CN111088201A - 一株重组丙酮丁醇梭菌及其构建方法与应用 - Google Patents
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Abstract
本发明公开了一株重组丙酮丁醇梭菌及其构建方法与应用，所述重组丙酮丁醇梭菌是通过抑制丙酮丁醇梭菌(为CGMCC No.5234)中CA_C2341基因的表达得到的,与现有技术相比，本发明具有如下优势：(1)本发明构建的丙酮丁醇梭菌CA_C2341基因突变工程菌株，这种工程菌株在发酵时生物被膜的形成能力提高。(2)本发明提供了一种通过失活相关基因提高产丁醇梭菌生物被膜形成能力的方法，通过此方法得到的工程菌株在发酵过程中，当以葡糖糖为碳源，在100mL厌氧瓶中发酵时，生物被膜产量提高约20％，丁醇产量提高约18％。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一株重组丙酮丁醇梭菌及其构建方法与应用，尤其是在提高产丁醇梭菌生物被膜形成能力中的应用。
背景技术
生物被膜是细菌在某些固体载体上通过自身分泌胞外聚合物质附着于固体载体表面的具有特殊微结构的细菌群落，细菌吸附于惰性物体如生物医学材料或机体粘膜表面后，分泌多糖基质、纤维蛋白、脂蛋白等胞外基质，使细菌相互粘连并将其自身克隆聚集缠绕其中。生物被膜的形成是细菌为适应自然环境而采取的一种策略，其形成可以分为五个过程：初始粘附期、对数生长期、时空立体发展期、扩散与瓦解期。
丁醇发酵，也被称为ABE(丙酮，丁醇和乙醇)发酵，曾经是世界第二大生物技术产业。以丙酮丁醇梭菌作为发酵菌种，对淀粉质、纸浆废液、糖蜜和废弃农作物秸秆等进行发酵，利用廉价易得的原料大大降低了成本，并且发酵制备的生物丁醇具有高能量密度、低挥发性、低吸水性以及低腐蚀的特性，能广泛应用于工业和能源领域。然而，在发酵过程中，产物丁醇对细胞的毒性作用，生产强度低，原料成本高等因素限制了其产业化的过程。丙酮丁醇梭菌能吸附在特定载体上，大量聚集后分泌胞外聚合物质，以生物被膜的形式生存。利用细胞固定化形成生物被膜进行发酵可以提高效率、降低成本、增强细胞耐受性。
生物被膜主要由细胞与胞外聚合物质组成，胞外聚合物质主要包括多糖、蛋白质、核酸和脂质等。这些聚合物种类繁多，与生物被膜的形成息息相关，能对细菌进行保护作用，且与代谢具有密切的关系。目前的一些研究表明，生物被膜还能用于固定材料，3D打印材料等领域。因此，需要开发一种提高产丁醇梭菌生物被膜形成能力的方法，以提高生物被膜的产量。
发明内容
发明目的：本发明所要解决的技术问题是针对现有技术的不足，提供一株重组丙酮丁醇梭菌。
本发明还要解决的问题的是提供上述重组丙酮丁醇梭菌的其构建方法。
本发明最后要解决的问题是提供上述重组丙酮丁醇梭菌的应用，尤其是在提高产丁醇梭菌生物被膜形成能力中的应用。
为了解决上述技术问题，本发明公开了一株重组丙酮丁醇梭菌，其是通过对丙酮丁醇梭菌进行基因改造而制得的。相对于野生型丙酮丁醇梭菌而言，其抑制了所述丙酮丁醇梭菌中CA_C2341基因的表达；其中，所述的丙酮丁醇梭菌(clostridiumacetobutylicum)为CGMCC No.5234，该菌株于中国普通微生物菌种保藏管理中心进行保藏，该菌株的具体信息在申请号为201210075094.X的中国专利中详细公开。
其中，所述的抑制其基因表达是指在该菌株中CA_C2341基因失去编码正常蛋白质的功能，将丙酮丁醇梭菌基因组上的CA_C2341基因删除一段DNA序列，抑制其正常功能；CA_C2341基因编码一种胶原蛋白酶，具有胶原酶蛋白结构域。其中，所述抑制或者降低胶原蛋白酶的活性通过敲除其部分DNA片段来实现，具体的为Crispr-Cas9n敲除技术。
其中，所述CA_C2341基因的核苷酸序列如SEQ ID NO.1所示。
上述重组丙酮丁醇梭菌的构建方法也在本发明的保护范围内，具体为：构建含有同源重组片段的基因敲除质粒pNICKclos2.0-2341，将基因敲除质粒pNICKclos2.0-2341甲基化后，电转进入丙酮丁醇梭菌CGMCC No.5234中，在红霉素平板上筛选得到CA_C2341基因失活的菌株，其核苷酸序列如SEQ ID NO.25所示；并将得到的菌株进行厌氧发酵。
其中，所述基因敲除质粒pNICKclos2.0-2341的构建方法为在CA_C2341基因上选择要删除的240bp，并在这240bp内选择3个不同的20bp靶向识别序列，通过overlap和一步克隆试剂盒将质粒pNICKclos2.0质粒上sgRNA区域内20bp靶向识别序列替换成CA_C2341基因上所选取的20bp识别序列位点，并将后面的同源臂序列替换成所选择要删除的240bp上下游1000bp的同源臂序列，得到pNICKclos2.0-2341。
其中，所述基因敲除质粒pNICKclos2.0-2341的构建方法具体包括如下步骤：
(1)sgRNA片段的合成：分析CA_C2341基因的核苷酸序列SEQ ID NO.1，在CA_C2341基因中要删除的240bp，其核苷酸序列如SEQ ID NO.18所示，片段中选择3个合适的PAM前20bp的靶向识别序列(其核苷酸序列如SEQ ID NO.19～21所示)，设计引物；以pNICKclos2.0质粒载体为模板分别，以SEQ ID NO.3和SEQ ID NO.6、SEQ ID NO.4和SEQ IDNO.6、SEQ ID NO.5和SEQ ID NO.6所示的核苷酸序列为引物，分别PCR扩增，得到sgRNA-2341-1片段，其核苷酸序列如SEQ ID NO.13所示；sgRNA-2341-3片段，其核苷酸序列如SEQID NO.14所示；sgRNA-2341-3片段，其核苷酸序列如SEQ ID NO.15所示；
(2)上下游同源臂片段的合成：以丙酮丁醇梭菌基因组为模板，以SEQ ID NO.7和SEQ ID NO.8、SEQ ID NO.9和SEQ ID NO.10所示的核苷酸序列为引物，分别PCR扩增，得到上游同源臂upHA，其核苷酸序列如SEQ ID NO.16所示；和下游同源臂dnHA，其核苷酸序列如SEQ ID NO.17所示；
(3)sgRNA片段和同源臂片段的整合：
(3a)通过重叠延伸PCR的技术，以sgRNA-2341-1片段和上游同源臂upHA片段为模板，以SEQ ID NO.3和SEQ ID NO.8所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-1-upHA片段；以sgRNA-2341-2片段和上游同源臂upHA片段为模板，以SEQ ID NO.4和SEQ IDNO.8所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-2-upHA片段；以sgRNA-2341-3片段和上游同源臂upHA片段为模板，以SEQ ID NO.5和SEQ ID NO.8所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-3-upHA片段；
(3b)通过重叠延伸PCR的技术，以sgRNA-2341-1-upHA片段和下游同源臂dnHA片段为模板，以SEQ ID NO.3和SEQ ID NO.10所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-1-upHA片段；以sgRNA-2341-2-upHA片段和下游同源臂dnHA片段为模板，以SEQ IDNO.4和SEQ ID NO.10所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-2-upHA片段；以sgRNA-2341-3-upHA片段和下游同源臂dnHA片段为模板，以SEQ ID NO.5和SEQ ID NO.10所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-3-upHA片段；
(4)构建质粒：将peI/XhoI双酶切后的载体pNICKclos2.0片段分别与sgRNA-2341-1-upHA片段、sgRNA-2341-2-upHA片段和sgRNA-2341-3-upHA片段进行重组反应，即得基因敲除质粒pNICKclos2.0-2341-1(其核苷酸序列如SEQ ID NO.22所示)，pNICKclos2.0-2341-2(其核苷酸序列如SEQ ID NO.23所示)，pNICKclos2.0-2341-3(其核苷酸序列如SEQID NO.24所示)。
其中，所述pNICKclos2.0基因的核苷酸序列如SEQ ID NO.2所示。
其中，所述的甲基化为将基因敲除质粒pNICKclos2.0-2341转化进入含有pAN2质粒的大肠杆菌E.coli Top10感受态细胞，筛选阳性克隆，即为甲基化的基因敲除质粒；其中，所述的pAN2质粒含有枯草芽抱杆菌噬菌体基因，能编码甲基转移酶，能实现外源质粒在大肠杆菌中的甲基化。
上述重组丙酮丁醇梭菌在生物被膜固定化催化体系中的应用也在本发明的保护范围之内；其中，所述重组丙酮丁醇梭菌是通过抑制或者降低CA_C2341胶原蛋白酶的活性来实现提高丙酮丁醇梭菌生物被膜形成能力。
其中，所述的应用包括以重组丙酮丁醇梭菌为发酵菌株，发酵制备丁醇。
其中，所述发酵培养基中各组分的浓度为葡萄糖60g/L，乙酸铵2.2g/L，磷酸二氢钾0.5g/L，磷酸氢二钾0.5g/L，氯化钠0.01g/L，七水合硫酸镁0.2g/L，七水合硫酸亚铁0.01g/L，一水合硫酸锰0.01g/L，溶剂为水。
其中，发酵的条件为37℃静置发酵60～96h，优选37℃静置发酵72h。
有益效果：与现有技术相比，本发明具有如下优势：
(1)本发明构建的丙酮丁醇梭菌CA_C2341基因突变工程菌株，这种工程菌株在发酵时生物被膜的形成能力提高。
(2)本发明提供了一种通过失活相关基因提高产丁醇梭菌生物被膜形成能力的方法，通过此方法得到的工程菌株在发酵过程中，当以葡糖糖为碳源，在100mL厌氧瓶中发酵时，生物被膜产量提高约20％，丁醇产量提高约18％。
附图说明
图1为sgRNA片段的PCR扩增产物电泳图。泳道1-5为sgRNA-2341-1片段，泳道6-10为sgRNA-2341-2片段，泳道11-15为sgRNA-2341-3片段；泳道16为DNA 1K bp Marker。
图2为上下游同源臂的PCR扩增产物电泳图，其中泳道3为DNA 2K bp Marker；泳道1-2为上游同源臂upHA片段，泳道4-5为下游同源臂dnHA片段。
图3为sgRNA与上下游同源臂三片段整合结果PCR扩增产物电泳图，其中泳道9为DNA 5K bp Marker；泳道1-3为sgRNA-2341-1-HA，泳道4-6为sgRNA-2341-2-HA，泳道7-8为sgRNA-2341-3-HA。
图4为一步克隆构建pNICKclos2.0-2341质粒电泳图，其中泳道1-3为pNICKclos2.0-2341-1质粒，泳道4-6为pNICKclos2.0-2341-2质粒，泳道7-8为pNICKclos2.0-2341-3质粒，泳道9为DNA 10K bp Marker。
图5为pNICKclos2.0-2341质粒SpeI/XhoI双酶切验证结果PCR扩增产物图。泳道1，3为pNICKclos2.0-2341质粒，泳道2、4、5为SpeI/XhoI双酶切结果，泳道6为DNA 10k bpMarker。
图6为敲除转化子菌落PCR扩增产物电泳图，其中泳道2，4，6，7，8，9，11，13，18，19电泳结果表明存在正确突变，泳道1,12为DNA 2K bp Marker。
图7为含正确条带的菌落平板划线后取单菌进行菌落PCR扩增产物电泳图，其中泳道8、10、17、19、21、22为正确突变结果，泳道1，12为DNA 2000bp Marker。
图8为质粒丢失结果图，左边为P2无抗平板，右边为P2红霉素抗性平板。
图9为丙酮丁醇梭菌B3野生菌发酵产物时序曲线。
图10为丙酮丁醇梭菌CA_C2341突变菌发酵产物时序曲线。
图11为生物被膜定量结果。
具体实施方式
根据下述实施例，可以更好地理解本发明。然而，本领域的技术人员容易理解，实施例所描述的内容仅用于说明本发明，而不应当也不会限制权利要求书中所详细描述的本发明。
以下实施例中如无特别说明，所用酶及质粒均为购买所得。
实施例1：敲除质粒pNICKclos2.0-2341的构建方法。
1、sgRNA片段的合成：
在丙酮丁醇梭菌的CA_C2730基因中要删除的240bp中间选取3个合适的PAM前20bp的靶向识别序列，设计引物sgRNA-1UP，sgRNA-2UP，sgRNA-3UP，sgRNA-DN(序列见表1)。使用sgRNA-1UP/sgRNA-DN作为上下游引物，pNICKclos2.0为模板，PCR得到sgRNA-2341-1片段，其核苷酸序列如SEQ ID NO.13所示。使用sgRNA-2UP/sgRNA-DN作为上下游引物，pNICKclos2.0为模板，PCR得到sgRNA-2341-2片段，其核苷酸序列如SEQ ID NO.14所示。使用sgRNA-3UP/sgRNA-DN作为上下游引物，pNICKclos2.0为模板，PCR得到sgRNA-2341-3片段，其核苷酸序列如SEQ ID NO.15所示(图1)。
表1引物序列表
2、上下游同源臂片段的合成：
使用pNICKclos2.0-2341质粒进行基因敲除时，cas9n蛋白造成的DNA单链切口需要进行同源重组进行修复，设计引物2341HA-1，2341HA-2，2341HA-3，2341HA-4(序列见表1)。使用2341HA-1/2341HA-2作为上下游引物，丙酮丁醇梭菌基因组为模板，PCR得到上游同源臂upHA，其核苷酸序列如SEQ ID NO.16所示，使用2341HA-3/2341HA-4作为上下游引物，丙酮丁醇梭菌基因组为模板，PCR得到下游同源臂dnHA，其核苷酸序列如SEQ ID NO.17所示(图2)。
3、sgRNA片段和同源臂片段的整合：
使用sgRNA-1UP/2341HA-2作为上下游引物，sgRNA-2341-1片段和上游同源臂upHA作为模板，进行overlap得到sgRNA-2341-1-upHA片段。
使用sgRNA-2UP/2341HA-2作为上下游引物，sgRNA-2341-2片段和上游同源臂upHA作为模板，进行overlap得到sgRNA-2341-2-upHA片段。
使用sgRNA-3UP/2341HA-2作为上下游引物，sgRNA-2341-3片段和上游同源臂upHA作为模板，进行overlap得到sgRNA-2341-3-upHA片段。
之后再使用sgRNA-1UP/2341HA-4作为上下游引物，sgRNA-2341-1-upHA片段和下游同源臂dnHA作为模板，进行overlap得到sgRNA-2341-1-HA片段。
使用sgRNA-2UP/2341HA-4作为上下游引物，sgRNA-2341-2-upHA片段和下游同源臂dnHA作为模板，进行overlap得到sgRNA-2341-2-HA片段。
使用sgRNA-3UP/2341HA-4作为上下游引物，sgRNA-2341-3-upHA片段和下游同源臂dnHA作为模板，进行overlap得到sgRNA-2341-3-HA片段。
根据图3的电泳图结果，泳道1-3为sgRNA-2341-1-HA片段大小为2000bp左右，泳道4-6为sgRNA-2341-2-HA片段大小为2000bp左右，泳道7-8为sgRNA-2341-3-HA片段大小为2000bp左右。
4、一步克隆构建质粒：
使用SpeI/XhoI对pNICKclos2.0进行双酶切，得到线性化载体pNICKclos2.0，使用一步克隆试剂盒(VAZYME)对线性化载体pNICKclos2.0与sgRNA-2341-1-HA、sgRNA-2341-2-HA、sgRNA-2341-3-HA分别进行重组反应后，将构建好的敲除质粒转化进大肠杆菌E.coliDH5a感受态中扩增，涂布到含50ug/mL氨苄霉素的LB平板上，37℃培养12h，挑取转化子接到液体LB培养基中(含有30ug/mL红霉素)、37℃、200rpm培养12h，提取重组质粒(AXYGEN质粒提取试剂盒)，测序验证，获得CA_C2341敲除质粒pNICKclos2.0-2341-1，pNICKclos2.0-2341-2，pNICKclos2.0-2341-3。
其中，pNICKclos2.0质粒赠自中国科学院上海生命科学研究院。
根据图4的电泳图结果，泳道1-3为pNICKclos2.0-2341-1质粒电泳图，大小为10kbp左右，泳道4-6为pNICKclos2.0-2341-1质粒电泳图，大小为10kbp左右，泳道7-8为pNICKclos2.0-2341-1质粒电泳图，大小为10kbp左右。对重组质粒进行SpeⅠ/XhoⅠ双酶切验证，根据图5的电泳图泳道2，4，5结果显示均切下一条2000bp左右条带，与预期结构一致，结合测序结果表明质粒构建成功。
实施例2：丙酮丁醇梭菌CA_C2341基因突变菌的构建方法：
1、CA_C2341敲除质粒的甲基化：
由于丙酮丁醇梭菌中含有限制性内切酶Cac824Ⅰ，可以切割未甲基化的外源DNA，因此在将敲除质粒转化进丙酮丁醇梭菌中之前需要对敲除质粒pNICKclos2.0-2341进行甲基化修饰。具体步骤为将敲除质粒pNICKclos2.0-2341(包括：pNICKclos2.0-2341-1，pNICKclos2.0-2341-2，pNICKclos2.0-2341-3)转化进大肠杆菌E.coli Top10感受态(含pAN2质粒，该质粒有一个枯草芽抱杆菌噬菌体基因，能编码甲基转移酶，能实现外源质粒在大肠杆菌中的甲基化)中扩增，涂布到30ug/mL红霉素和10ug/mL四环素(pAN2质粒具有四环素抗性)与的LB平板上，37℃培养12h，挑取转化子接到液体LB培养基中(含有30ug/mL红霉素和10ug/mL四环素)、37℃、200rpm培养12h，提取甲基化的敲除质粒。
2、电转化甲基化敲除质粒至丙酮丁醇梭菌：
在厌氧条件下，取在2xYTG培养基生长至对数期(OD600＝1.1)的丙酮丁醇梭菌培养液，在4℃、4000rpm离心10min弃去上清，加入足量预冷的电转缓冲液EPB(270mM蔗糖，5mM磷酸盐缓冲液，pH 7.4)，洗涤两次，并用2.3mL EPB重悬。然后取570μL加入4mm电转杯放置在冰浴中冷却，并加入20μL甲基化质粒，冰浴放置2min。2.0kV电压，25uF电容进行电转化。随后将电转液加入到5mL 37℃的2xYTG培养基中复苏培养4h，离心收集细胞100uL并将细胞涂布于含有30ug/mL红霉素的P2平板中。
3、筛选CA_C2341突变菌株：
挑取转化子，使用引物CA_C2341-F、CA_C2341-R(序列见表1)，对转化子进行菌落PCR验证，筛选出基因组上CA_C2341基因被删除240bp的突变株(突变后，PCR扩增出基因条带比野生型小大约240bp)。挑取含正确突变条带的菌落，进行平板划线后再进行菌落PCR验证。
根据图6敲除转化子菌落PCR扩增产物电泳图，其中泳道2，4，6，7，8，9，11，13，18，19出现两条条带，说明其菌落中存在正确突变，将含正确条带的菌落平板划线后挑单菌再进行菌落PCR验证，根据图7电泳图，其中泳道8，10，17，19，21，22出现比野生型条带小240bp左右的单一条带，表明纯化出突变菌株。
将正确的突变株传代三次，同时涂布在含有红霉素抗性和无抗的P2固体培养基上，筛选出质粒丢失的突变株(在含红霉素抗性平板上不能生长的突变株)。
根据图8，将纯化后的突变菌株同时涂在含有红霉素抗性和无抗的P2固体培养基上，结果显示在含红霉素抗性的平板上不能生长，表明质粒丢失成功，将质粒丢失后的突变菌株扩培保藏进行下一步的实验。
实施例3：丙酮丁醇梭菌CA_C2341突变菌的培养和发酵方法。
P2液体培养基：酵母粉3g/L，蛋白胨5g/L，葡萄糖10g/L，乙酸铵2g/L，氯化钠3g/L，七水合硫酸镁3g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，七水合硫酸亚铁0.1g/L，其余为水。
P2平板培养基：酵母粉3g/L，蛋白胨5g/L，葡萄糖10g/L，乙酸铵2g/L，氯化钠3g/L，七水合硫酸镁3g/L，磷酸二氢钾1g/L，磷酸氢二钾1g/L，七水合硫酸亚铁0.1g/L，酵母粉3.5g/L，其余为水。
发酵培养基：葡萄糖60g/L，乙酸铵2.2g/L，磷酸二氢钾0.5g/L，磷酸氢二钾0.5g/L，氯化钠0.01g/L，七水合硫酸镁0.2g/L，七水合硫酸亚铁0.01g/L，一水合硫酸锰0.01g/L，其余为水。
将实例2中构建的CA_C2341基因突变菌和丙酮丁醇梭菌B3野生菌接种至P2平板培养基中，放置于厌氧箱中于37℃，培养时间12h。将平板培养的CA_C2341基因突变菌与丙酮丁醇梭菌B3野生菌转接至P2液体培养基中，于37℃静置培养至OD600＝2.0后将其按照接种量10％接种于终体积为50mL的发酵培养基中，置于37℃静置发酵72h，每隔12h取样2mL。
实施例4：气相色谱法测定发酵产物
色谱条件如下：HP-INNOWax毛细管色谱柱，PEG-20M，60m×0.25mm×0.5μm，担体为硅藻土。柱温采用程序升温法：初始70℃，保持0.5min后以20℃·min-1的速度升至190℃，保持0.5min，柱流量1.0mL·min-1，尾吹流速29mL·min-1，氢气流速30mL·min-1，空气流速300mL·min-1。前进样口温度180℃，FID温度220℃，进样量1.0μL，分流比90：1。实施例3中每隔12h取的样品在气相检测前先离心然后取上清1～1.5mL过0.22μm滤膜后备用。
检测结果如图9和图10。图9为丙酮丁醇梭菌B3野生菌发酵产物时序曲线；图10丙酮丁醇梭菌CA_C2341突变菌发酵产物时序曲线。从图中可以看出，相比于丙酮丁醇梭菌B3野生菌，本发明中抑制CA_C2341基因的表达后得到的重组丙酮丁醇梭菌发酵得到丁醇的产量提高约18％。
实施例5：结晶紫染色法定量测定生物被膜
(1)取丙酮丁醇梭菌B3野生菌和丙酮丁醇梭菌CA_C2341基因突变菌株起始发酵液200uL于96孔板中，置于37℃厌氧培养20h后取出，上下翻转倾倒发酵液。
(2)将孔板温和地置于去离子水中，轻轻摇晃后倒出液体，除去未附着的细胞，重复两次。
(3)向加入了发酵液的孔内加入200uL的0.3％结晶紫溶液染色15min后，用去离子水洗净。
(4)室温晾干后每孔加入33％的乙酸溶解结晶紫溶液，室温放置15min。
(5)取一新的96孔板，将溶解后的结晶紫溶液转移至新的孔板，以33％乙酸作为空白，在酶标仪中于570nm处对吸光度进行定量。
图11为其检测结果，左边为丙酮丁醇梭菌B3野生菌生物被膜量，右边为丙酮丁醇梭菌CA_C2341基因突变菌株生物被膜量。从图中可以清晰的看出，相比于丙酮丁醇梭菌B3野生菌，本发明中抑制CA_C2341基因的表达后得到的重组丙酮丁醇梭菌发酵产丁醇的过程中生物被膜产量提高约20％。
本发明提供了一株重组丙酮丁醇梭菌及其构建方法与应用的思路及方法，具体实现该技术方案的方法和途径很多，以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。本实施例中未明确的各组成部分均可用现有技术加以实现。
序列表
<110> 南京工业大学
<120> 一株重组丙酮丁醇梭菌及其构建方法与应用
<160> 25
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2364
<212> DNA
<213> 1(CA_C2341)
<400> 1
atgaataagg ttgaattgct agcaccagca ggcagtattg agagcatata tgcagctgtt 60
caaaacggtg ctgatgcagt ttatgtaggt ggaagtaggt tttcagctag agcttatgct 120
gaaaatttta atgatgaaat tatggaaaaa gcagttgatt attgtcacga ttatggagta 180
aggctatacg taacagtaaa tacgcttata aaacaagatg agataaaaga agttattaga 240
tatatagaat ttctttataa aataggagta gatgcactta tagttcaaga tactgctata 300
ttgcaactta ttaaaagata cgttcctgat tttgaaatac atgcatcaac tcagatgaca 360
attcacaatg cattaggagc tgtgtattat aaagaaagag gctttaaaag aattgtgctt 420
tcgagggagt tgtccttaag ggaaattgaa tacatatcta aagaacttaa tatagaaaca 480
gaggtttttg ttcatggtgc attatgtgta tgttattctg gacagtgtct tatgagttct 540
ataataggtg gtagaagtgg taatagagga agatgtgctc aaccatgtag acaaagttat 600
acacttattg ataaaagcag tttggaaagt aaaaaaggat attttctaag taccaaggat 660
atgtgtactg tggatatttt agataggctc attgataccg gaacttcttc cttaaagata 720
gagggaagaa tgaaaaagcc tgaatatgca ggaggagttg tttcatcata tagaaaagcc 780
attgataata agcttaatgg agactataaa gaagagaaag caaagctttt aaagcttttt 840
aatagagagg gtttttcaag agcatatctt ttgggcaata caggaaaaga tatgatggct 900
tataaattcc ctaaaaatac aggggttttt atagggaagg taataagtga tggaagtata 960
gttcttaaag aaagtttaaa cttaaaggat ggagttagaa atgatgtcaa gggtactgtt 1020
gtgtcaaaga tacttcttga tggggttgaa gttaaagagg ctaagccagg agaaaaggtg 1080
aaaatatttc ctcgctttta caagtctgga gattatatat acaagactct tgatttaaag 1140
cttactcgtg aaattgagga aagttataaa aaggatttta agagaaaagt atatttagat 1200
ttagaaattg aatttaaaat cggagagcca tttaagatta ctgcaagttt ttcaagagta 1260
aatgatttag gggcaaatga aaaaaatagg gctattagta tacaagtttt aggggatatg 1320
gtttgcaagg ctgaaaaaag tcctgtaaag agagaaaaag tagaggaaca tttaaggaaa 1380
acaggaggta ctccttttga ggtcagaaac atagagttta cctgctttga tgaaggatat 1440
ataccgatat ctgctttgaa ttcaataagg agagaagttc tagaaaaatt tcaaaatagt 1500
attcagaaaa gctataaaag agaagctaat ttaaagtttg aaggaataaa gatttctaag 1560
aggtcagaag ttaaaatccc agagatcatg gttttagttt cgaataagga gcagtatctt 1620
gcagttaaag aagagggatt taagaacata atagttgact tatttaaaag aaaatgtgac 1680
ttgaagctta aggaacttaa ggatgtttat attaaggttc ctaatgtaat taaagaagag 1740
tttaacgata tatgcaataa aatagatgaa aatatagaaa acattaaagg catagtaacg 1800
gttaatgcag gaataataaa taaatataaa ggtctagtta atataatagg agactataaa 1860
ggaaatatat ttaatagcga agcattagag ttttataagg aagatctaga tggtttttgc 1920
attagcacag agttaagtaa aaatgaaata aaggcattga ataaatgtaa atgcatggat 1980
acccaaatgc ttatatatgg taaaatagaa gcaatggtaa gtgagtattg tcctatagga 2040
agtgtttttg gaggtaaatc ttctaaaagc aattgccaag gtacatgcat gaaatcagac 2100
tttgtactta aagataagat gggagtatct tttgatgtgg taacagacaa atattgcaga 2160
agtcatatat ataatccatt accattaaat cttataggca atttagatga aataaagaat 2220
ttaggcgtga attcttatag aatagatttt attgatgaaa atagagaaag tgttaaaaga 2280
gttctttcat ggatgaaaaa tggtaagatt gatggagaat tcagtggttt tacaagagga 2340
agttataaaa gaggagttga ataa 2364
<210> 2
<211> 11729
<212> DNA
<213> 2(pNICKclos2.0)
<400> 2
cgataattta cagaaaagaa aattatagaa tttagtatga ttaattatac tcatttatga 60
atgtttaatt gaatacaaaa aaaaatactt gttatgtatt caattacggg ttaaaatata 120
gacaagttga aaaatttaat aaaaaaataa gtcctcagct cttatatatt aagctaccaa 180
cttagtatat aagccaaaac ttaaatgtgc taccaacaca tcaagccgtt agagaactct 240
atctatagca atatttcaaa tgtaccgaca tacaagagaa acattaacta tatatattca 300
atttatgaga ttatcttaac agatataaat gtaaattgca ataagtaaga tttagaagtt 360
tatagccttt gtgtattgga agcagtacgc aaaggctttt ttatttgata aaaattagaa 420
gtatatttat tttttcataa ttaatttatg aaaatgaaag ggggtgagca aagtgacaga 480
ggaaagcagt atcttatcaa ataacaaggt attagcaata tcattattga ctttagcagt 540
aaacattatg acttttatag tgcttgtagc taagtagtac gaaaggggga gctttaaaaa 600
gctccttgga atacatagaa ttcataaatt aatttatgaa aagaagggcg tatatgaaaa 660
cttgtaaaaa ttgcaaagag tttattaaag atactgaaat atgcaaaata cattcgttga 720
tgattcatga taaaacagta gcaacctatt gcagtaaata caatgagtca agatgtttac 780
ataaagggaa agtccaatgt attaattgtt caaagatgaa ccgatatgga tggtgtgcca 840
taaaaatgag atgttttaca gaggaagaac agaaaaaaga acgtacatgc attaaatatt 900
atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa aaagaaaaaa taatttattt 960
attaatttaa tattgagagt gccgacacag tatgcactaa aaaatatatc tgtggtgtag 1020
tgagccgata caaaaggata gtcactcgca ttttcataat acatcttatg ttatgattat 1080
gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa agagttcggg gtagggttaa 1140
gcatagttga ggcaactaaa caatcaagct aggatatgca gtagcagacc gtaaggtcgt 1200
tgtttaggtg tgttgtaata catacgctat taagatgtaa aaatacggat accaatgaag 1260
ggaaaagtat aatttttgga tgtagtttgt ttgttcatct atgggcaaac tacgtccaaa 1320
gccgtttcca aatctgctaa aaagtatatc ctttctaaaa tcaaagtcaa gtatgaaatc 1380
ataaataaag tttaattttg aagttattat gatattatgt ttttctatta aaataaatta 1440
agtatataga atagtttaat aatagtatat acttaatgtg ctgcagttga cagctagctc 1500
agtcctaggt ataatactag tcgagttaga cataatagtg agttttagag ctagaaatag 1560
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 1620
tttgcggccg caaaacctgg ttctaatgga attgtttact taccatatct tatgggagaa 1680
agaacacctc atatagaccc taatgtaaaa ggtgcatttt taggagtatc actaataaat 1740
aaccatgatg attttgtacg tagtatatta gaaggtgtta gctttagttt gaaaaactgc 1800
cttgatttaa tcgaagacat gaatgtaaac attgatgaaa taagagtaag tggcggtgga 1860
gcagaaagtg ctgtatggag acaaatatta gctgatattt tccaatattc attaactaca 1920
gttaaagcat ctgaaggcgg tgcgcttgga gttgcaatat tagcaggggt tggagcagga 1980
atctatgatt ctgtagaaga tgcttgtgat agaatcgtaa aaggaaacaa gaaagtttct 2040
ccaaatgaag aattaaaaga agtatactca aaagtatatg aaacatataa ttcagcttat 2100
ccaagaataa aagatattta aatatattaa agatagaaat caatcatctt agttaagtaa 2160
ttgtataatt aattaagggt gtgtagtagt atcgaatttt agcaaaaaaa tcatatagtt 2220
ttttaaatac tttaacaaaa ataataatag tgatattatt taagaaaaag ctactattac 2280
tgataatagt cgagaggagt attttaaagt ttgatagaag taaatcacag taaaataaaa 2340
gaaactaaca gaaaaaagat aatagcatta ttattaaaga aaaatgagat caccaaatta 2400
gatatatcta gaatattaga tataagtata acaacagttt ctacaaatat tacagaattg 2460
aaaagcgaag gtattgtaga agatgtaaga tcgttggaat ctacaggagg aagaaaagca 2520
attgctatta agttgaatga aaactgtaga tattcaattg gagtcgcact aacaccgaat 2580
catataaaga tatcattagt aaatataaag aaaaaagtta ttgaaagtat gctgcagtgg 2640
aatagagaac ataataaatt tacttaatga aaatatagat ttgttaatga agaaatataa 2700
tttatgctct gcaaatttat taggaatagg gatttcatta cctggaaccg tagattttaa 2760
agaaggcata ataaagtatt catatttgtt aggcgccaaa gattttaatt taaaggaaaa 2820
gttcgaatac ttagacattc cagtatatgt agataatgaa gcaaatttat ctgcttacta 2880
cgagttttta aataaaagag atatactaag aaatctatta tatgtatcaa taacagaagg 2940
tttaggcctt ggtataataa taaatggaaa aatttacaga ggtgacaata actcatcagg 3000
agaattaggt catactaaga tagcaattga tggaaaaaaa tgtaagtgtg gagctagagg 3060
ctgtttagaa gcatacacat ctatgaattc tctgattgat agttataatg aagctaatag 3120
ttcaaatatt agtgatattg atgaatttga agaaagatat aatcaaaatg acaaagatgc 3180
tcatgatgta ttaaatgatt atttaaaaac attgtcttta ggtctatcga atttggttat 3240
gatatttgat cctagcagca tagtaatagg tggagatata aataatttac ttaatgataa 3300
gatagatatg ctgaaaaaag aggtttataa agataattta tttacaaatg aaaataattg 3360
cagtataagt attgcaagtt ttaaagaatc ttatctacta ggagctgcta tgatgcctat 3420
agaagaattc ttagaaatta aataatggag tggtgaaagc tgctctatta atttaaaaac 3480
ttattaaata aatttttaaa atatattttt aaagttatgt aagcatagct atattaaggg 3540
gggtcttaat ctttgattgg aactccagat taagacaagc cttttatata aaactaaata 3600
tctatttatt tcatattaga tttatgttct tgattttctc gagtttttaa caaaatatat 3660
tgataaaaat aataatagtg ggtataatta agttgttaga gaaaacgtat aaattaggga 3720
taaactatgg aacttatgaa atagattgaa atggtttatc tgttaccccg tatcaaaatt 3780
taggaggtta gttagaggat ccccgggatg gataagaaat actcaatagg cttagctatc 3840
ggcacaaata gcgtcggatg ggcggtgatc actgatgaat ataaggttcc gtctaaaaag 3900
ttcaaggttc tgggaaatac agaccgccac agtatcaaaa aaaatcttat aggggctctt 3960
ttatttgaca gtggagagac agcggaagcg actcgtctca aacggacagc tcgtagaagg 4020
tatacacgtc ggaagaatcg tatttgttat ctacaggaga ttttttcaaa tgagatggcg 4080
aaagtagatg atagtttctt tcatcgactt gaagagtctt ttttggtgga agaagacaag 4140
aagcatgaac gtcatcctat ttttggaaat atagtagatg aagttgctta tcatgagaaa 4200
tatccaacta tctatcatct gcgaaaaaaa ttggtagatt ctactgataa agcggatttg 4260
cgcttaatct atttggcctt agcgcatatg attaagtttc gtggtcattt tttgattgag 4320
ggagatttaa atcctgataa tagtgatgtg gacaaactat ttatccagtt ggtacaaacc 4380
tacaatcaat tatttgaaga aaaccctatt aacgcaagtg gagtagatgc taaagcgatt 4440
ctttctgcac gattgagtaa atcaagacga ttagaaaatc tcattgctca gctccccggt 4500
gagaagaaaa atggcttatt tgggaatctc attgctttgt cattgggttt gacccctaat 4560
tttaaatcaa attttgattt ggcagaagat gctaaattac agctttcaaa agatacttac 4620
gatgatgatt tagataattt attggcgcaa attggagatc aatatgctga tttgtttttg 4680
gcagctaaga atttatcaga tgctatttta ctttcagata tcctaagagt aaatactgaa 4740
ataactaagg ctcccctatc agcttcaatg attaaacgct acgatgaaca tcatcaagac 4800
ttgactcttt taaaagcttt agttcgacaa caacttccag aaaagtataa agaaatcttt 4860
tttgatcaat caaaaaacgg atatgcaggt tatattgatg ggggagctag ccaagaagaa 4920
ttttataaat ttatcaaacc aattttagaa aaaatggatg gtactgagga attattggtg 4980
aaactaaatc gtgaagattt gctgcgcaag caacggacct ttgacaacgg ctctattccc 5040
catcaaattc acttgggtga gctgcatgct attttgagaa gacaagaaga cttttatcca 5100
tttttaaaag acaatcgtga gaagattgaa aaaatcttga cttttcgaat tccttattat 5160
gttggtccat tggcgcgtgg caatagtcgt tttgcatgga tgactcggaa gtctgaagaa 5220
acaattaccc catggaattt tgaagaagtt gtcgataaag gtgcttcagc tcaatcattt 5280
attgaacgca tgacaaactt tgataaaaat cttccaaatg aaaaagtact accaaaacat 5340
agtttgcttt atgagtattt tacggtttat aacgaattga caaaggtcaa atatgttact 5400
gaaggaatgc gaaaaccagc atttctttca ggtgaacaga agaaagccat tgttgattta 5460
ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa aagaagatta tttcaaaaaa 5520
atagaatgtt ttgatagtgt tgaaatttca ggagttgaag atagatttaa tgcttcatta 5580
ggtacctacc atgatttgct aaaaattatt aaagataaag attttttgga taatgaagaa 5640
aatgaagata tcttagagga tattgtttta acattgacct tatttgaaga tagggagatg 5700
attgaggaaa gacttaaaac atatgctcac ctctttgatg ataaggtgat gaaacagctt 5760
aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa aattgattaa tggtattagg 5820
gataagcaat ctggcaaaac aatattagat tttttgaaat cagatggttt tgccaatcgc 5880
aattttatgc agctgatcca tgatgatagt ttgacattta aagaagacat tcaaaaagca 5940
caagtgtctg gacaaggcga tagtttacat gaacatattg caaatttagc tggtagccct 6000
gctattaaaa aaggtatttt acagactgta aaagttgttg atgaattggt caaagtaatg 6060
gggcggcata agccagaaaa tatcgttatt gaaatggcac gtgaaaatca gacaactcaa 6120
aagggccaga aaaattcgcg agagcgtatg aaacgaatcg aagaaggtat caaagaatta 6180
ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc aattgcaaaa tgaaaagctc 6240
tatctctatt atctccaaaa tggaagagac atgtatgtgg accaagaatt agatattaat 6300
cgtttaagtg attatgatgt cgatcacatt gttccacaaa gtttccttaa agacgattca 6360
atagacaata aggtcttaac gcgttctgat aaaaatcgtg gtaaatcgga taacgttcca 6420
agtgaagaag tagtcaaaaa gatgaaaaac tattggagac aacttctaaa cgccaagtta 6480
atcactcaac gtaagtttga taatttaacg aaagctgaac gtggaggttt gagtgaactt 6540
gataaagctg gttttatcaa acgccaattg gttgaaactc gccaaatcac taagcatgtg 6600
gcacaaattt tggatagtcg catgaatact aaatacgatg aaaatgataa acttattcga 6660
gaggttaaag tgattacctt aaaatctaaa ttagtttctg acttccgaaa agatttccaa 6720
ttctataaag tacgtgagat taacaattac catcatgccc atgatgcgta tctaaatgcc 6780
gtcgttggaa ctgctttgat taagaaatat ccaaaacttg aatcggagtt tgtctatggt 6840
gattataaag tttatgatgt tcgtaaaatg attgctaagt ctgagcaaga aataggcaaa 6900
gcaaccgcaa aatatttctt ttactctaat atcatgaact tcttcaaaac agaaattaca 6960
cttgcaaatg gagagattcg caaacgccct ctaatcgaaa ctaatgggga aactggagaa 7020
attgtctggg ataaagggcg agattttgcc acagtgcgca aagtattgtc catgccccaa 7080
gtcaatattg tcaagaaaac agaagtacag acaggcggat tctccaagga gtcaatttta 7140
ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag actgggatcc aaaaaaatat 7200
ggtggttttg atagtccaac ggtagcttat tcagtcctag tggttgctaa ggtggaaaaa 7260
gggaaatcga agaagttaaa atccgttaaa gagttactag ggatcacaat tatggaaaga 7320
agttcctttg aaaaaaatcc gattgacttt ttagaagcta aaggatataa ggaagttaaa 7380
aaagacttaa tcattaaact acctaaatat agtctttttg agttagaaaa cggtcgtaaa 7440
cggatgctgg ctagtgccgg agaattacaa aaaggaaatg agctggctct gccaagcaaa 7500
tatgtgaatt ttttatattt agctagtcat tatgaaaagt tgaagggtag tccagaagat 7560
aacgaacaaa aacaattgtt tgtggagcag cataagcatt atttagatga gattattgag 7620
caaatcagtg aattttctaa gcgtgttatt ttagcagatg ccaatttaga taaagttctt 7680
agtgcatata acaaacatag agacaaacca atacgtgaac aagcagaaaa tattattcat 7740
ttatttacgt tgacgaatct tggagctccc gctgctttta aatattttga tacaacaatt 7800
gatcgtaaac gatatacgtc tacaaaagaa gttttagatg ccactcttat ccatcaatcc 7860
atcactggtc tttatgaaac acgcattgat ttgagtcagc taggaggtga ctgagcggcc 7920
gctctagagg gggtaccgag ctcgaattca ctggccgtcg ttttacaacg tcgtgactgg 7980
gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg 8040
cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc 8100
gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata 8160
tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg 8220
ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa 8280
gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc 8340
gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg 8400
gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 8460
tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 8520
caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc 8580
ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa 8640
gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt 8700
aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt 8760
ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc 8820
atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg 8880
gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg 8940
gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac 9000
atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca 9060
aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta 9120
actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat 9180
aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa 9240
tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc agatggtaag 9300
ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat 9360
agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt 9420
tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg 9480
aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga 9540
gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta 9600
atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa 9660
gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact 9720
gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca 9780
tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt 9840
accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg 9900
ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag 9960
cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta 10020
agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat 10080
ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg 10140
tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc 10200
ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc tgtggataac 10260
cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac cgagcgcagc 10320
gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt 10380
tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc gggcagtgag 10440
cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt acactttatg 10500
cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac aggaaacagc 10560
tatgaccatg attacgccaa gctttggcta acacacacgc cattccaacc aatagttttc 10620
tcggcataaa gccatgctct gacgcttaaa tgcactaatg ccttaaaaaa acattaaagt 10680
ctaacacact agacttattt acttcgtaat taagtcgtta aaccgtgtgc tctacgacca 10740
aaagtataaa acctttaaga actttctttt ttcttgtaaa aaaagaaact agataaatct 10800
ctcatatctt ttattcaata atcgcatcag attgcagtat aaatttaacg atcactcatc 10860
atgttcatat ttatcagagc tcgtgctata attatactaa ttttataagg aggaaaaaat 10920
aaagagggtt ataatgaacg agaaaaatat aaaacacagt caaaacttta ttacttcaaa 10980
acataatata gataaaataa tgacaaatat aagattaaat gaacatgata atatctttga 11040
aatcggctca ggaaaagggc attttaccct tgaattagta cagaggtgta atttcgtaac 11100
tgccattgaa atagaccata aattatgcaa aactacagaa aataaacttg ttgatcacga 11160
taatttccaa gttttaaaca aggatatatt gcagtttaaa tttcctaaaa accaatccta 11220
taaaatattt ggtaatatac cttataacat aagtacggat ataatacgca aaattgtttt 11280
tgatagtata gctgatgaga tttatttaat cgtggaatac gggtttgcta aaagattatt 11340
aaatacaaaa cgctcattgg cattattttt aatggcagaa gttgatattt ctatattaag 11400
tatggttcca agagaatatt ttcatcctaa acctaaagtg aatagctcac ttatcagatt 11460
aaatagaaaa aaatcaagaa tatcacacaa agataaacag aagtataatt atttcgttat 11520
gaaatgggtt aacaaagaat acaagaaaat atttacaaaa aatcaattta acaattcctt 11580
aaaacatgca ggaattgacg atttaaacaa tattagcttt gaacaattct tatctctttt 11640
caatagctat aaattattta ataagtaagt taagggatgc ataaactgca tcccttaact 11700
tgtttttcgt gtacctattt tttgtgaat 11729
<210> 3
<211> 65
<212> DNA
<213> 3(sgRNA-1UP)
<400> 3
gctcagtcct aggtataata ctagttatct ttaaggaaga agttcgtttt agagctagaa 60
atagc 65
<210> 4
<211> 65
<212> DNA
<213> 4(sgRNA-2UP)
<400> 4
gctcagtcct aggtataata ctagtaactt cttccttaaa gataggtttt agagctagaa 60
atagc 65
<210> 5
<211> 65
<212> DNA
<213> 5(sgRNA-3UP)
<400> 5
gctcagtcct aggtataata ctagtgccat tgataataag cttaagtttt agagctagaa 60
atagc 65
<210> 6
<211> 42
<212> DNA
<213> 6(sgRNA-DN)
<400> 6
cattagcaca gagttaagta agcggccgca aaaaagcacc ga 42
<210> 7
<211> 42
<212> DNA
<213> 7(2341HA-1)
<400> 7
tcggtgcttt tttgcggccg cttacttaac tctgtgctaa tg 42
<210> 8
<211> 40
<212> DNA
<213> 8(2341HA-2)
<400> 8
gataggctca ttgataccgt aataagtgat ggaagtatag 40
<210> 9
<211> 39
<212> DNA
<213> 9(2341HA-3)
<400> 9
ctatacttcc atcacttatt acggtatcaa tgagcctat 39
<210> 10
<211> 45
<212> DNA
<213> 10(2341HA-4)
<400> 10
caatatattt tgttaaaaac tcgagttgat atatttgatc cacct 45
<210> 11
<211> 21
<212> DNA
<213> 11(CA_C2341-F)
<400> 11
ctatacttcc atcacttatt a 21
<210> 12
<211> 18
<212> DNA
<213> 12(CA_C2341-R)
<400> 12
gtgaagaaat atctgaag 18
<210> 13
<211> 156
<212> DNA
<213> 13(sgRNA-2341-1)
<400> 13
gctcagtcct aggtataata ctagttatct ttaaggaaga agttcgtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120
cttttttgcg gccgcttact taactctgtg ctaatg 156
<210> 14
<211> 156
<212> DNA
<213> 14(sgRNA-2341-2)
<400> 14
gctcagtcct aggtataata ctagtaactt cttccttaaa gataggtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120
cttttttgcg gccgcttact taactctgtg ctaatg 156
<210> 15
<211> 156
<212> DNA
<213> 15(sgRNA-2341-3)
<400> 15
gctcagtcct aggtataata ctagtgccat tgataataag cttaagtttt agagctagaa 60
atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac cgagtcggtg 120
cttttttgcg gccgcttact taactctgtg ctaatg 156
<210> 16
<211> 1000
<212> DNA
<213> 上游同源臂(UpHA)
<400> 16
ttacttaact ctgtgctaat gcaaaaacca tctagatctt ccttataaaa ctctaatgct 60
tcgctattaa atatatttcc tttatagtct cctattatat taactagacc tttatattta 120
tttattattc ctgcattaac cgttactatg cctttaatgt tttctatatt ttcatctatt 180
ttattgcata tatcgttaaa ctcttcttta attacattag gaaccttaat ataaacatcc 240
ttaagttcct taagcttcaa gtcacatttt cttttaaata agtcaactat tatgttctta 300
aatccctctt ctttaactgc aagatactgc tccttattcg aaactaaaac catgatctct 360
gggattttaa cttctgacct cttagaaatc tttattcctt caaactttaa attagcttct 420
cttttatagc ttttctgaat actattttga aatttttcta gaacttctct ccttattgaa 480
ttcaaagcag atatcggtat atatccttca tcaaagcagg taaactctat gtttctgacc 540
tcaaaaggag tacctcctgt tttccttaaa tgttcctcta ctttttctct ctttacagga 600
cttttttcag ccttgcaaac catatcccct aaaacttgta tactaatagc cctatttttt 660
tcatttgccc ctaaatcatt tactcttgaa aaacttgcag taatcttaaa tggctctccg 720
attttaaatt caatttctaa atctaaatat acttttctct taaaatcctt tttataactt 780
tcctcaattt cacgagtaag ctttaaatca agagtcttgt atatataatc tccagacttg 840
taaaagcgag gaaatatttt caccttttct cctggcttag cctctttaac ttcaacccca 900
tcaagaagta tctttgacac aacagtaccc ttgacatcat ttctaactcc atcctttaag 960
tttaaacttt ctttaagaac tatacttcca tcacttatta 1000
<210> 17
<211> 1000
<212> DNA
<213> 下游同源臂(DnHA)
<400> 17
cggtatcaat gagcctatct aaaatatcca cagtacacat atccttggta cttagaaaat 60
atcctttttt actttccaaa ctgcttttat caataagtgt ataactttgt ctacatggtt 120
gagcacatct tcctctatta ccacttctac cacctattat agaactcata agacactgtc 180
cagaataaca tacacataat gcaccatgaa caaaaacctc tgtttctata ttaagttctt 240
tagatatgta ttcaatttcc cttaaggaca actccctcga aagcacaatt cttttaaagc 300
ctctttcttt ataatacaca gctcctaatg cattgtgaat tgtcatctga gttgatgcat 360
gtatttcaaa atcaggaacg tatcttttaa taagttgcaa tatagcagta tcttgaacta 420
taagtgcatc tactcctatt ttataaagaa attctatata tctaataact tcttttatct 480
catcttgttt tataagcgta tttactgtta cgtatagcct tactccataa tcgtgacaat 540
aatcaactgc tttttccata atttcatcat taaaattttc agcataagct ctagctgaaa 600
acctacttcc acctacataa actgcatcag caccgttttg aacagctgca tatatgctct 660
caatactgcc tgctggtgct agcaattcaa ccttattcat aagtacctcc aaaattatta 720
aattttatat aactatgata ttatccacta atcctcttac tatgtatggt cctataacta 780
ttatacataa agtatctatt attacaaata aaagttataa ggtaaatttt attgcaaaat 840
aataaggagg gtttcccctc catttactta tattaatttt taaatactaa aagttaatct 900
ttatctaatt ctccaatctc ttcatttaag tattgatgtg aacaattttc atcctctggt 960
aaagcaaaat cctctttttg aggtggatca aatatatcaa 1000
<210> 18
<211> 240
<212> DNA
<213> 18(CA_C2341)
<400> 18
ccttccctat aaaaacccct gtatttttag ggaatttata agccatcata tcttttcctg 60
tattgcccaa aagatatgct cttgaaaaac cctctctatt aaaaagcttt aaaagctttg 120
ctttctcttc tttatagtct ccattaagct tattatcaat ggcttttcta tatgatgaaa 180
caactcctcc tgcatattca ggctttttca ttcttccctc tatctttaag gaagaagttc 240
<210> 19
<211> 20
<212> DNA
<213> 识别序列1(20bp)
<400> 19
tatctttaag gaagaagttc 20
<210> 20
<211> 20
<212> DNA
<213> 识别序列2(20bp)
<400> 20
aacttcttcc ttaaagatag 20
<210> 21
<211> 20
<212> DNA
<213> 识别序列3(20bp)
<400> 21
gccattgata ataagcttaa 20
<210> 22
<211> 11723
<212> DNA
<213> 基因敲除质粒1(NICKclos2.0-2341 -1)
<400> 22
cgataattta cagaaaagaa aattatagaa tttagtatga ttaattatac tcatttatga 60
atgtttaatt gaatacaaaa aaaaatactt gttatgtatt caattacggg ttaaaatata 120
gacaagttga aaaatttaat aaaaaaataa gtcctcagct cttatatatt aagctaccaa 180
cttagtatat aagccaaaac ttaaatgtgc taccaacaca tcaagccgtt agagaactct 240
atctatagca atatttcaaa tgtaccgaca tacaagagaa acattaacta tatatattca 300
atttatgaga ttatcttaac agatataaat gtaaattgca ataagtaaga tttagaagtt 360
tatagccttt gtgtattgga agcagtacgc aaaggctttt ttatttgata aaaattagaa 420
gtatatttat tttttcataa ttaatttatg aaaatgaaag ggggtgagca aagtgacaga 480
ggaaagcagt atcttatcaa ataacaaggt attagcaata tcattattga ctttagcagt 540
aaacattatg acttttatag tgcttgtagc taagtagtac gaaaggggga gctttaaaaa 600
gctccttgga atacatagaa ttcataaatt aatttatgaa aagaagggcg tatatgaaaa 660
cttgtaaaaa ttgcaaagag tttattaaag atactgaaat atgcaaaata cattcgttga 720
tgattcatga taaaacagta gcaacctatt gcagtaaata caatgagtca agatgtttac 780
ataaagggaa agtccaatgt attaattgtt caaagatgaa ccgatatgga tggtgtgcca 840
taaaaatgag atgttttaca gaggaagaac agaaaaaaga acgtacatgc attaaatatt 900
atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa aaagaaaaaa taatttattt 960
attaatttaa tattgagagt gccgacacag tatgcactaa aaaatatatc tgtggtgtag 1020
tgagccgata caaaaggata gtcactcgca ttttcataat acatcttatg ttatgattat 1080
gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa agagttcggg gtagggttaa 1140
gcatagttga ggcaactaaa caatcaagct aggatatgca gtagcagacc gtaaggtcgt 1200
tgtttaggtg tgttgtaata catacgctat taagatgtaa aaatacggat accaatgaag 1260
ggaaaagtat aatttttgga tgtagtttgt ttgttcatct atgggcaaac tacgtccaaa 1320
gccgtttcca aatctgctaa aaagtatatc ctttctaaaa tcaaagtcaa gtatgaaatc 1380
ataaataaag tttaattttg aagttattat gatattatgt ttttctatta aaataaatta 1440
agtatataga atagtttaat aatagtatat acttaatgtg ctgcagttga cagctagctc 1500
agtcctaggt ataatactag ttatctttaa ggaagaagtt cgttttagag ctagaaatag 1560
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 1620
tttgcggccg cttacttaac tctgtgctaa tgcaaaaacc atctagatct tccttataaa 1680
actctaatgc ttcgctatta aatatatttc ctttatagtc tcctattata ttaactagac 1740
ctttatattt atttattatt cctgcattaa ccgttactat gcctttaatg ttttctatat 1800
tttcatctat tttattgcat atatcgttaa actcttcttt aattacatta ggaaccttaa 1860
tataaacatc cttaagttcc ttaagcttca agtcacattt tcttttaaat aagtcaacta 1920
ttatgttctt aaatccctct tctttaactg caagatactg ctccttattc gaaactaaaa 1980
ccatgatctc tgggatttta acttctgacc tcttagaaat ctttattcct tcaaacttta 2040
aattagcttc tcttttatag cttttctgaa tactattttg aaatttttct agaacttctc 2100
tccttattga attcaaagca gatatcggta tatatccttc atcaaagcag gtaaactcta 2160
tgtttctgac ctcaaaagga gtacctcctg ttttccttaa atgttcctct actttttctc 2220
tctttacagg acttttttca gccttgcaaa ccatatcccc taaaacttgt atactaatag 2280
ccctattttt ttcatttgcc cctaaatcat ttactcttga aaaacttgca gtaatcttaa 2340
atggctctcc gattttaaat tcaatttcta aatctaaata tacttttctc ttaaaatcct 2400
ttttataact ttcctcaatt tcacgagtaa gctttaaatc aagagtcttg tatatataat 2460
ctccagactt gtaaaagcga ggaaatattt tcaccttttc tcctggctta gcctctttaa 2520
cttcaacccc atcaagaagt atctttgaca caacagtacc cttgacatca tttctaactc 2580
catcctttaa gtttaaactt tctttaagaa ctatacttcc atcacttatt acggtatcaa 2640
tgagcctatc taaaatatcc acagtacaca tatccttggt acttagaaaa tatccttttt 2700
tactttccaa actgctttta tcaataagtg tataactttg tctacatggt tgagcacatc 2760
ttcctctatt accacttcta ccacctatta tagaactcat aagacactgt ccagaataac 2820
atacacataa tgcaccatga acaaaaacct ctgtttctat attaagttct ttagatatgt 2880
attcaatttc ccttaaggac aactccctcg aaagcacaat tcttttaaag cctctttctt 2940
tataatacac agctcctaat gcattgtgaa ttgtcatctg agttgatgca tgtatttcaa 3000
aatcaggaac gtatctttta ataagttgca atatagcagt atcttgaact ataagtgcat 3060
ctactcctat tttataaaga aattctatat atctaataac ttcttttatc tcatcttgtt 3120
ttataagcgt atttactgtt acgtatagcc ttactccata atcgtgacaa taatcaactg 3180
ctttttccat aatttcatca ttaaaatttt cagcataagc tctagctgaa aacctacttc 3240
cacctacata aactgcatca gcaccgtttt gaacagctgc atatatgctc tcaatactgc 3300
ctgctggtgc tagcaattca accttattca taagtacctc caaaattatt aaattttata 3360
taactatgat attatccact aatcctctta ctatgtatgg tcctataact attatacata 3420
aagtatctat tattacaaat aaaagttata aggtaaattt tattgcaaaa taataaggag 3480
ggtttcccct ccatttactt atattaattt ttaaatacta aaagttaatc tttatctaat 3540
tctccaatct cttcatttaa gtattgatgt gaacaatttt catcctctgg taaagcaaaa 3600
tcctcttttt gaggtggatc aaatatatca actcgagttt ttaacaaaat atattgataa 3660
aaataataat agtgggtata attaagttgt tagagaaaac gtataaatta gggataaact 3720
atggaactta tgaaatagat tgaaatggtt tatctgttac cccgtatcaa aatttaggag 3780
gttagttaga ggatccccgg gatggataag aaatactcaa taggcttagc tatcggcaca 3840
aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag 3900
gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt 3960
gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag aaggtataca 4020
cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta 4080
gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga caagaagcat 4140
gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca 4200
actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta 4260
atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat 4320
ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat 4380
caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct 4440
gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag 4500
aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa 4560
tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat 4620
gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct 4680
aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact 4740
aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact 4800
cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat 4860
caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat 4920
aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta 4980
aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa 5040
attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta tccattttta 5100
aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt 5160
ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt 5220
accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa 5280
cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg 5340
ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga 5400
atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc 5460
aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa 5520
tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc 5580
taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa 5640
gatatcttag aggatattgt tttaacattg accttatttg aagataggga gatgattgag 5700
gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt 5760
cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag 5820
caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt 5880
atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa agcacaagtg 5940
tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt 6000
aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg 6060
cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc 6120
cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt 6180
cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc 6240
tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta 6300
agtgattatg atgtcgatca cattgttcca caaagtttcc ttaaagacga ttcaatagac 6360
aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa 6420
gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact 6480
caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa 6540
gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa 6600
attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt 6660
aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat 6720
aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt 6780
ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat 6840
aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc 6900
gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca 6960
aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc 7020
tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat 7080
attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa 7140
agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt 7200
tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa 7260
tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc 7320
tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac 7380
ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg 7440
ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg 7500
aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa 7560
caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc 7620
agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca 7680
tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt 7740
acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt 7800
aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact 7860
ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactgagc ggccgctcta 7920
gagggggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 7980
cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 8040
agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 8100
cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 8160
actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 8220
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 8280
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 8340
cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 8400
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 8460
taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 8520
tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 8580
gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 8640
gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 8700
cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 8760
tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 8820
tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 8880
atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 8940
ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 9000
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 9060
gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 9120
gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 9180
gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 9240
gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 9300
cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 9360
atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 9420
tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 9480
ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 9540
gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 9600
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 9660
ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 9720
ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 9780
gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 9840
ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 9900
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 9960
ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 10020
agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 10080
agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 10140
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 10200
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 10260
accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 10320
gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 10380
attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 10440
gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 10500
gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 10560
catgattacg ccaagctttg gctaacacac acgccattcc aaccaatagt tttctcggca 10620
taaagccatg ctctgacgct taaatgcact aatgccttaa aaaaacatta aagtctaaca 10680
cactagactt atttacttcg taattaagtc gttaaaccgt gtgctctacg accaaaagta 10740
taaaaccttt aagaactttc ttttttcttg taaaaaaaga aactagataa atctctcata 10800
tcttttattc aataatcgca tcagattgca gtataaattt aacgatcact catcatgttc 10860
atatttatca gagctcgtgc tataattata ctaattttat aaggaggaaa aaataaagag 10920
ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt caaaacataa 10980
tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct ttgaaatcgg 11040
ctcaggaaaa gggcatttta cccttgaatt agtacagagg tgtaatttcg taactgccat 11100
tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc acgataattt 11160
ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat cctataaaat 11220
atttggtaat ataccttata acataagtac ggatataata cgcaaaattg tttttgatag 11280
tatagctgat gagatttatt taatcgtgga atacgggttt gctaaaagat tattaaatac 11340
aaaacgctca ttggcattat ttttaatggc agaagttgat atttctatat taagtatggt 11400
tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca gattaaatag 11460
aaaaaaatca agaatatcac acaaagataa acagaagtat aattatttcg ttatgaaatg 11520
ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt ccttaaaaca 11580
tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc ttttcaatag 11640
ctataaatta tttaataagt aagttaaggg atgcataaac tgcatccctt aacttgtttt 11700
tcgtgtacct attttttgtg aat 11723
<210> 23
<211> 11723
<212> DNA
<213> 基因敲除质粒2(NICKclos2.0-2341 -2)
<400> 23
cgataattta cagaaaagaa aattatagaa tttagtatga ttaattatac tcatttatga 60
atgtttaatt gaatacaaaa aaaaatactt gttatgtatt caattacggg ttaaaatata 120
gacaagttga aaaatttaat aaaaaaataa gtcctcagct cttatatatt aagctaccaa 180
cttagtatat aagccaaaac ttaaatgtgc taccaacaca tcaagccgtt agagaactct 240
atctatagca atatttcaaa tgtaccgaca tacaagagaa acattaacta tatatattca 300
atttatgaga ttatcttaac agatataaat gtaaattgca ataagtaaga tttagaagtt 360
tatagccttt gtgtattgga agcagtacgc aaaggctttt ttatttgata aaaattagaa 420
gtatatttat tttttcataa ttaatttatg aaaatgaaag ggggtgagca aagtgacaga 480
ggaaagcagt atcttatcaa ataacaaggt attagcaata tcattattga ctttagcagt 540
aaacattatg acttttatag tgcttgtagc taagtagtac gaaaggggga gctttaaaaa 600
gctccttgga atacatagaa ttcataaatt aatttatgaa aagaagggcg tatatgaaaa 660
cttgtaaaaa ttgcaaagag tttattaaag atactgaaat atgcaaaata cattcgttga 720
tgattcatga taaaacagta gcaacctatt gcagtaaata caatgagtca agatgtttac 780
ataaagggaa agtccaatgt attaattgtt caaagatgaa ccgatatgga tggtgtgcca 840
taaaaatgag atgttttaca gaggaagaac agaaaaaaga acgtacatgc attaaatatt 900
atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa aaagaaaaaa taatttattt 960
attaatttaa tattgagagt gccgacacag tatgcactaa aaaatatatc tgtggtgtag 1020
tgagccgata caaaaggata gtcactcgca ttttcataat acatcttatg ttatgattat 1080
gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa agagttcggg gtagggttaa 1140
gcatagttga ggcaactaaa caatcaagct aggatatgca gtagcagacc gtaaggtcgt 1200
tgtttaggtg tgttgtaata catacgctat taagatgtaa aaatacggat accaatgaag 1260
ggaaaagtat aatttttgga tgtagtttgt ttgttcatct atgggcaaac tacgtccaaa 1320
gccgtttcca aatctgctaa aaagtatatc ctttctaaaa tcaaagtcaa gtatgaaatc 1380
ataaataaag tttaattttg aagttattat gatattatgt ttttctatta aaataaatta 1440
agtatataga atagtttaat aatagtatat acttaatgtg ctgcagttga cagctagctc 1500
agtcctaggt ataatactag taacttcttc cttaaagata ggttttagag ctagaaatag 1560
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 1620
tttgcggccg cttacttaac tctgtgctaa tgcaaaaacc atctagatct tccttataaa 1680
actctaatgc ttcgctatta aatatatttc ctttatagtc tcctattata ttaactagac 1740
ctttatattt atttattatt cctgcattaa ccgttactat gcctttaatg ttttctatat 1800
tttcatctat tttattgcat atatcgttaa actcttcttt aattacatta ggaaccttaa 1860
tataaacatc cttaagttcc ttaagcttca agtcacattt tcttttaaat aagtcaacta 1920
ttatgttctt aaatccctct tctttaactg caagatactg ctccttattc gaaactaaaa 1980
ccatgatctc tgggatttta acttctgacc tcttagaaat ctttattcct tcaaacttta 2040
aattagcttc tcttttatag cttttctgaa tactattttg aaatttttct agaacttctc 2100
tccttattga attcaaagca gatatcggta tatatccttc atcaaagcag gtaaactcta 2160
tgtttctgac ctcaaaagga gtacctcctg ttttccttaa atgttcctct actttttctc 2220
tctttacagg acttttttca gccttgcaaa ccatatcccc taaaacttgt atactaatag 2280
ccctattttt ttcatttgcc cctaaatcat ttactcttga aaaacttgca gtaatcttaa 2340
atggctctcc gattttaaat tcaatttcta aatctaaata tacttttctc ttaaaatcct 2400
ttttataact ttcctcaatt tcacgagtaa gctttaaatc aagagtcttg tatatataat 2460
ctccagactt gtaaaagcga ggaaatattt tcaccttttc tcctggctta gcctctttaa 2520
cttcaacccc atcaagaagt atctttgaca caacagtacc cttgacatca tttctaactc 2580
catcctttaa gtttaaactt tctttaagaa ctatacttcc atcacttatt acggtatcaa 2640
tgagcctatc taaaatatcc acagtacaca tatccttggt acttagaaaa tatccttttt 2700
tactttccaa actgctttta tcaataagtg tataactttg tctacatggt tgagcacatc 2760
ttcctctatt accacttcta ccacctatta tagaactcat aagacactgt ccagaataac 2820
atacacataa tgcaccatga acaaaaacct ctgtttctat attaagttct ttagatatgt 2880
attcaatttc ccttaaggac aactccctcg aaagcacaat tcttttaaag cctctttctt 2940
tataatacac agctcctaat gcattgtgaa ttgtcatctg agttgatgca tgtatttcaa 3000
aatcaggaac gtatctttta ataagttgca atatagcagt atcttgaact ataagtgcat 3060
ctactcctat tttataaaga aattctatat atctaataac ttcttttatc tcatcttgtt 3120
ttataagcgt atttactgtt acgtatagcc ttactccata atcgtgacaa taatcaactg 3180
ctttttccat aatttcatca ttaaaatttt cagcataagc tctagctgaa aacctacttc 3240
cacctacata aactgcatca gcaccgtttt gaacagctgc atatatgctc tcaatactgc 3300
ctgctggtgc tagcaattca accttattca taagtacctc caaaattatt aaattttata 3360
taactatgat attatccact aatcctctta ctatgtatgg tcctataact attatacata 3420
aagtatctat tattacaaat aaaagttata aggtaaattt tattgcaaaa taataaggag 3480
ggtttcccct ccatttactt atattaattt ttaaatacta aaagttaatc tttatctaat 3540
tctccaatct cttcatttaa gtattgatgt gaacaatttt catcctctgg taaagcaaaa 3600
tcctcttttt gaggtggatc aaatatatca actcgagttt ttaacaaaat atattgataa 3660
aaataataat agtgggtata attaagttgt tagagaaaac gtataaatta gggataaact 3720
atggaactta tgaaatagat tgaaatggtt tatctgttac cccgtatcaa aatttaggag 3780
gttagttaga ggatccccgg gatggataag aaatactcaa taggcttagc tatcggcaca 3840
aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag 3900
gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt 3960
gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag aaggtataca 4020
cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta 4080
gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga caagaagcat 4140
gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca 4200
actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta 4260
atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat 4320
ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat 4380
caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct 4440
gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag 4500
aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa 4560
tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat 4620
gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct 4680
aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact 4740
aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact 4800
cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat 4860
caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat 4920
aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta 4980
aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa 5040
attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta tccattttta 5100
aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt 5160
ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt 5220
accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa 5280
cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg 5340
ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga 5400
atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc 5460
aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa 5520
tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc 5580
taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa 5640
gatatcttag aggatattgt tttaacattg accttatttg aagataggga gatgattgag 5700
gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt 5760
cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag 5820
caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt 5880
atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa agcacaagtg 5940
tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt 6000
aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg 6060
cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc 6120
cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt 6180
cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc 6240
tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta 6300
agtgattatg atgtcgatca cattgttcca caaagtttcc ttaaagacga ttcaatagac 6360
aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa 6420
gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact 6480
caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa 6540
gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa 6600
attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt 6660
aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat 6720
aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt 6780
ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat 6840
aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc 6900
gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca 6960
aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc 7020
tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat 7080
attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa 7140
agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt 7200
tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa 7260
tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc 7320
tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac 7380
ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg 7440
ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg 7500
aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa 7560
caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc 7620
agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca 7680
tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt 7740
acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt 7800
aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact 7860
ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactgagc ggccgctcta 7920
gagggggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 7980
cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 8040
agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 8100
cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 8160
actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 8220
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 8280
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 8340
cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 8400
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 8460
taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 8520
tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 8580
gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 8640
gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 8700
cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 8760
tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 8820
tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 8880
atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 8940
ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 9000
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 9060
gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 9120
gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 9180
gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 9240
gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 9300
cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 9360
atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 9420
tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 9480
ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 9540
gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 9600
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 9660
ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 9720
ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 9780
gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 9840
ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 9900
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 9960
ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 10020
agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 10080
agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 10140
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 10200
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 10260
accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 10320
gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 10380
attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 10440
gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 10500
gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 10560
catgattacg ccaagctttg gctaacacac acgccattcc aaccaatagt tttctcggca 10620
taaagccatg ctctgacgct taaatgcact aatgccttaa aaaaacatta aagtctaaca 10680
cactagactt atttacttcg taattaagtc gttaaaccgt gtgctctacg accaaaagta 10740
taaaaccttt aagaactttc ttttttcttg taaaaaaaga aactagataa atctctcata 10800
tcttttattc aataatcgca tcagattgca gtataaattt aacgatcact catcatgttc 10860
atatttatca gagctcgtgc tataattata ctaattttat aaggaggaaa aaataaagag 10920
ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt caaaacataa 10980
tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct ttgaaatcgg 11040
ctcaggaaaa gggcatttta cccttgaatt agtacagagg tgtaatttcg taactgccat 11100
tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc acgataattt 11160
ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat cctataaaat 11220
atttggtaat ataccttata acataagtac ggatataata cgcaaaattg tttttgatag 11280
tatagctgat gagatttatt taatcgtgga atacgggttt gctaaaagat tattaaatac 11340
aaaacgctca ttggcattat ttttaatggc agaagttgat atttctatat taagtatggt 11400
tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca gattaaatag 11460
aaaaaaatca agaatatcac acaaagataa acagaagtat aattatttcg ttatgaaatg 11520
ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt ccttaaaaca 11580
tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc ttttcaatag 11640
ctataaatta tttaataagt aagttaaggg atgcataaac tgcatccctt aacttgtttt 11700
tcgtgtacct attttttgtg aat 11723
<210> 24
<211> 11723
<212> DNA
<213> 基因敲除质粒3(NICKclos2.0-2341-3)
<400> 24
cgataattta cagaaaagaa aattatagaa tttagtatga ttaattatac tcatttatga 60
atgtttaatt gaatacaaaa aaaaatactt gttatgtatt caattacggg ttaaaatata 120
gacaagttga aaaatttaat aaaaaaataa gtcctcagct cttatatatt aagctaccaa 180
cttagtatat aagccaaaac ttaaatgtgc taccaacaca tcaagccgtt agagaactct 240
atctatagca atatttcaaa tgtaccgaca tacaagagaa acattaacta tatatattca 300
atttatgaga ttatcttaac agatataaat gtaaattgca ataagtaaga tttagaagtt 360
tatagccttt gtgtattgga agcagtacgc aaaggctttt ttatttgata aaaattagaa 420
gtatatttat tttttcataa ttaatttatg aaaatgaaag ggggtgagca aagtgacaga 480
ggaaagcagt atcttatcaa ataacaaggt attagcaata tcattattga ctttagcagt 540
aaacattatg acttttatag tgcttgtagc taagtagtac gaaaggggga gctttaaaaa 600
gctccttgga atacatagaa ttcataaatt aatttatgaa aagaagggcg tatatgaaaa 660
cttgtaaaaa ttgcaaagag tttattaaag atactgaaat atgcaaaata cattcgttga 720
tgattcatga taaaacagta gcaacctatt gcagtaaata caatgagtca agatgtttac 780
ataaagggaa agtccaatgt attaattgtt caaagatgaa ccgatatgga tggtgtgcca 840
taaaaatgag atgttttaca gaggaagaac agaaaaaaga acgtacatgc attaaatatt 900
atgcaaggag ctttaaaaaa gctcatgtaa agaagagtaa aaagaaaaaa taatttattt 960
attaatttaa tattgagagt gccgacacag tatgcactaa aaaatatatc tgtggtgtag 1020
tgagccgata caaaaggata gtcactcgca ttttcataat acatcttatg ttatgattat 1080
gtgtcggtgg gacttcacga cgaaaaccca caataaaaaa agagttcggg gtagggttaa 1140
gcatagttga ggcaactaaa caatcaagct aggatatgca gtagcagacc gtaaggtcgt 1200
tgtttaggtg tgttgtaata catacgctat taagatgtaa aaatacggat accaatgaag 1260
ggaaaagtat aatttttgga tgtagtttgt ttgttcatct atgggcaaac tacgtccaaa 1320
gccgtttcca aatctgctaa aaagtatatc ctttctaaaa tcaaagtcaa gtatgaaatc 1380
ataaataaag tttaattttg aagttattat gatattatgt ttttctatta aaataaatta 1440
agtatataga atagtttaat aatagtatat acttaatgtg ctgcagttga cagctagctc 1500
agtcctaggt ataatactag tgccattgat aataagctta agttttagag ctagaaatag 1560
caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag tcggtgcttt 1620
tttgcggccg cttacttaac tctgtgctaa tgcaaaaacc atctagatct tccttataaa 1680
actctaatgc ttcgctatta aatatatttc ctttatagtc tcctattata ttaactagac 1740
ctttatattt atttattatt cctgcattaa ccgttactat gcctttaatg ttttctatat 1800
tttcatctat tttattgcat atatcgttaa actcttcttt aattacatta ggaaccttaa 1860
tataaacatc cttaagttcc ttaagcttca agtcacattt tcttttaaat aagtcaacta 1920
ttatgttctt aaatccctct tctttaactg caagatactg ctccttattc gaaactaaaa 1980
ccatgatctc tgggatttta acttctgacc tcttagaaat ctttattcct tcaaacttta 2040
aattagcttc tcttttatag cttttctgaa tactattttg aaatttttct agaacttctc 2100
tccttattga attcaaagca gatatcggta tatatccttc atcaaagcag gtaaactcta 2160
tgtttctgac ctcaaaagga gtacctcctg ttttccttaa atgttcctct actttttctc 2220
tctttacagg acttttttca gccttgcaaa ccatatcccc taaaacttgt atactaatag 2280
ccctattttt ttcatttgcc cctaaatcat ttactcttga aaaacttgca gtaatcttaa 2340
atggctctcc gattttaaat tcaatttcta aatctaaata tacttttctc ttaaaatcct 2400
ttttataact ttcctcaatt tcacgagtaa gctttaaatc aagagtcttg tatatataat 2460
ctccagactt gtaaaagcga ggaaatattt tcaccttttc tcctggctta gcctctttaa 2520
cttcaacccc atcaagaagt atctttgaca caacagtacc cttgacatca tttctaactc 2580
catcctttaa gtttaaactt tctttaagaa ctatacttcc atcacttatt acggtatcaa 2640
tgagcctatc taaaatatcc acagtacaca tatccttggt acttagaaaa tatccttttt 2700
tactttccaa actgctttta tcaataagtg tataactttg tctacatggt tgagcacatc 2760
ttcctctatt accacttcta ccacctatta tagaactcat aagacactgt ccagaataac 2820
atacacataa tgcaccatga acaaaaacct ctgtttctat attaagttct ttagatatgt 2880
attcaatttc ccttaaggac aactccctcg aaagcacaat tcttttaaag cctctttctt 2940
tataatacac agctcctaat gcattgtgaa ttgtcatctg agttgatgca tgtatttcaa 3000
aatcaggaac gtatctttta ataagttgca atatagcagt atcttgaact ataagtgcat 3060
ctactcctat tttataaaga aattctatat atctaataac ttcttttatc tcatcttgtt 3120
ttataagcgt atttactgtt acgtatagcc ttactccata atcgtgacaa taatcaactg 3180
ctttttccat aatttcatca ttaaaatttt cagcataagc tctagctgaa aacctacttc 3240
cacctacata aactgcatca gcaccgtttt gaacagctgc atatatgctc tcaatactgc 3300
ctgctggtgc tagcaattca accttattca taagtacctc caaaattatt aaattttata 3360
taactatgat attatccact aatcctctta ctatgtatgg tcctataact attatacata 3420
aagtatctat tattacaaat aaaagttata aggtaaattt tattgcaaaa taataaggag 3480
ggtttcccct ccatttactt atattaattt ttaaatacta aaagttaatc tttatctaat 3540
tctccaatct cttcatttaa gtattgatgt gaacaatttt catcctctgg taaagcaaaa 3600
tcctcttttt gaggtggatc aaatatatca actcgagttt ttaacaaaat atattgataa 3660
aaataataat agtgggtata attaagttgt tagagaaaac gtataaatta gggataaact 3720
atggaactta tgaaatagat tgaaatggtt tatctgttac cccgtatcaa aatttaggag 3780
gttagttaga ggatccccgg gatggataag aaatactcaa taggcttagc tatcggcaca 3840
aatagcgtcg gatgggcggt gatcactgat gaatataagg ttccgtctaa aaagttcaag 3900
gttctgggaa atacagaccg ccacagtatc aaaaaaaatc ttataggggc tcttttattt 3960
gacagtggag agacagcgga agcgactcgt ctcaaacgga cagctcgtag aaggtataca 4020
cgtcggaaga atcgtatttg ttatctacag gagatttttt caaatgagat ggcgaaagta 4080
gatgatagtt tctttcatcg acttgaagag tcttttttgg tggaagaaga caagaagcat 4140
gaacgtcatc ctatttttgg aaatatagta gatgaagttg cttatcatga gaaatatcca 4200
actatctatc atctgcgaaa aaaattggta gattctactg ataaagcgga tttgcgctta 4260
atctatttgg ccttagcgca tatgattaag tttcgtggtc attttttgat tgagggagat 4320
ttaaatcctg ataatagtga tgtggacaaa ctatttatcc agttggtaca aacctacaat 4380
caattatttg aagaaaaccc tattaacgca agtggagtag atgctaaagc gattctttct 4440
gcacgattga gtaaatcaag acgattagaa aatctcattg ctcagctccc cggtgagaag 4500
aaaaatggct tatttgggaa tctcattgct ttgtcattgg gtttgacccc taattttaaa 4560
tcaaattttg atttggcaga agatgctaaa ttacagcttt caaaagatac ttacgatgat 4620
gatttagata atttattggc gcaaattgga gatcaatatg ctgatttgtt tttggcagct 4680
aagaatttat cagatgctat tttactttca gatatcctaa gagtaaatac tgaaataact 4740
aaggctcccc tatcagcttc aatgattaaa cgctacgatg aacatcatca agacttgact 4800
cttttaaaag ctttagttcg acaacaactt ccagaaaagt ataaagaaat cttttttgat 4860
caatcaaaaa acggatatgc aggttatatt gatgggggag ctagccaaga agaattttat 4920
aaatttatca aaccaatttt agaaaaaatg gatggtactg aggaattatt ggtgaaacta 4980
aatcgtgaag atttgctgcg caagcaacgg acctttgaca acggctctat tccccatcaa 5040
attcacttgg gtgagctgca tgctattttg agaagacaag aagactttta tccattttta 5100
aaagacaatc gtgagaagat tgaaaaaatc ttgacttttc gaattcctta ttatgttggt 5160
ccattggcgc gtggcaatag tcgttttgca tggatgactc ggaagtctga agaaacaatt 5220
accccatgga attttgaaga agttgtcgat aaaggtgctt cagctcaatc atttattgaa 5280
cgcatgacaa actttgataa aaatcttcca aatgaaaaag tactaccaaa acatagtttg 5340
ctttatgagt attttacggt ttataacgaa ttgacaaagg tcaaatatgt tactgaagga 5400
atgcgaaaac cagcatttct ttcaggtgaa cagaagaaag ccattgttga tttactcttc 5460
aaaacaaatc gaaaagtaac cgttaagcaa ttaaaagaag attatttcaa aaaaatagaa 5520
tgttttgata gtgttgaaat ttcaggagtt gaagatagat ttaatgcttc attaggtacc 5580
taccatgatt tgctaaaaat tattaaagat aaagattttt tggataatga agaaaatgaa 5640
gatatcttag aggatattgt tttaacattg accttatttg aagataggga gatgattgag 5700
gaaagactta aaacatatgc tcacctcttt gatgataagg tgatgaaaca gcttaaacgt 5760
cgccgttata ctggttgggg acgtttgtct cgaaaattga ttaatggtat tagggataag 5820
caatctggca aaacaatatt agattttttg aaatcagatg gttttgccaa tcgcaatttt 5880
atgcagctga tccatgatga tagtttgaca tttaaagaag acattcaaaa agcacaagtg 5940
tctggacaag gcgatagttt acatgaacat attgcaaatt tagctggtag ccctgctatt 6000
aaaaaaggta ttttacagac tgtaaaagtt gttgatgaat tggtcaaagt aatggggcgg 6060
cataagccag aaaatatcgt tattgaaatg gcacgtgaaa atcagacaac tcaaaagggc 6120
cagaaaaatt cgcgagagcg tatgaaacga atcgaagaag gtatcaaaga attaggaagt 6180
cagattctta aagagcatcc tgttgaaaat actcaattgc aaaatgaaaa gctctatctc 6240
tattatctcc aaaatggaag agacatgtat gtggaccaag aattagatat taatcgttta 6300
agtgattatg atgtcgatca cattgttcca caaagtttcc ttaaagacga ttcaatagac 6360
aataaggtct taacgcgttc tgataaaaat cgtggtaaat cggataacgt tccaagtgaa 6420
gaagtagtca aaaagatgaa aaactattgg agacaacttc taaacgccaa gttaatcact 6480
caacgtaagt ttgataattt aacgaaagct gaacgtggag gtttgagtga acttgataaa 6540
gctggtttta tcaaacgcca attggttgaa actcgccaaa tcactaagca tgtggcacaa 6600
attttggata gtcgcatgaa tactaaatac gatgaaaatg ataaacttat tcgagaggtt 6660
aaagtgatta ccttaaaatc taaattagtt tctgacttcc gaaaagattt ccaattctat 6720
aaagtacgtg agattaacaa ttaccatcat gcccatgatg cgtatctaaa tgccgtcgtt 6780
ggaactgctt tgattaagaa atatccaaaa cttgaatcgg agtttgtcta tggtgattat 6840
aaagtttatg atgttcgtaa aatgattgct aagtctgagc aagaaatagg caaagcaacc 6900
gcaaaatatt tcttttactc taatatcatg aacttcttca aaacagaaat tacacttgca 6960
aatggagaga ttcgcaaacg ccctctaatc gaaactaatg gggaaactgg agaaattgtc 7020
tgggataaag ggcgagattt tgccacagtg cgcaaagtat tgtccatgcc ccaagtcaat 7080
attgtcaaga aaacagaagt acagacaggc ggattctcca aggagtcaat tttaccaaaa 7140
agaaattcgg acaagcttat tgctcgtaaa aaagactggg atccaaaaaa atatggtggt 7200
tttgatagtc caacggtagc ttattcagtc ctagtggttg ctaaggtgga aaaagggaaa 7260
tcgaagaagt taaaatccgt taaagagtta ctagggatca caattatgga aagaagttcc 7320
tttgaaaaaa atccgattga ctttttagaa gctaaaggat ataaggaagt taaaaaagac 7380
ttaatcatta aactacctaa atatagtctt tttgagttag aaaacggtcg taaacggatg 7440
ctggctagtg ccggagaatt acaaaaagga aatgagctgg ctctgccaag caaatatgtg 7500
aattttttat atttagctag tcattatgaa aagttgaagg gtagtccaga agataacgaa 7560
caaaaacaat tgtttgtgga gcagcataag cattatttag atgagattat tgagcaaatc 7620
agtgaatttt ctaagcgtgt tattttagca gatgccaatt tagataaagt tcttagtgca 7680
tataacaaac atagagacaa accaatacgt gaacaagcag aaaatattat tcatttattt 7740
acgttgacga atcttggagc tcccgctgct tttaaatatt ttgatacaac aattgatcgt 7800
aaacgatata cgtctacaaa agaagtttta gatgccactc ttatccatca atccatcact 7860
ggtctttatg aaacacgcat tgatttgagt cagctaggag gtgactgagc ggccgctcta 7920
gagggggtac cgagctcgaa ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac 7980
cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat 8040
agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg 8100
cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg catatggtgc 8160
actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca cccgccaaca 8220
cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagctgtg 8280
accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga 8340
cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat aatggtttct 8400
tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc 8460
taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa 8520
tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt 8580
gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct 8640
gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc 8700
cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta 8760
tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac 8820
tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc 8880
atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac 8940
ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg 9000
gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac 9060
gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc 9120
gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt 9180
gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga 9240
gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc 9300
cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag 9360
atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca 9420
tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc 9480
ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca 9540
gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc 9600
tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta 9660
ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt 9720
ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc 9780
gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg 9840
ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg 9900
tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag 9960
ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc 10020
agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat 10080
agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg 10140
gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc 10200
tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt 10260
accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca 10320
gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg 10380
attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac 10440
gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg 10500
gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac 10560
catgattacg ccaagctttg gctaacacac acgccattcc aaccaatagt tttctcggca 10620
taaagccatg ctctgacgct taaatgcact aatgccttaa aaaaacatta aagtctaaca 10680
cactagactt atttacttcg taattaagtc gttaaaccgt gtgctctacg accaaaagta 10740
taaaaccttt aagaactttc ttttttcttg taaaaaaaga aactagataa atctctcata 10800
tcttttattc aataatcgca tcagattgca gtataaattt aacgatcact catcatgttc 10860
atatttatca gagctcgtgc tataattata ctaattttat aaggaggaaa aaataaagag 10920
ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt caaaacataa 10980
tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct ttgaaatcgg 11040
ctcaggaaaa gggcatttta cccttgaatt agtacagagg tgtaatttcg taactgccat 11100
tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc acgataattt 11160
ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat cctataaaat 11220
atttggtaat ataccttata acataagtac ggatataata cgcaaaattg tttttgatag 11280
tatagctgat gagatttatt taatcgtgga atacgggttt gctaaaagat tattaaatac 11340
aaaacgctca ttggcattat ttttaatggc agaagttgat atttctatat taagtatggt 11400
tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca gattaaatag 11460
aaaaaaatca agaatatcac acaaagataa acagaagtat aattatttcg ttatgaaatg 11520
ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt ccttaaaaca 11580
tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc ttttcaatag 11640
ctataaatta tttaataagt aagttaaggg atgcataaac tgcatccctt aacttgtttt 11700
tcgtgtacct attttttgtg aat 11723
<210> 25
<211> 2124
<212> DNA
<213> 失活的菌株(Inactivated strain)
<400> 25
atgaataagg ttgaattgct agcaccagca ggcagtattg agagcatata tgcagctgtt 60
caaaacggtg ctgatgcagt ttatgtaggt ggaagtaggt tttcagctag agcttatgct 120
gaaaatttta atgatgaaat tatggaaaaa gcagttgatt attgtcacga ttatggagta 180
aggctatacg taacagtaaa tacgcttata aaacaagatg agataaaaga agttattaga 240
tatatagaat ttctttataa aataggagta gatgcactta tagttcaaga tactgctata 300
ttgcaactta ttaaaagata cgttcctgat tttgaaatac atgcatcaac tcagatgaca 360
attcacaatg cattaggagc tgtgtattat aaagaaagag gctttaaaag aattgtgctt 420
tcgagggagt tgtccttaag ggaaattgaa tacatatcta aagaacttaa tatagaaaca 480
gaggtttttg ttcatggtgc attatgtgta tgttattctg gacagtgtct tatgagttct 540
ataataggtg gtagaagtgg taatagagga agatgtgctc aaccatgtag acaaagttat 600
acacttattg ataaaagcag tttggaaagt aaaaaaggat attttctaag taccaaggat 660
atgtgtactg tggatatttt agataggctc attgataccg taataagtga tggaagtata 720
gttcttaaag aaagtttaaa cttaaaggat ggagttagaa atgatgtcaa gggtactgtt 780
gtgtcaaaga tacttcttga tggggttgaa gttaaagagg ctaagccagg agaaaaggtg 840
aaaatatttc ctcgctttta caagtctgga gattatatat acaagactct tgatttaaag 900
cttactcgtg aaattgagga aagttataaa aaggatttta agagaaaagt atatttagat 960
ttagaaattg aatttaaaat cggagagcca tttaagatta ctgcaagttt ttcaagagta 1020
aatgatttag gggcaaatga aaaaaatagg gctattagta tacaagtttt aggggatatg 1080
gtttgcaagg ctgaaaaaag tcctgtaaag agagaaaaag tagaggaaca tttaaggaaa 1140
acaggaggta ctccttttga ggtcagaaac atagagttta cctgctttga tgaaggatat 1200
ataccgatat ctgctttgaa ttcaataagg agagaagttc tagaaaaatt tcaaaatagt 1260
attcagaaaa gctataaaag agaagctaat ttaaagtttg aaggaataaa gatttctaag 1320
aggtcagaag ttaaaatccc agagatcatg gttttagttt cgaataagga gcagtatctt 1380
gcagttaaag aagagggatt taagaacata atagttgact tatttaaaag aaaatgtgac 1440
ttgaagctta aggaacttaa ggatgtttat attaaggttc ctaatgtaat taaagaagag 1500
tttaacgata tatgcaataa aatagatgaa aatatagaaa acattaaagg catagtaacg 1560
gttaatgcag gaataataaa taaatataaa ggtctagtta atataatagg agactataaa 1620
ggaaatatat ttaatagcga agcattagag ttttataagg aagatctaga tggtttttgc 1680
attagcacag agttaagtaa aaatgaaata aaggcattga ataaatgtaa atgcatggat 1740
acccaaatgc ttatatatgg taaaatagaa gcaatggtaa gtgagtattg tcctatagga 1800
agtgtttttg gaggtaaatc ttctaaaagc aattgccaag gtacatgcat gaaatcagac 1860
tttgtactta aagataagat gggagtatct tttgatgtgg taacagacaa atattgcaga 1920
agtcatatat ataatccatt accattaaat cttataggca atttagatga aataaagaat 1980
ttaggcgtga attcttatag aatagatttt attgatgaaa atagagaaag tgttaaaaga 2040
gttctttcat ggatgaaaaa tggtaagatt gatggagaat tcagtggttt tacaagagga 2100
agttataaaa gaggagttga ataa 2124
Claims (10)
1.一株重组丙酮丁醇梭菌，其特征在于，抑制所述丙酮丁醇梭菌中CA_C2341基因的表达；其中，所述的丙酮丁醇梭菌(clostridium acetobutylicum)为CGMCC No.5234。
2.根据权利要求1所述的重组丙酮丁醇梭菌，其特征在于，所述CA_C2341基因的核苷酸序列如SEQ ID NO.1所示。
3.权利要求1或2所述重组丙酮丁醇梭菌的构建方法，其特征在于，构建含有同源重组片段的基因敲除质粒pNICKclos2.0-2341，将基因敲除质粒pNICKclos2.0-2341甲基化后，电转进入丙酮丁醇梭菌CGMCC No.5234中，筛选得到CA_C2341基因失活的菌株；
其中，所述的基因敲除质粒pNICKclos2.0-2341为pNICKclos2.0-2341-1、pNICKclos2.0-2341-2和pNICKclos2.0-2341-3。
4.根据权利要求3所述的构建方法，其特征在于，所述基因敲除质粒pNICKclos2.0-2341的构建方法，包括如下步骤：
(1)sgRNA片段的合成：以pNICKclos2.0质粒为模板分别，以SEQ ID NO.3和SEQ IDNO.6、SEQ ID NO.4和SEQ ID NO.6、SEQ ID NO.5和SEQ ID NO.6所示的核苷酸序列为引物，分别PCR扩增，得到sgRNA-2341-1片段、sgRNA-2341-3片段、sgRNA-2341-3片段；
(2)上下游同源臂片段的合成：以丙酮丁醇梭菌基因组为模板，以SEQ ID NO.7和SEQID NO.8、SEQ ID NO.9和SEQ ID NO.10所示的核苷酸序列为引物，分别PCR扩增，得到上游同源臂upHA和下游同源臂dnHA；
(3)sgRNA片段和同源臂片段的整合：
(3a)通过重叠延伸PCR的技术，以sgRNA-2341-1片段和上游同源臂upHA片段为模板，以SEQ ID NO.3和SEQ ID NO.8所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-1-upHA片段；以sgRNA-2341-2片段和上游同源臂upHA片段为模板，以SEQ ID NO.4和SEQ ID NO.8所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-2-upHA片段；以sgRNA-2341-3片段和上游同源臂upHA片段为模板，以SEQ ID NO.5和SEQ ID NO.8所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-3-upHA片段；
(3b)通过重叠延伸PCR的技术，以sgRNA-2341-1-upHA片段和下游同源臂dnHA片段为模板，以SEQ ID NO.3和SEQ ID NO.10所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-1-upHA片段；以sgRNA-2341-2-upHA片段和下游同源臂dnHA片段为模板，以SEQ ID NO.4和SEQID NO.10所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-2-upHA片段；以sgRNA-2341-3-upHA片段和下游同源臂dnHA片段为模板，以SEQ ID NO.5和SEQ ID NO.10所示的核苷酸序列为引物，PCR扩增得到sgRNA-2341-3-upHA片段；
(4)构建质粒：将SpeI/XhoI双酶切后的载体pNICKclos2.0片段分别与sgRNA-2341-1-upHA片段、sgRNA-2341-2-upHA片段和sgRNA-2341-3-upHA片段进行重组反应，即得基因敲除质粒pNICKclos2.0-2341-1，pNICKclos2.0-2341-2，pNICKclos2.0-2341-3。
5.根据权利要求4所述的构建方法，其特征在于，所述pNICKclos2.0质粒的核苷酸序列如SEQ ID NO.2所示。
6.根据权利要求3所述的构建方法，其特征在于，所述的甲基化为将基因敲除质粒pNICKclos2.0-2341转化进入含有pAN2质粒的大肠杆菌E.coli Top10感受态细胞，筛选阳性克隆，即为甲基化的基因敲除质粒。
7.权利要求1所述重组丙酮丁醇梭菌在生物被膜固定化催化体系中的应用。
8.根据权利要求7所述的应用，其特征在于，以重组丙酮丁醇梭菌为发酵菌株，发酵制备丁醇。
9.根据权利要求8所述的应用，其特征在于，所述发酵培养基中各组分的浓度为葡萄糖60g/L，乙酸铵2.2g/L，磷酸二氢钾0.5g/L，磷酸氢二钾0.5g/L，氯化钠0.01g/L，七水合硫酸镁0.2g/L，七水合硫酸亚铁0.01g/L，一水合硫酸锰0.01g/L，溶剂为水。
10.根据权利要求8所述的应用，其特征在于，发酵的条件为37℃静置发酵60～96h。
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| CN113234653A (zh) * | 2021-04-22 | 2021-08-10 | 中国科学院青岛生物能源与过程研究所 | 一种提高合成气发酵产乙醇含量的突变株和利用突变株的应用 |
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