tggaaattgg caatgccaac aacctgatta atcgctataa tcagcagcag 1500

agcgataaaa gccagaaact gaaactgttt aaaacgctgc ataagcagat tggttgtggt 1560

gatcgtggtg gttttattcc gagcattaaa ggtgaagaag atctgcgcga acgtctgcaa 1620

gaaattaaga ataacagcat cgaatacttt gaaaacatca acgacttcat cgagtacctg 1680

aaaaatcacg agaactacga aaatgtgtat tggagcgaca aagccattaa taccattagc 1740

agcaaatatt tcagcgactg gctgaacctg aagaaagaaa tttggggtaa acgtgatcgc 1800

aaaggcaatc tgaaagatga agaaaccaaa attccgcgtg cagtgcagct gaaagatctg 1860

ctggaaaatc tggataaaat caccgattgg aaactggaag gtcgtctgtt taaactgagc 1920

ctgtttgaga atggtcgtaa agccaaaaag ctgcagcaag aggatctgaa caaattcaat 1980

aaaaacaaaa tcgaaaacga gctggaaatc gagaaactgc agattattga acagaatccg 2040

agtccgtttc aggcactgct gaatatgatc tttgcagaca ttaaatccaa agagagcgca 2100

tttctggaaa gccgcatttt tgaaattagc gatttcgtgc acaacgagga caagcagatc 2160

attaaacagt ggctggatag cattctggcc attaaccaga ttatcaaata ctggcgtgtg 2220

aaagatacct ttggcaccga aggcaccctg gatgaaaaac tgaaaaatat catttatagc 2280

gagaaaaacc cgacgcgctt ttatgatatt attcgcaatt acctgaccaa gaaaccgcag 2340

gatgagctga ataaactgaa gctgaatttt gagaatagca ccctggcaca aggtctggat 2400

gttaataaag aaaaggataa cttttgcatt atcctgcgcg acgataaaca gaaccagtat 2460

ctgggtattc tgaacagcaa gaataagaac atcttcgaga tcgatcagaa cgaggatatc 2520

tatcaggatg atggtttagg ttggagcaag atgatgtata aactgattcc gggtgcaagc 2580

aaaacactgc cgaaaatctt tttctcaaaa cgctggaccg aaaataatcc gacaccggat 2640

gaaatcagca agattaaaaa gggcgaaacc ttcaagaaag gcgacaattt cattaaacgc 2700

gatctgcatg aactgatcaa cttctataaa gccaaccttg agaaatatcc gagcgttaat 2760

gaaagctggg caaaactgtt catcttcaac tttagcgata ccaaaaccta tgagagcatc 2820

gatcagtttt ataacgaggt ggataaacag ggctataagg tgagctttat cagcatcaat 2880

aagaataccc tggacaactt tatcgacaaa gagaagctgt acctgtttca gattaagaac 2940

aaggacaaca atctggacaa aggcgaaaag aaacagagca acaaaaatct gcatagcatc 3000

tattgggaag ccatttttgg taaagcactg aacaaaccga aactgaatgg tggcgcagaa 3060

attttctatc gtccggcact gagtgaaaag aaaattagtg aactgaaaat caaagataaa 3120

aacggcaaga acattatcat cattaaaaac tatcgctata gcaaagacaa attcattttt 3180

cactgcccga tcacgctgaa ctttagtgca aaaagtagca aactgaacga cgagatcaac 3240

gatcatatca agaacaagaa agaattttgc tttatgggca tcgatcgcgg tgaaaaacat 3300

ctggcatatt atagcctggt gaatcagaac ggcaaaatcc ttgataaagg tcagggcacc 3360

ctgaatctgc cgtttgttga taaagatggt aacaagcgtt gcatcaaaac cgagaaatac 3420

ttcgaagagg ataagaaaga aaacgaaaag tggaaaccgc gtattattga ttgtccggat 3480

tataactgtc tgctggatgc acgtgccagc aatcgtgatc tggcacgcaa aaattggcag 3540

accattggta caatcaaaga actgaaagaa ggctatatca gccaggtggt tcgtaaaatt 3600

gttgatctgg cgattgaaaa caacgccttt attgttctgg aaaacctgaa cattggtttt 3660

aaacgtggtc gccagaagat cgaaaaacag gtttatcaga agctggaact ggcactggca 3720

cgtaaactga attttctggt tgataaaaag gccatcattg gtgaagttgg tagcgttacc 3780

aaagcactgc agctgacccc tccggttaat aactttggtg atattggtgg caaaagccag 3840

tttggcatta tgttttatac caaagcggat tacaccagcc agaccgatcc ggttaccggt 3900

tggcgtaaaa gcatttatct gaaacgcggt ccggaagatt acatcaaaga tcagatcctg 3960

ggtaacaaaa acaaaaacat tgaaccggca tttgaggaca tttgctttga tggtcaggat 4020

tattgcttca cctacattaa caaaaatacc ggcaagaaat ggaccctgta cagcagtaaa 4080

aatggtaaaa gcctggatcg ctatcatcgt gaactggtgt atgaaaacag cgataagaaa 4140

tggctgccga agaaacagga tgtgctggaa atgctgaata acctgtttga aggcttcgac 4200

aagaaaaagt ctctgctgaa acagcttgaa acgaagaatc cgaataaaac aggtgaacat 4260

ccggcatggg aaagtctgcg ctttaccatt gatctgattc agcagattcg taacaccggg 4320

attaaagaac gtgatgagga tttcattctg agtccggttc gtgacaaaaa gggtgatcat 4380

tttgatagcc gtgaagcaag tccggatctg ccgaatagcg gtgatgcaaa tggtgcatac 4440

aatattgcac gcaagggcat tattatggcc aaacatatcg aaaaaggcta ctttctgtat 4500

atcagcgacg aagaatggga tgcatggctg gcaggcgaag aatgttggaa tcgttgggct 4560

gagaaaaata ccaaaagcct gctgaagaac aactattaat ggtctagagg tcgaaattca 4620

aattgtgagc ggataacaat ttgaattttc tgtatgaggt tttgctaaac aactttcaac 4680

agtttcagtg gagtgagaat agaaaggaac aactaaagga attgcgaata ataatttttt 4740

cacgttgaaa atctccaaaa aaaaaggctc caaaaggagc ctttaattgt atcggtttat 4800

cagcttgctt tcgaggtgaa ttttgaccct ctagcgaaaa tgcaagagca aagacgaaaa 4860

catgccacac atgaggaata ccgattctct cattaacata ttcaggccag ttatctgggc 4920

ttaaaagcag aagtccaacc cagataacga tcatatacat ggttctctcc agaggttcat 4980

tactgaacac tcgtccgaga ataacgagtg gatcccctcc aattcgccct atagtgagtc 5040

gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 5100

tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 5160

ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggaaattgta 5220

agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc attttttaac 5280

caataggccg actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg 5340

tgcccttaaa cgcctggtgc tacgcctgaa taagtgataa taagcggatg aatggcagaa 5400

attcgaaagc aaattcgacc cggtcgtcgg ttcagggcag ggtcgttaaa tagccgctta 5460

tgtctattgc tggtttaccg gtttattgac taccggaagc agtgtgaccg tgtgcttctc 5520

aaatgcctga ggccagtttg ctcaggctct ccccgtggag gtaataattg acgatatgat 5580

catttattct gcctcccaga gcctgataaa aacggtgaat ccgttagcga ggtgccgccg 5640

gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag 5700

acaaggtata gggcggcgag gcggctacag ccgatagtct ggaacagcgc acttacgggt 5760

tgctgcgcaa cccaagtgct accggcgcgg cagcgtgacc cgtgtcggcg gctccaacgg 5820

ctcgccatcg tccagaaaac acggctcatc gggcatcggc aggcgctgct gcccgcgccg 5880

ttcccattcc tccgtttcgg tcaaggctgg caggtctggt tccatgcccg gaatgccggg 5940

ctggctgggc ggctcctcgc cggggccggt cggtagttgc tgctcgcccg gatacagggt 6000

cgggatgcgg cgcaggtcgc catgccccaa cagcgattcg tcctggtcgt cgtgatcaac 6060

caccacggcg gcactgaaca ccgacaggcg caactggtcg cggggctggc cccacgccac 6120

gcggtcattg accacgtagg ccgacacggt gccggggccg ttgagcttca cgacggagat 6180

ccagcgctcg gccaccaagt ccttgactgc gtattggacc gtccgcaaag aacgtccgat 6240

gagcttggaa agtgtctttt ggctgaccac cacggcgttc tggtggccca tctgcgccac 6300

gaggtgatgc agcagcattg ccgccgtggg tttcctcgca ataagcccgg cccacgcctc 6360

atgcgctttg cgttccgttt gcacccagtg accgggcttg ttcttggctt gaatgccgat 6420

ttctctggac tgcgtggcca tgcttatctc catgcggtag ggtgccgcac ggttgcggca 6480

ccatgcgcaa tcagctgcaa cttttcggca gcgcgacaac aattatgcgt tgcgtaaaag 6540

tggcagtcaa ttacagattt tctttaacct acgcaatgag ctattgcggg gggtgccgca 6600

atgagctgtt gcgtaccccc cttttttaag ttgttgattt ttaagtcttt cgcatttcgc 6660

cctatatcta gttctttggt gcccaaagaa gggcacccct gcggggttcc cccacgcctt 6720

cggcgcggct ccccctccgg caaaaagtgg cccctccggg gcttgttgat cgactgcgcg 6780

gccttcggcc ttgcccaagg tggcgctgcc cccttggaac ccccgcactc gccgccgtga 6840

ggctcggggg gcaggcgggc gggcttcgcc ttcgactgcc cccactcgca taggcttggg 6900

tcgttccagg cgcgtcaagg ccaagccgct gcgcggtcgc tgcgcgagcc ttgacccgcc 6960

ttccacttgg tgtccaaccg gcaagcgaag cgcgcaggcc gcaggccgga ggcttttccc 7020

cagagaaaat taaaaaaatt gatggggcaa ggccgcaggc cgcgcagttg gagccggtgg 7080

gtatgtggtc gaaggctggg tagccggtgg gcaatccctg tggtcaagct cgtgggcagg 7140

cgcagcctgt ccatcagctt gtccagcagg gttgtccacg ggccgagcga agcgagccag 7200

ccggtggccg ctcgcggcca tcgtccacat atccacgggc tggcaaggga gcgcagcgac 7260

cgcgcagggc gaagcccgga gagcaagccc gtagggcgcc gcagccgccg taggcggtca 7320

cgactttgcg aagcaaagtc tagtgagtat actcaagcat tgagtggccc gccggaggca 7380

ccgccttgcg ctgcccccgt cgagccggtt ggacaccaaa agggaggggc aggcatggcg 7440

gcatacgcga tcatgcgatg caagaagctg gcgaaaatgg gcaacgtggc ggccagtctc 7500

aagcacgcct accgcgagcg cgagacgccc aacgctgacg ccagcaggac gccagagaac 7560

gagcactggg cggccagcag caccgatgaa gcgatgggcc gactgcgcga gttgctgcca 7620

gagaagcggc gcaaggacgc tgtgttggcg gtcgagtacg tcatgacggc cagcccggaa 7680

tggtggaagt cggccagcca agaacagcag gcggcgttct tcgagaaggc gcacaagtgg 7740

ctggcggaca agtacggggc ggatcgcatc gtgacggcca gcatccaccg tgacgaaacc 7800

agcccgcaca tgaccgcgtt cgtggtgccg ctgacgcagg acggcaggct gtcggccaag 7860

gagttcatcg gcaacaaagc gcagatgacc cgcgaccaga ccacgtttgc ggccgctgtg 7920

gccgatctag ggctgcaacg gggcatcgag ggcagcaagg cacgtcacac gcgcattcag 7980

gcgttctacg aggccctgga gcggccacca gtgggccacg tcaccatcag cccgcaagcg 8040

gtcgagccac gcgcctatgc accgcaggga ttggccgaaa agctgggaat ctcaaagcgc 8100

gttgagacgc cggaagccgt ggccgaccgg ctgacaaaag cggttcggca ggggtatgag 8160

cctgccctac aggccgccgc aggagcgcgt gagatgcgca agaaggccga tcaagcccaa 8220

gagacggccc gagaccttcg ggagcgcctg aagcccgttc tggacgccct ggggccgttg 8280

aatcgggata tgcaggccaa ggccgccgcg atcatcaagg ccgtgggcga aaagctgctg 8340

acggaacagc gggaagtcca gcgccagaaa caggcccagc gccagcagga acgcgggcgc 8400

gcacatttcc ccgaaaagtg ccacctgaac cccagagtcc cgctcagaag aactcgtcaa 8460

gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc gataccgtaa agcacgagga 8520

agcggtcagc ccattcgccg ccaagctctt cagcaatatc acgggtagcc aacgctatgt 8580

cctgatagcg gtccgccaca cccagccggc cacagtcgat gaatccagaa aagcggccat 8640

tttccaccat gatattcggc aagcaggcat cgccatgggt cacgacgaga tcctcgccgt 8700

cgggcatccg cgccttgagc ctggcgaaca gttcggctgg cgcgagcccc tgatgctctt 8760

cgtccagatc atcctgatcg acaagaccgg cttccatccg agtacgtgct cgctcgatgc 8820

gatgtttcgc ttggtggtcg aatgggcagg tagccggatc aagcgtatgc agccgccgca 8880

ttgcatcagc catgatggat actttctcgg caggagcaag gtgagatgac aggagatcct 8940

gccccggcac ttcgcccaat agcagccagt cccttcccgc ttcagtgaca acgtcgagca 9000

cagctgcgca aggaacgccc gtcgtggcca gccacgatag ccgcgctgcc tcgtcttgga 9060

gttcattcag ggcaccggac aggtcggtct tgacaaaaag aaccgggcgc ccctgcgctg 9120

acagccggaa cacggcggca tcagagcagc cgattgtctg ttgtgcccag tcatagccga 9180

atagcctctc cacccaagcg gccggagaac ctgcgtgcaa tccatcttgt tcaatcatgc 9240

gaaacgatcc tcatcctgtc tcttgatcag atcttgatcc cctgcgccat cagatccttg 9300

gcggcaagaa agccatccag tttactttgc agggcttccc aaccttacca gagggcgccc 9360

cagctggcaa ttccggttcg cttgctgtcc ataaaaccgc ccagtctagc tatcgccatg 9420

taagcccact gcaagctacc tgctttctct ttgcgcttgc gttttccctt gtccagatag 9480

cccagtagct gacattcatc cggggtcagc accgtttctg cggactggct ttctacgtgt 9540

tccgcttcct ttagcagccc ttgcgccctg agtgcttgcg gcagcgtgaa gctagctgca 9600

taatgtgcct gtcaaatgga cgaagcaggg attctgcaaa ccctatgcta ctccgtcaag 9660

ccgtcaattg tctgattcgt taccaattat gacaacttga cggctacatc attcactttt 9720

tcttcacaac cggcacggaa ctcgctcggg ctggccccgg tgcatttttt aaatacccgc 9780

gagaaataga gttgatcgtc aaaaccaaca ttgcgaccga cggtggcgat aggcatccgg 9840

gtggtgctca aaagcagctt cgcctggctg atacgttggt cctcgcgcca gcttaagacg 9900

ctaatcccta actgctggcg gaaaagatgt gacagacgcg acggcgacaa gcaaacatgc 9960

tgtgcgacgc tggcgatatc aaaattgctg tctgccaggt gatcgctgat gtactgacaa 10020

gcctcgcgta cccgattatc catcggtgga tggagcgact cgttaatcgc ttccatgcgc 10080

cgcagtaaca attgctcaag cagatttatc gccagcagct ccgaatagcg cccttcccct 10140

tgcccggcgt taatgatttg cccaaacagg tcgctgaaat gcggctggtg cgcttcatcc 10200

gggcgaaaga accccgtatt ggcaaatatt gacggccagt taagccattc atgccagtag 10260

gcgcgcggac gaaagtaaac ccactggtga taccattcgc gagcctccgg atgacgaccg 10320

tagtgatgaa tctctcctgg cgggaacagc aaaatatcac ccggtcggca aacaaattct 10380

cgtccctgat ttttcaccac cccctgaccg cgaatggtga gattgagaat ataacctttc 10440

attcccagcg gtcggtcgat aaaaaaatcg agataaccgt tggcct 10486

<210> 64

<211> 10261

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC14-Ec载体系统

<400> 64

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgagccag aacaacatca aagaaaagag catctttgac 480

gaatttacca acaaatatag cctgcagaaa accctgcgtt ttgaactgcg tccggttctg 540

aataccgagc agatgctgac cgatagcggt attatcaaac tggatgaaaa acgcaaactg 600

aactatgaga aaaccaaacc gtttctgaat cgcctgcatc aagaatttgt taccgaaagc 660

ctgaatggtg ttcgtctgaa aagtctggat ggttatgcag ttctgtatgc caattggaag 720

aaaagcatcg ataagaaaga aaaggacgca gcctataaag tgctggaaaa gaaagaactg 780

gaaatccgcc aagaaattgt ggtgctgttt gatgaaaaag ccgttgaatg gattggtaaa 840

ctgcctgcag atgttaaaaa gccgaagaaa ccgaattatg aattcctgtt tgaaccggca 900

attttcagca tcctgaagaa aaagtatagt gatgaagttg gcaccaccat tgatgaggaa 960

agcatttttg atagctggga taaatggacc gcctattttg gcaaattttt cgaaacccgc 1020

aagaacttct ataaaagtga tggtaaagca accgcagttg caacccgtat tgttaatgaa 1080

aatctgcgtc gcttttgtga tgatgtgagc acctttgaaa acatccagag caaaattgat 1140

ctgagtccgc tggaaaaaga attcgatgtt agcctgaaaa aggtgttcga tatccagcat 1200

tataatcagt gtctgaatca gagcggtatc gatgcattta ataccctgtt aggtggtgaa 1260

gtgcatgaaa atggtgagaa aatcaaaggc atcaacgagt atatcaacga acatcgtcag 1320

aaaaccggtg aaaaactgac ccgtctgaag aaactggata aacaaattgg cagcgacaaa 1380

gagaacttca tcgatctgat tgaaaccgat gaacagctga aaaccacact ggttaccttt 1440

attgcaaacg ccaaagagaa agttgatctg ctggataaaa gcgttagcta tctgaccaaa 1500

gataccgatg ttaaactgag cggtattttc tttcgcaaag aagccattaa taccattacg 1560

cgtcgttggt ttgttagcca cgagaaaatt agtgatgcac tggttagcgc attcagcgat 1620

aaaaacgtta aattcgatca gaagcgcgaa gagtataaat tcccggattt tatcagctgg 1680

catgtgattc agaatgcagt ggaaaaactg gcatcagatg gtgaagaaat ttggaagaag 1740

tattatctgg aagaagagaa actgagcctg ctggacaaaa ccccgtggca gcagtttctg 1800

accgtttttg aatgtgaata caacaacctg aaaagcaaag gccatgaaag cgaaggtcgt 1860

agctttaccg aactggttca ggatattgaa agtctgctga aaacggatac cctggatcgt 1920

aatgatcatg tgaccgaaat catcaaaagc tttagcgatc gtgtgctgaa catttatcgt 1980

ttcgcaaaat acttcgccct ggataaatca tgccagtgga atccggatgg tctggataca 2040

gatgattttt atgttgccta tgaacagttc tatagcgacg gctatgaaaa gatcgtgaaa 2100

gtgtatgata aagtgcgcaa ctacatgacc aagaaaccgt ttaatcagga caaatggaag 2160

ctgaattttg aaaatccgac actggcaaat ggttgggaca aaaacaaaga aacagacaac 2220

accgcaatta ttctgcgtcg tgcaggtcgt tattacctgg cagttatgga acgtggtcat 2280

aatacgctgt ttaaaaagat tccgatgagc agcagcggtt atcagaaaat gacctataaa 2340

ctgtttccgg atccgagtaa aatgatgccg aaagtttgtt ttagcaagaa gggccttgaa 2400

ttctttaaac cgagcgcaga aatcatgcgc atttacaaaa atggcgaatt caaaaagggc 2460

gataccttta gcctgagcag catgcatgtt ctgattgact tttataagaa cgccctgaaa 2520

acctatgatg gctggaccat gtatgatttc agcaatctga aaaagaccag cgagtatacc 2580

gaaaacatcg gcgaatttta tcgtgatgtt gcagaaagcg gctaccagat taactttgat 2640

tatatcgccg aacagtatat cgaggatgcc aataaagaag gtaaactgta cctgtttgag 2700

atccacaaca aagactggaa tctgaaagat ggtgcaatta aaaccggtag caaaaatgca 2760

cacaccctgt attttgaaca ggtgttttca gatgaaaacg cgcagaacaa ttttgtggtg 2820

aaactgaatg gcgaagccga actgtttttc cgtccggcaa ccagcaccga gaaactgggt 2880

aatcattatg atagcaaagg taacgtggtg accaaaaata agcgttatgc ccatgacaaa 2940

atgttctttc atgttccggt tacactgaat cgtaccgcac cggatgcacg caaatttaac 3000

cagagcgtta atgtttttct ggcgaataat ccggatacca acattattgg tattgaccgt 3060

ggcgaaaaac atctggcata tctgagcgtt attaaccaga aaggtgacat cctgaaaatc 3120

aagagcctga acaaaatcga ggtgaaagat aaagatggca acgtgatcaa agaagatgat 3180

tacgcaaaac tgctggaaga tcgtgccaaa aatcgtgaat cagcacgtcg tgattggaaa 3240

agcgttgagc agattaaaga tcttaaaaag ggctacatta gcaacgtggt tcgtgaaatt 3300

gcagatctgg tgattaaata caatgccatc gtggtgtttg aggatctgaa tatgcgcttt 3360

aaacaggttc gtggtggcat tgaaaaatcg gtttatcagc agttagaaaa ggccctgatt 3420

gataagctga acttcctggt ggataaaaat gaactggatc cgcagaaagc aggtcatatt 3480

ctgcatgcat atcagctgac cgcaccgttt gaaaccttta aagatatggg taaacagacc 3540

ggtgtgctgt tttataccca ggcagaatac accagccaga cagatccggt taccggcttt 3600

cgtaaaaatg tttatctgag caatagcgcc accgtggaaa agattaaagc ctttgttgaa 3660

atgttcgatg tgatcggctg ggacgataaa ctgaaaagct attatttcaa gtataacccg 3720

gtgaacttcg tggaaaccaa gtttaaagag aacaccttca gcaaagattg ggtgatttat 3780

gcaaatgtgc ctcgcattaa acgcgaacgc aaaaatggtt attgggaagc aaccgttgtt 3840

aacccgaatg aagaatttct gaagctgttc aaagagtggg atttcgataa catctacgtc 3900

gaggacatta aagaacaaat tttccagatg ttcgaagagg gtcgcctgga tggcaccaaa 3960

gaatttgatg gcaaaaaccg caacttttgg cacagcttca tttttctgtt taacctgatg 4020

ctgcaggttc gtaatagcac cgcaacacag tataaaaagg atgaggatgg caacattatc 4080

gaaaccgttg aaggtgtgga ttttattgcc agtccggtgt ttccgttctt taccaccgat 4140

ggtggtgatt ttaccgaagg ttgtgtgaat ctggcaaaac tggaagataa atttgtgggt 4200

agcaacgccg ataaagaacg cttcaagaaa gagtttaatg gtgatgcaaa tggtgcgtat 4260

aatatcgcac gtaaaggcat tattatgctg aacaatatca aaaacaaccc cgaaaaaccg 4320

gacctgtttg tgagcaaaaa ggattgggat aagtttgcac aggccaacca gtaatggtct 4380

agaggtcgaa attcaaattg tgagcggata acaatttgaa ttttctgtat gaggttttgc 4440

taaacaactt tcaacagttt cagtggagtg agaatagaaa ggaacaacta aaggaattgc 4500

gaataataat tttttcacgt tgaaaatctc caaaaaaaaa ggctccaaaa ggagccttta 4560

attgtatcgg tttatcagct tgctttcgag gtgaattttg accctctagc gaaaatgcaa 4620

gagcaaagac gaaaacatgc cacacatgag gaataccgat tctctcatta acatattcag 4680

gccagttatc tgggcttaaa agcagaagtc caacccagat aacgatcata tacatggttc 4740

tctccagagg ttcattactg aacactcgtc cgagaataac gagtggatcc cctccaattc 4800

gccctatagt gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg 4860

ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg 4920

gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg 4980

cgaatggaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc 5040

agctcatttt ttaaccaata ggccgactgc gatgagtggc agggcggggc gtaatttttt 5100

taaggcagtt attggtgccc ttaaacgcct ggtgctacgc ctgaataagt gataataagc 5160

ggatgaatgg cagaaattcg aaagcaaatt cgacccggtc gtcggttcag ggcagggtcg 5220

ttaaatagcc gcttatgtct attgctggtt taccggttta ttgactaccg gaagcagtgt 5280

gaccgtgtgc ttctcaaatg cctgaggcca gtttgctcag gctctccccg tggaggtaat 5340

aattgacgat atgatcattt attctgcctc ccagagcctg ataaaaacgg tgaatccgtt 5400

agcgaggtgc cgccggcttc cattcaggtc gaggtggccc ggctccatgc accgcgacgc 5460

aacgcgggga ggcagacaag gtatagggcg gcgaggcggc tacagccgat agtctggaac 5520

agcgcactta cgggttgctg cgcaacccaa gtgctaccgg cgcggcagcg tgacccgtgt 5580

cggcggctcc aacggctcgc catcgtccag aaaacacggc tcatcgggca tcggcaggcg 5640

ctgctgcccg cgccgttccc attcctccgt ttcggtcaag gctggcaggt ctggttccat 5700

gcccggaatg ccgggctggc tgggcggctc ctcgccgggg ccggtcggta gttgctgctc 5760

gcccggatac agggtcggga tgcggcgcag gtcgccatgc cccaacagcg attcgtcctg 5820

gtcgtcgtga tcaaccacca cggcggcact gaacaccgac aggcgcaact ggtcgcgggg 5880

ctggccccac gccacgcggt cattgaccac gtaggccgac acggtgccgg ggccgttgag 5940

cttcacgacg gagatccagc gctcggccac caagtccttg actgcgtatt ggaccgtccg 6000

caaagaacgt ccgatgagct tggaaagtgt cttttggctg accaccacgg cgttctggtg 6060

gcccatctgc gccacgaggt gatgcagcag cattgccgcc gtgggtttcc tcgcaataag 6120

cccggcccac gcctcatgcg ctttgcgttc cgtttgcacc cagtgaccgg gcttgttctt 6180

ggcttgaatg ccgatttctc tggactgcgt ggccatgctt atctccatgc ggtagggtgc 6240

cgcacggttg cggcaccatg cgcaatcagc tgcaactttt cggcagcgcg acaacaatta 6300

tgcgttgcgt aaaagtggca gtcaattaca gattttcttt aacctacgca atgagctatt 6360

gcggggggtg ccgcaatgag ctgttgcgta cccccctttt ttaagttgtt gatttttaag 6420

tctttcgcat ttcgccctat atctagttct ttggtgccca aagaagggca cccctgcggg 6480

gttcccccac gccttcggcg cggctccccc tccggcaaaa agtggcccct ccggggcttg 6540

ttgatcgact gcgcggcctt cggccttgcc caaggtggcg ctgccccctt ggaacccccg 6600

cactcgccgc cgtgaggctc ggggggcagg cgggcgggct tcgccttcga ctgcccccac 6660

tcgcataggc ttgggtcgtt ccaggcgcgt caaggccaag ccgctgcgcg gtcgctgcgc 6720

gagccttgac ccgccttcca cttggtgtcc aaccggcaag cgaagcgcgc aggccgcagg 6780

ccggaggctt ttccccagag aaaattaaaa aaattgatgg ggcaaggccg caggccgcgc 6840

agttggagcc ggtgggtatg tggtcgaagg ctgggtagcc ggtgggcaat ccctgtggtc 6900

aagctcgtgg gcaggcgcag cctgtccatc agcttgtcca gcagggttgt ccacgggccg 6960

agcgaagcga gccagccggt ggccgctcgc ggccatcgtc cacatatcca cgggctggca 7020

agggagcgca gcgaccgcgc agggcgaagc ccggagagca agcccgtagg gcgccgcagc 7080

cgccgtaggc ggtcacgact ttgcgaagca aagtctagtg agtatactca agcattgagt 7140

ggcccgccgg aggcaccgcc ttgcgctgcc cccgtcgagc cggttggaca ccaaaaggga 7200

ggggcaggca tggcggcata cgcgatcatg cgatgcaaga agctggcgaa aatgggcaac 7260

gtggcggcca gtctcaagca cgcctaccgc gagcgcgaga cgcccaacgc tgacgccagc 7320

aggacgccag agaacgagca ctgggcggcc agcagcaccg atgaagcgat gggccgactg 7380

cgcgagttgc tgccagagaa gcggcgcaag gacgctgtgt tggcggtcga gtacgtcatg 7440

acggccagcc cggaatggtg gaagtcggcc agccaagaac agcaggcggc gttcttcgag 7500

aaggcgcaca agtggctggc ggacaagtac ggggcggatc gcatcgtgac ggccagcatc 7560

caccgtgacg aaaccagccc gcacatgacc gcgttcgtgg tgccgctgac gcaggacggc 7620

aggctgtcgg ccaaggagtt catcggcaac aaagcgcaga tgacccgcga ccagaccacg 7680

tttgcggccg ctgtggccga tctagggctg caacggggca tcgagggcag caaggcacgt 7740

cacacgcgca ttcaggcgtt ctacgaggcc ctggagcggc caccagtggg ccacgtcacc 7800

atcagcccgc aagcggtcga gccacgcgcc tatgcaccgc agggattggc cgaaaagctg 7860

ggaatctcaa agcgcgttga gacgccggaa gccgtggccg accggctgac aaaagcggtt 7920

cggcaggggt atgagcctgc cctacaggcc gccgcaggag cgcgtgagat gcgcaagaag 7980

gccgatcaag cccaagagac ggcccgagac cttcgggagc gcctgaagcc cgttctggac 8040

gccctggggc cgttgaatcg ggatatgcag gccaaggccg ccgcgatcat caaggccgtg 8100

ggcgaaaagc tgctgacgga acagcgggaa gtccagcgcc agaaacaggc ccagcgccag 8160

caggaacgcg ggcgcgcaca tttccccgaa aagtgccacc tgaaccccag agtcccgctc 8220

agaagaactc gtcaagaagg cgatagaagg cgatgcgctg cgaatcggga gcggcgatac 8280

cgtaaagcac gaggaagcgg tcagcccatt cgccgccaag ctcttcagca atatcacggg 8340

tagccaacgc tatgtcctga tagcggtccg ccacacccag ccggccacag tcgatgaatc 8400

cagaaaagcg gccattttcc accatgatat tcggcaagca ggcatcgcca tgggtcacga 8460

cgagatcctc gccgtcgggc atccgcgcct tgagcctggc gaacagttcg gctggcgcga 8520

gcccctgatg ctcttcgtcc agatcatcct gatcgacaag accggcttcc atccgagtac 8580

gtgctcgctc gatgcgatgt ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg 8640

tatgcagccg ccgcattgca tcagccatga tggatacttt ctcggcagga gcaaggtgag 8700

atgacaggag atcctgcccc ggcacttcgc ccaatagcag ccagtccctt cccgcttcag 8760

tgacaacgtc gagcacagct gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg 8820

ctgcctcgtc ttggagttca ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg 8880

ggcgcccctg cgctgacagc cggaacacgg cggcatcaga gcagccgatt gtctgttgtg 8940

cccagtcata gccgaatagc ctctccaccc aagcggccgg agaacctgcg tgcaatccat 9000

cttgttcaat catgcgaaac gatcctcatc ctgtctcttg atcagatctt gatcccctgc 9060

gccatcagat ccttggcggc aagaaagcca tccagtttac tttgcagggc ttcccaacct 9120

taccagaggg cgccccagct ggcaattccg gttcgcttgc tgtccataaa accgcccagt 9180

ctagctatcg ccatgtaagc ccactgcaag ctacctgctt tctctttgcg cttgcgtttt 9240

cccttgtcca gatagcccag tagctgacat tcatccgggg tcagcaccgt ttctgcggac 9300

tggctttcta cgtgttccgc ttcctttagc agcccttgcg ccctgagtgc ttgcggcagc 9360

gtgaagctag ctgcataatg tgcctgtcaa atggacgaag cagggattct gcaaacccta 9420

tgctactccg tcaagccgtc aattgtctga ttcgttacca attatgacaa cttgacggct 9480

acatcattca ctttttcttc acaaccggca cggaactcgc tcgggctggc cccggtgcat 9540

tttttaaata cccgcgagaa atagagttga tcgtcaaaac caacattgcg accgacggtg 9600

gcgataggca tccgggtggt gctcaaaagc agcttcgcct ggctgatacg ttggtcctcg 9660

cgccagctta agacgctaat ccctaactgc tggcggaaaa gatgtgacag acgcgacggc 9720

gacaagcaaa catgctgtgc gacgctggcg atatcaaaat tgctgtctgc caggtgatcg 9780

ctgatgtact gacaagcctc gcgtacccga ttatccatcg gtggatggag cgactcgtta 9840

atcgcttcca tgcgccgcag taacaattgc tcaagcagat ttatcgccag cagctccgaa 9900

tagcgccctt ccccttgccc ggcgttaatg atttgcccaa acaggtcgct gaaatgcggc 9960

tggtgcgctt catccgggcg aaagaacccc gtattggcaa atattgacgg ccagttaagc 10020

cattcatgcc agtaggcgcg cggacgaaag taaacccact ggtgatacca ttcgcgagcc 10080

tccggatgac gaccgtagtg atgaatctct cctggcggga acagcaaaat atcacccggt 10140

cggcaaacaa attctcgtcc ctgatttttc accaccccct gaccgcgaat ggtgagattg 10200

agaatataac ctttcattcc cagcggtcgg tcgataaaaa aatcgagata accgttggcc 10260

t 10261

<210> 65

<211> 10156

<212> DNA

<213> 人工序列

<220>

<223> CRISPR/BMC15-Ec载体系统

<400> 65

caatcggcgt taaacccgcc accagatggg cattaaacga gtatcccggc agcaggggat 60

cattttgcgc ttcagccata cttttcatac tcccgccatt cagagaagaa accaattgtc 120

catattgcat cagacattgc cgtcactgcg tcttttactg gctcttctcg ctaaccaaac 180

cggtaacccc gcttattaaa agcattctgt aacaaagcgg gaccaaagcc atgacaaaaa 240

cgcgtaacaa aagtgtctat aatcacggca gaaaagtcca cattgattat ttgcacggcg 300

tcacactttg ctatgccata gcatttttat ccataagatt agcggatcct acctgacgct 360

ttttatcgca actctctact gtttctccat acccgttttt tggtaccggg ccccccctcg 420

agtttatttt aggaggcaaa aatgaatccg acacagaccg ataaaacccc gagcaaaccg 480

tttgaaaaat tcaccaatct gtactgcctg agcaaaaccc tgcgttttga actgaaaccg 540

attggtaaaa cccagaagat tctggaagat aacaaagtgt tcgagaacga taaaaagcgt 600

gccaaaagct atgaagaggc caaaaagtat ttcaacaaac tgcaccgcga atttatcgat 660

gaaagcctga aaaacattac cctgagcaac aatctgatcg agaaattcga gaaaaagtac 720

ctgacctgga aaaacagcaa aaacaaagat aatagcaccg agctgaaaaa gagcgcaaaa 780

cgtctgcgta ttgttatcct ggaaagcttt aataagaaag ccaacgaatg gaacagcgag 840

tatagcaatc aggtgaaaaa cgagaagaag aaaaagaaaa tccaagaaat caccggcatc 900

gacctgtttt tcaaagttga agtgtttgac ttcctgatcc acaaatatcc ggaagtgcag 960

attaatggcg aaagcatttt tagcccgttc aacaaattta gcggctactt caaaaagttt 1020

cacgaaaccc gcaaaaactt ctataaagat gatggcacca gcaccgcaat tccgacacgt 1080

attattgatg tgaatctgga aaagtttctg gaaaacaagg acatctacta taccaaatac 1140

tttcagaaat acaacagcat cttcaacaaa gaagaaaccg acatcttcaa actggaatcc 1200

ttcaaaaatt gtctgaccca gagccagatc gacaaataca atgaaagcat tgcaaccctg 1260

aaaagcaaga ttaataacct gcgtcagaat aacccggaag tgaataaaca tgatctgccg 1320

ttcttcaaag aactgtttcg tcagattctg ggccagccga ttaagaaaga aacagaacag 1380

gataacttca tcgagatcct gaccaatgat gaagtttttc cggtgctgca gaaaaacatc 1440

gatgagaacg aactgtatat tccgaaagca gataccctgt ttaaagagtt tctgaaatcc 1500

cagatccaag agacaaacga gtataacatc aacgaaattt atgtggccag ccgctttatt 1560

aacagcatta gcaataattg gtttgccgag tgggatacca ttattaacct gctgcgtacc 1620

gaactgaaaa tcaaacagaa tcagaaaaag ctgccggatt ttatcagcat tgccagtctg 1680

aaacgtgtac tgcagaaatc acaggatgaa attgatgcca aagacctgtt ccgcaacaat 1740

tatgaaaacc tgtttgaatc caccaccgac ttctacaaaa tcttcctgaa aatctgggag 1800

cttgagttca acgacaacat caaaaagtac aacctggaaa ccgaaaacat ccgcaaaatc 1860

atcatcgagg ataaaaagta tctgccgaac aagaaaagca tcctgaaaaa tggtgaaacc 1920

ggcattatcc acaacgagaa aattctggat tatgcacaga gcgcactgaa catttatcag 1980

atgatgaaat acttcagcct ggaaaagggt aaagaacgtg aatggaatcc ggatggtctg 2040

aatgaagata ccacaggtgg tttttatgac gatttcaata aatactacca gaatgtgaac 2100

acctggaagt attttaacga attccgcaac tacctgacca aaaagccgta taaaaccgac 2160

aaaatcaagc tgtattttgg ccacaaaagc ctgttaggtg gctttaccga aagcaaaacg 2220

gaaaaatcaa ataacggcac ccagtatggt gcatatctgc tgcgcaaaaa gcatggttta 2280

ggcggttttg attattatct gggtattagc accgatccgc atctgatgag ctattttgat 2340

ccgattgatg atagcggtga tagcgaatat gaacgcctga actattatca ggttctgacc 2400

cgtaccattt atggtccgag ttatgaaggt gattatgagc tggataagaa aaacctgagc 2460

gaaatcgaaa tcatcaagaa gattaaacgc agcctgtcct attataccag ccgtgttaaa 2520

aagatccagg acatcatcaa caacaactat gaatcagtgc gcgatatcca caaagatatt 2580

accgatgtgc tgaaagaatt cggcaccatc tttgattata aggtgatcac gaatagccag 2640

atccagaaag cctttaattg cgacaaaggc ttttacctgt tcgagatcta tagcaaggat 2700

tttagcaaag agaaaggcga caagagcaag aacagtaaag ataatctgca taccacctac 2760

ttcaagagcc tgatggatcg taaacagagc acctttgatt taggtagcgg tgaaattttc 2820

tttcgcgaga aaagcgttca gagcgaaatt gatagcatgc gcaaaaccaa aaacaagatc 2880

acccgtttta aacgctacac caaaaatctg atccagttca atctgagcat cacgctgaat 2940

aataactgta ccgaagttcc gcagaataaa aacgcacgta aagcgtttat caacaacttc 3000

aatatcgagc tgagcaagaa actgctgacc aataatagcg atattaacat tattggcatc 3060

gatcgcggtg agaaacatct ggcatattat agcgtgattg atcagcagag caatattctg 3120

gaaacaggca gctttaacaa aattcaagaa cgcaaagatc gtgagccgac cgattatcaa 3180

cagaaactgg ataaaattca gaaagatcgc gattggcagc gtaaaagttg gcaagaaatt 3240

agcaacatca aggacctgaa aaagggctat attagccagg tggtgtatga aattagtaaa 3300

ctggtgaaga agtataacgc catcatcgtt tttgaggatc tgaacattgg ctttaaacgt 3360

ggtcgttttg caattgagaa acaggtgtat caaaatctgg aactgagcct ggcaaagaaa 3420

ctgaattatc tggttttcaa agatgccaac gaaggtgaaa gtggccatta tctgaaagca 3480

tatcagctga ccagtccggt taataacttt caggatattg gtaaacagtg cggcatcatt 3540

ttctatattc cggcaagcta taccagcgca atttgtccga gctgtggttt tcacaaaaac 3600

attccgacca gcattaaaaa gctggccaag aacaaagagt tcgtcgaaaa atttgtgatc 3660

acgtacgagc tgaagaagga tcgtttctat ttcggctaca agatcaacga tttctataac 3720

agcaatctgc aggacaacgt gatcttttat agcaatgtgg aacgcctgcg ttacaaacgc 3780

aataaagata accgtagtgg tgaagtgcaa gagcgtctgc cgaatgaaga actgaagaaa 3840

ctgtttgaac agaaccacat caactacaaa gacaatccgc agattagcgg tcagatcaaa 3900

aatcagaaac ttgacaacga aaagttttac aaaccgctga tctatgaaat cagcctgatt 3960

ctgcagctgc gtaatagcaa aaccgttaaa agcgaagatg gcacgattaa caccaatatt 4020

aaccgcgatt tcattagctg tccggcatgt tattttcaca gcgagaataa tctgatgaac 4080

ctgccgaata aatacaaagg cggtaaaaag ttcgaattta atggtgatgc aaacggtgcc 4140

tataacattg cccgtaaagg tattctgctg ctgaataaac tgaacaacat taaagacatc 4200

gagaagatcg agtacaacga cctgaatatc agccaagagg attgggataa ttttgtcaaa 4260

aacccgtaat ggtctagagg tcgaaattca aattgtgagc ggataacaat ttgaattttc 4320

tgtatgaggt tttgctaaac aactttcaac agtttcagtg gagtgagaat agaaaggaac 4380

aactaaagga attgcgaata ataatttttt cacgttgaaa atctccaaaa aaaaaggctc 4440

caaaaggagc ctttaattgt atcggtttat cagcttgctt tcgaggtgaa ttttgaccct 4500

ctagcgaaaa tgcaagagca aagacgaaaa catgccacac atgaggaata ccgattctct 4560

cattaacata ttcaggccag ttatctgggc ttaaaagcag aagtccaacc cagataacga 4620

tcatatacat ggttctctcc agaggttcat tactgaacac tcgtccgaga ataacgagtg 4680

gatcccctcc aattcgccct atagtgagtc gtattacgcg cgctcactgg ccgtcgtttt 4740

acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc 4800

ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt 4860

gcgcagcctg aatggcgaat ggaaattgta agcgttaata ttttgttaaa attcgcgtta 4920

aatttttgtt aaatcagctc attttttaac caataggccg actgcgatga gtggcagggc 4980

ggggcgtaat ttttttaagg cagttattgg tgcccttaaa cgcctggtgc tacgcctgaa 5040

taagtgataa taagcggatg aatggcagaa attcgaaagc aaattcgacc cggtcgtcgg 5100

ttcagggcag ggtcgttaaa tagccgctta tgtctattgc tggtttaccg gtttattgac 5160

taccggaagc agtgtgaccg tgtgcttctc aaatgcctga ggccagtttg ctcaggctct 5220

ccccgtggag gtaataattg acgatatgat catttattct gcctcccaga gcctgataaa 5280

aacggtgaat ccgttagcga ggtgccgccg gcttccattc aggtcgaggt ggcccggctc 5340

catgcaccgc gacgcaacgc ggggaggcag acaaggtata gggcggcgag gcggctacag 5400

ccgatagtct ggaacagcgc acttacgggt tgctgcgcaa cccaagtgct accggcgcgg 5460

cagcgtgacc cgtgtcggcg gctccaacgg ctcgccatcg tccagaaaac acggctcatc 5520

gggcatcggc aggcgctgct gcccgcgccg ttcccattcc tccgtttcgg tcaaggctgg 5580

caggtctggt tccatgcccg gaatgccggg ctggctgggc ggctcctcgc cggggccggt 5640

cggtagttgc tgctcgcccg gatacagggt cgggatgcgg cgcaggtcgc catgccccaa 5700

cagcgattcg tcctggtcgt cgtgatcaac caccacggcg gcactgaaca ccgacaggcg 5760

caactggtcg cggggctggc cccacgccac gcggtcattg accacgtagg ccgacacggt 5820

gccggggccg ttgagcttca cgacggagat ccagcgctcg gccaccaagt ccttgactgc 5880

gtattggacc gtccgcaaag aacgtccgat gagcttggaa agtgtctttt ggctgaccac 5940

cacggcgttc tggtggccca tctgcgccac gaggtgatgc agcagcattg ccgccgtggg 6000

tttcctcgca ataagcccgg cccacgcctc atgcgctttg cgttccgttt gcacccagtg 6060

accgggcttg ttcttggctt gaatgccgat ttctctggac tgcgtggcca tgcttatctc 6120

catgcggtag ggtgccgcac ggttgcggca ccatgcgcaa tcagctgcaa cttttcggca 6180

gcgcgacaac aattatgcgt tgcgtaaaag tggcagtcaa ttacagattt tctttaacct 6240

acgcaatgag ctattgcggg gggtgccgca atgagctgtt gcgtaccccc cttttttaag 6300

ttgttgattt ttaagtcttt cgcatttcgc cctatatcta gttctttggt gcccaaagaa 6360

gggcacccct gcggggttcc cccacgcctt cggcgcggct ccccctccgg caaaaagtgg 6420

cccctccggg gcttgttgat cgactgcgcg gccttcggcc ttgcccaagg tggcgctgcc 6480

cccttggaac ccccgcactc gccgccgtga ggctcggggg gcaggcgggc gggcttcgcc 6540

ttcgactgcc cccactcgca taggcttggg tcgttccagg cgcgtcaagg ccaagccgct 6600

gcgcggtcgc tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg gcaagcgaag 6660

cgcgcaggcc gcaggccgga ggcttttccc cagagaaaat taaaaaaatt gatggggcaa 6720

ggccgcaggc cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg tagccggtgg 6780

gcaatccctg tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg 6840

gttgtccacg ggccgagcga agcgagccag ccggtggccg ctcgcggcca tcgtccacat 6900

atccacgggc tggcaaggga gcgcagcgac cgcgcagggc gaagcccgga gagcaagccc 6960

gtagggcgcc gcagccgccg taggcggtca cgactttgcg aagcaaagtc tagtgagtat 7020

actcaagcat tgagtggccc gccggaggca ccgccttgcg ctgcccccgt cgagccggtt 7080

ggacaccaaa agggaggggc aggcatggcg gcatacgcga tcatgcgatg caagaagctg 7140

gcgaaaatgg gcaacgtggc ggccagtctc aagcacgcct accgcgagcg cgagacgccc 7200

aacgctgacg ccagcaggac gccagagaac gagcactggg cggccagcag caccgatgaa 7260

gcgatgggcc gactgcgcga gttgctgcca gagaagcggc gcaaggacgc tgtgttggcg 7320

gtcgagtacg tcatgacggc cagcccggaa tggtggaagt cggccagcca agaacagcag 7380

gcggcgttct tcgagaaggc gcacaagtgg ctggcggaca agtacggggc ggatcgcatc 7440

gtgacggcca gcatccaccg tgacgaaacc agcccgcaca tgaccgcgtt cgtggtgccg 7500

ctgacgcagg acggcaggct gtcggccaag gagttcatcg gcaacaaagc gcagatgacc 7560

cgcgaccaga ccacgtttgc ggccgctgtg gccgatctag ggctgcaacg gggcatcgag 7620

ggcagcaagg cacgtcacac gcgcattcag gcgttctacg aggccctgga gcggccacca 7680

gtgggccacg tcaccatcag cccgcaagcg gtcgagccac gcgcctatgc accgcaggga 7740

ttggccgaaa agctgggaat ctcaaagcgc gttgagacgc cggaagccgt ggccgaccgg 7800

ctgacaaaag cggttcggca ggggtatgag cctgccctac aggccgccgc aggagcgcgt 7860

gagatgcgca agaaggccga tcaagcccaa gagacggccc gagaccttcg ggagcgcctg 7920

aagcccgttc tggacgccct ggggccgttg aatcgggata tgcaggccaa ggccgccgcg 7980

atcatcaagg ccgtgggcga aaagctgctg acggaacagc gggaagtcca gcgccagaaa 8040

caggcccagc gccagcagga acgcgggcgc gcacatttcc ccgaaaagtg ccacctgaac 8100

cccagagtcc cgctcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat 8160

cgggagcggc gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt 8220

cagcaatatc acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc 8280

cacagtcgat gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat 8340

cgccatgggt cacgacgaga tcctcgccgt cgggcatccg cgccttgagc ctggcgaaca 8400

gttcggctgg cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg 8460

cttccatccg agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg 8520

tagccggatc aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg 8580

caggagcaag gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt 8640

cccttcccgc ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca 8700

gccacgatag ccgcgctgcc tcgtcttgga gttcattcag ggcaccggac aggtcggtct 8760

tgacaaaaag aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc 8820

cgattgtctg ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac 8880

ctgcgtgcaa tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatcag 8940

atcttgatcc cctgcgccat cagatccttg gcggcaagaa agccatccag tttactttgc 9000

agggcttccc aaccttacca gagggcgccc cagctggcaa ttccggttcg cttgctgtcc 9060

ataaaaccgc ccagtctagc tatcgccatg taagcccact gcaagctacc tgctttctct 9120

ttgcgcttgc gttttccctt gtccagatag cccagtagct gacattcatc cggggtcagc 9180

accgtttctg cggactggct ttctacgtgt tccgcttcct ttagcagccc ttgcgccctg 9240

agtgcttgcg gcagcgtgaa gctagctgca taatgtgcct gtcaaatgga cgaagcaggg 9300

attctgcaaa ccctatgcta ctccgtcaag ccgtcaattg tctgattcgt taccaattat 9360

gacaacttga cggctacatc attcactttt tcttcacaac cggcacggaa ctcgctcggg 9420

ctggccccgg tgcatttttt aaatacccgc gagaaataga gttgatcgtc aaaaccaaca 9480

ttgcgaccga cggtggcgat aggcatccgg gtggtgctca aaagcagctt cgcctggctg 9540

atacgttggt cctcgcgcca gcttaagacg ctaatcccta actgctggcg gaaaagatgt 9600

gacagacgcg acggcgacaa gcaaacatgc tgtgcgacgc tggcgatatc aaaattgctg 9660

tctgccaggt gatcgctgat gtactgacaa gcctcgcgta cccgattatc catcggtgga 9720

tggagcgact cgttaatcgc ttccatgcgc cgcagtaaca attgctcaag cagatttatc 9780

gccagcagct ccgaatagcg cccttcccct tgcccggcgt taatgatttg cccaaacagg 9840

tcgctgaaat gcggctggtg cgcttcatcc gggcgaaaga accccgtatt ggcaaatatt 9900

gacggccagt taagccattc atgccagtag gcgcgcggac gaaagtaaac ccactggtga 9960

taccattcgc gagcctccgg atgacgaccg tagtgatgaa tctctcctgg cgggaacagc 10020

aaaatatcac ccggtcggca aacaaattct cgtccctgat ttttcaccac cccctgaccg 10080

cgaatggtga gattgagaat ataacctttc attcccagcg gtcggtcgat aaaaaaatcg 10140

agataaccgt tggcct 10156

Claims

1.一种编码RNA引导的DNA核酸内切酶的核酸分子，其是

(a)编码包含SEQ ID NO:9、1至5、7、8和10至15中任一项的氨基酸序列或由SEQ ID NO:9、1至5、7、8和10至15中任一项的氨基酸序列组成的RNA引导的DNA核酸内切酶的核酸分子；

(b)包含SEQ ID NO:24、16至20、22、23和25至30中任一项的核苷酸序列或由SEQ IDNO:24、16至20、22、23和25至30中任一项的核苷酸序列组成的核酸分子；

(c)编码氨基酸序列与(a)的氨基酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的RNA引导的DNA核酸内切酶的核酸分子；

(d)包含与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列或由与(b)的核苷酸序列至少70％相同、优选至少80％相同、更优选至少90％相同和最优选至少95％相同的核苷酸序列组成的核酸分子；

(e)相对于(d)的核酸分子简并的核酸分子；或

(f)对应于(a)至(d)中任一项的核酸分子的核酸分子，其中T被U替代。

2.权利要求1所述的核酸分子，其中所述核酸分子可操作地连接至对于所述核酸分子是天然的或异源的启动子。

3.权利要求1或2所述的核酸分子，其中所述核酸分子针对真核细胞中、优选植物细胞或动物细胞中的表达进行了密码子优化。

4.一种载体，其编码权利要求1至3中任一项所述的核酸分子。

5.一种宿主细胞，其包含权利要求1至3中任一项所述的核酸分子或转化、转导或转染有权利要求4所述的载体。

6.权利要求5所述的宿主细胞，其中所述宿主细胞是真核细胞或原核细胞，优选是植物细胞或动物细胞。

7.一种植物、种子或植物的一部分、或者动物，其包含权利要求1至3中任一项所述的核酸分子或转化、转导或转染有权利要求4所述的载体，所述植物的一部分不是单个植物细胞。

8.一种产生RNA引导的DNA核酸内切酶的方法，其包含培养权利要求5或6所述的宿主细胞并分离所产生的RNA引导的DNA核酸内切酶。

9.一种RNA引导的DNA核酸内切酶，其由权利要求1至3中任一项所述的核酸分子编码。

10.一种组合物，其包含权利要求1至3中任一项所述的核酸分子，权利要求4所述的载体，权利要求5或6所述的宿主细胞，权利要求7所述的植物、种子、细胞的一部分或动物，权利要求9所述的RNA引导的DNA核酸内切酶或它们的组合。

11.权利要求10所述的组合物，其中所述组合物是药物组合物或诊断组合物。

12.权利要求1至3中任一项所述的核酸分子，权利要求4所述的载体，权利要求5或6所述的宿主细胞，权利要求7所述的植物、种子、细胞的一部分或动物，权利要求9所述的RNA引导的DNA核酸内切酶或它们的组合，其用于通过修饰受试者或植物的基因组中靶位点的核苷酸序列而治疗所述受试者或植物的疾病的用途。

13.一种修饰细胞的基因组中靶位点的核苷酸序列的方法，包括将如下引入所述细胞：

(i)靶向DNA的RNA或编码靶向DNA的RNA的DNA多核苷酸，其中所述靶向DNA的RNA包含：

(a)包含与靶DNA中的序列互补的核苷酸序列的第一片段；和

(b)与权利要求9所述的RNA引导的DNA核酸内切酶相互作用的第二片段；和

(ii)权利要求9所述的RNA引导的DNA核酸内切酶，或权利要求1至3中任一项所述的编码RNA引导的DNA核酸内切酶的核酸分子，或权利要求4所述的载体，其中所述RNA引导的DNA核酸内切酶包含：

(a)与所述靶向DNA的RNA相互作用的RNA结合部分；和

(b)表现出定点酶活性的活性部分。

14.权利要求13所述的方法，其中所述细胞不是编码所述RNA引导的DNA核酸内切酶的基因的天然宿主。

15.权利要求13或14所述的方法，其中在将所述RNA引导的DNA核酸内切酶和所述靶向DNA的RNA直接引入所述细胞的情况下，它们以核糖核蛋白复合物(RNP)的形式引入。