CN108060175B - 诱导型酵母转化重组系统的构建及其应用 - Google Patents

诱导型酵母转化重组系统的构建及其应用 Download PDF

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CN108060175B
CN108060175B CN201711364673.5A CN201711364673A CN108060175B CN 108060175 B CN108060175 B CN 108060175B CN 201711364673 A CN201711364673 A CN 201711364673A CN 108060175 B CN108060175 B CN 108060175B
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汪洋
苏会娟
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Jiangsu Gansan Biotechnology Co ltd
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Abstract

本发明公开了一种诱导型酵母转化重组系统的构建及其在大片段DNA捕捉中的应用。所述的iTAR载体含有PGal1‑V10、I‑SceI、BleR、CEN6、ARSH4、URA3、同源臂1、同源臂2及同源臂间的I‑SceI酶切位点。iTAR载体可以通过半乳糖诱导Gal1‑V10启动子，产生I‑SceI酶，作用于I‑SceI酶切位点，使iTAR载体自身线性化，将大片段DNA转化到含iTAR载体的酿酒酵母中，实现空载体的降解和酵母体内的同源重组，达到克隆大片段DNA的目的。本发明的iTAR克隆方法简单、高效，能够快速、准确地从基因组中获得所需要的大片段DNA而无酶切位点的限制，转化效率可达73％左右。

Description

诱导型酵母转化重组系统的构建及其应用
技术领域
本发明涉及诱导型酵母转化重组系统的构建方法及其在捕捉线性大片段DNA中的应用，即利用iTAR克隆技术捕捉线性大片段DNA，具体涉及一种将具有同源序列的线性大片段DNA转入含有iTAR载体的酿酒酵母中，完成DNA的组装，属于生物技术领域。
背景技术
微生物能够产生许多具有生物活性的次级代谢产物，其中包括抗生素(Polymyxin)、抗癌药物(埃博霉素)、杀虫剂(阿维菌素)和免疫抑制剂(雷帕霉素)。合成这些次级代谢产物的基因簇一般都大于10kb，有的甚至超过100kb。然而传统的PCR克隆方法已不再能满足人们的需求，因此，迫切需要开发一种能高效捕捉大基因簇片段的方法。
目前，捕捉大基因簇的方法主要有基因组文库和同源重组策略，基因组文库主要包括Cosmid文库(Eiglmeier K,et al.Use of an ordered cosmid library to deducethe genomic organization of Mycobacterium leprae[J].Molecular microbiology,1993,7(2):197-206)，BAC文库(Choi S,et al.Construction and characterization ofa bacterial artificial chromosome library ofArabidopsis thaliana[J].PlantMolecular Biology Reporter,1995,13(2):124-128.)和YAC文库(Anand R,etal.A3.5genome equivalent multi access YAC library:construction,characterisation,screening and storage[J].Nucleic acids research,1990,18(8):1951-1956).，同源重组主要有RecE重组克隆(Gillen J R,et al.Genetic analysis ofthe RecE pathway of genetic recombination in Escherichia coli K-12[J].Journalof bacteriology,1981,145(1):521-532.)和TAR(Transformation-AssociatedRecombination)克隆(Li Y,et al.Directed natural product biosynthesis genecluster capture and expression in the model bacterium Bacillus subtilis[J].Scientific reports,2015,5.)。基因组文库因其具备容量大和易操作等优点，大片段基因组文库被广泛应用于动物、植物和微生物的基因组研究中，然而基因文库的随机性和筛选困难等缺陷限制了其应用范围。RecE重组克隆是利用噬菌体RecE将两条含有同源末端的线性DNA重组成环状质粒，由于该系统需要目的基因簇两端含有特异的酶切位点，因而限制了其使用。TAR克隆是基于转化介导的同源重组，没有酶切位点的限制，可以方便有效地对复杂、较大的DNA片段进行克隆。但传统的TAR克隆方法是将线性化的载体和线性化的大片段DNA同时转入酿酒酵母，然后利用酿酒酵母自身的同源重组机制，将大片段DNA克隆下来。由于两个DNA片段的共转化和酿酒酵母自身的修复机制，导致TAR克隆的效率仅为2％，因此，TAR克隆的应用也受到了一定的限制。
发明内容
针对现有的TAR克隆效率低的问题，本发明提供一种诱导型酵母转化重组系统，即iTAR克隆系统，利用半乳糖诱导Gal1-V10启动子控制基因I-SceI表达I-SceI酶，作用于I-SceI酶切位点，在酿酒酵母体内实现iTAR载体的线性化，进而实现大片段DNA简单、高效的克隆。
本发明的技术方案如下：
本发明提供一种重组载体pSHTB10，为将质粒pSH-SceI中的colE1、AmpR和PGal1基因元件替换成pSWB中的oriV、trfA和BleR DNA片段和Gal1-V10DNA合成片段，形成全长序列为SEQ ID NO.32的质粒pSHTB10；所述的质粒pSH-SceI是将质粒pSH47中的Cre元件替换成I-SceI基因片段；所述的质粒pSWB是将质粒pDGICZ中的Phbs-BleR基因元件插入到质粒pSW-2中。
本发明还提供基于上述重组载体pSHTB10的iTAR载体，为将质粒pSHTB10用不破坏其功能元件的限制性内切酶酶切，通过在酶切位点处插入与目标线性大片段DNA两端具有同源性的两个同源臂并在同源臂间插入I-SceI酶切位点构建而成。
进一步地，本发明提供一种基于上述质粒pSHTB10的iTAR载体pSHTB10-HR12，为将pSHTB10用限制性内切酶FspI酶切，在该酶切位点处插入同源臂HR1、同源臂HR2和同源臂间的I-SceI酶切位点，形成全长序列为SEQ ID NO.33的iTAR载体pSHTB10-HR12。
同样地，本发明还提供一种基于上述质粒pSHTB10的iTAR载体pSHTB10-HR34，为将pSHTB10用限制性内切酶FspI酶切，在该酶切位点处插入同源臂HR3、同源臂HR4和同源臂间的I-SceI酶切位点，形成全长序列为SEQ ID NO.34的iTAR载体pSHTB10-HR34。
本发明提供上述重组载体pSHTB10的构建方法，包括引物设计、基因克隆和载体构建，具体为通过合成Gal1-V10基因片段，设计引物从质粒pSWB上PCR扩增oriV、trfA和BleR目标片段，限制性内切酶PmlI和SacI酶切质粒pSH-SceI，将Gal1-V10、oriV、trfA、BleR和pSH-SceI目的片段连接，得到质粒pSHTB10；所述的质粒pSH-SceI的构建方法是通过PCR扩增I-SceI DNA序列，将其与限制性内切酶ClaI酶切的pSH47目的片段连接，得到质粒pSH-SceI；所述的质粒pSWB的构建方法是通过限制性内切酶BamHI酶切质粒pDGICZ和pSW-2，将其目的片段连接，得到质粒pSWB。
本发明还提供了上述iTAR载体的构建方法，包括引物设计、基因克隆和载体构建，具体为通过合成引物PCR扩增相应的两个同源臂片段及I-SceI酶切位点，与经不破坏pSHTB10功能元件的限制性内切酶酶切回收的目的DNA片段连接，得到相应的iTAR载体。
进一步地，本发明提供上述iTAR载体pSHTB10-HR12的构建方法，包括引物设计、基因克隆和载体构建，通过合成引物PCR扩增HR1和HR2DNA片段，与经限制性内切酶FspI酶切的pSHTB10片段连接，得到iTAR载体pSHTB10-HR12。
同样地，本发明提供上述iTAR载体pSHTB10-HR34的构建方法，包括引物设计、基因克隆和载体构建，通过合成引物PCR扩增HR3和HR4DNA片段，与经限制性内切酶FspI酶切的pSHTB10片段连接，得到iTAR载体pSHTB10-HR34。
本发明进一步提供上述iTAR载体在捕捉大片段DNA中的应用，具体应用方法如下：
步骤1：针对目标线性大片段DNA的两端设计合成相应DNA片段，用体外转录的方式获得gRNA；
步骤2：利用Cas9核酸酶和gRNA可以特异性切割DNA的特性得到目标线性DNA；
步骤3：将iTAR载体转化到酿酒酵母中，通过将目标线性DNA片段转化至经半乳糖诱导的且含iTAR载体的酿酒酵母中，筛选阳性克隆，得到相应的捕捉载体；
步骤4：将捕捉载体转移至相应的模式菌株中使其表达，实现外源大片段DNA的克隆和表达。
本发明构建一种能高效捕捉DNA大片段的克隆载体，不需要化学合成DNA片段，只需利用半乳糖诱导iTAR载体就能在酿酒酵母体内实现自然界中任何生物体DNA的克隆。本发明在TAR克隆的基础上，利用半乳糖诱导Gal1-V10启动子产生I-SceI酶作用于I-SceI酶切位点，实现在酵母体内iTAR载体自身线性化，降低了空载体的污染，使转化效率提高到了73％左右。
附图说明
图1是pSH-SceI载体的酶切验证图，M表示marker，a表示BamHI酶切，b表示PstI酶切。
图2是pSH-SceI的质粒图谱。
图3是pSWB载体的酶切验证图，M表示marker，a表示XbaI酶切，b表示HindIII酶切。
图4是pSWB的质粒图谱。
图5是pSHTB10质粒的酶切验证图，M表示marker，a表示FspI酶切，b表示PstI酶切。
图6是pSHTB10的质粒图谱。
图7是pSHTB10-HR12质粒的酶切验证图，M表示marker，a表示SpeI酶切，b表示NcoI酶切。
图8是pSHTB10-HR12的质粒图谱。
图9是pSHTB10-HR34质粒的酶切验证图，M表示marker，a表示KpnI酶切，b表示ScaI酶切。
图10是pSHTB10-HR34的质粒图谱。
图11是重组菌株E.coli BL21-hygR的PCR验证图。
图12是BY4742/pSHTB10-HR12捕捉含hygR元件的大片段DNA后平板上转化子的生长情况，a表示涂布在SG-URA平板上的转化子生长情况，b表示涂布在含200μg/mL潮霉素的SG-URA平板上的转化子生长情况。
图13是iTAR载体pSHTB10-HR12捕捉含hygR元件的DNA大片段后的酶切验证图，M表示marker，a表示PciI酶切。
图14是iTAR载体pSHTB10-HR34捕捉polymyxin基因簇片段后的酶切验证图，M表示marker，a表示NcoI酶切。
图15是Bacillus subtilis 168::pSHTB10和Bacillus subtilis 168::pmx的抑菌活性测试，a表示Bacillus subtilis 168::pSHTB10的发酵液的抑菌测试，b表示Bacillus subtilis 168::pmx的发酵液的抑菌测试。
具体实施方式
为了进一步阐述本发明，下面结合实施例和附图加以说明。
实施例一：iTAR载体的构建
1.重组载体pSH-SceI的构建，包括以下步骤：
(1)引物设计：人工设计合成特异性引物序列。
SceIFor:5'-cgatatcaagcttatggaattgtgagcggataacaattcatgcatatgaaaaacatcaa-3'；
SceIRev:5'-catccttagcgccgtaaatcaatttatttcaggaaagtttcgg-3'；
(2)PCR扩增：PCR反应体系配置如表一所示。
试剂名称 储备液浓度 加入PCR反应体系的体积
5×Q5Reaction Buffer 5× 10μL
5×Q5High GC Enhancer 5× 10μL
dNTPs 10mmol/L 1μL
Forward Primer 10μmol/L 2.5μL
Reverse Primer 10μmol/L 2.5μL
Q5High-Fidelity DNA Polymerase 5U/μL 0.5μL
DNA模板 50ng/μL 2μL
ddH<sub>2</sub>O 加ddH<sub>2</sub>O至终体积50μL
PCR程序是98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸，30个循环；最后72℃延伸10min。所述DNA模板是pSW-2，引物是SceIFor/SceIRev，延伸时间是30s。将PCR产物I-SceI通过琼脂糖凝胶电泳进行分离，回收的I-SceI DNA片段是长度为776bp的核酸片段。
(3)载体构建：将质粒pSH47用限制性内切酶ClaI酶切，回收线性DNA片段，并将其与I-SceI用Gibson Assembly的方式进行连接，并将连接产物转化至E.coli DH10B感受态细胞中，挑取阳性克隆验证，获得克隆载体pSH-SceI。pSH47质粒用限制性内切酶ClaI酶切后的片段大小分别为871bp和6108bp，回收的目的片段是长度为6108bp的DNA片段。pSH-SceI重组质粒用BamHI酶切后片段大小为6844bp，用PstI酶切后片段大小分别为3166bp和3678bp，验证图如图1所示：M表示marker，a表示BamHI酶切，b表示PstI酶切。构建完成的pSH-SceI质粒图谱如图2所示。
2.重组载体pSWB的构建，包括以下步骤：
(1)质粒酶切：将pDGICZ质粒DNA用限制性内切酶BamHI酶切，酶切后的片段大小分别为434bp、2033bp和8248bp，回收长度为434bp的Phbs-BleR DNA片段，再用限制性内切酶BamHI酶切pSW-2质粒DNA，回收线性化DNA片段pSW-2的长度为7457bp。
(2)载体构建：将经步骤2回收的Phbs-BleR和pSW-2线性DNA片段以摩尔比3:1的比例进行连接，在T4连接酶的作用下，于16℃连接2h，然后将连接产物转化至E.coli DH10B感受态细胞中，挑取阳性克隆验证，获得克隆载体pSWB。pSWB质粒用XbaI酶切后片段大小为7891bp，用HindIII酶切后片段大小分别为1438bp和6453bp，验证图如图3所示：M表示marker，a表示XbaI酶切，b表示HindIII酶切。构建完成的pSWB质粒图谱如图4所示。
3.质粒pSHTB10的构建，包括以下步骤：
(1)Minigene合成：
Gal1-V10:5'-gatccactagttctagaatccggggttttttctccttgacgtttaagtatagaggcatattgacaattttttgttgatacttttattacgtttgaataagaagtaatacaaaccgaaaacgttgaaagtattagttaaagtggttatgcagtttctgcatttatatatctgttaatagatcaaaaatcatcgcttcgctaattaattaccccagaaataaggctaaaaaactaatcgcattatcatcctatggttg-3'；
(2)引物设计：
vtbFor:5'-ggcttctaatccgtactagagctcactacagcagagccatgtag-3'；
vtbRev:5'-ttataattatttttatagcacgtgatgtacaaactgccgaac-3'；
pSHTB10For:5'-cgcattatcatcctatggttg-3'；
pSHTB10Rev:5'-ggattctagaactagtggatc-3'；
(3)PCR扩增：PCR反应体系是5×Q5PCR buffer 10μL，5×enhancer 10μL，dNTPs(10mmol/L)1μL，引物(10μmol/L)各2.5μL，质粒DNA(约50ng/μL)2μL，Q5DNA聚合酶(5U/μL)0.5μL，加ddH2O至50μL。PCR程序是98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸，30个循环；最后72℃延伸10min。所述DNA模板分别是pSWB和pSHTB，引物分别是vtbFor/vtbRev和pSHTB10For/pSHTB10Rev，延伸时间分别是2min和4min。将PCR产物通过琼脂糖凝胶电泳进行分离，回收的目的DNA片段vtb的长度为3291bp的核酸片段，pSHTB10的长度为7712bp的核酸片段。
(4)载体构建：质粒pSH-SceI经限制性内切酶PmlI和SacI酶切，回收目标DNA片段，并将其与步骤(3)回收的vtb DNA片段以Gibson Assembly的方式连接，并将连接产物转化至E.coli DH10B感受态细胞中，挑取阳性克隆验证，获得克隆载体pSHTB。再将步骤(1)合成的Gal1-V10与步骤(3)回收的pSHTB10DNA片段以Gibson Assembly的方式连接，并将连接产物转化至E.coli DH10B感受态细胞中，挑取阳性克隆验证，获得克隆载体pSHTB10。pSH-SceI质粒用限制性内切酶pmlI和SacI酶切后的片段大小分别为2220bp和4624bp，回收的目的片段是长度为4624bp的DNA片段。pSHTB10质粒用FspI酶切后片段大小为7871bp，用PstI酶切后片段大小分别为3166bp和4705bp，验证图如图5所示：M表示marker，a表示FspI酶切，b表示PstI酶切。构建完成的pSHTB10质粒图谱如图6所示。
4.iTAR载体pSHTB10-HR12的构建，包括以下步骤：
(1)引物设计：
HR1For:5'-cattcgccattcaggctgcgtgcgctatgaccgggtattg-3'；
HR1Rev:5'-attaccctgttatccctagtaatgtgttcatccatcatcg-3'；
HR2For:5'-tagggataacagggtaatcggtaatggtgagtttgctg-3'；
HR2Rev:5'-gcccttcccaacagttgcgcatcactttccgtaccag-3'；
(2)PCR扩增：PCR反应体系是5×Q5PCR buffer 10μL，5×enhancer 10μL，dNTPs(10mmol/L)1μL，引物(10μmol/L)各2.5μL，质粒DNA(约500ng/μL)2μL，Q5DNA聚合酶(5U/μL)0.5μL，加ddH2O至50μL。PCR程序是98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸，30个循环；最后72℃延伸10min。所述DNA模板均是E.coli BL21(DE3)基因组DNA，引物分别是HR1For/HR1Rev和HR2For/HR2Rev，延伸时间均是45s。将PCR产物通过琼脂糖凝胶电泳进行分离，回收的目的DNA片段HR1的长度为1013bp的核酸片段，HR2的长度为1008bp的核酸片段。
(3)载体构建：质粒pSHTB10经限制性内切酶FspI酶切，回收线性DNA片段，并将其与步骤(2)回收的HR1和HR2DNA片段以Gibson Assembly的方式连接，并将连接产物转化至E.coli DH10B感受态细胞中，挑取阳性克隆验证，获得克隆载体pSHTB10-HR12。pSHTB10质粒用限制性内切酶FspI酶切后的片段大小为7871bp。pSHTB10-HR12质粒用SpeI酶切后片段大小为9910bp，用NcoI酶切后片段大小分别为1989bp和7921bp，验证图如图7所示：M表示marker，a表示SpeI酶切，b表示NcoI酶切。构建完成的pSHTB10-HR12质粒图谱如图8所示。
5.iTAR载体pSHTB10-HR34的构建，包括以下步骤：
(1)引物设计：
HR3For:5'-caaataaggagtgtcaagaatggcttttgaaaaagaaac-3'；
HR3Rev:5'-attaccctgttatccctactccagaatgtagcggatgc-3'；
HR4For:5'-tagggataacagggtaataagtgaaacgaagagcatcg-3'；
HR4Rev:5'-caatgcatgtttctgagatctacggcatacacccaagcc-3'；
EctBFor:5'-ggcttgggtgtatgccgtagatctcagaaacatgcattg-3'；
EctBRev:5'-gcccttcccaacagttgctacagcgtaaccaagttgc-3'；
HRamyEFor:5'-cattcgccattcaggctgcggttgaagcggttatgcttg-3'；
HRamyERev:5'-gtttctttttcaaaagccattcttgacactccttatttg-3'；
(2)PCR扩增：PCR反应体系是5×Q5PCR buffer 10μL，5×enhancer 10μL，dNTPs(10mmol/L)1μL，引物(10μmol/L)各2.5μL，质粒DNA(约500ng/μL)2μL，Q5DNA聚合酶(5U/μL)0.5μL，加ddH2O至50μL。PCR程序是98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸，30个循环；最后72℃延伸10min。合成HR3、HR4和EctB DNA片段的模板均是Paenibacilluspolymyxa SQR-21基因组DNA，合成HRamyE DNA片段的模板是Bacillus subtilis 168基因组DNA。引物分别是HR3For/HR3Rev、HR4For/HR4Rev、EctBFor/EctBRev和HRamyEFor/HRamyERev，延伸时间分别是45s、45s、1min和45s。将PCR产物通过琼脂糖凝胶电泳进行分离，回收的目的DNA片段HR3的长度为1090bp的核酸片段，HR4的长度为1136bp的核酸片段，EctB的长度为1605bp的核酸片段，HRamyE的长度为1083bp的核酸片段。
(3)载体构建：质粒pSHTB10经限制性内切酶FspI酶切，回收线性DNA片段，并将其与步骤(2)回收的HR3、HR4、EctB和HRAmyE DNA片段以Gibson Assembly的方式连接，并将连接产物转化至E.coli DH10B感受态细胞中，挑取阳性克隆验证，获得克隆载体pSHTB10-HR34。pSHTB10-HR34质粒用KpnI酶切后片段大小为12653bp，用ScaI酶切后片段大小分别为3707bp和8946bp，验证图如图9所示：M表示marker，a表示KpnI酶切，b表示ScaI酶切。构建完成的pSHTB10-HR34质粒图谱如图10所示。
琼脂糖凝胶电泳参见《分子克隆实验指南》中琼脂糖凝胶电泳的方法，胶回收采用qiagen胶回收试剂盒，大肠杆菌化学转化法参见《分子克隆实验指南》中氯化钙制备和感受态转化大肠杆菌的方法。所述酶切方法参照限制性内切酶说明书，连接体系和方法参见Gibson Assembly和T4连接酶说明书。
实施例二：一种iTAR载体pSHTB10-HR12在捕捉含hygR元件的DNA大片段中的应用，包括以下步骤：
1.重组菌株E.coli BL21-hygR的构建
(1)引物设计：
TE7For:5'-cagtgctgacaatacaaccgttcaggctgtagcaagtacgagcttgctcctgatc-3'；
TE7Rev:5'-gtatccagcagcctgattcggctgagtgaacatcagttattcctttgccctcggac-3'；
P1:5'-gagcgtcaacttaaagctg-3'；
P2:5'-gaagtggtacggcgatgcgc-3'；
P3:5'-gtcgatgcgacgcaatcgtc-3'；
P4:5'-gtccgactctaagatgtcac-3'；
(2)PCR扩增：PCR反应体系是5×Q5PCR buffer 10μL，5×enhancer 10μL，dNTPs(10mmol/L)1μL，引物(10μmol/L)各2.5μL，质粒DNA(约50ng/μL)2μL，Q5DNA聚合酶(5U/μL)0.5μL，加ddH2O至50μL。PCR程序是98℃预变性30s；98℃变性10s，55℃退火30s，72℃延伸，30个循环；最后72℃延伸10min。所述DNA模板是pPICZαA-hygR，引物是TE7For/TE7Rev，延伸时间是1min。将PCR产物通过琼脂糖凝胶电泳进行分离，回收的TE7DNA片段是长度为1734bp的核酸片段。
(3)E.coli BL21/pKD46感受态细胞制备：于新鲜平板上接种E.coli BL21/pKD46单克隆，30℃培养过夜，然后1％转接到20mL含100μg/mL氨苄青霉素的新鲜LB培养基中，在30℃、200rpm的条件下培养至OD600到0.2，添加20％的阿拉伯糖200μL，再培养到OD600至0.5；冰浴10min后，在4000rpm，4℃下离心5min收集细胞。然后用冰浴的0.1mol/L的氯化钙处理20min；在4000rpm，4℃下离心5min收集细胞。最后，悬浮在1mL含15％甘油的0.1mol/L氯化钙中。
(4)菌株构建：将步骤(2)回收的TE7DNA片段转化至步骤(3)的E.coli BL21/pKD46感受态细胞中，涂布在含100μg/mL潮霉素的LB平板上，在37℃培养箱中培养16h，挑取单克隆验证，获得重组菌株E.coli BL21-hygR。重组菌株E.coli BL21-hygR通过PCR的方式验证，验证引物P1/P2和P3/P4。重组菌株E.coli BL21-hygR PCR扩增片段大小分别为489bp和800bp，验证图如图11所示：M表示marker，a表示由引物P1/P2扩增，b表示由引物P3/P4扩增。
2.含hygR元件的DNA大片段的酶切与克隆
(1)Minigene合成：
S1:5'-gactgacactgataatacgactcactataggcgtgcgatagcccatagaagttttagagctagaaatagcaagtt-3'；
S2:5'-gactgacactgataatacgactcactatagggtggcccccaccatcggcagttttagagctagaaatagcaagtt-3'；
(2)E.coli BL21-hygR基因组酶切：体外转录DNA片段S1和S2，回收得到gRNA-1和gRNA-2。将BL21-hygR基因组DNA与Cas9酶，gRNA-1和gRNA-2混合，酶切体系是BL21-hygR基因组DNA(约25μg/μL)20μL，Cas9酶(70pmol)1μL，gRNA-1(5μg)1μL，gRNA-2(5μg)1μL，Cas9缓冲液4μL，加ddH2O至40μL，37℃过夜酶切，回收酶切产物。
(3)BY4742/pSHTB10-HR12感受态细胞制备：将iTAR载体pSHTB10-HR12转化到酿酒酵母BY4742中，涂布于SD-URA平板，挑取阳性克隆于28℃培养过夜，然后1％转接到50mLYPD培养基中，在28℃，200rpm的条件下培养至OD600到0.4，将菌体用无菌水洗涤两次，重新悬浮在含2％半乳糖的50mL YEP-lactate培养基中，在28℃，200rpm的条件下培养2h。3000g离心5min，收集细胞。然后用25mL无菌水悬浮细胞，3000g离心5min。再用25mL无菌水悬浮细胞，3000g离心5min。最后，悬浮细胞在500μL无菌水中，以每管100μL分装感受态细胞。
(4)捕捉大片段：将纯化后的酶切产物(1μg)转化至经半乳糖诱导的BY4742/pSHTB10-HR12感受态细胞中，分别取100μL转化液涂布在SG-URA平板上和含200μg/mL潮霉素SG-URA平板，在28℃培养箱中培养4～5天，挑取阳性克隆验证，获得捕捉质粒pSHTB10-HygR，如图12所示，a表示涂布在在SG-URA平板上的转化子生长情况，b表示涂布在含200μg/mL潮霉素的SG-URA平板上的转化子生长情况。从图12中可以看出，在SG-URA平板上的转化子的数目为33个，在含200μg/mL潮霉素的SG-URA平板上的转化子的数目为24个。经过三次重复试验，得出iTAR载体捕捉含hygR的大片段DNA的阳性克隆率为73.2％。pSHTB10-HygR用PciI酶切后的片段大小分别是2082bp、3027bp、4622bp、5827bp、6928bp、16660bp和19759bp，验证图如图13所示：M表示marker，a表示PciI酶切。
细菌DNA提取方法参见《精编分子生物学实验指南》中从细菌中制备基因组DNA的方法，酵母菌转化方法参见《精编分子生物学实验指南》中乙酸锂转化方法，所述DNA转录采用NEB体外转录试剂盒，酶切方法参照限制性内切酶说明书，酶切产物用QIAamp试剂盒纯化。
实施例三：一种iTAR载体pSHTB10-HR34在捕捉polymyxin基因簇中的应用，包括以下步骤：
(1)Minigene合成：
S3:5'-gactgacactgataatacgactcactataggatatctaaaacgtgcgccggttttagagctagaaatagcaagtt-3'；
S4:5'-gactgacactgataatacgactcactataggcttttaccgatgaagagaggttttagagctagaaatagcaagtt-3'；
(2)Paenibacillus polymyxa SQR-21基因组酶切：体外转录DNA片段S3和S4，回收得到gRNA-3和gRNA-4。将SQR-21基因组DNA与Cas9酶，gRNA-3和gRNA-4混合，酶切体系是SQR-21基因组DNA(约25μg/μL)20μL，Cas9酶(70pmol)1μL，gRNA-1(5μg)1μL，gRNA-2(5μg)1μL，Cas9缓冲液4μL，加ddH2O至40μL，37℃过夜酶切，回收酶切产物。
(3)polymyxin基因簇捕捉：将纯化后的酶切产物(1μg)转化至经半乳糖诱导的BY4742/pSHTB10-HR34感受态细胞中，涂布SG-URA平板，在28℃培养箱中培养4～5天，挑取阳性克隆验证，获得捕捉质粒pSHTB10-pmx。pSHTB10-pmx用NcoI酶切后的片段大小分别是116bp、1767bp、2953bp、3245bp、4995bp、17228bp和19829bp，验证图如图14所示：M表示marker，a表示NcoI酶切。
(4)polymyxin表达：将质粒pSHTB10和pSHTB10-pmx分别电转化到Bacillussubtilis 168感受态中，涂布在含25μg/ml博来霉素的LB平板上，挑取阳性克隆，得到重组菌株Bacillus subtilis168::pSHTB10和Bacillus subtilis 168::pmx。挑取Bacillussubtilis 168::pSHTB10和Bacillus subtilis168::pmx单菌落分别接种于10mL GSC培养基中，于37℃培养24h，然后1％分别转接到50mL GSC培养基中，于30℃培养24h。
(5)抑菌活性测试：将质粒pCMrfp转化到E.coli DH10B感受态细胞中，挑取阳性克隆于10mL LB培养基中，过夜培养。取300μL培养液均匀涂布在含0.2％阿拉伯糖的LB平板上，待平板晾干，分别取经步骤(4)培养的Bacillus subtilis 168::pSHTB10和Bacillussubtilis 168::pmx发酵液各5μL滴在平板上，过夜培养，测试其抑菌活性。活性测试如图15所示，a表示Bacillus subtilis 168::pSHTB10的发酵液的抑菌测试，b表示Bacillussubtilis 168::pmx的发酵液的抑菌测试。从图15可以看出，Bacillus subtilis168::pSHTB10作为对照，没有产生抑菌圈，而Bacillus subtilis 168::pmx产生了抑菌圈，由此说明，利用iTAR克隆技术成功捕获了polymyxin基因簇，通过将polymyxin基因簇转移至Bacillus subtilis 168基因组上的方式，使该基因簇成功表达，产生了抑菌物质，抑制了E.coli DH10B/pCMrfp，故此产生了抑菌圈的效果。
细菌DNA提取方法参见《精编分子生物学实验指南》中从细菌中制备基因组DNA的方法，酵母菌转化方法参见《精编分子生物学实验指南》中乙酸锂转化方法，枯草芽孢杆菌转化方法参见《分子克隆实验指南》中的电击转化方法，所述DNA转录采用NEB体外转录试剂盒，酶切方法参照限制性内切酶说明书，酶切产物用QIAamp试剂盒纯化。
序列表
<110> 南京理工大学
<120> 诱导型酵母转化重组系统的构建及其应用
<141> 2017-12-12
<160> 34
<170> SIPOSequenceListing 1.0
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<211> 59
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<213> 人工序列(Artificial Sequence)
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<221> misc_feature
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<400> 2
cgatatcaag cttatggaat tgtgagcgga taacaattca tgcatatgaa aaacatcaa 59
<210> 3
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<223> primer
<400> 16
caatgcatgt ttctgagatc tacggcatac acccaagcc 39
<210> 17
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(39)
<223> primer
<400> 17
ggcttgggtg tatgccgtag atctcagaaa catgcattg 39
<210> 18
<211> 37
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(37)
<223> primer
<400> 18
gcccttccca acagttgcta cagcgtaacc aagttgc 37
<210> 19
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(39)
<223> primer
<400> 19
cattcgccat tcaggctgcg gttgaagcgg ttatgcttg 39
<210> 20
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(39)
<223> primer
<400> 20
gtttcttttt caaaagccat tcttgacact ccttatttg 39
<210> 21
<211> 55
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(55)
<223> primer
<400> 21
cagtgctgac aatacaaccg ttcaggctgt agcaagtacg agcttgctcc tgatc 55
<210> 22
<211> 56
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(56)
<223> primer
<400> 22
gtatccagca gcctgattcg gctgagtgaa catcagttat tcctttgccc tcggac 56
<210> 23
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(19)
<223> primer
<400> 23
gagcgtcaac ttaaagctg 19
<210> 24
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> primer
<400> 24
gaagtggtac ggcgatgcgc 20
<210> 25
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> primer
<400> 25
gtcgatgcga cgcaatcgtc 20
<210> 26
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(20)
<223> primer
<400> 26
gtccgactct aagatgtcac 20
<210> 27
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(75)
<223> Synthetic DNA
<400> 27
gactgacact gataatacga ctcactatag gcgtgcgata gcccatagaa gttttagagc 60
tagaaatagc aagtt 75
<210> 28
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(75)
<223> Synthetic DNA
<400> 28
gactgacact gataatacga ctcactatag ggtggccccc accatcggca gttttagagc 60
tagaaatagc aagtt 75
<210> 29
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(75)
<223> Synthetic DNA
<400> 29
gactgacact gataatacga ctcactatag gatatctaaa acgtgcgccg gttttagagc 60
tagaaatagc aagtt 75
<210> 30
<211> 75
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(75)
<223> Synthetic DNA
<400> 30
gactgacact gataatacga ctcactatag gcttttaccg atgaagagag gttttagagc 60
tagaaatagc aagtt 75
<210> 31
<211> 6844
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(6844)
<223> plasmid
<400> 31
agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg 60
tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt 120
cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg 180
tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg tactgagagt 240
gcaccatacc acagcttttc aattcaattc atcatttttt ttttattctt ttttttgatt 300
tcggtttctt tgaaattttt ttgattcggt aatctccgaa cagaaggaag aacgaaggaa 360
ggagcacaga cttagattgg tatatatacg catatgtagt gttgaagaaa catgaaattg 420
cccagtattc ttaacccaac tgcacagaac aaaaacctgc aggaaacgaa gataaatcat 480
gtcgaaagct acatataagg aacgtgctgc tactcatcct agtcctgttg ctgccaagct 540
atttaatatc atgcacgaaa agcaaacaaa cttgtgtgct tcattggatg ttcgtaccac 600
caaggaatta ctggagttag ttgaagcatt aggtcccaaa atttgtttac taaaaacaca 660
tgtggatatc ttgactgatt tttccatgga gggcacagtt aagccgctaa aggcattatc 720
cgccaagtac aattttttac tcttcgaaga cagaaaattt gctgacattg gtaatacagt 780
caaattgcag tactctgcgg gtgtatacag aatagcagaa tgggcagaca ttacgaatgc 840
acacggtgtg gtgggcccag gtattgttag cggtttgaag caggcggcag aagaagtaac 900
aaaggaacct agaggccttt tgatgttagc agaattgtca tgcaagggct ccctatctac 960
tggagaatat actaagggta ctgttgacat tgcgaagagc gacaaagatt ttgttatcgg 1020
ctttattgct caaagagaca tgggtggaag agatgaaggt tacgattggt tgattatgac 1080
acccggtgtg ggtttagatg acaagggaga cgcattgggt caacagtata gaaccgtgga 1140
tgatgtggtc tctacaggat ctgacattat tattgttgga agaggactat ttgcaaaggg 1200
aagggatgct aaggtagagg gtgaacgtta cagaaaagca ggctgggaag catatttgag 1260
aagatgcggc cagcaaaact aaaaaactgt attataagta aatgcatgta tactaaactc 1320
acaaattaga gcttcaattt aattatatca gttattaccc tatgcggtgt gaaataccgc 1380
acagatgcgt aaggagaaaa taccgcatca ggaaattgta aacgttaata ttttgttaaa 1440
attcgcgtta aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa 1500
aatcccttat aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa 1560
caagagtcca ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca 1620
gggcgatggc ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg 1680
taaagcacta aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc 1740
ggcgaacgtg gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc 1800
aagtgtagcg gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca 1860
gggcgcgtcg cgccattcgc cattcaggct gcgcaactgt tgggaagggc gatcggtgcg 1920
ggcctcttcg ctattacgcc agctggcgaa agggggatgt gctgcaaggc gattaagttg 1980
ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg acggccagtg agcgcgcgta 2040
atacgactca ctatagggcg aattgggtac cggccgcaaa ttaaagcctt cgagcgtccc 2100
aaaaccttct caagcaaggt tttcagtata atgttacatg cgtacacgcg tctgtacaga 2160
aaaaaaagaa aaatttgaaa tataaataac gttcttaata ctaacataac tataaaaaaa 2220
taaataggga cctagacttc aggttgtcta actccttcct tttcggttag agcggatgtg 2280
gggggagggc gtgaatgtaa gcgtgacata actaattaca tgactcgagg tcgacaattc 2340
caaccttacc caagagttcg ccaaactcag acatcacttt angcaaaacc gcgccgtgct 2400
tcttcctcgg tggcattcat cacgaaatgt tcagcactac gcatactttt gacaggaaac 2460
gcaacggata ttgagtcaat atcaggcatt ctatcgctca gctttacagt gacaatgacg 2520
gctggcgact gaatattagt gcttacagac agcactacat attttccgtc gatgttgaaa 2580
tcctttctca tatgtcacca taaatatcaa ataattatag caatcattta cgcgttaatg 2640
gctaatcgcc atcttccagc aggcgcacca ttgcccctgt ttcactatcc agggtacgga 2700
tatagttcat gacaatattt acattggtcc agccaccagc ttgcatgatc tccggtattg 2760
aaactccagc gcgggccata tctcgcgcgg ctccgacacg ggcactgtgt ccagaccagg 2820
ccaggtatct ctgaccagag tcatccttag cgccgtaaat caatttattt caggaaagtt 2880
tcggaggaga tagtgttcgg cagtttgtac atcatctgcg ggatcaggta cggtttgatc 2940
aggttgtaga agatcaggta agacatagaa tcgatgtaga tgatcggttt gtttttgttg 3000
atttttacgt aacagttcag ttggaatttg ttacgcagac ccttaaccag gtattctact 3060
tcttcgaaag tgaaagactg ggtgttcagt acgatcgatt tgttggtaga gtttttgttg 3120
taatcccatt taccaccatc atccatgaac cagtatgcca gagacatcgg ggtcaggtag 3180
ttttcaacca ggttgttcgg gatggttttt ttgttgttaa cgatgaacag gttagccagt 3240
ttgttgaaag cttggtgttt gaaagtctgg gcgccccagg tgattaccag gttacccagg 3300
tggttaacac gttctttttt gtgcggcggg gacagtaccc actgatcgta cagcagacat 3360
acgtggtcca tgtatgcttt gtttttccac tcgaactgca tacagtaggt tttaccttca 3420
tcacgagaac ggatgtaagc atcacccagg atcagaccga tacctgcttc gaactgttcg 3480
atgttcagtt cgatcagctg ggatttgtat tctttcagca gtttagagtt cggacccagg 3540
ttcattacct ggtttttttt gatgtttttc atatgcatga attgttatcc gctcacaatt 3600
ccataagctt gatatcgaat tcctgcagcc cgggggatcc actagttcta gaatccgggg 3660
ttttttctcc ttgacgttaa agtatagagg tatattaaca attttttgtt gatactttta 3720
ttacatttga ataagaagta atacaaaccg aaaatgttga aagtattagt taaagtggtt 3780
atgcagtttt tgcatttata tatctgttaa tagatcaaaa atcatcgctt cgctgattaa 3840
ttaccccaga aataaggcta aaaaactaat cgcattatca tcctatggtt gttaatttga 3900
ttcgttcatt tgaaggtttg tggggccagg ttactgccaa tttttcctct tcataaccat 3960
aaaagctagt attgtagaat ctttattgtt cggagcagtg cggcgcgagg cacatctgcg 4020
tttcaggaac gcgaccggtg aagacgagga cgcacggagg agagtcttcc ttcggagggc 4080
tgtcacccgc tcggcggctt ctaatccgta ctagagctcc agcttttgtt ccctttagtg 4140
agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 4200
tccgctcaca attccacaca acataggagc cggaagcata aagtgtaaag cctggggtgc 4260
ctaatgagtg aggtaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 4320
aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 4380
tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 4440
gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 4500
cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 4560
gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 4620
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 4680
ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 4740
cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 4800
ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 4860
cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 4920
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 4980
gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct 5040
gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 5100
tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 5160
agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 5220
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 5280
atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 5340
cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 5400
actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 5460
aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 5520
cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 5580
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 5640
cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 5700
ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 5760
cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 5820
ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 5880
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 5940
ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 6000
aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 6060
gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 6120
gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 6180
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 6240
catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 6300
atttccccga aaagtgccac ctgggtcctt ttcatcacgt gctataaaaa taattataat 6360
ttaaattttt taatataaat atataaatta aaaatagaaa gtaaaaaaag aaattaaaga 6420
aaaaatagtt tttgttttcc gaagatgtaa aagactctag ggggatcgcc aacaaatact 6480
accttttatc ttgctcttcc tgctctcagg tattaatgcc gaattgtttc atcttgtctg 6540
tgtagaagac cacacacgaa aatcctgtga ttttacattt tacttatcgt taatcgaatg 6600
tatatctatt taatctgctt ttcttgtcta ataaatatat atgtaaagta cgctttttgt 6660
tgaaattttt taaacctttg tttatttttt tttcttcatt ccgtaactct tctaccttct 6720
ttatttactt tctaaaatcc aaatacaaaa cataaaaata aataaacaca gagtaaattc 6780
ccaaattatt ccatcattaa aagatacgag gcgcgtgtaa gttacaggca agcgatccgt 6840
ccta 6844
<210> 32
<211> 7891
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(7891)
<223> plasmid
<400> 32
cagatcccct ttatccgcca attcgtcgcc gcatgccccg acagcacgaa cttctggtgt 60
tctcgcttct taaaaagaac gtcttcgttc tgcttggcgt tatttttgct tggaaaagtg 120
gtcactgatt gcaaaaagga tggcgcaacg tggcaatggg ggtaacccgt atacgcatca 180
cgtcgagatg cattttcatc gacttggcgc ctttctacat cacaccaagc agcccacatt 240
aaaataagag aaccgtgaac tatggatttt tgcttattga acgagaaaag tcagatcttc 300
gtccacgccg agccctatgc agtctccgat tatgttaacc agtatgtcgg tacgcactct 360
attcgcctgc ccaagggcgg gcccccggca ggcaggctgc accacagaat cttcggatgc 420
ctcgacctgt gtcgaatcag ctacggcggt agcgtgaggg taatctcgcc tggattagag 480
acctgttatc atctgcaaat aatactcaaa ggccattgcc tgtggcgtgg ccatggccag 540
gagcactatt ttgcgccggg cgaactattg ctgctcaatc cggatgacca agccgacctg 600
acctattcag aagattgcga gaaatttatc gttaaattgc cctcagtggt ccttgatcgg 660
gcatgcagtg acaacaattg gcacaagccg agggagggta tccgtttcgc cgcgcgacac 720
aatctccagc aactcgatgg ctttatcaat ctactcgggt tagtttgtga cgaagcggaa 780
catacaaagt cgatgcctcg ggtccaagag cactatgcgg ggatcatcgc ttccaagctg 840
ctcgaaatgc tgggcagcaa tgtcagccgt gaaattttca gcaaaggtaa cccgtctttc 900
gagcgagtcg ttcaattcat tgaggagaat ctcaaacgga atatcagcct tgagcggtta 960
gcggagctgg cgatgatgag tccacgctcg ctctacaatt tgttcgagaa gcatgccggc 1020
accacgccga agaactacat ccgcaaccgc aagctcgaaa gcatccgcgc ctgcttgaac 1080
gatcccagtg ccaatgtgcg tagtataact gagatagccc tagactacgg cttcttacat 1140
ttgggacgct tcgctgaaaa ctataggagc gcgttcggcg agttgccttc cgacaccctg 1200
cgtcaatgca aaaaggaagt ggcttgatta cgaacgtagc cgaagaaggg atgggttggc 1260
atcgcccggc tttcttagac actctccaag ctctgaaata gcgttttaca aactccactg 1320
gctatagtgc cggcgtttgc ccgcgctctc cgtggccgcg tcccaatgca ggtcgggttc 1380
ctcgacccga agtgcccttg ggcatctcca cttcagcgtc tgaccttgct ggccagttcg 1440
ccgccgatgc cgaagtggcc cttggccatc tccgtgataa tcactcgcac gctggtcagc 1500
ggcgcatcca gggagcgcga gatggcctcg ctgacttccc gaatgagggt ttccttctgc 1560
tcgtcgctgc ggccttcaag gatgtggatc tgctggccga aagggggatg tgctgcaagg 1620
cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt 1680
gaattaattc ttgaagacga aagggcctcg tgatacgcct atttttatag gttaatgtca 1740
tgataataat ggtttcttag agcttacggc cagcctcgca gagcaggatt cccgttgagc 1800
accgccaggt gcgaataagg gacagtgaag aaggaacacc cgctcgcggg tgggcctact 1860
tcacctatcc tgcccggctg acgccgttgg atacaccaag gaaagtctac acgaaccctt 1920
tggcaaaatc ctgtatatcg tgcgaaaaag gatggatata ccgaaaaaat cgctataatg 1980
accccgaagc agggttatgc agcggaaaag atccgtcgat cgacccaggt ggcacttttc 2040
ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 2100
cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 2160
gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 2220
ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 2280
tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 2340
aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgtg 2400
ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 2460
agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 2520
gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 2580
gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 2640
gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 2700
tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 2760
ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 2820
cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 2880
gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 2940
cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 3000
tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 3060
aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 3120
aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 3180
gatcttcttg agatcctttt tttctgcggg ggatcaggac cgctgccgga gcgcaaccca 3240
ctcactacag cagagccatg tagggccgcc ggcgttgtgg ataccacgcg gaaaacttgg 3300
ccctcactga cagatgaggg gcggacgttg acacttgagg ggccgactca cccggcgcgg 3360
cgttgacaga tgaggggcag gctcgatttc ggccggcgac gtggagctgg ccagcctcgc 3420
aaatcggcga aaacgcctga ttttacgcga gtttcccaca gatgatgtgg acaagcctgg 3480
ggataagtgc cctgcggtat tgacacttga ggggcgcgac tactgacaga tgaggggcgc 3540
gatccttgac acttgagggg cagagtgatg acagatgagg ggcgcaccta ttgacatttg 3600
aggggctgtc cacaggcaga aaatccagca tttgcaaggg tttccgcccg tttttcggcc 3660
accgctaacc tgtcttttaa cctgctttta aaccaatatt tataaacctt gtttttaacc 3720
agggctgcgc cctggcgcgt gaccgcgcac gccgaagggg ggtgcccccc cttctcgaac 3780
cctcccggcc cgctaacgcg gcaccccatc cccccagggg ctgcgcccct cggccgcgaa 3840
cgacctcacc ccaaaaatgg cagccacgta gaaagccagt ccgcagaaac ggtgctgacc 3900
ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa 3960
gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc 4020
aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 4080
aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gatcgacgga 4140
tcgatccggg gaattaattc cggggcaatc ccgcaaggag ggtgaatgaa tcggacgttt 4200
gaccggaagg catacaggca agaactgatc gacgcggggt tttccgccga ggatgccgaa 4260
accatcgcaa gccgcaccgt catgcgtgcg ccccgcgaaa ccttccagtc cgtcggctcg 4320
atggtccagc aagctacggc caagatcgag cgcgacagcg tgcaactggc tccccctgcc 4380
ctgcccgcgc catcggccgc cgtggagcgt tcgcgtcgtc tcgaacagga ggcggcaggt 4440
ttggcgaagt cgatgaccat cgacacgcga ggaactatga cgaccaagaa gcgaaaaacc 4500
gccggcgagg acctggcaaa acaggtcagc gaggccaagc aggccgcgtt gctgaaacac 4560
acgaagcagc agatcaagga aatgcagctt tccttgttcg atattgcgcc gtggccggac 4620
acgatgcgag cgatgccaaa cgacacggcc cgctctgccc tgttcaccac gcgcaacaag 4680
aaaatcccgc gcgaggcgct gcaaaacaag gtcattttcc acgtcaacaa ggacgtgaag 4740
atcacctaca ccggcgtcga gctgcgggcc gacgatgacg aactggtgtg gcagcaggtg 4800
ttggagtacg cgaagcgcac ccctatcggc gagccgatca ccttcacgtt ctacgagctt 4860
tgccaggacc tgggctggtc gatcaatggc cggtattaca cgaaggccga ggaatgcctg 4920
tcgcgcctac aggcgacggc gatgggcttc acgtccgacc gcgttgggca cctggaatcg 4980
gtgtcgctgc tgcaccgctt ccgcgtcctg gaccgtggca agaaaacgtc ccgttgccag 5040
gtcctgatcg acgaggaaat cgtcgtgctg tttgctggcg accactacac gaaattcaca 5100
tgggagaagt accgcaagct gtcgccgacg gcccgacgga tgttcgacta tttcagctcg 5160
caccgggagc cgtacccgct caagctggaa accttccgcc tcatgtgcgg atcggattcc 5220
acccgcgtga agaagtggcg cgagcaggtc ggcgaagcct gcgaagagtt gcgaggcagc 5280
ggcctggtgg aacacgcctg ggtcaatgat gacctggtgc attgcaaacg ctagggcctt 5340
gtggggtcag ttccggctgg gggttcagca gccaccggca tgcaagcttg ctagagggtc 5400
agctttatgc ttgtaaaccg ttttgtgaaa aaatttttaa aataaaaaag gggacctcta 5460
gggtccccaa ttaattagta atataatcta ttaaaggtca ttcaaaaggt catccaccgg 5520
atcaattccc ctgctcgcgc aggctgggtg ccaagctctc gggtaacatc aaggcccgat 5580
ccttggtagt cggcaaataa tgtctaacaa ttcgttcaag ccgacgccgc ttcgcggcgc 5640
ggcttaactc aagcgttaga tgcactaagc acataattgc tcacagccaa actatcaggt 5700
caagtctgct tttattattt ttaagcgtgc ataataagcc ctacacaaat tgggagatat 5760
atcatgaaag gctggctttt tcttgttatc gcaatagttg gcgaagtaat cgcaacatcc 5820
gcattaaaat ctagcgaggg ctttactaag ctgatccggt ggatgacctt ttgaatgacc 5880
tttaatagat tatattacta attaattggg gaccctagag gtcccctttt ttattttaaa 5940
aattttttca caaaacggtt tacaagcata aagctgactc tagctagagg atccttgata 6000
tggcttttta tatgtgttac tctacataca gaaaggagga actaaatatg gccaagttga 6060
ccagtgccgt tccggtgctc accgcgcgcg acgtcgccgg agcggtcgag ttctggaccg 6120
accggctcgg gttctcccgg gacttcgtgg aggacgactt cgccggtgtg gtccgggacg 6180
acgtgaccct gttcatcagc gcggtccagg accaggtggt gccggacaac accctggcct 6240
gggtgtgggt gcgcggcctg gacgagctgt acgccgagtg gtcggaggtc gtgtccacga 6300
acttccggga cgcctccggg ccggccatga ccgagatcgg cgagcagccg tgggggcggg 6360
agttcgccct gcgcgacccg gccggcaact gcgtgcactt cgtggccgag gagcaggact 6420
gaggatcccc tcgacttatt atttcaggaa agtttcggag gagatagtgt tcggcagttt 6480
gtacatcatc tgcgggatca ggtacggttt gatcaggttg tagaagatca ggtaagacat 6540
agaatcgatg tagatgatcg gtttgttttt gttgattttt acgtaacagt tcagttggaa 6600
tttgttacgc agacccttaa ccaggtattc tacttcttcg aaagtgaaag actgggtgtt 6660
cagtacgatc gatttgttgg tagagttttt gttgtaatcc catttaccac catcatccat 6720
gaaccagtat gccagagaca tcggggtcag gtagttttca accaggttgt tcgggatggt 6780
ttttttgttg ttaacgatga acaggttagc cagtttgttg aaagcttggt gtttgaaagt 6840
ctgggcgccc caggtgatta ccaggttacc caggtggtta acacgttctt ttttgtgcgg 6900
cggggacagt acccactgat cgtacagcag acatacgtgg tccatgtatg ctttgttttt 6960
ccactcgaac tgcatacagt aggttttacc ttcatcacga gaacggatgt aagcatcacc 7020
caggatcaga ccgatacctg cttcgaactg ttcgatgttc agttcgatca gctgggattt 7080
gtattctttc agcagtttag agttcggacc caggttcatt acctggtttt ttttgatgtt 7140
tttcatatgt tcatgactcc attattattg tttctgttgc ataaagccta aggggtaggc 7200
ctttctagag atagccattt tttgcactcc tgtatccgct tcttgcaagg ctggacttat 7260
ccctatcaaa ccggacacgt ttatgctggt tatgctggct gcatggggca ttagttgggg 7320
agccagattt gtcatggagc aggccgttct gctttatgga tcaggaaaaa actatttgtt 7380
cttcagtcat ggtactgttc tgatgtacct gctgtgtgtt ttcctggtat accgccgttg 7440
gatagctccg ctaccggtcg ttggtcagat caagctaatt cccatgcatg gtgaaaacgg 7500
gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg 7560
gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt 7620
caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt 7680
attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt 7740
gaacactatc ccatatcacc agctcaccgt ctttcattgc catacgtaat tacggatgag 7800
cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct 7860
ttacggtctt taaaaaggcc gtaatatcca g 7891
<210> 33
<211> 7871
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(7871)
<223> plasmid
<400> 33
atcacgtgct ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa 60
atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag 120
actctagggg gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat 180
taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt 240
tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata 300
aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt 360
cttcattccg taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat 420
aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg 480
cgtgtaagtt acaggcaagc gatccgtcct aagaaaccat tattatcatg acattaacct 540
ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa 600
acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 660
gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact 720
atgcggcatc agagcagatt gtactgagag tgcaccatac cacagctttt caattcaatt 780
catcattttt tttttattct tttttttgat ttcggtttct ttgaaatttt tttgattcgg 840
taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 900
gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 960
caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 1020
ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 1080
acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 1140
taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 1200
agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag 1260
acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 1320
gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 1380
gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 1440
cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 1500
ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 1560
gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 1620
acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta 1680
ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 1740
acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 1800
tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 1860
agttattacc ctatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 1920
aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1980
cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 2040
agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 2100
ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 2160
cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 2220
gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 2280
aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 2340
ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 2400
tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga 2460
aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac 2520
gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc gaattgggta 2580
ccggccgcaa attaaagcct tcgagcgtcc caaaaccttc tcaagcaagg ttttcagtat 2640
aatgttacat gcgtacacgc gtctgtacag aaaaaaaaga aaaatttgaa atataaataa 2700
cgttcttaat actaacataa ctataaaaaa ataaataggg acctagactt caggttgtct 2760
aactccttcc ttttcggtta gagcggatgt ggggggaggg cgtgaatgta agcgtgacat 2820
aactaattac atgactcgag gtcgacaatt ccaaccttac ccaagagttc gccaaactca 2880
gacatcactt tangcaaaac cgcgccgtgc ttcttcctcg gtggcattca tcacgaaatg 2940
ttcagcacta cgcatacttt tgacaggaaa cgcaacggat attgagtcaa tatcaggcat 3000
tctatcgctc agctttacag tgacaatgac ggctggcgac tgaatattag tgcttacaga 3060
cagcactaca tattttccgt cgatgttgaa atcctttctc atatgtcacc ataaatatca 3120
aataattata gcaatcattt acgcgttaat ggctaatcgc catcttccag caggcgcacc 3180
attgcccctg tttcactatc cagggtacgg atatagttca tgacaatatt tacattggtc 3240
cagccaccag cttgcatgat ctccggtatt gaaactccag cgcgggccat atctcgcgcg 3300
gctccgacac gggcactgtg tccagaccag gccaggtatc tctgaccaga gtcatcctta 3360
gcgccgtaaa tcaatttatt tcaggaaagt ttcggaggag atagtgttcg gcagtttgta 3420
catcatctgc gggatcaggt acggtttgat caggttgtag aagatcaggt aagacataga 3480
atcgatgtag atgatcggtt tgtttttgtt gatttttacg taacagttca gttggaattt 3540
gttacgcaga cccttaacca ggtattctac ttcttcgaaa gtgaaagact gggtgttcag 3600
tacgatcgat ttgttggtag agtttttgtt gtaatcccat ttaccaccat catccatgaa 3660
ccagtatgcc agagacatcg gggtcaggta gttttcaacc aggttgttcg ggatggtttt 3720
tttgttgtta acgatgaaca ggttagccag tttgttgaaa gcttggtgtt tgaaagtctg 3780
ggcgccccag gtgattacca ggttacccag gtggttaaca cgttcttttt tgtgcggcgg 3840
ggacagtacc cactgatcgt acagcagaca tacgtggtcc atgtatgctt tgtttttcca 3900
ctcgaactgc atacagtagg ttttaccttc atcacgagaa cggatgtaag catcacccag 3960
gatcagaccg atacctgctt cgaactgttc gatgttcagt tcgatcagct gggatttgta 4020
ttctttcagc agtttagagt tcggacccag gttcattacc tggttttttt tgatgttttt 4080
catatgcatg aattgttatc cgctcacaat tccataagct tgatatcgaa ttcctgcagc 4140
ccgggggatc cactagttct agaatccggg gttttttctc cttgacgttt aagtatagag 4200
gcatattgac aattttttgt tgatactttt attacgtttg aataagaagt aatacaaacc 4260
gaaaacgttg aaagtattag ttaaagtggt tatgcagttt ctgcatttat atatctgtta 4320
atagatcaaa aatcatcgct tcgctaatta attaccccag aaataaggct aaaaaactaa 4380
tcgcattatc atcctatggt tgttaatttg attcgttcat ttgaaggttt gtggggccag 4440
gttactgcca atttttcctc ttcataacca taaaagctag tattgtagaa tctttattgt 4500
tcggagcagt gcggcgcgag gcacatctgc gtttcaggaa cgcgaccggt gaagacgagg 4560
acgcacggag gagagtcttc cttcggaggg ctgtcacccg ctcggcggct tctaatccgt 4620
actagagctc actacagcag agccatgtag ggccgccggc gttgtggata ccacgcggaa 4680
aacttggccc tcactgacag atgaggggcg gacgttgaca cttgaggggc cgactcaccc 4740
ggcgcggcgt tgacagatga ggggcaggct cgatttcggc cggcgacgtg gagctggcca 4800
gcctcgcaaa tcggcgaaaa cgcctgattt tacgcgagtt tcccacagat gatgtggaca 4860
agcctgggga taagtgccct gcggtattga cacttgaggg gcgcgactac tgacagatga 4920
ggggcgcgat ccttgacact tgaggggcag agtgatgaca gatgaggggc gcacctattg 4980
acatttgagg ggctgtccac aggcagaaaa tccagcattt gcaagggttt ccgcccgttt 5040
ttcggccacc gctaacctgt cttttaacct gcttttaaac caatatttat aaaccttgtt 5100
tttaaccagg gctgcgccct ggcgcgtgac cgcgcacgcc gaaggggggt gccccccctt 5160
ctcgaaccct cccggcccgc taacgcggca ccccatcccc ccaggggctg cgcccctcgg 5220
ccgcgaacga cctcacccca aaaatggcag ccacgtagaa agccagtccg cagaaacggt 5280
gctgaccccg gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa 5340
agagaaagca ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat 5400
ggacagcaag cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct 5460
gcaaagtaaa ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat 5520
cgacggatcg atccggggaa ttaattccgg ggcaatcccg caaggagggt gaatgaatcg 5580
gacgtttgac cggaaggcat acaggcaaga actgatcgac gcggggtttt ccgccgagga 5640
tgccgaaacc atcgcaagcc gcaccgtcat gcgtgcgccc cgcgaaacct tccagtccgt 5700
cggctcgatg gtccagcaag ctacggccaa gatcgagcgc gacagcgtgc aactggctcc 5760
ccctgccctg cccgcgccat cggccgccgt ggagcgttcg cgtcgtctcg aacaggaggc 5820
ggcaggtttg gcgaagtcga tgaccatcga cacgcgagga actatgacga ccaagaagcg 5880
aaaaaccgcc ggcgaggacc tggcaaaaca ggtcagcgag gccaagcagg ccgcgttgct 5940
gaaacacacg aagcagcaga tcaaggaaat gcagctttcc ttgttcgata ttgcgccgtg 6000
gccggacacg atgcgagcga tgccaaacga cacggcccgc tctgccctgt tcaccacgcg 6060
caacaagaaa atcccgcgcg aggcgctgca aaacaaggtc attttccacg tcaacaagga 6120
cgtgaagatc acctacaccg gcgtcgagct gcgggccgac gatgacgaac tggtgtggca 6180
gcaggtgttg gagtacgcga agcgcacccc tatcggcgag ccgatcacct tcacgttcta 6240
cgagctttgc caggacctgg gctggtcgat caatggccgg tattacacga aggccgagga 6300
atgcctgtcg cgcctacagg cgacggcgat gggcttcacg tccgaccgcg ttgggcacct 6360
ggaatcggtg tcgctgctgc accgcttccg cgtcctggac cgtggcaaga aaacgtcccg 6420
ttgccaggtc ctgatcgacg aggaaatcgt cgtgctgttt gctggcgacc actacacgaa 6480
attcacatgg gagaagtacc gcaagctgtc gccgacggcc cgacggatgt tcgactattt 6540
cagctcgcac cgggagccgt acccgctcaa gctggaaacc ttccgcctca tgtgcggatc 6600
ggattccacc cgcgtgaaga agtggcgcga gcaggtcggc gaagcctgcg aagagttgcg 6660
aggcagcggc ctggtggaac acgcctgggt caatgatgac ctggtgcatt gcaaacgcta 6720
gggccttgtg gggtcagttc cggctggggg ttcagcagcc accggcatgc aagcttgcta 6780
gagggtcagc tttatgcttg taaaccgttt tgtgaaaaaa tttttaaaat aaaaaagggg 6840
acctctaggg tccccaatta attagtaata taatctatta aaggtcattc aaaaggtcat 6900
ccaccggatc aattcccctg ctcgcgcagg ctgggtgcca agctctcggg taacatcaag 6960
gcccgatcct tggtagtcgg caaataatgt ctaacaattc gttcaagccg acgccgcttc 7020
gcggcgcggc ttaactcaag cgttagatgc actaagcaca taattgctca cagccaaact 7080
atcaggtcaa gtctgctttt attattttta agcgtgcata ataagcccta cacaaattgg 7140
gagatatatc atgaaaggct ggctttttct tgttatcgca atagttggcg aagtaatcgc 7200
aacatccgca ttaaaatcta gcgagggctt tactaagctg atccggtgga tgaccttttg 7260
aatgaccttt aatagattat attactaatt aattggggac cctagaggtc ccctttttta 7320
ttttaaaaat tttttcacaa aacggtttac aagcataaag ctgactctag ctagaggatc 7380
cttgatatgg ctttttatat gtgttactct acatacagaa aggaggaact aaatatggcc 7440
aagttgacca gtgccgttcc ggtgctcacc gcgcgcgacg tcgccggagc ggtcgagttc 7500
tggaccgacc ggctcgggtt ctcccgggac ttcgtggagg acgacttcgc cggtgtggtc 7560
cgggacgacg tgaccctgtt catcagcgcg gtccaggacc aggtggtgcc ggacaacacc 7620
ctggcctggg tgtgggtgcg cggcctggac gagctgtacg ccgagtggtc ggaggtcgtg 7680
tccacgaact tccgggacgc ctccgggccg gccatgaccg agatcggcga gcagccgtgg 7740
gggcgggagt tcgccctgcg cgacccggcc ggcaactgcg tgcacttcgt ggccgaggag 7800
caggactgag gatcccctcg acttattatt tcaggaaagt ttcggaggag atagtgttcg 7860
gcagtttgta c 7871
<210> 34
<211> 9910
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(9910)
<223> plasmid
<400> 34
atcacgtgct ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa 60
atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag 120
actctagggg gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat 180
taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt 240
tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata 300
aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt 360
cttcattccg taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat 420
aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg 480
cgtgtaagtt acaggcaagc gatccgtcct aagaaaccat tattatcatg acattaacct 540
ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa 600
acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 660
gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact 720
atgcggcatc agagcagatt gtactgagag tgcaccatac cacagctttt caattcaatt 780
catcattttt tttttattct tttttttgat ttcggtttct ttgaaatttt tttgattcgg 840
taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 900
gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 960
caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 1020
ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 1080
acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 1140
taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 1200
agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag 1260
acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 1320
gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 1380
gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 1440
cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 1500
ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 1560
gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 1620
acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta 1680
ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 1740
acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 1800
tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 1860
agttattacc ctatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 1920
aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1980
cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 2040
agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 2100
ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 2160
cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 2220
gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 2280
aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 2340
ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 2400
tgcgtgcgct atgaccgggt attgcaaatt gaagaaagct ctgatgatgg tgatgaatat 2460
gactattcac ccgcaaaaga agagtgggta attaccgcag cgaacgcgaa accgcaatgc 2520
gatattattc atgaagcctg gcagagcagg gctgttatcc gctatgacat ggcagtgccg 2580
ctcaatttgt cagaacgcag cgcccggcaa tccactggca gagtaggggt ggtgttggtt 2640
gtcactctta gtcataacag caggcgtatt gatgtggata tcaatcttga taaccaggct 2700
gacgatcatc gccttcgtgt cctggtccct acacctttta acaccgacag tgttctggca 2760
gatacgcagt ttggttcgct aacgcgcccc gtgaacgaca gtgcaatgaa caactggcag 2820
caagaaggct ggaaagaagc gccggttccg gtatggaata tgctcaacta tgttgcctta 2880
caggaagggc gtaacggcat ggctgtcttt agcgaagggt tacgtgaatt tgaagtcatc 2940
ggtgaagaga agaaaacctt tgccattacg ttgctgcgtg gcgtgggctt actgggcaaa 3000
gaagatctgc ttttaaggcc tgggcggcct tcgggaatta aaatgccagt cccggactca 3060
caactacgtg gtctgctttc ttgtcgccta agtttattga gttataccgg tacgccaacc 3120
gccgctggtg tagctcagca ggcgcgagca tggctgactc cagtacagtg ttacaacaaa 3180
atcccatggg atgtgatgaa gctcaacaaa gccggattca acgtgccgga aagttatagt 3240
ttgttgaaaa tgcccccagt gggatgcctg ataagcgcac ttaagaaagc tgaagaccga 3300
caagaagtga ttttacggct gtttaatccg gctgaatcag caacctgtga tgcgactgtt 3360
gctttcagtc gcgaggtgat ttcttgctca gaaacgatga tggatgaaca cattactagg 3420
gataacaggg taatcggtaa tggtgagttt gctgcggttg cagaaattac cgtcaccgcc 3480
agttaatccg gagagtcagc gatgttcctg aaaaccgaat catttgaaca taacggtgtg 3540
accgtcacgc tttctgaact gtcagccctg cagcgcattg agcatctcgc cctgatgaaa 3600
cggcaggcag aacaggcgga gtcagacagc aaccggaagt ttactgtgga agacgccatc 3660
agaaccggcg cgtttctggt ggcgatgtcc ctgtggcata accatccgca gaagacgcag 3720
atgccgtcca tgaatgaagc cgttaaacag attgagcagg aagtgcttac cacctggccc 3780
acggaggcaa tttctcatgc tgaaaacgtg gtgtaccggc tgtctggtat gtatgagttt 3840
gtggtgaata atgcccctga acagacagag gacgccgggc ccgcagagcc tgtttctgcg 3900
ggaaagtgtt cgacggtgag ctgagttttg ccctgaaact ggcgcgtgag atggggcgac 3960
ccgactggcg tgccatgctt gccgggatgt catccacgga gtatgccgac tggcaccgct 4020
tttacagtac ccattatttt catgatgttc tgctggatat gcacttttcc gggctgacgt 4080
acaccgtgct cagcctgttt ttcagcgatc cggatatgca tccgctggat ttcagtctgc 4140
tgaaccggcg cgaggctgac gaagagcctg aagatgatgt gctgatgcag aaagcggcag 4200
ggcttgccgg aggtgtccgc tttggcccgg acgggaatga agttatcccc gcttccccgg 4260
atgtggcgga catgacggag gatgacgtaa tgctgatgac agtatcagaa gggatcgcag 4320
gaggagtccg gtatggctga accggtaggc gatctggtcg ttgatttgag tctggatgcg 4380
gccagatttg acgagcagat ggccagagtc aggcgtcatt tttctggtac ggaaagtgat 4440
gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa 4500
agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg 4560
ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac 4620
cggccgcaaa ttaaagcctt cgagcgtccc aaaaccttct caagcaaggt tttcagtata 4680
atgttacatg cgtacacgcg tctgtacaga aaaaaaagaa aaatttgaaa tataaataac 4740
gttcttaata ctaacataac tataaaaaaa taaataggga cctagacttc aggttgtcta 4800
actccttcct tttcggttag agcggatgtg gggggagggc gtgaatgtaa gcgtgacata 4860
actaattaca tgactcgagg tcgacaattc caaccttacc caagagttcg ccaaactcag 4920
acatcacttt angcaaaacc gcgccgtgct tcttcctcgg tggcattcat cacgaaatgt 4980
tcagcactac gcatactttt gacaggaaac gcaacggata ttgagtcaat atcaggcatt 5040
ctatcgctca gctttacagt gacaatgacg gctggcgact gaatattagt gcttacagac 5100
agcactacat attttccgtc gatgttgaaa tcctttctca tatgtcacca taaatatcaa 5160
ataattatag caatcattta cgcgttaatg gctaatcgcc atcttccagc aggcgcacca 5220
ttgcccctgt ttcactatcc agggtacgga tatagttcat gacaatattt acattggtcc 5280
agccaccagc ttgcatgatc tccggtattg aaactccagc gcgggccata tctcgcgcgg 5340
ctccgacacg ggcactgtgt ccagaccagg ccaggtatct ctgaccagag tcatccttag 5400
cgccgtaaat caatttattt caggaaagtt tcggaggaga tagtgttcgg cagtttgtac 5460
atcatctgcg ggatcaggta cggtttgatc aggttgtaga agatcaggta agacatagaa 5520
tcgatgtaga tgatcggttt gtttttgttg atttttacgt aacagttcag ttggaatttg 5580
ttacgcagac ccttaaccag gtattctact tcttcgaaag tgaaagactg ggtgttcagt 5640
acgatcgatt tgttggtaga gtttttgttg taatcccatt taccaccatc atccatgaac 5700
cagtatgcca gagacatcgg ggtcaggtag ttttcaacca ggttgttcgg gatggttttt 5760
ttgttgttaa cgatgaacag gttagccagt ttgttgaaag cttggtgttt gaaagtctgg 5820
gcgccccagg tgattaccag gttacccagg tggttaacac gttctttttt gtgcggcggg 5880
gacagtaccc actgatcgta cagcagacat acgtggtcca tgtatgcttt gtttttccac 5940
tcgaactgca tacagtaggt tttaccttca tcacgagaac ggatgtaagc atcacccagg 6000
atcagaccga tacctgcttc gaactgttcg atgttcagtt cgatcagctg ggatttgtat 6060
tctttcagca gtttagagtt cggacccagg ttcattacct ggtttttttt gatgtttttc 6120
atatgcatga attgttatcc gctcacaatt ccataagctt gatatcgaat tcctgcagcc 6180
cgggggatcc actagttcta gaatccgggg ttttttctcc ttgacgttta agtatagagg 6240
catattgaca attttttgtt gatactttta ttacgtttga ataagaagta atacaaaccg 6300
aaaacgttga aagtattagt taaagtggtt atgcagtttc tgcatttata tatctgttaa 6360
tagatcaaaa atcatcgctt cgctaattaa ttaccccaga aataaggcta aaaaactaat 6420
cgcattatca tcctatggtt gttaatttga ttcgttcatt tgaaggtttg tggggccagg 6480
ttactgccaa tttttcctct tcataaccat aaaagctagt attgtagaat ctttattgtt 6540
cggagcagtg cggcgcgagg cacatctgcg tttcaggaac gcgaccggtg aagacgagga 6600
cgcacggagg agagtcttcc ttcggagggc tgtcacccgc tcggcggctt ctaatccgta 6660
ctagagctca ctacagcaga gccatgtagg gccgccggcg ttgtggatac cacgcggaaa 6720
acttggccct cactgacaga tgaggggcgg acgttgacac ttgaggggcc gactcacccg 6780
gcgcggcgtt gacagatgag gggcaggctc gatttcggcc ggcgacgtgg agctggccag 6840
cctcgcaaat cggcgaaaac gcctgatttt acgcgagttt cccacagatg atgtggacaa 6900
gcctggggat aagtgccctg cggtattgac acttgagggg cgcgactact gacagatgag 6960
gggcgcgatc cttgacactt gaggggcaga gtgatgacag atgaggggcg cacctattga 7020
catttgaggg gctgtccaca ggcagaaaat ccagcatttg caagggtttc cgcccgtttt 7080
tcggccaccg ctaacctgtc ttttaacctg cttttaaacc aatatttata aaccttgttt 7140
ttaaccaggg ctgcgccctg gcgcgtgacc gcgcacgccg aaggggggtg cccccccttc 7200
tcgaaccctc ccggcccgct aacgcggcac cccatccccc caggggctgc gcccctcggc 7260
cgcgaacgac ctcaccccaa aaatggcagc cacgtagaaa gccagtccgc agaaacggtg 7320
ctgaccccgg atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa 7380
gagaaagcag gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg 7440
gacagcaagc gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg 7500
caaagtaaac tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagatc 7560
gacggatcga tccggggaat taattccggg gcaatcccgc aaggagggtg aatgaatcgg 7620
acgtttgacc ggaaggcata caggcaagaa ctgatcgacg cggggttttc cgccgaggat 7680
gccgaaacca tcgcaagccg caccgtcatg cgtgcgcccc gcgaaacctt ccagtccgtc 7740
ggctcgatgg tccagcaagc tacggccaag atcgagcgcg acagcgtgca actggctccc 7800
cctgccctgc ccgcgccatc ggccgccgtg gagcgttcgc gtcgtctcga acaggaggcg 7860
gcaggtttgg cgaagtcgat gaccatcgac acgcgaggaa ctatgacgac caagaagcga 7920
aaaaccgccg gcgaggacct ggcaaaacag gtcagcgagg ccaagcaggc cgcgttgctg 7980
aaacacacga agcagcagat caaggaaatg cagctttcct tgttcgatat tgcgccgtgg 8040
ccggacacga tgcgagcgat gccaaacgac acggcccgct ctgccctgtt caccacgcgc 8100
aacaagaaaa tcccgcgcga ggcgctgcaa aacaaggtca ttttccacgt caacaaggac 8160
gtgaagatca cctacaccgg cgtcgagctg cgggccgacg atgacgaact ggtgtggcag 8220
caggtgttgg agtacgcgaa gcgcacccct atcggcgagc cgatcacctt cacgttctac 8280
gagctttgcc aggacctggg ctggtcgatc aatggccggt attacacgaa ggccgaggaa 8340
tgcctgtcgc gcctacaggc gacggcgatg ggcttcacgt ccgaccgcgt tgggcacctg 8400
gaatcggtgt cgctgctgca ccgcttccgc gtcctggacc gtggcaagaa aacgtcccgt 8460
tgccaggtcc tgatcgacga ggaaatcgtc gtgctgtttg ctggcgacca ctacacgaaa 8520
ttcacatggg agaagtaccg caagctgtcg ccgacggccc gacggatgtt cgactatttc 8580
agctcgcacc gggagccgta cccgctcaag ctggaaacct tccgcctcat gtgcggatcg 8640
gattccaccc gcgtgaagaa gtggcgcgag caggtcggcg aagcctgcga agagttgcga 8700
ggcagcggcc tggtggaaca cgcctgggtc aatgatgacc tggtgcattg caaacgctag 8760
ggccttgtgg ggtcagttcc ggctgggggt tcagcagcca ccggcatgca agcttgctag 8820
agggtcagct ttatgcttgt aaaccgtttt gtgaaaaaat ttttaaaata aaaaagggga 8880
cctctagggt ccccaattaa ttagtaatat aatctattaa aggtcattca aaaggtcatc 8940
caccggatca attcccctgc tcgcgcaggc tgggtgccaa gctctcgggt aacatcaagg 9000
cccgatcctt ggtagtcggc aaataatgtc taacaattcg ttcaagccga cgccgcttcg 9060
cggcgcggct taactcaagc gttagatgca ctaagcacat aattgctcac agccaaacta 9120
tcaggtcaag tctgctttta ttatttttaa gcgtgcataa taagccctac acaaattggg 9180
agatatatca tgaaaggctg gctttttctt gttatcgcaa tagttggcga agtaatcgca 9240
acatccgcat taaaatctag cgagggcttt actaagctga tccggtggat gaccttttga 9300
atgaccttta atagattata ttactaatta attggggacc ctagaggtcc ccttttttat 9360
tttaaaaatt ttttcacaaa acggtttaca agcataaagc tgactctagc tagaggatcc 9420
ttgatatggc tttttatatg tgttactcta catacagaaa ggaggaacta aatatggcca 9480
agttgaccag tgccgttccg gtgctcaccg cgcgcgacgt cgccggagcg gtcgagttct 9540
ggaccgaccg gctcgggttc tcccgggact tcgtggagga cgacttcgcc ggtgtggtcc 9600
gggacgacgt gaccctgttc atcagcgcgg tccaggacca ggtggtgccg gacaacaccc 9660
tggcctgggt gtgggtgcgc ggcctggacg agctgtacgc cgagtggtcg gaggtcgtgt 9720
ccacgaactt ccgggacgcc tccgggccgg ccatgaccga gatcggcgag cagccgtggg 9780
ggcgggagtt cgccctgcgc gacccggccg gcaactgcgt gcacttcgtg gccgaggagc 9840
aggactgagg atcccctcga cttattattt caggaaagtt tcggaggaga tagtgttcgg 9900
cagtttgtac 9910
<210> 35
<211> 12653
<212> DNA
<213> 人工序列(Artificial Sequence)
<220>
<221> misc_feature
<222> (1)..(12653)
<223> plasmid
<400> 35
atcacgtgct ataaaaataa ttataattta aattttttaa tataaatata taaattaaaa 60
atagaaagta aaaaaagaaa ttaaagaaaa aatagttttt gttttccgaa gatgtaaaag 120
actctagggg gatcgccaac aaatactacc ttttatcttg ctcttcctgc tctcaggtat 180
taatgccgaa ttgtttcatc ttgtctgtgt agaagaccac acacgaaaat cctgtgattt 240
tacattttac ttatcgttaa tcgaatgtat atctatttaa tctgcttttc ttgtctaata 300
aatatatatg taaagtacgc tttttgttga aattttttaa acctttgttt attttttttt 360
cttcattccg taactcttct accttcttta tttactttct aaaatccaaa tacaaaacat 420
aaaaataaat aaacacagag taaattccca aattattcca tcattaaaag atacgaggcg 480
cgtgtaagtt acaggcaagc gatccgtcct aagaaaccat tattatcatg acattaacct 540
ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg tttcggtgat gacggtgaaa 600
acctctgaca catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga 660
gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc tggcttaact 720
atgcggcatc agagcagatt gtactgagag tgcaccatac cacagctttt caattcaatt 780
catcattttt tttttattct tttttttgat ttcggtttct ttgaaatttt tttgattcgg 840
taatctccga acagaaggaa gaacgaagga aggagcacag acttagattg gtatatatac 900
gcatatgtag tgttgaagaa acatgaaatt gcccagtatt cttaacccaa ctgcacagaa 960
caaaaacctg caggaaacga agataaatca tgtcgaaagc tacatataag gaacgtgctg 1020
ctactcatcc tagtcctgtt gctgccaagc tatttaatat catgcacgaa aagcaaacaa 1080
acttgtgtgc ttcattggat gttcgtacca ccaaggaatt actggagtta gttgaagcat 1140
taggtcccaa aatttgttta ctaaaaacac atgtggatat cttgactgat ttttccatgg 1200
agggcacagt taagccgcta aaggcattat ccgccaagta caatttttta ctcttcgaag 1260
acagaaaatt tgctgacatt ggtaatacag tcaaattgca gtactctgcg ggtgtataca 1320
gaatagcaga atgggcagac attacgaatg cacacggtgt ggtgggccca ggtattgtta 1380
gcggtttgaa gcaggcggca gaagaagtaa caaaggaacc tagaggcctt ttgatgttag 1440
cagaattgtc atgcaagggc tccctatcta ctggagaata tactaagggt actgttgaca 1500
ttgcgaagag cgacaaagat tttgttatcg gctttattgc tcaaagagac atgggtggaa 1560
gagatgaagg ttacgattgg ttgattatga cacccggtgt gggtttagat gacaagggag 1620
acgcattggg tcaacagtat agaaccgtgg atgatgtggt ctctacagga tctgacatta 1680
ttattgttgg aagaggacta tttgcaaagg gaagggatgc taaggtagag ggtgaacgtt 1740
acagaaaagc aggctgggaa gcatatttga gaagatgcgg ccagcaaaac taaaaaactg 1800
tattataagt aaatgcatgt atactaaact cacaaattag agcttcaatt taattatatc 1860
agttattacc ctatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc 1920
aggaaattgt aaacgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct 1980
cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa gaatagaccg 2040
agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag aacgtggact 2100
ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt gaaccatcac 2160
cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac cctaaaggga 2220
gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag gaagggaaga 2280
aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg cgcgtaacca 2340
ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc gcgccattcg ccattcaggc 2400
tgcggttgaa gcggttatgc ttggagaagg ggtcgtcaac tgtgtgaacc cgacatccgg 2460
cgttctcatg gcggtgcttg ccgccagcgg tattccgtat gtcaagtggc tgcggtttat 2520
ggtgccgctt gctctgattt ggttcttgat cgggcttgtc tttatcgtga tcggagtcat 2580
gatcaattgg gggccgtttt aacgattgct gcccgccggc ttgtacggcg ggcttttgag 2640
ttattcattg cagaagcgca ggctgttatt gtaacatgta agccataagc cattcgtaaa 2700
agtgcgggag gaaggtcatg aataatctgc gtaatagact ttcaggcgtg aatgggaaaa 2760
ataagagagt aaaagaaaaa gaacaaaaaa tctggtcgga gattgggatg atagcgggag 2820
catttgcgct gcttgatgtg atcatccgcg gcattatgtt tgaatttccg tttaaagaat 2880
gggctgcaag ccttgtgttt ttgttcatca ttatcttata ttactgcatc agggctgcgg 2940
catccggaat gctcatgccg agaatagaca ccaaagaaga actgcaaaaa cgggtgaagc 3000
agcagcgaat agaatcaatt gcggtcgcct ttgcggtagt ggtgcttacg atgtacgaca 3060
gggggattcc ccatacattc ttcgcttggc tgaaaatgat tcttcttttt atcgtctgcg 3120
gcggcgttct gtttctgctt cggtatgtga ttgtgaagct ggcttacaga agagcggtaa 3180
aagaagaaat aaaaaagaaa tcatcttttt tgtttggaaa gcgagggaag cgttcacagt 3240
ttcgggcagc tttttttata ggaacattga tttgtattca ctctgccaag ttgttttgat 3300
agagtgattg tgataatttt aaatgtaagc gttaacaaaa ttctccagtc ttcacatcgg 3360
tttgaaagga ggaagcggaa gaatgaagta agagggattt ttgactccga agtaagtctt 3420
caaaaaatca aataaggagt gtcaagaatg gcttttgaaa aagaaacgtt gttttggaac 3480
gaaaaattcg gtagcgacga ttataccttg acgcggctac cttacagcaa agctccaagc 3540
tccctgacgc ccattatgac aaccgtcggt ggaacgcttt cggaggaagt ggcgcagcgc 3600
gtccttcaaa tgagcaaggg cgctcctctg gccactttta tgattttgct cgcaggtgtt 3660
cagtcactct tgcataaata tacaggcgct tctgacattc tggtcggcat gccggttgta 3720
cggaaaccga cggagacgcg ccgatccgtt aatcatacgt tcattttgaa aaactcgctt 3780
tcggcgggtg cgacttttaa aacgcttctg agcgagctga ggacttcctt gccggaagcg 3840
attcagcatc aacatattcc attcttgaaa atgacggaga agctggatct gcaatatgca 3900
gacgggatac ccgccgtcca tacgctagta tcccttaagg aactgcatct ggacgaaatt 3960
gggcaaaacg tggtcacgga ttgttccttt gaattcagct taacaggcgg ggcgatacag 4020
ctagcgctgt catataacga gcacttatat gactccgagt tcatgactcg ggtcgtaggc 4080
catctgaatc gcctgctgac cgtggggctt cacgagttgg agcttgacat cgtgcgtgtg 4140
gacatgctgt cggaggacga gaaatttcaa ttgttgcaaa gctttaacga taccgagaag 4200
gactatcccc gtgatcggac gattcatcaa ctcgtggagg agcaggcgaa gcgggtgccc 4260
gaagcgacgg cggttgtctt tgagggtcgg cggctttcgt acgcggagct gaacgaacgg 4320
gcgaaccggc tggcgcggac gctgcgatcg gtcggtgtgc tgcccaatca gctggtaggc 4380
ttgatggcca ggagatcact ggagacggtc gttggcatac tggcggttct gaaagctggc 4440
ggtgcctacg tgccgatcga cccggaatac ccggaagaac gcatccgcta cattctggag 4500
tagggataac agggtaataa gtgaaacgaa gagcatcgta gacggctttg tgcgcgtctt 4560
tgatttggag cggccgccgc tgctgcgtgt gggcttagtc gaatgggaag cggaacggca 4620
tctgctcatg ctggacattc atcatatcgt cacggatggc atgtcgatgg gcatcttcgt 4680
cgaagagctg ctgcgcttgt ataacggtga gactctggaa ccgcttcgga ttcaatacaa 4740
ggaattcgcc gcttggcagc agtcagaacc tgtaaaagag cggctgaaac gtcaggaagc 4800
ctactggctg gacgtgctgg aaggcgaact gccgacgctt gaactgccta cggactttgt 4860
cagacctgcc gctcgcagct ttgagggaga tgtgctgcct ttcagcatcg acaagcagat 4920
gaccgacagc ttgcagcgca tcgccgatga gaacggtgcc accctttata tggtgttatt 4980
ggcggcctat tcaatcctgc tcagcaagta ctcgggacaa gaagacttca ttgtaggcat 5040
gccggtttcg ggccgcacac atgccgacct ggagccgctc atcggaatgt ttgtcaacac 5100
tttggcgatt cgccattatc cgtccgggga gaaggcgttc ctcgcttatt tgaacgaagt 5160
caaagaaacg atgctggggg cctacgacca ccaggattat ccgttcgagg agcttgtgaa 5220
aaagctgcag gtgccgcgag atcaaagccg caatcctgta ttcgatgtca tgtttgctct 5280
ggaaaccaag gaagataacg ttcaaagctt cggggatatc aagatcgaat cttatccgga 5340
aactcatacg gtttcccaat ttgatcttac cttggtcatt tcgttgctgg atgagggaat 5400
gaacgggcag tttgaatatg ccaccaagtt gtttacgcgc aatctgatcg acaatttcgc 5460
tcaggacctg ctcgtaatca tcacccaaat ttgcgaacag ccttcggtgc tgttgaagga 5520
tatttccctg aacgggcaat ccgaacagga gcaagatgtg ctagaggcca ttgatattat 5580
tttctaatcc cttacccggc ttgggtgtat gccgtagatc tcagaaacat gcattgactt 5640
taatgaaaat tatggtattt tattacatag gagttgacat cctctgacag acctcttaga 5700
aaaccccact atttaatctt ctattcatct tactctcaat gtgcatctct gttgtattct 5760
tttgtactct atatgttctt gtgatcgtac tcaagatttt atggtgtgga tatcccattg 5820
tgaattcaga ctgtctactc attttagatt acatatttac actttttttg ctttccatgc 5880
ttgtattttt caaaaatcag gtcagcggga ggacgaatat ttatgaacac attcgaaaca 5940
ctggagtcca atgtaaggtc ttattgcagg agttttccag ttgttttcaa caaagcgaaa 6000
aacgatgttt tgtacacgga ggcaggagag ggatatattg actttttcgc cggtgcaggg 6060
gctctaaatt atgggcacaa taatgacttt atgaaaaacc ggttactaga ttatttaacc 6120
tccgatcgga tcatgcatgg tctggatatg tacaccacgg ccaagcaaga gtttatagag 6180
tctttttccg agcgtattct ccggcctaag ggcttgaatt ataagcttca attttgcgga 6240
ccgacgggaa ccaatgcggt ggaagcagca ctgaaacttg cgcgtaaggt gaaaaaaaga 6300
aatggtgttt ttgcttttat gggcgcgttc cacggtatgt cgctcggtag tttatcgatt 6360
accagcaata actccatgcg agagagcgca ggagtcccac tgaataatgt tacctttata 6420
ccttataaca gcacatttaa cggaatggat accattttgt acatggaaca gcttttaaca 6480
gacactcatt ccggtgtgga gaagcctgct gcgattattt tggaaacgat acaggctgaa 6540
ggcggtatta atattgcaga taacgaatgg ctgcgtgatt tgcgacagct ttgcgatgat 6600
catgatattt tgctcattgt tgacgatatt caggtcggct gcggccgagt tggttcgttc 6660
ttctcgtttg agcgagcagg catcgtgcct gatatggttg tcttatccaa gtcgatcagt 6720
ggatacggtc tgccaatgtc cttgttgctg cttaagccag aactggatat ttggagtcct 6780
ggtgaacata acggcacctt ccgtggaaat cagctagcgt ttgtcggagc aaaggcggcg 6840
ctggagttca gagacacggt tgggttggaa gcccaagtga aggaaaaaga agcttttgtt 6900
cagcagttct tgcgtgaaca tatccaaaca atggatccac ttattgaaat ccgcggcttg 6960
ggactgatct ggggaattga tgtgacccat ctgggagaag ctttcgccaa ggaggttgca 7020
actctctgct ttagtaaggg gcttattatt gagcgagctg ggcgtaacga tacggtactt 7080
aaaattatgc cagcgttaac gataagtatg gagaacttga gcaagggctg caatattatt 7140
aaagaaagca tggctcaggt aaccagcaac ttggttacgc tgtaagcaac tgttgggaag 7200
ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa 7260
ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca 7320
gtgagcgcgc gtaatacgac tcactatagg gcgaattggg taccggccgc aaattaaagc 7380
cttcgagcgt cccaaaacct tctcaagcaa ggttttcagt ataatgttac atgcgtacac 7440
gcgtctgtac agaaaaaaaa gaaaaatttg aaatataaat aacgttctta atactaacat 7500
aactataaaa aaataaatag ggacctagac ttcaggttgt ctaactcctt ccttttcggt 7560
tagagcggat gtggggggag ggcgtgaatg taagcgtgac ataactaatt acatgactcg 7620
aggtcgacaa ttccaacctt acccaagagt tcgccaaact cagacatcac tttangcaaa 7680
accgcgccgt gcttcttcct cggtggcatt catcacgaaa tgttcagcac tacgcatact 7740
tttgacagga aacgcaacgg atattgagtc aatatcaggc attctatcgc tcagctttac 7800
agtgacaatg acggctggcg actgaatatt agtgcttaca gacagcacta catattttcc 7860
gtcgatgttg aaatcctttc tcatatgtca ccataaatat caaataatta tagcaatcat 7920
ttacgcgtta atggctaatc gccatcttcc agcaggcgca ccattgcccc tgtttcacta 7980
tccagggtac ggatatagtt catgacaata tttacattgg tccagccacc agcttgcatg 8040
atctccggta ttgaaactcc agcgcgggcc atatctcgcg cggctccgac acgggcactg 8100
tgtccagacc aggccaggta tctctgacca gagtcatcct tagcgccgta aatcaattta 8160
tttcaggaaa gtttcggagg agatagtgtt cggcagtttg tacatcatct gcgggatcag 8220
gtacggtttg atcaggttgt agaagatcag gtaagacata gaatcgatgt agatgatcgg 8280
tttgtttttg ttgattttta cgtaacagtt cagttggaat ttgttacgca gacccttaac 8340
caggtattct acttcttcga aagtgaaaga ctgggtgttc agtacgatcg atttgttggt 8400
agagtttttg ttgtaatccc atttaccacc atcatccatg aaccagtatg ccagagacat 8460
cggggtcagg tagttttcaa ccaggttgtt cgggatggtt tttttgttgt taacgatgaa 8520
caggttagcc agtttgttga aagcttggtg tttgaaagtc tgggcgcccc aggtgattac 8580
caggttaccc aggtggttaa cacgttcttt tttgtgcggc ggggacagta cccactgatc 8640
gtacagcaga catacgtggt ccatgtatgc tttgtttttc cactcgaact gcatacagta 8700
ggttttacct tcatcacgag aacggatgta agcatcaccc aggatcagac cgatacctgc 8760
ttcgaactgt tcgatgttca gttcgatcag ctgggatttg tattctttca gcagtttaga 8820
gttcggaccc aggttcatta cctggttttt tttgatgttt ttcatatgca tgaattgtta 8880
tccgctcaca attccataag cttgatatcg aattcctgca gcccggggga tccactagtt 8940
ctagaatccg gggttttttc tccttgacgt ttaagtatag aggcatattg acaatttttt 9000
gttgatactt ttattacgtt tgaataagaa gtaatacaaa ccgaaaacgt tgaaagtatt 9060
agttaaagtg gttatgcagt ttctgcattt atatatctgt taatagatca aaaatcatcg 9120
cttcgctaat taattacccc agaaataagg ctaaaaaact aatcgcatta tcatcctatg 9180
gttgttaatt tgattcgttc atttgaaggt ttgtggggcc aggttactgc caatttttcc 9240
tcttcataac cataaaagct agtattgtag aatctttatt gttcggagca gtgcggcgcg 9300
aggcacatct gcgtttcagg aacgcgaccg gtgaagacga ggacgcacgg aggagagtct 9360
tccttcggag ggctgtcacc cgctcggcgg cttctaatcc gtactagagc tcactacagc 9420
agagccatgt agggccgccg gcgttgtgga taccacgcgg aaaacttggc cctcactgac 9480
agatgagggg cggacgttga cacttgaggg gccgactcac ccggcgcggc gttgacagat 9540
gaggggcagg ctcgatttcg gccggcgacg tggagctggc cagcctcgca aatcggcgaa 9600
aacgcctgat tttacgcgag tttcccacag atgatgtgga caagcctggg gataagtgcc 9660
ctgcggtatt gacacttgag gggcgcgact actgacagat gaggggcgcg atccttgaca 9720
cttgaggggc agagtgatga cagatgaggg gcgcacctat tgacatttga ggggctgtcc 9780
acaggcagaa aatccagcat ttgcaagggt ttccgcccgt ttttcggcca ccgctaacct 9840
gtcttttaac ctgcttttaa accaatattt ataaaccttg tttttaacca gggctgcgcc 9900
ctggcgcgtg accgcgcacg ccgaaggggg gtgccccccc ttctcgaacc ctcccggccc 9960
gctaacgcgg caccccatcc ccccaggggc tgcgcccctc ggccgcgaac gacctcaccc 10020
caaaaatggc agccacgtag aaagccagtc cgcagaaacg gtgctgaccc cggatgaatg 10080
tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag caggtagctt 10140
gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca agcgaaccgg 10200
aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta aactggatgg 10260
ctttcttgcc gccaaggatc tgatggcgca ggggatcaag atcgacggat cgatccgggg 10320
aattaattcc ggggcaatcc cgcaaggagg gtgaatgaat cggacgtttg accggaaggc 10380
atacaggcaa gaactgatcg acgcggggtt ttccgccgag gatgccgaaa ccatcgcaag 10440
ccgcaccgtc atgcgtgcgc cccgcgaaac cttccagtcc gtcggctcga tggtccagca 10500
agctacggcc aagatcgagc gcgacagcgt gcaactggct ccccctgccc tgcccgcgcc 10560
atcggccgcc gtggagcgtt cgcgtcgtct cgaacaggag gcggcaggtt tggcgaagtc 10620
gatgaccatc gacacgcgag gaactatgac gaccaagaag cgaaaaaccg ccggcgagga 10680
cctggcaaaa caggtcagcg aggccaagca ggccgcgttg ctgaaacaca cgaagcagca 10740
gatcaaggaa atgcagcttt ccttgttcga tattgcgccg tggccggaca cgatgcgagc 10800
gatgccaaac gacacggccc gctctgccct gttcaccacg cgcaacaaga aaatcccgcg 10860
cgaggcgctg caaaacaagg tcattttcca cgtcaacaag gacgtgaaga tcacctacac 10920
cggcgtcgag ctgcgggccg acgatgacga actggtgtgg cagcaggtgt tggagtacgc 10980
gaagcgcacc cctatcggcg agccgatcac cttcacgttc tacgagcttt gccaggacct 11040
gggctggtcg atcaatggcc ggtattacac gaaggccgag gaatgcctgt cgcgcctaca 11100
ggcgacggcg atgggcttca cgtccgaccg cgttgggcac ctggaatcgg tgtcgctgct 11160
gcaccgcttc cgcgtcctgg accgtggcaa gaaaacgtcc cgttgccagg tcctgatcga 11220
cgaggaaatc gtcgtgctgt ttgctggcga ccactacacg aaattcacat gggagaagta 11280
ccgcaagctg tcgccgacgg cccgacggat gttcgactat ttcagctcgc accgggagcc 11340
gtacccgctc aagctggaaa ccttccgcct catgtgcgga tcggattcca cccgcgtgaa 11400
gaagtggcgc gagcaggtcg gcgaagcctg cgaagagttg cgaggcagcg gcctggtgga 11460
acacgcctgg gtcaatgatg acctggtgca ttgcaaacgc tagggccttg tggggtcagt 11520
tccggctggg ggttcagcag ccaccggcat gcaagcttgc tagagggtca gctttatgct 11580
tgtaaaccgt tttgtgaaaa aatttttaaa ataaaaaagg ggacctctag ggtccccaat 11640
taattagtaa tataatctat taaaggtcat tcaaaaggtc atccaccgga tcaattcccc 11700
tgctcgcgca ggctgggtgc caagctctcg ggtaacatca aggcccgatc cttggtagtc 11760
ggcaaataat gtctaacaat tcgttcaagc cgacgccgct tcgcggcgcg gcttaactca 11820
agcgttagat gcactaagca cataattgct cacagccaaa ctatcaggtc aagtctgctt 11880
ttattatttt taagcgtgca taataagccc tacacaaatt gggagatata tcatgaaagg 11940
ctggcttttt cttgttatcg caatagttgg cgaagtaatc gcaacatccg cattaaaatc 12000
tagcgagggc tttactaagc tgatccggtg gatgaccttt tgaatgacct ttaatagatt 12060
atattactaa ttaattgggg accctagagg tccccttttt tattttaaaa attttttcac 12120
aaaacggttt acaagcataa agctgactct agctagagga tccttgatat ggctttttat 12180
atgtgttact ctacatacag aaaggaggaa ctaaatatgg ccaagttgac cagtgccgtt 12240
ccggtgctca ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg 12300
ttctcccggg acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg 12360
ttcatcagcg cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg 12420
cgcggcctgg acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac 12480
gcctccgggc cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg 12540
cgcgacccgg ccggcaactg cgtgcacttc gtggccgagg agcaggactg aggatcccct 12600
cgacttatta tttcaggaaa gtttcggagg agatagtgtt cggcagtttg tac 12653

Claims (9)

1.重组载体pSHTB10，其特征在于，为将质粒pSH-SceI中的colE1、AmpR和PGal1基因元件替换成pSWB中的oriV、trfA和BleRDNA片段和Gal1-V10 DNA合成片段，形成全长序列为SEQID NO.32的质粒pSHTB10；所述的质粒pSH-SceI是将质粒pSH47中的Cre元件替换成I-SceI基因片段；所述的质粒pSWB是将质粒pDGICZ中的Phbs-BleR基因元件插入到质粒pSW-2中。
2.基于如权利要求1所述的重组载体pSHTB10的iTAR载体，其特征在于，为将质粒pSHTB10用限制性内切酶FspI酶切，通过在酶切位点处插入与目标线性大片段DNA两端具有同源性的两个同源臂并在同源臂间插入I-SceI酶切位点构建而成。
3.如权利要求2所述的iTAR载体，其特征在于，所述的iTAR载体为pSHTB10-HR12，为将pSHTB10用限制性内切酶FspI酶切，在该酶切位点处插入同源臂HR1、同源臂HR2和同源臂间的I-SceI酶切位点，形成全长序列为SEQ ID NO.33的iTAR载体pSHTB10-HR12。
4.如权利要求2所述的iTAR载体，其特征在于，所述的iTAR载体为pSHTB10-HR34，为将pSHTB10用限制性内切酶FspI酶切，在该酶切位点处插入同源臂HR3、同源臂HR4和同源臂间的I-SceI酶切位点，形成全长序列为SEQ ID NO.34的iTAR载体pSHTB10-HR34。
5.如权利要求1所述的重组载体pSHTB10的构建方法，其特征在于，包括引物设计、基因克隆和载体构建，具体为通过合成Gal1-V10基因片段，设计引物从质粒pSWB上PCR扩增oriV、trfA和BleR目标片段，限制性内切酶PmlI和SacI酶切质粒pSH-SceI，将Gal1-V10、oriV、trfA、BleR和pSH-SceI目的片段连接，得到质粒pSHTB10；所述的质粒pSH-SceI的构建方法是通过PCR扩增I-SceIDNA序列，将其与限制性内切酶ClaI酶切的pSH47目的片段连接，得到质粒pSH-SceI；所述的质粒pSWB的构建方法是通过限制性内切酶BamHI酶切质粒pDGICZ和pSW-2，将其目的片段连接，得到质粒pSWB。
6.如权利要求2所述的iTAR载体的构建方法，其特征在于，包括引物设计、基因克隆和载体构建，具体为通过合成引物PCR扩增相应的两个同源臂片段及I-SceI酶切位点，与经不破坏pSHTB10功能元件的限制性内切酶酶切回收的目的DNA片段连接，得到相应的iTAR载体。
7.如权利要求3所述的iTAR载体的构建方法，其特征在于，包括引物设计、基因克隆和载体构建，具体为通过合成引物PCR扩增HR1和HR2 DNA片段，与经限制性内切酶FspI酶切的pSHTB10片段连接，得到iTAR载体pSHTB10-HR12。
8.如权利要求4所述的iTAR载体的构建方法，其特征在于，包括引物设计、基因克隆和载体构建，具体为通过合成引物PCR扩增HR3和HR4 DNA片段，与经限制性内切酶FspI酶切的pSHTB10片段连接，得到iTAR载体pSHTB10-HR34。
9.如权利要求2～4所述的iTAR载体在捕捉大片段DNA中的应用，其特征在于，具体应用方法如下：
步骤1：针对目标线性大片段DNA的两端设计合成相应DNA片段，用体外转录的方式获得gRNA；
步骤2：利用Cas9核酸酶和gRNA可以特异性切割DNA的特性得到目标线性DNA；
步骤3：将iTAR载体转化到酿酒酵母中，通过将目标线性DNA片段转化至经半乳糖诱导的且含iTAR载体的酿酒酵母中，筛选阳性克隆，得到相应的捕捉载体；
步骤4：将捕捉载体转移至相应的模式菌株中使其表达，实现外源大片段DNA的克隆和表达。
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