一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统及应用
阅读说明:本技术 一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统及应用 (Dual-function system for simultaneous gene knockout and gene expression inhibition of corynebacterium glutamicum and application ) 是由 徐志南 连佳长 刘伟 杨修亮 于 2019-08-21 设计创作,主要内容包括:本发明公开了一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统及应用。本发明构建的CRISPR/Cas12a系统的载体,包括Cas12a蛋白基因和crRNA的编码DNA。本发明构建的CRISPR/Cas12a基因载体能够对谷氨酸棒杆菌基因组同时进行基因敲除和基因表达抑制,具有实验周期短、节省时间和效率高等优点。(The invention discloses a bifunctional system for simultaneous gene knockout and gene expression inhibition of corynebacterium glutamicum and application thereof. The vector of the CRISPR/Cas12a system constructed by the invention comprises a Cas12a protein gene and a coding DNA of crRNA. The CRISPR/Cas12a gene vector constructed by the invention can simultaneously carry out gene knockout and gene expression inhibition on a corynebacterium glutamicum genome, and has the advantages of short experimental period, time saving, high efficiency and the like.)
技术领域
本发明涉及基因编辑领域,具体涉及一种谷氨酸棒杆菌同时基因敲除与基因表达抑制的CRISPR/Cas12a系统及其应用。
背景技术
CRISPR/Cas(Clustered regularly interspaced short palindromic repeats,CRISPR)系统作为细菌和古细菌的获得性免疫系统,通过RNA介导,特异性的切割外源遗传物质,用以对抗入侵的病毒。近年来,该系统被成功的应用到大肠杆菌、拟南芥、酿酒酵母以及小鼠等物种中,实现快速高效的基因敲除、基因***和基因沉默。
CRISPR/Cas12a属于V-A型CRISPR系统。该系统发挥基因编辑的作用建立在CRISPRRNA(crRNA)与Cas12a蛋白形成的蛋白核酸复合体,在crRNA 的引导下,识别富含T的PAM位点(TTTN)的3’端下游序列;而对目的片段的切割是由crRNA上长度为20bp的向导序列与目的基因上的特定序列互补,招募Cas12a蛋白对目的基因进行切割。而当crRNA的长度小于17bp时,Cas12a 蛋白仍然能够结合到DNA上,但缺失了DNA切割的能力。
谷氨酸棒杆菌(Corynebacterium glutamicum)是革兰氏阳性菌,普遍认为其安全,无致病性,被广泛的用于工业生产谷氨酸,赖氨酸等氨基酸,以及其他应用于食品、饲料及医药的化学品。
早前,谷氨酸棒杆菌的菌株主要通过诱变育种的方法获得高产工业菌。随着全基因组测序的发展,利用基因工程的方法定向改造谷氨酸棒杆菌基因组越来越受到重视。目前,除了pK18mobsacB/pK19mobsacB介导的同源重组法外, CRISPR/Cas的方法也被用于谷氨酸棒杆菌。由于谷氨酸棒杆菌对外源DNA的排斥和消化能力较强,且同源重组酶活力低下,从而导致谷氨酸棒杆菌转化效率与同源重组修复效率低下。虽然单一功能的CRISPR系统(如基因敲除和基因抑制)已经实现,但基因组上多位点同时操作却难以成功。
发明内容
针对上述问题,本发明提供了一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统,其具有较高的基因敲除与基因抑制效率。为此,本发明采用以下技术方案:
一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统,其特征在于:
所述的双功能系统包含cas12a表达载体和crRNA表达载体;
所述cas12a表达载体包括cas12a序列和启动子序列,所述cas12a序列如SEQ IDNo.1所示,所述启动子序列如SEQ ID No.2所示,并连接在cas12a序列的起始密码子ATG前;
所述的crRNA表达载体包含同源修复序列,crRNA1和crRNA2序列,所述 crRNA1序列如SEQ ID No.3所示,crRNA2序列如SEQ ID No.4所述,所述 crRNA1序列为18~24bp的引导序列、与目的基因的N18~N24序列相同,所述 crRNA2序列为13~16bp的引导序列,与目的基因的N13~N16序列相同。
进一步的,所述cas12a表达载体的核苷酸序列如SEQ ID No.5所示。
进一步的,所述cas12a表达载体由以下方法制备得到:a.合成cas12a基因序列;以谷氨酸棒杆菌基因组为模板,PCR扩增启动子Ptuf序列;以pEC-XK99E 质粒为模板,扩增卡那霉素抗性基因KanR,谷氨酸棒杆菌复制子与大肠杆菌复制子元件以及终止子rrnB;b.利用同源重组技术,同源重组连接Ptuf启动子,cas12a 基因,rrnB终止子元件,KanR抗性基因,谷氨酸棒杆菌与大肠杆菌复制子元件,构建成所述cas12a表达载体。
进一步的,所述的crRNA表达载体包含的同源修复序列,由敲除的目的基因的上游序列和目的基因下游序列连接形成,所述的目的基因上游序列、目的基因下游序列长度为300bp~1500bp。
进一步的,a.合成crRNA1序列,以pXMJ19质粒为模板,扩增CmR抗性基因,谷氨酸棒杆菌与大肠杆菌复制子元件;构建同源修复序列,并在同源修复序列的5’端引入XhoI、PstI、BamHI、XbaI、EcoRI、HindIII酶切位点;b.利用同源重组技术,同源重组连接crRNA1序列,CmR抗性基因,谷氨酸棒杆菌与大肠杆菌复制子元件以及同源修复序列得到psg-A1-HR载体;c.合成crRNA2序列,并在crRNA2两端引入XhoI、PstI、BamHI、XbaI、EcoRI、HindIII中两个酶切识别序列;d.使用选中的两个限制性内切酶酶切psg-A1-HR质粒和crRNA2序列,纯化后进行连接,得到所述用于同时基因敲除和基因表达抑制的crRNA表达载体。
进一步地,上述的同时基因敲除及基因表达抑制的双功能系统在谷氨酸棒杆菌中的应用,所述应用的具体操作如下:a.将cas12a表达载体转入谷氨酸棒杆菌,培养、筛选,获得转化子;b.将crRNA表达质粒转入含有cas12a表达载体的谷氨酸棒杆菌中,培养、筛选,获得转化子。
本发明构建的基因组中同时基因敲除与基因抑制的CRISPR/Cas12a系统,利用人工设计的两个crRNA结合cas12a蛋白对基因靶点位置分别进行基因敲除和基因抑制;其中进行基因敲除的靶点位置DNA双链被切割,形成双链断裂缺口,同时提供同源修复序列,以达到基因敲除的目的;其中进行基因抑制的靶点, crRNA与cas12a蛋白核酸复合体结合在靶标位点DNA上,不引起DNA双链的切割,但阻止基因的转录,从而达到基因表达抑制的目的;其中Ptuf启动子能够使得Cas12a蛋白在谷氨酸棒杆菌中持续表达。
实验证明,利用本发明构建的同时基因敲除与基因抑制的CRISPR/Cas12a 系统,对谷氨酸棒杆菌基因组进行同时地基因敲除和基因表达抑制,具有周期短,效率高等优点。
附图说明
图1为pEC-XK99E的质粒图谱。
图2为cas12a表达载体的质粒图谱。
图3为pXMJ19的质粒图谱。
图4为psg-A1-HR的质粒图谱。
图5为crRNA表达载体的质粒图谱。
图6为基因组同时基因敲除和基因表达抑制的设计示意图。
图7为aecD基因敲除后基因型验证结果(A)以及aecD敲除菌mcbR基因表达抑制结果(B)。M代表Marker,1-20代表随机挑选的单菌落; Cg-ΔaecD-Control代表aecD基因敲除菌作为对照;Cg-ΔaecD-mcbRi代表aecD 基因敲除菌中加入基因抑制质粒作为对照;Cg-aecDd-mcbRi代表使用本发明的 CRISPR/Cas12a系统构建的同时aecD敲除以及mcbR表达抑制菌株。
图8为aecD基因敲除与mcbR基因抑制菌株半胱氨酸产量。WT代表野生型C.glutamicum;Cg-ΔaecD代表aecD基因敲除菌;Cg-ΔaecD-mcbRi代表aecD 基因敲除菌中加入基因抑制质粒作为对照;Cg-aecDd-mcbRi代表使用本发明的 CRISPR/Cas12a系统构建的同时aecD敲除以及mcbR表达抑制菌株。
具体实施方式
下述实施例或应用例中的实验方法,如无特殊说明,均为常规方法。
下述实施例或应用例中所用的材料、试剂、基因合成等,如无特殊说明,均可从商业途径获得。
下述实施例或应用例中所用载体骨架pEC-XK99E及pXMJ19均购自 Addgene。
下述实施例或应用例中所用大肠杆菌MC1061购自唯地生物公司。
下述实施例或应用例中所用谷氨酸棒杆菌C.glutamicum B253购买自中国工业微生物菌种保藏中心(CICC)。
下述实施例或应用例中所用PrimeSTAR Max DNA polymerase、限制性内切酶与T4连接酶购买自TaKaRa公司。
下述实施例或应用例中所用pEASY-Uni Seamless Cloning and Assembly Kit购买自TransGen公司。
下述实施例或应用例中大肠杆菌的培养基使用LB培养基,培养基配方为:蛋白胨10g、酵母提取物5g、NaCl 10g,去离子水1L。
下述实施例或应用例中谷氨酸棒杆菌的培养基使用BHISG培养基,培养基配方为:脑心浸液37g、D-山梨醇91g、葡萄糖10g,去离子水1L。
下述实施例或应用例中谷氨酸棒杆菌的半胱氨酸合成培养基使用MC培养基,培养基配方为:50葡萄糖,25g玉米浆,15g(NH4)2SO4,1g K2HPO4,1g KH2PO4,1g MgSO4·7H2O,5gNa2S2O3,10mg FeSO4·7H2O,10mg MnSO4·H2O,1 mg ZnSO4·7H2O,0.2mg CuSO4,0.02mgNiCl2,0.2mg生物素,0.2mg硫胺素和 20g CaCO3,去离子水1L。
实施例1:一种谷氨酸棒杆菌基因组同时基因敲除和基因表达抑制的 CRISPR/Cas12a系统
1、cas12a表达载体
cas12a表达载体,其制备过程如下:、
a.以谷氨酸棒杆菌基因组为模板扩增Ptuf启动子序列,合成cas12a序列;Ptuf启动子序列和cas12a序列分别如SEQ ID No.2和SEQ ID No.1所示,以质粒 pEC-XK99E(图谱如图1所示)为模板,扩增带有rrnB、大肠杆菌谷氨酸棒杆菌复制子以及卡那霉素抗性基因的骨架;
b利用同源重组技术,同源重组连接上述片段,得到所述cas12a表达载体。
PCR反应体系如下:50μL反应体系中含有PrimeSTAR Max DNA polymerase 25μL,上游引物与下游引物各2μL,模板1μL以及ddH2O 20μL。其中,用于Ptuf启动子扩增的引物为Ptuf-For(SEQ ID No.7)和Ptuf-Rev(SEQ ID No.8);用于 cas12a扩增的引物为cas12a-For(SEQ ID No.9)和cas12a-Rev(SEQ ID No.10);用于pEC-XK99E质粒骨架扩增的引物为pEC-For(SEQ ID No.11)和pEC-Rev (SEQ ID No.12)。Ptuf启动子扩增PCR循环程序为预变性98℃3min,变性98℃ 10s,退火55℃15s,延伸72℃5s,循环31次,延伸72℃5min,保存4℃。cas12a扩增PCR循环程序为预变性98℃3min,变性98℃10s,退火53℃15s,延伸72℃40s,循环31次,延伸72℃5min,保存4℃。pEC-XK99E骨架扩增PCR循环程序为预变性98℃3min,变性98℃10s,退火60℃5s,延伸72 ℃1min,循环31次,延伸72℃5min,保存4℃。PCR得到的片段经PCR产物纯化试剂盒纯化回收,回收后的三个片段按0.03pmol:0.03pmol:0.03pmol 的比例加入pEASY-Uni Seamless Cloning and Assembly Kit反应体系中,加入 ddH2O使得最终反应体系为10μL,50℃温育60min。热击转化E.coli MC1061 感受态细胞;均匀涂布50μg/mL卡那霉素的LB固体培养基上,37℃倒置培养 16h;挑选阳性克隆子,提取质粒即为cas12a表达载体。
2.crRNA表达载体
crRNA表达载体,其制备过程如下:
a.合成crRNA1序列;以pXMJ19质粒为模板,扩增含CmR抗性基因,谷氨酸棒杆菌与大肠杆菌复制子元件的骨架;根据基因敲除的不同目的基因,构建同源修复序列,目的基因上游和下游序列长度均为300~1000bp,并在上游同源修复序列的5’端引入XhoI、PstI、BamHI、XbaI、EcoRI、HindIII酶切位点;
b.利用同源重组技术,同源重组连接crRNA1序列,以pXMJ19为模板扩增的骨架序列以及含有酶切位点的上游同源修复序列和下游同源修复序列按照 0.03pmol:0.03pmol:0.03pmol:0.03pmol的比例加入pEASY-Uni Seamless Cloning and Assembly Kit反应体系中,加入ddH2O使得最终反应体系为10μL, 50℃温育60min。热击转化E.coli MC1061感受态细胞;均匀涂布34μg/mL氯霉素的LB固体培养基上,37℃倒置培养16h;挑选阳性克隆子,提取质粒即为psg-A1-HR载体;
c.合成crRNA2序列,根据psg-A1-HR载体上的序列,在crRNA2两端设计引入XhoI、PstI、BamHI、XbaI、EcoRI、HindIII中的两个酶切识别序列;
d.使用选中的两个限制性内切酶酶切psg-A1-HR质粒和crRNA2序列,酶切体系为50μL,其中加入待酶切片段2μg,两个内切酶各2.5μL,使用ddH2O补足体积为50μL,37℃温育30min;1%琼脂糖凝胶电泳,切胶回收,胶回收试剂盒纯化;psg-A1-HR质粒与crRNA2序列片段按0.3pmol:0.03pmol的比例,加入2.5μL 10×T4Ligase Buffer,1μL T4 Ligase,加入ddH2O补足体积为25μL,25 ℃温育60min。热击转化E.coli MC1061感受态细胞;均匀涂布34μg/mL氯霉素的LB固体培养基上,37℃倒置培养16h;挑选阳性克隆子,提取质粒即为用于同时基因敲除和基因表达抑制的crRNA表达载体。
3.基因组同时基因敲除与基因表达抑制
基因组同时基因敲除与基因表达抑制过程如下:
a.提取cas12a表达质粒并转化谷氨酸棒杆菌,培养、筛选,获得转化子;
b.提取crRNA表达质粒并转化含有cas12a表达载体的谷氨酸棒杆菌,培养、筛选,获得转化子。
基因组同时基因敲除与基因表达抑制包括基因组同时基因敲除与基因表达抑制以及基因组同时基因敲入与基因表达抑制,如图6所示,基因A为待基因敲除基因,基因B为待基因表达抑制基因。
应用例1:基因组上同时aecD基因敲除与mcbR基因表达抑制
一种谷氨酸棒杆菌aecD与mcbR基因同时敲除和抑制的CRISPR/Cas12a系统,包括cas12a载体构建与crRNA载体构建。
1.Cas12a表达载体
Cas12a表达载体的制备方法如实施例1中所示。
Cas12a表达载体的质粒图谱如图2所示,其核苷酸序列如SEQ ID No.5所示,其中5516-5694位的核苷酸序列为谷氨酸棒杆菌中Ptuf启动子;5695-9597位的核苷酸序列为cas12a序列;207-412位核苷酸序列为rrnB终止子序列。
2.crRNA表达载体
crRNA表达载体制备方法如下:
a.制备载体psg-A1-HR
根据谷氨酸棒杆菌基因组序列(NCBI Reference Sequence:CP010451),针对aecD基因寻找5’-TTTN-(N20)-3’结构,针对靶点序列设计的crRNA序列如下所示:
靶点序列为:
TTTATGAAGGCACCCATGTGGTTG,对应序列表中SEQ ID No.13。
crRNA1-aecD序列为:
CCACAGGGTAGCTGGTAGTTTGAAAATCAACGCCGTTGCCCTTAGGAT TCAGTAACTGGCACATTTTGTAATGCGCTAGATCTGTGTGCTCAGTCTTCCA GGCTGCTTATCACAGTGAAAGCAAAACCAATTCGTGGCTGCGAAAGTCGTA GCCACCACGAAGTCCAGGAGGACATACATAATTTCTACTCTTGTAGATTGA AGGCACCCATGTGGTTGCATCAAATAAAACGAAAGGCTCAGTCGAAAGAC TGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGA CAAAT,对应序列表中SED ID No.14;
其中,crRNA1-aecD序列交由基因公司合成;
以谷氨酸棒杆菌基因组为模板,使用引物aecD-SY-For(SEQ ID No.15)和 aecD-SY-Rev(SEQ ID No.16)扩增aecD基因上游同源臂;使用引物aecD-XY-For (SEQ ID No.17)和aecD-XY-Rev(SEQ ID No.18)扩增aecD基因下游同源臂;以pXMJ19质粒(质粒图谱如图3所示)模板,使用引物p19-For(SEQ ID No.19) 和p19-Rev(SEQ ID No.20)扩增含有氯霉素抗性基因,大肠杆菌和谷氨酸棒杆菌复制子的骨架;aecD基因上游同源臂扩增PCR循环程序为预变性98℃3min,变性98℃10s,退火53℃15s,延伸72℃10s,循环31次,延伸72℃5min,保存4℃。aecD基因下游同源臂扩增PCR循环程序为预变性98℃3min,变性 98℃10s,退火55℃15s,延伸72℃15s,循环31次,延伸72℃5min,保存4℃。pXMJ19骨架扩增PCR循环程序为预变性98℃3min,变性98℃10s,退火55℃15s,延伸72℃1min,循环31次,延伸72℃5min,保存4℃。PCR 得到的片段经PCR产物纯化试剂盒纯化回收,回收后的三个片段按0.03pmol:0.03pmol:0.03pmol的比例加入pEASY-Uni Seamless Cloning and Assembly Kit 反应体系中,加入ddH2O使得最终反应体系为10μL,50℃温育60min。热击转化E.coli MC1061感受态细胞;均匀涂布34μg/mL氯霉素的LB固体培养基上,37℃倒置培养16h;挑选阳性克隆子,提取质粒即为psg-A1-HR表达载体,图谱如图4所示。
b.制备crRNA表达载体
根据谷氨酸棒杆菌基因组序列(NCBI Reference Sequence:CP010451),针对mcbR基因寻找5’-TTTN-(N16)-3’结构,针对靶点序列设计的crRNA序列如下所示:
靶点序列为:
TTTCACCACAGAAGGTATTC,对应序列表中SEQ ID No.21。
crRNA2-mcbR序列为:
CCATGCCATAACAGGAATGTTCCTTTCGAAAATTGAGGAAGCCTTATGC CCTACAACCCTACTTAGCTGCCAATTATTCCGGGCTTGTGACCCGCTACCCA ATAAATAGGTGGGCTGAAAAATTTCGTTGCAATATCAACAAAAAGGCCTAT CATTGGGAAGTGTCGCACCAAGTACTTTTGCGAAGCGCCATCTGACGGATT TTCAAAAGATGTATATGCTCGGTGCGGAAACCTACGAAAGGATTTTTTACCC TAATTTCTACTCTTGTAGATACCACAGAAGGTATTCCATCAAATAAAACGAA AGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCCTGAGTAGGACAAAT,对应序列表中SED ID No.22。
crRNA2-mcbR序列交由基因公司合成;以合成的crRNA2片段为模板,使用含有PstI和XhoI酶切位点的引物crRNA2-For(SED ID No.23)和crRNA2-Rev (SED ID No.24)扩增,PCR循环程序为预变性98℃3min,变性98℃10s,退火55℃15s,延伸72℃10s,循环31次,延伸72℃5min,保存4℃。PCR 产物通过PCR产物纯化试剂盒纯化回收。
将crRNA2的PCR产物片段和psg-A1-HR质粒分别使用PstI和XhoI酶切。酶切体系为50μL,其中加入待酶切片段2μg,两个内切酶各2.5μL,使用ddH2O 补足体积为50μL,37℃温育30min;1%琼脂糖凝胶电泳,切胶回收,胶回收试剂盒纯化;psg-A1-HR质粒与crRNA2序列片段按0.3pmol:0.03pmol的比例,加入2.5μL 10×T4Ligase Buffer,1μL T4 Ligase,加入ddH2O补足体积为25μL, 25℃温育60min。热击转化E.coli MC1061感受态细胞;均匀涂布34μg/mL氯霉素的LB固体培养基上,37℃倒置培养16h;挑选阳性克隆子,提取质粒即为用于同时基因敲除和基因表达抑制的crRNA表达载体。
靶标aecD与mcbR的crRNA表达载体的质粒图谱如图5所示。其核苷酸序列如SED IDNo.6所示。其中,7760-110位核苷酸序列为crRNA1序列,其中7760-7938位核苷酸为crRNA1序列的启动子Ptuf序列;5452-5832位核苷酸为 crRNA2序列,其中5452-5706位核苷酸为crRNA2序列的启动子Psod序列, 5473-5832位核苷酸为crRNA2序列的rrnBT1终止子序列;6813-7759位核苷酸为aecD基因下游同源臂序列;5839-6812位核苷酸为aecD基因上游同源臂序列。
2.基因组上同时aecD基因敲除与mcbR基因表达抑制具体操作流程:
a.将cas12a表达质粒从E.coli中提出,转化谷氨酸棒杆菌感受态,电击转化条件为2500V,6ms,恢复培养后涂布于含有卡那霉素的BHISG固体培养基, 30℃培养36h。
b.挑取平板上的单菌落,利用cas12a引物:cas12a-YZ-For(SEQ ID No.25) 和cas12a-YZ-Rev(SEQ ID No.26)验证cas12a质粒是否正确转入;
c.将crRNA表达质粒从E.coli中提出,转化含有cas12a表达质粒的谷氨酸棒杆菌,电击转化条件为2500V,6ms,恢复培养后涂布于含有卡那霉素和氯霉素的BHISG固体培养基,30℃培养36h。
d.从平板上挑取单菌落,使用引物aecD-YZ-For(SEQ ID No.27)和aecD-YZ-Rev(SEQ ID No.28)验证aecD是否成功敲除,基因型验证结果如图7 所示;
e.挑取aecD基因已经验证被敲除的单菌落,接种至BHISG培养基中培养16 h,再转接MC培养基中,培养至对数中期,取菌体用于检测mcbR基因的表达,结果如图7所示,mcbR基因的表达得到成功的抑制。
f.将再MC培养基中培养35h的发酵液上清用于检测半胱氨酸含量,发现基因组上aecD基因敲除与mcbR基因的抑制能够增强谷氨酸棒杆菌合成半胱氨酸的能力。
实验证明,利用本发明构建的同时基因敲除和基因表达抑制的 CRISPR/Cas12a系统,对谷氨酸棒杆菌的基因组进行改造,具有周期短,效率高的特点,能够快速成功用于多靶点代谢改造谷氨酸棒杆菌。
质粒的除去,把得到的改造菌株在不加抗生素的培养基中培养,cas12a表达质粒与crRNA表达质粒均可除去。
SEQ ID No.1
cas12a基因序列
atgtccatctaccaagagtttgtgaataaatactccctgtccaagaccctccgttttgagctgatcccccaaggcaagaccctcgaaaacatcaa ggcacgcggcctcatcctggatgacgaaaagcgcgctaaggattacaagaaggcaaagcagatcatcgacaagtaccaccagttcttcatc gaagagatcctgtcctccgtgtgcatctccgaggacctgctccagaactactccgatgtctacttcaagctcaagaagtccgatgacgataacc tgcagaaggacttcaagtccgctaaggataccatcaagaagcagatctccgaatacatcaaggattccgagaagttcaagaacctcttcaacc agaacctgatcgacgcaaagaagggccaggaatccgatctcatcctgtggctcaagcagtccaaggataacggcatcgagctcttcaaggc caactccgacatcaccgacatcgatgaagctctggagatcatcaagtccttcaagggctggaccacctacttcaagggcttccacgaaaaccg caagaacgtgtactcctccaacgatatcccaacctctatcatctaccgcatcgtcgacgataacctgccaaagttcctcgaaaacaaggcaaag tacgagtccctgaaggataaggccccagaagctatcaactacgagcagatcaagaaggacctggccgaagagctcaccttcgacatcgatta caagacctctgaagtgaaccagcgcgtcttctccctcgatgaagtgttcgagatcgccaacttcaacaactacctgaaccagtccggcatcacc aagttcaacaccatcatcggcggcaagttcgtcaacggcgaaaacaccaagcgcaagggcatcaacgagtacatcaacctctactcccagc agatcaacgataagaccctgaagaagtacaagatgtccgtgctcttcaagcagatcctgtccgacaccgaatccaagtccttcgtcatcgacaa gctggaggacgattccgatgtggtcaccaccatgcagtccttctacgaacagatcgcagccttcaagaccgtggaagagaagtccatcaagg agaccctctccctgctcttcgacgatctgaaggctcagaagctggatctctccaagatctacttcaagaacgacaagtccctgaccgatctctcc cagcaggtcttcgacgattactccgtgatcggcaccgcagtcctggaatacatcacccagcagatcgccccaaagaacctcgataacccatc caagaaggaacaggagctgatcgccaagaagaccgaaaaggctaagtacctgtccctcgagaccatcaagctggctctcgaagagttcaac aagcaccgcgacatcgataagcagtgccgcttcgaagagatcctcgcaaacttcgctgcaatcccaatgatcttcgacgaaatcgcacagaa caaggataacctggcccagatctccatcaagtaccagaaccagggcaagaaggatctgctccaggcctccgctgaggacgatgtgaaggc aatcaaggacctgctcgatcagaccaacaacctgctccacaagctgaagatcttccacatctcccagtccgaagacaaggccaacatcctcg acaaggatgagcacttctacctggtgttcgaagagtgctacttcgaactcgctaacatcgtcccactgtacaacaagatccgcaactacatcac ccagaagccatactccgatgaaaagttcaagctcaacttcgagaactccaccctggcaaacggctgggacaagaacaaggaaccagataac accgccatcctcttcatcaaggacgataagtactacctgggcgtgatgaacaagaagaacaacaagatcttcgacgataaggccatcaagga aaacaagggcgagggctacaagaagatcgtgtacaagctgctcccaggcgctaacaagatgctcccaaaggtcttcttctccgcaaagtcca tcaagttctacaacccatccgaagatatcctgcgcatccgcaaccactccacccacaccaagaacggctccccacagaagggctacgaaaa gttcgagttcaacatcgaagactgccgcaagttcatcgatttctacaagcagtccatctccaagcacccagagtggaaggacttcggcttccgc ttctccgatacccagcgctacaactccatcgatgaattctaccgcgaagtggagaaccagggctacaagctgaccttcgaaaacatctccgag tcctacatcgattccgtggtcaaccagggcaagctgtacctcttccagatctacaacaaggacttctccgcttactccaagggccgcccaaacc tgcacaccctctactggaaggcactcttcgacgaacgcaacctgcaggatgtggtctacaagctcaacggcgaagcagagctgttctaccgc aagcagtccatcccaaagaagatcacccacccagccaaggaagcaatcgccaacaagaacaaggataacccaaagaaggaatccgtgttc gagtacgacctgatcaaggataagcgcttcaccgaggacaagttcttcttccactgcccaatcaccatcaacttcaagtcctccggcgccaaca agttcaacgatgaaatcaacctgctcctgaaggagaaggctaacgacgtgcacatcctgtccatcgatcgcggcgaacgccacctcgcctactacaccctggtcgacggcaagggcaacatcatcaagcaggacaccttcaacatcatcggcaacgatcgcatgaagaccaactaccacgaca agctggccgctatcgagaaggaccgcgattccgctcgcaaggattggaagaagatcaacaacatcaaggaaatgaaggaaggctacctctc ccaggtggtccacgaaatcgctaagctggtgatcgagtacaacgcaatcgtggtcttcgaagacctgaacttcggcttcaagcgcggccgctt caaggtggagaagcaggtctaccagaagctggaaaagatgctcatcgagaagctgaactacctcgtgttcaaggacaacgaattcgataag accggcggcgtcctccgtgcataccagctgaccgccccattcgagaccttcaagaagatgggcaagcagaccggcatcatctactacgtgc cagctggcttcacctctaagatctgcccagtgaccggcttcgtcaaccagctctacccaaagtacgaatccgtctccaagtcccaggagttcttc tccaagttcgacaagatctgctacaacctggataagggctacttcgaattctccttcgactacaagaacttcggcgataaggcagccaagggca agtggaccatcgcatccttcggctcccgcctcatcaacttccgcaactccgacaagaaccacaactgggatacccgcgaagtgtacccaacc aaggaactggagaagctcctgaaggattactccatcgaatacggccacggcgagtgcatcaaggctgcaatctgcggcgaatccgacaagaagttcttcgcaaagctgacctctgtgctcaacaccatcctgcagatgcgcaactccaagaccggcaccgagctggattacctcatctccccagt ggccgacgtcaacggcaacttcttcgattcccgccaggctccaaagaacatgccacaggacgctgatgcaaacggcgcctaccacatcggt ctgaagggtctcatgctcctgggtcgcatcaagaacaaccaggaaggcaagaagctgaatctcgtcattaagaacgaagaatactttgaatttg tccagaaccgcaataac
SEQ ID No.2
启动子序列
ccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatgcgctagatctgtgtgctcagtctt ccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtccaggaggacataca
SEQ ID No.3
crRNA1序列
ccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatgcgctagatctgtgtgctcagtctt ccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtccaggaggacatacataatttctact cttgtagatcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaa at
SEQ ID No.4
crRNA2序列
gatcccataacggttctggcaaatattctgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcggataacaattt cacacaggaaacagaccatgccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatgc gctagatctgtgtgctcagtcttccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtccag gaggacatacataatttctactcttgtagatcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtga acgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcactcgagc
SEQ ID No.5
cas12a表达载体序列
ccttggctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggc ggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcg agagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcc tgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctttttgtttatttttctaaatacattcaaatatgtatcc gctcatgaattaattccgctagatgacgtgcggcttcgacctcctgggcgtggcgcttgttggcgcgctcgcggctggctgcggcacgacac gcgtctgagcagtattttgcgcgccgtcctcgtgggtcaggccggggtgggatcaggccaccgcagtaggcgcagctgatgcgatcctcca ctactgcgcgtcctcctggcgctgccgagcacgcagctcgtcggccagctcttcaaggtcggccacaagcgtttctaggtcgctcgcggcac ttgcccagtcgcgtgatgctggcgcgtctgtcgtatcgagggcgcggaaaaatccgatcaccgtttttaaatcgacggcggcatcgagtgcgt cggactccagcgcgacatcggagagatccaccgctgatgcttcaggccagttttggtacttcgtcgtgaaggtcatgacaccattataacgaa cgttcgttaaaaattctagccccaattctgataatttcttccggcactcctgcgaaaacctgcgagacttcttgcccagaaaaaacgccaagcgc agcggttaccgcactttttttccaggtgatttcaccctgaccagcgaagcggcactttagtgcatgaggtgtgcccctggtttcccctctttggagggttcaacccaaaaaagcacacaagcaaaaatgaaaatcatcatgagcaagttggtgcgaagcagcaacgcgctagctccaaaaaggtctc caggatctcgaggagatttttgagggggagggagtcgaggaagagccagagcagaaggcggggaaccgttctctgccgacagcgtgagc cccccttaaaaatcaggccggggaggaaccggggagggatcagagctaggagcgagacaccctaaagggggggaaccgttttctgctga cggtgtttcgtttattagttttcagcccgtggatagcggagggtgagggcaagtgagagccagagcaaggacgggacccctaaagggggga accgttttctgctgacggtgtttcgtttattagttttcagcccgtggacggccgcgtttagcttccattccaagtgcctttctgacttgttggatgcgc ctttcactgacacctagttcgcctgcaagctcacgagtcgagggatcagcaaccgattgagaacgggcatccaggatcgcagttttgacgcga agttcgagcaactcgcctgtcatttctcggcgtttgtttgcttccgctaatcgctgtcgcgtctcctgcgcatacttactttctgggtcagcccatctg cgtgcattcgatgtagctgcgccccgtcgccccatcgtcgctagagctttccgccctcggctgctctgcgtttccacccgacgagcagggacg actggctggcctttagccacgtagccgcgcacacgacgcgccatcgtcaggcgatcacgcatggcgggaagatccggctcccggccgtct gcaccgaccgcctgggcaacgttgtacgccacttcatacgcgtcgatgatcttggcatcttttaggcgctcaccagcagctttgagctggtatcc cacggtcaacgcgtggcgaaacgcggtctcgtcgcgcgctcgctctggatttgtccagagcactcgcacgccgtcgatcaggtcgccggac gcgtccagggcgctcggcaggctcgcgtccaaaatcgctagcgccttggcttctgcggtggcgcgttgtgccgcttcaatgcgggcgcgtcc gctggaaaagtcctgctcaatgtactttttcggcttctgtgatccggtcatcgttcgagcaatctccattaggtcggccagccgatccacacgatc atgctggcagtgccatttataggctgtcggatcgtctgagacgtgcagcggccaccggctcagcctatgcgaaaaagcctggtcagcgccga aaacacgagtcatttcttccgtcgttgcagccagcaggcgcatatttgggctggttttacctgctgcggcatacaccgggtcaatgagccagat gagctggcatttcccgctcagcggattcacgccgatccaagccggcgctttttctaggcgtgcccatttctctaaaatcgcgtagacctgcggg tttacgtgctcaatcttcccgccggcctggtggctgggcacatcgatgtcaagcacgatcaccgcggcatgttgcgcgtgcgtcagcgcaacgtactggcaccgcgtcagcgcttttgagccagcccggtagagctttggttgggtttcgccggtatccgggtttttaatccaggcgctcgcgaaatc tcttgtcttgctgccctggaagctttcgcgtcccaggtgagcgagcagttcgcggcgatcttctgccgtccagccgcgtgagccgcagcgcat agcttcggggtgggtgtcgaacagatcggcggacaatttccacgcgctagctgtgactgtgtcctgcggatcggctagagtcatgtcttgagt gctttctcccagctgatgactgggggttagccgacgccctgtgagttcccgctcacggggcgttcaactttttcaggtatttgtgcagcttatcgtgttttcttcgtaaatgaacgcttaactaccttgttaaacgtggcaaataggcaggattgatggggatctagcttcacgctgccgcaagcactcag ggcgcaagggctgctaaaggaagcggaacacgtagaaagccagtccgcagaaacggtgctgaccccggatgaatgtcagctactgggct atctggacaagggaaaacgcaagcgcaaagagaaagcaggtagcttgcagtgggcttacatggcgatagctagactgggcggttttatgga cagcaagcgaaccggaattgccagctggggcgccctctggtaaggttgggaagccctgcaaagtaaactggatggctttcttgccgccaag gatctgatggcgcaggggatcaagatctgatcaagagacaggatgaggatcgtttcgcatgattgaacaagatggattgcacgcaggttctcc ggccgcttgggtggagaggctattcggctatgactgggcacaacagacaatcggctgctctgatgccgccgtgttccggctgtcagcgcagg ggcgcccggttctttttgtcaagaccgacctgtccggtgccctgaatgaactccaagacgaggcagcgcggctatcgtggctggccacgacg ggcgttccttgcgcagctgtgctcgacgttgtcactgaagcgggaagggactggctgctattgggcgaagtgccggggcaggatctcctgtc atctcaccttgctcctgccgagaaagtatccatcatggctgatgcaatgcggcggctgcatacgcttgatccggctacctgcccattcgaccaccaagcgaaacatcgcatcgagcgagcacgtactcggatggaagccggtcttgtcgatcaggatgatctggacgaagagcatcaggggctc gcgccagccgaactgttcgccaggctcaaggcgcggatgcccgacggcgaggatctcgtcgtgacccatggcgatgcctgcttgccgaat atcatggtggaaaatggccgcttttctggattcatcgactgtggccggctgggtgtggcggaccgctatcaggacatagcgttggctacccgtg atattgctgaagagcttggcggcgaatgggctgaccgcttcctcgtgctttacggtatcgccgctcccgattcgcagcgcatcgccttctatcgc cttcttgacgagttcttctgagcgggactctggggttcgcggaatcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccc cgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgt ttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagtta ggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcg aacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggt aagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctc tgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttg ctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagcc gaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacac cgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcg ccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccggga gctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttac gttgacaccatcgaatggtgcaccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatg cgctagatctgtgtgctcagtcttccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtcca ggaggacatacaatgtccatctaccaagagtttgtgaataaatactccctgtccaagaccctccgttttgagctgatcccccaaggcaagaccct cgaaaacatcaaggcacgcggcctcatcctggatgacgaaaagcgcgctaaggattacaagaaggcaaagcagatcatcgacaagtacca ccagttcttcatcgaagagatcctgtcctccgtgtgcatctccgaggacctgctccagaactactccgatgtctacttcaagctcaagaagtccg atgacgataacctgcagaaggacttcaagtccgctaaggataccatcaagaagcagatctccgaatacatcaaggattccgagaagttcaaga acctcttcaaccagaacctgatcgacgcaaagaagggccaggaatccgatctcatcctgtggctcaagcagtccaaggataacggcatcgag ctcttcaaggccaactccgacatcaccgacatcgatgaagctctggagatcatcaagtccttcaagggctggaccacctacttcaagggcttccacgaaaaccgcaagaacgtgtactcctccaacgatatcccaacctctatcatctaccgcatcgtcgacgataacctgccaaagttcctcgaaaa caaggcaaagtacgagtccctgaaggataaggccccagaagctatcaactacgagcagatcaagaaggacctggccgaagagctcacctt cgacatcgattacaagacctctgaagtgaaccagcgcgtcttctccctcgatgaagtgttcgagatcgccaacttcaacaactacctgaaccag tccggcatcaccaagttcaacaccatcatcggcggcaagttcgtcaacggcgaaaacaccaagcgcaagggcatcaacgagtacatcaacc tctactcccagcagatcaacgataagaccctgaagaagtacaagatgtccgtgctcttcaagcagatcctgtccgacaccgaatccaagtcctt cgtcatcgacaagctggaggacgattccgatgtggtcaccaccatgcagtccttctacgaacagatcgcagccttcaagaccgtggaagaga agtccatcaaggagaccctctccctgctcttcgacgatctgaaggctcagaagctggatctctccaagatctacttcaagaacgacaagtccct gaccgatctctcccagcaggtcttcgacgattactccgtgatcggcaccgcagtcctggaatacatcacccagcagatcgccccaaagaacct cgataacccatccaagaaggaacaggagctgatcgccaagaagaccgaaaaggctaagtacctgtccctcgagaccatcaagctggctctc gaagagttcaacaagcaccgcgacatcgataagcagtgccgcttcgaagagatcctcgcaaacttcgctgcaatcccaatgatcttcgacgaa atcgcacagaacaaggataacctggcccagatctccatcaagtaccagaaccagggcaagaaggatctgctccaggcctccgctgaggac gatgtgaaggcaatcaaggacctgctcgatcagaccaacaacctgctccacaagctgaagatcttccacatctcccagtccgaagacaaggc caacatcctcgacaaggatgagcacttctacctggtgttcgaagagtgctacttcgaactcgctaacatcgtcccactgtacaacaagatccgc aactacatcacccagaagccatactccgatgaaaagttcaagctcaacttcgagaactccaccctggcaaacggctgggacaagaacaagg aaccagataacaccgccatcctcttcatcaaggacgataagtactacctgggcgtgatgaacaagaagaacaacaagatcttcgacgataag gccatcaaggaaaacaagggcgagggctacaagaagatcgtgtacaagctgctcccaggcgctaacaagatgctcccaaaggtcttcttctc cgcaaagtccatcaagttctacaacccatccgaagatatcctgcgcatccgcaaccactccacccacaccaagaacggctccccacagaag ggctacgaaaagttcgagttcaacatcgaagactgccgcaagttcatcgatttctacaagcagtccatctccaagcacccagagtggaaggac ttcggcttccgcttctccgatacccagcgctacaactccatcgatgaattctaccgcgaagtggagaaccagggctacaagctgaccttcgaaa acatctccgagtcctacatcgattccgtggtcaaccagggcaagctgtacctcttccagatctacaacaaggacttctccgcttactccaagggc cgcccaaacctgcacaccctctactggaaggcactcttcgacgaacgcaacctgcaggatgtggtctacaagctcaacggcgaagcagagc tgttctaccgcaagcagtccatcccaaagaagatcacccacccagccaaggaagcaatcgccaacaagaacaaggataacccaaagaaggaatccgtgttcgagtacgacctgatcaaggataagcgcttcaccgaggacaagttcttcttccactgcccaatcaccatcaacttcaagtcctcc ggcgccaacaagttcaacgatgaaatcaacctgctcctgaaggagaaggctaacgacgtgcacatcctgtccatcgatcgcggcgaacgcc acctcgcctactacaccctggtcgacggcaagggcaacatcatcaagcaggacaccttcaacatcatcggcaacgatcgcatgaagaccaa ctaccacgacaagctggccgctatcgagaaggaccgcgattccgctcgcaaggattggaagaagatcaacaacatcaaggaaatgaagga aggctacctctcccaggtggtccacgaaatcgctaagctggtgatcgagtacaacgcaatcgtggtcttcgaagacctgaacttcggcttcaa gcgcggccgcttcaaggtggagaagcaggtctaccagaagctggaaaagatgctcatcgagaagctgaactacctcgtgttcaaggacaac gaattcgataagaccggcggcgtcctccgtgcataccagctgaccgccccattcgagaccttcaagaagatgggcaagcagaccggcatca tctactacgtgccagctggcttcacctctaagatctgcccagtgaccggcttcgtcaaccagctctacccaaagtacgaatccgtctccaagtcc caggagttcttctccaagttcgacaagatctgctacaacctggataagggctacttcgaattctccttcgactacaagaacttcggcgataaggc agccaagggcaagtggaccatcgcatccttcggctcccgcctcatcaacttccgcaactccgacaagaaccacaactgggatacccgcgaa gtgtacccaaccaaggaactggagaagctcctgaaggattactccatcgaatacggccacggcgagtgcatcaaggctgcaatctgcggcg aatccgacaagaagttcttcgcaaagctgacctctgtgctcaacaccatcctgcagatgcgcaactccaagaccggcaccgagctggattacc tcatctccccagtggccgacgtcaacggcaacttcttcgattcccgccaggctccaaagaacatgccacaggacgctgatgcaaacggcgcc taccacatcggtctgaagggtctcatgctcctgggtcgcatcaagaacaaccaggaaggcaagaagctgaatctcgtcattaagaacgaaga atactttgaatttgtccagaaccgcaataactaa
SEQ ID No.6
crRNA表达载体序列
SEQ ID No.7
引物Ptuf-For序列
catcgaatggtgcaccacagggtagctggtagt
SEQ ID No.8
引物Ptuf-Rev序列
tagatggacattgtatgtcctcctggacttc
SEQ ID No.9
引物cas12a-For序列
caggaggacatacaatgtccatctaccaagagt
SEQ ID No.10
引物cas12a-Rev序列
aaaacagccaaggttagttattgcggttctggac
SEQ ID No.11
引物PEC-For序列
gcaataactaaccttggctgttttggcggatg
SEQ ID No.12
引物PEC-Rev序列
ctaccctgtggtgcaccattcgatggtgtcaac
SEQ ID No.13
aecD靶点序列
tttatgaaggcacccatgtggttg
SEQ ID No.14
crRNA1-aecD序列
ccacagggtagctggtagtttgaaaatcaacgccgttgcccttaggattcagtaactggcacattttgtaatgcgctagatctgtgtgctcagtctt ccaggctgcttatcacagtgaaagcaaaaccaattcgtggctgcgaaagtcgtagccaccacgaagtccaggaggacatacataatttctact cttgtagattgaaggcacccatgtggttgcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaat
SEQ ID No.15
引物aecD-SY-For序列
gagctcgaattcacttacctgcagtctagactcgagcgatctttgtagtcgtaacg
SEQ ID No.16
引物aecD-SY-Rev序列
gaccttcacgccaagctttttgctcaatggcaagcttcag
SEQ ID No.17
引物aecD-XY-For序列
ctgaagcttgccattgagcaaaaagcttggcgtgaaggtc
SEQ ID No.18
引物aecD-XY-Rev序列
actaccagctaccctgtggcgagcccaaaccaagataaac
SEQ ID No.19
引物p19-For序列
ctctcctgagtaggacaaatccgccgggagcggatttgaac
SEQ ID No.20
引物p19-Rev序列
ctgcaggtaagtgaattcgagctcggtacccatggtctgtttcctgtgtg
SEQ ID No.21
mcbR靶点序列
tttcaccacagaaggtattc
SEQ ID No.22
crRNA2-mcbR序列
ccatgccataacaggaatgttcctttcgaaaattgaggaagccttatgccctacaaccctacttagctgccaattattccgggcttgtgacccgct acccaataaataggtgggctgaaaaatttcgttgcaatatcaacaaaaaggcctatcattgggaagtgtcgcaccaagtacttttgcgaagcgc catctgacggattttcaaaagatgtatatgctcggtgcggaaacctacgaaaggattttttaccctaatttctactcttgtagataccacagaaggt attccatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaat
SEQ ID No.23
引物crRNA2-For序列
aactgcagccatgccataacaggaatgttcc
SEQ ID No.24
引物crRNA2-Rev序列
ccgctcgagatttgtcctactcaggagag
SEQ ID No.25
引物cas12a-YZ-For序列
ATGTCCATCTACCAAGAGTT
SEQ ID No.26
引物cas12a-YZ-Rev序列
TTAGTTATTGCGGTTCTGGA
SEQ ID No.27
引物aecD-YZ-For序列
CAACTTCACTTCGGGCTCCT
SEQ ID No.28
引物aecD-YZ-Rev序列
TAAGTCCACTTGAGACGGA
序列表
<110> 山东金城生物药业有限公司
<120> 一种谷氨酸棒杆菌同时基因敲除及基因表达抑制的双功能系统及应用
<160> 28
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3900
<212> DNA
<213> Corynebacterium glutamicum
<400> 1
atgtccatct accaagagtt tgtgaataaa tactccctgt ccaagaccct ccgttttgag 60
ctgatccccc aaggcaagac cctcgaaaac atcaaggcac gcggcctcat cctggatgac 120
gaaaagcgcg ctaaggatta caagaaggca aagcagatca tcgacaagta ccaccagttc 180
ttcatcgaag agatcctgtc ctccgtgtgc atctccgagg acctgctcca gaactactcc 240
gatgtctact tcaagctcaa gaagtccgat gacgataacc tgcagaagga cttcaagtcc 300
gctaaggata ccatcaagaa gcagatctcc gaatacatca aggattccga gaagttcaag 360
aacctcttca accagaacct gatcgacgca aagaagggcc aggaatccga tctcatcctg 420
tggctcaagc agtccaagga taacggcatc gagctcttca aggccaactc cgacatcacc 480
gacatcgatg aagctctgga gatcatcaag tccttcaagg gctggaccac ctacttcaag 540
ggcttccacg aaaaccgcaa gaacgtgtac tcctccaacg atatcccaac ctctatcatc 600
taccgcatcg tcgacgataa cctgccaaag ttcctcgaaa acaaggcaaa gtacgagtcc 660
ctgaaggata aggccccaga agctatcaac tacgagcaga tcaagaagga cctggccgaa 720
gagctcacct tcgacatcga ttacaagacc tctgaagtga accagcgcgt cttctccctc 780
gatgaagtgt tcgagatcgc caacttcaac aactacctga accagtccgg catcaccaag 840
ttcaacacca tcatcggcgg caagttcgtc aacggcgaaa acaccaagcg caagggcatc 900
aacgagtaca tcaacctcta ctcccagcag atcaacgata agaccctgaa gaagtacaag 960
atgtccgtgc tcttcaagca gatcctgtcc gacaccgaat ccaagtcctt cgtcatcgac 1020
aagctggagg acgattccga tgtggtcacc accatgcagt ccttctacga acagatcgca 1080
gccttcaaga ccgtggaaga gaagtccatc aaggagaccc tctccctgct cttcgacgat 1140
ctgaaggctc agaagctgga tctctccaag atctacttca agaacgacaa gtccctgacc 1200
gatctctccc agcaggtctt cgacgattac tccgtgatcg gcaccgcagt cctggaatac 1260
atcacccagc agatcgcccc aaagaacctc gataacccat ccaagaagga acaggagctg 1320
atcgccaaga agaccgaaaa ggctaagtac ctgtccctcg agaccatcaa gctggctctc 1380
gaagagttca acaagcaccg cgacatcgat aagcagtgcc gcttcgaaga gatcctcgca 1440
aacttcgctg caatcccaat gatcttcgac gaaatcgcac agaacaagga taacctggcc 1500
cagatctcca tcaagtacca gaaccagggc aagaaggatc tgctccaggc ctccgctgag 1560
gacgatgtga aggcaatcaa ggacctgctc gatcagacca acaacctgct ccacaagctg 1620
aagatcttcc acatctccca gtccgaagac aaggccaaca tcctcgacaa ggatgagcac 1680
ttctacctgg tgttcgaaga gtgctacttc gaactcgcta acatcgtccc actgtacaac 1740
aagatccgca actacatcac ccagaagcca tactccgatg aaaagttcaa gctcaacttc 1800
gagaactcca ccctggcaaa cggctgggac aagaacaagg aaccagataa caccgccatc 1860
ctcttcatca aggacgataa gtactacctg ggcgtgatga acaagaagaa caacaagatc 1920
ttcgacgata aggccatcaa ggaaaacaag ggcgagggct acaagaagat cgtgtacaag 1980
ctgctcccag gcgctaacaa gatgctccca aaggtcttct tctccgcaaa gtccatcaag 2040
ttctacaacc catccgaaga tatcctgcgc atccgcaacc actccaccca caccaagaac 2100
ggctccccac agaagggcta cgaaaagttc gagttcaaca tcgaagactg ccgcaagttc 2160
atcgatttct acaagcagtc catctccaag cacccagagt ggaaggactt cggcttccgc 2220
ttctccgata cccagcgcta caactccatc gatgaattct accgcgaagt ggagaaccag 2280
ggctacaagc tgaccttcga aaacatctcc gagtcctaca tcgattccgt ggtcaaccag 2340
ggcaagctgt acctcttcca gatctacaac aaggacttct ccgcttactc caagggccgc 2400
ccaaacctgc acaccctcta ctggaaggca ctcttcgacg aacgcaacct gcaggatgtg 2460
gtctacaagc tcaacggcga agcagagctg ttctaccgca agcagtccat cccaaagaag 2520
atcacccacc cagccaagga agcaatcgcc aacaagaaca aggataaccc aaagaaggaa 2580
tccgtgttcg agtacgacct gatcaaggat aagcgcttca ccgaggacaa gttcttcttc 2640
cactgcccaa tcaccatcaa cttcaagtcc tccggcgcca acaagttcaa cgatgaaatc 2700
aacctgctcc tgaaggagaa ggctaacgac gtgcacatcc tgtccatcga tcgcggcgaa 2760
cgccacctcg cctactacac cctggtcgac ggcaagggca acatcatcaa gcaggacacc 2820
ttcaacatca tcggcaacga tcgcatgaag accaactacc acgacaagct ggccgctatc 2880
gagaaggacc gcgattccgc tcgcaaggat tggaagaaga tcaacaacat caaggaaatg 2940
aaggaaggct acctctccca ggtggtccac gaaatcgcta agctggtgat cgagtacaac 3000
gcaatcgtgg tcttcgaaga cctgaacttc ggcttcaagc gcggccgctt caaggtggag 3060
aagcaggtct accagaagct ggaaaagatg ctcatcgaga agctgaacta cctcgtgttc 3120
aaggacaacg aattcgataa gaccggcggc gtcctccgtg cataccagct gaccgcccca 3180
ttcgagacct tcaagaagat gggcaagcag accggcatca tctactacgt gccagctggc 3240
ttcacctcta agatctgccc agtgaccggc ttcgtcaacc agctctaccc aaagtacgaa 3300
tccgtctcca agtcccagga gttcttctcc aagttcgaca agatctgcta caacctggat 3360
aagggctact tcgaattctc cttcgactac aagaacttcg gcgataaggc agccaagggc 3420
aagtggacca tcgcatcctt cggctcccgc ctcatcaact tccgcaactc cgacaagaac 3480
cacaactggg atacccgcga agtgtaccca accaaggaac tggagaagct cctgaaggat 3540
tactccatcg aatacggcca cggcgagtgc atcaaggctg caatctgcgg cgaatccgac 3600
aagaagttct tcgcaaagct gacctctgtg ctcaacacca tcctgcagat gcgcaactcc 3660
aagaccggca ccgagctgga ttacctcatc tccccagtgg ccgacgtcaa cggcaacttc 3720
ttcgattccc gccaggctcc aaagaacatg ccacaggacg ctgatgcaaa cggcgcctac 3780
cacatcggtc tgaagggtct catgctcctg ggtcgcatca agaacaacca ggaaggcaag 3840
aagctgaatc tcgtcattaa gaacgaagaa tactttgaat ttgtccagaa ccgcaataac 3900
<210> 2
<211> 179
<212> DNA
<213> Corynebacterium glutamicum
<400> 2
ccacagggta gctggtagtt tgaaaatcaa cgccgttgcc cttaggattc agtaactggc 60
acattttgta atgcgctaga tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa 120
gcaaaaccaa ttcgtggctg cgaaagtcgt agccaccacg aagtccagga ggacataca 179
<210> 3
<211> 289
<212> DNA
<213> Corynebacterium glutamicum
<400> 3
ccacagggta gctggtagtt tgaaaatcaa cgccgttgcc cttaggattc agtaactggc 60
acattttgta atgcgctaga tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa 120
gcaaaaccaa ttcgtggctg cgaaagtcgt agccaccacg aagtccagga ggacatacat 180
aatttctact cttgtagatc atcaaataaa acgaaaggct cagtcgaaag actgggcctt 240
tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaat 289
<210> 4
<211> 445
<212> DNA
<213> Corynebacterium glutamicum
<400> 4
gatcccataa cggttctggc aaatattctg aaatgagctg ttgacaatta atcatccggc 60
tcgtataatg tgtggaattg tgagcggata acaatttcac acaggaaaca gaccatgcca 120
cagggtagct ggtagtttga aaatcaacgc cgttgccctt aggattcagt aactggcaca 180
ttttgtaatg cgctagatct gtgtgctcag tcttccaggc tgcttatcac agtgaaagca 240
aaaccaattc gtggctgcga aagtcgtagc caccacgaag tccaggagga catacataat 300
ttctactctt gtagatcatc aaataaaacg aaaggctcag tcgaaagact gggcctttcg 360
ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc cgggagcgga 420
tttgaacgtt gcgaagcact cgagc 445
<210> 5
<211> 9597
<212> DNA
<213> Corynebacterium glutamicum
<400> 5
ccttggctgt tttggcggat gagagaagat tttcagcctg atacagatta aatcagaacg 60
cagaagcggt ctgataaaac agaatttgcc tggcggcagt agcgcggtgg tcccacctga 120
ccccatgccg aactcagaag tgaaacgccg tagcgccgat ggtagtgtgg ggtctcccca 180
tgcgagagta gggaactgcc aggcatcaaa taaaacgaaa ggctcagtcg aaagactggg 240
cctttcgttt tatctgttgt ttgtcggtga acgctctcct gagtaggaca aatccgccgg 300
gagcggattt gaacgttgcg aagcaacggc ccggagggtg gcgggcagga cgcccgccat 360
aaactgccag gcatcaaatt aagcagaagg ccatcctgac ggatggcctt tttgcgtttc 420
tacaaactct ttttgtttat ttttctaaat acattcaaat atgtatccgc tcatgaatta 480
attccgctag atgacgtgcg gcttcgacct cctgggcgtg gcgcttgttg gcgcgctcgc 540
ggctggctgc ggcacgacac gcgtctgagc agtattttgc gcgccgtcct cgtgggtcag 600
gccggggtgg gatcaggcca ccgcagtagg cgcagctgat gcgatcctcc actactgcgc 660
gtcctcctgg cgctgccgag cacgcagctc gtcggccagc tcttcaaggt cggccacaag 720
cgtttctagg tcgctcgcgg cacttgccca gtcgcgtgat gctggcgcgt ctgtcgtatc 780
gagggcgcgg aaaaatccga tcaccgtttt taaatcgacg gcggcatcga gtgcgtcgga 840
ctccagcgcg acatcggaga gatccaccgc tgatgcttca ggccagtttt ggtacttcgt 900
cgtgaaggtc atgacaccat tataacgaac gttcgttaaa aattctagcc ccaattctga 960
taatttcttc cggcactcct gcgaaaacct gcgagacttc ttgcccagaa aaaacgccaa 1020
gcgcagcggt taccgcactt tttttccagg tgatttcacc ctgaccagcg aagcggcact 1080
ttagtgcatg aggtgtgccc ctggtttccc ctctttggag ggttcaaccc aaaaaagcac 1140
acaagcaaaa atgaaaatca tcatgagcaa gttggtgcga agcagcaacg cgctagctcc 1200
aaaaaggtct ccaggatctc gaggagattt ttgaggggga gggagtcgag gaagagccag 1260
agcagaaggc ggggaaccgt tctctgccga cagcgtgagc cccccttaaa aatcaggccg 1320
gggaggaacc ggggagggat cagagctagg agcgagacac cctaaagggg gggaaccgtt 1380
ttctgctgac ggtgtttcgt ttattagttt tcagcccgtg gatagcggag ggtgagggca 1440
agtgagagcc agagcaagga cgggacccct aaagggggga accgttttct gctgacggtg 1500
tttcgtttat tagttttcag cccgtggacg gccgcgttta gcttccattc caagtgcctt 1560
tctgacttgt tggatgcgcc tttcactgac acctagttcg cctgcaagct cacgagtcga 1620
gggatcagca accgattgag aacgggcatc caggatcgca gttttgacgc gaagttcgag 1680
caactcgcct gtcatttctc ggcgtttgtt tgcttccgct aatcgctgtc gcgtctcctg 1740
cgcatactta ctttctgggt cagcccatct gcgtgcattc gatgtagctg cgccccgtcg 1800
ccccatcgtc gctagagctt tccgccctcg gctgctctgc gtttccaccc gacgagcagg 1860
gacgactggc tggcctttag ccacgtagcc gcgcacacga cgcgccatcg tcaggcgatc 1920
acgcatggcg ggaagatccg gctcccggcc gtctgcaccg accgcctggg caacgttgta 1980
cgccacttca tacgcgtcga tgatcttggc atcttttagg cgctcaccag cagctttgag 2040
ctggtatccc acggtcaacg cgtggcgaaa cgcggtctcg tcgcgcgctc gctctggatt 2100
tgtccagagc actcgcacgc cgtcgatcag gtcgccggac gcgtccaggg cgctcggcag 2160
gctcgcgtcc aaaatcgcta gcgccttggc ttctgcggtg gcgcgttgtg ccgcttcaat 2220
gcgggcgcgt ccgctggaaa agtcctgctc aatgtacttt ttcggcttct gtgatccggt 2280
catcgttcga gcaatctcca ttaggtcggc cagccgatcc acacgatcat gctggcagtg 2340
ccatttatag gctgtcggat cgtctgagac gtgcagcggc caccggctca gcctatgcga 2400
aaaagcctgg tcagcgccga aaacacgagt catttcttcc gtcgttgcag ccagcaggcg 2460
catatttggg ctggttttac ctgctgcggc atacaccggg tcaatgagcc agatgagctg 2520
gcatttcccg ctcagcggat tcacgccgat ccaagccggc gctttttcta ggcgtgccca 2580
tttctctaaa atcgcgtaga cctgcgggtt tacgtgctca atcttcccgc cggcctggtg 2640
gctgggcaca tcgatgtcaa gcacgatcac cgcggcatgt tgcgcgtgcg tcagcgcaac 2700
gtactggcac cgcgtcagcg cttttgagcc agcccggtag agctttggtt gggtttcgcc 2760
ggtatccggg tttttaatcc aggcgctcgc gaaatctctt gtcttgctgc cctggaagct 2820
ttcgcgtccc aggtgagcga gcagttcgcg gcgatcttct gccgtccagc cgcgtgagcc 2880
gcagcgcata gcttcggggt gggtgtcgaa cagatcggcg gacaatttcc acgcgctagc 2940
tgtgactgtg tcctgcggat cggctagagt catgtcttga gtgctttctc ccagctgatg 3000
actgggggtt agccgacgcc ctgtgagttc ccgctcacgg ggcgttcaac tttttcaggt 3060
atttgtgcag cttatcgtgt tttcttcgta aatgaacgct taactacctt gttaaacgtg 3120
gcaaataggc aggattgatg gggatctagc ttcacgctgc cgcaagcact cagggcgcaa 3180
gggctgctaa aggaagcgga acacgtagaa agccagtccg cagaaacggt gctgaccccg 3240
gatgaatgtc agctactggg ctatctggac aagggaaaac gcaagcgcaa agagaaagca 3300
ggtagcttgc agtgggctta catggcgata gctagactgg gcggttttat ggacagcaag 3360
cgaaccggaa ttgccagctg gggcgccctc tggtaaggtt gggaagccct gcaaagtaaa 3420
ctggatggct ttcttgccgc caaggatctg atggcgcagg ggatcaagat ctgatcaaga 3480
gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag gttctccggc 3540
cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg gctgctctga 3600
tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca agaccgacct 3660
gtccggtgcc ctgaatgaac tccaagacga ggcagcgcgg ctatcgtggc tggccacgac 3720
gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg actggctgct 3780
attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg ccgagaaagt 3840
atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta cctgcccatt 3900
cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag ccggtcttgt 3960
cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac tgttcgccag 4020
gctcaaggcg cggatgcccg acggcgagga tctcgtcgtg acccatggcg atgcctgctt 4080
gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg gccggctggg 4140
tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg aagagcttgg 4200
cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg attcgcagcg 4260
catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg gttcgcggaa 4320
tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa 4380
agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa 4440
aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc 4500
cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt 4560
agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc 4620
tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac 4680
gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca 4740
gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg 4800
ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag 4860
gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt 4920
ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat 4980
ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc 5040
acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt 5100
gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag 5160
cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca 5220
tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatacactcc 5280
gctatcgcta cgtgactggg tcatggctgc gccccgacac ccgccaacac ccgctgacgc 5340
gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 5400
gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgaggc agcagatcaa 5460
ttcgcgcgcg aaggcgaagc ggcatgcatt tacgttgaca ccatcgaatg gtgcaccaca 5520
gggtagctgg tagtttgaaa atcaacgccg ttgcccttag gattcagtaa ctggcacatt 5580
ttgtaatgcg ctagatctgt gtgctcagtc ttccaggctg cttatcacag tgaaagcaaa 5640
accaattcgt ggctgcgaaa gtcgtagcca ccacgaagtc caggaggaca tacaatgtcc 5700
atctaccaag agtttgtgaa taaatactcc ctgtccaaga ccctccgttt tgagctgatc 5760
ccccaaggca agaccctcga aaacatcaag gcacgcggcc tcatcctgga tgacgaaaag 5820
cgcgctaagg attacaagaa ggcaaagcag atcatcgaca agtaccacca gttcttcatc 5880
gaagagatcc tgtcctccgt gtgcatctcc gaggacctgc tccagaacta ctccgatgtc 5940
tacttcaagc tcaagaagtc cgatgacgat aacctgcaga aggacttcaa gtccgctaag 6000
gataccatca agaagcagat ctccgaatac atcaaggatt ccgagaagtt caagaacctc 6060
ttcaaccaga acctgatcga cgcaaagaag ggccaggaat ccgatctcat cctgtggctc 6120
aagcagtcca aggataacgg catcgagctc ttcaaggcca actccgacat caccgacatc 6180
gatgaagctc tggagatcat caagtccttc aagggctgga ccacctactt caagggcttc 6240
cacgaaaacc gcaagaacgt gtactcctcc aacgatatcc caacctctat catctaccgc 6300
atcgtcgacg ataacctgcc aaagttcctc gaaaacaagg caaagtacga gtccctgaag 6360
gataaggccc cagaagctat caactacgag cagatcaaga aggacctggc cgaagagctc 6420
accttcgaca tcgattacaa gacctctgaa gtgaaccagc gcgtcttctc cctcgatgaa 6480
gtgttcgaga tcgccaactt caacaactac ctgaaccagt ccggcatcac caagttcaac 6540
accatcatcg gcggcaagtt cgtcaacggc gaaaacacca agcgcaaggg catcaacgag 6600
tacatcaacc tctactccca gcagatcaac gataagaccc tgaagaagta caagatgtcc 6660
gtgctcttca agcagatcct gtccgacacc gaatccaagt ccttcgtcat cgacaagctg 6720
gaggacgatt ccgatgtggt caccaccatg cagtccttct acgaacagat cgcagccttc 6780
aagaccgtgg aagagaagtc catcaaggag accctctccc tgctcttcga cgatctgaag 6840
gctcagaagc tggatctctc caagatctac ttcaagaacg acaagtccct gaccgatctc 6900
tcccagcagg tcttcgacga ttactccgtg atcggcaccg cagtcctgga atacatcacc 6960
cagcagatcg ccccaaagaa cctcgataac ccatccaaga aggaacagga gctgatcgcc 7020
aagaagaccg aaaaggctaa gtacctgtcc ctcgagacca tcaagctggc tctcgaagag 7080
ttcaacaagc accgcgacat cgataagcag tgccgcttcg aagagatcct cgcaaacttc 7140
gctgcaatcc caatgatctt cgacgaaatc gcacagaaca aggataacct ggcccagatc 7200
tccatcaagt accagaacca gggcaagaag gatctgctcc aggcctccgc tgaggacgat 7260
gtgaaggcaa tcaaggacct gctcgatcag accaacaacc tgctccacaa gctgaagatc 7320
ttccacatct cccagtccga agacaaggcc aacatcctcg acaaggatga gcacttctac 7380
ctggtgttcg aagagtgcta cttcgaactc gctaacatcg tcccactgta caacaagatc 7440
cgcaactaca tcacccagaa gccatactcc gatgaaaagt tcaagctcaa cttcgagaac 7500
tccaccctgg caaacggctg ggacaagaac aaggaaccag ataacaccgc catcctcttc 7560
atcaaggacg ataagtacta cctgggcgtg atgaacaaga agaacaacaa gatcttcgac 7620
gataaggcca tcaaggaaaa caagggcgag ggctacaaga agatcgtgta caagctgctc 7680
ccaggcgcta acaagatgct cccaaaggtc ttcttctccg caaagtccat caagttctac 7740
aacccatccg aagatatcct gcgcatccgc aaccactcca cccacaccaa gaacggctcc 7800
ccacagaagg gctacgaaaa gttcgagttc aacatcgaag actgccgcaa gttcatcgat 7860
ttctacaagc agtccatctc caagcaccca gagtggaagg acttcggctt ccgcttctcc 7920
gatacccagc gctacaactc catcgatgaa ttctaccgcg aagtggagaa ccagggctac 7980
aagctgacct tcgaaaacat ctccgagtcc tacatcgatt ccgtggtcaa ccagggcaag 8040
ctgtacctct tccagatcta caacaaggac ttctccgctt actccaaggg ccgcccaaac 8100
ctgcacaccc tctactggaa ggcactcttc gacgaacgca acctgcagga tgtggtctac 8160
aagctcaacg gcgaagcaga gctgttctac cgcaagcagt ccatcccaaa gaagatcacc 8220
cacccagcca aggaagcaat cgccaacaag aacaaggata acccaaagaa ggaatccgtg 8280
ttcgagtacg acctgatcaa ggataagcgc ttcaccgagg acaagttctt cttccactgc 8340
ccaatcacca tcaacttcaa gtcctccggc gccaacaagt tcaacgatga aatcaacctg 8400
ctcctgaagg agaaggctaa cgacgtgcac atcctgtcca tcgatcgcgg cgaacgccac 8460
ctcgcctact acaccctggt cgacggcaag ggcaacatca tcaagcagga caccttcaac 8520
atcatcggca acgatcgcat gaagaccaac taccacgaca agctggccgc tatcgagaag 8580
gaccgcgatt ccgctcgcaa ggattggaag aagatcaaca acatcaagga aatgaaggaa 8640
ggctacctct cccaggtggt ccacgaaatc gctaagctgg tgatcgagta caacgcaatc 8700
gtggtcttcg aagacctgaa cttcggcttc aagcgcggcc gcttcaaggt ggagaagcag 8760
gtctaccaga agctggaaaa gatgctcatc gagaagctga actacctcgt gttcaaggac 8820
aacgaattcg ataagaccgg cggcgtcctc cgtgcatacc agctgaccgc cccattcgag 8880
accttcaaga agatgggcaa gcagaccggc atcatctact acgtgccagc tggcttcacc 8940
tctaagatct gcccagtgac cggcttcgtc aaccagctct acccaaagta cgaatccgtc 9000
tccaagtccc aggagttctt ctccaagttc gacaagatct gctacaacct ggataagggc 9060
tacttcgaat tctccttcga ctacaagaac ttcggcgata aggcagccaa gggcaagtgg 9120
accatcgcat ccttcggctc ccgcctcatc aacttccgca actccgacaa gaaccacaac 9180
tgggataccc gcgaagtgta cccaaccaag gaactggaga agctcctgaa ggattactcc 9240
atcgaatacg gccacggcga gtgcatcaag gctgcaatct gcggcgaatc cgacaagaag 9300
ttcttcgcaa agctgacctc tgtgctcaac accatcctgc agatgcgcaa ctccaagacc 9360
ggcaccgagc tggattacct catctcccca gtggccgacg tcaacggcaa cttcttcgat 9420
tcccgccagg ctccaaagaa catgccacag gacgctgatg caaacggcgc ctaccacatc 9480
ggtctgaagg gtctcatgct cctgggtcgc atcaagaaca accaggaagg caagaagctg 9540
aatctcgtca ttaagaacga agaatacttt gaatttgtcc agaaccgcaa taactaa 9597
<210> 6
<211> 7958
<212> DNA
<213> Corynebacterium glutamicum
<400> 6
tgaaggcacc catgtggttg catcaaataa aacgaaaggc tcagtcgaaa gactgggcct 60
ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat ccgccgggag 120
cggatttgaa cgttgcgaag caacggcccg gagggtggcg ggcaggacgc ccgccataaa 180
ctgccaggca tcaaattaag cagaaggcca tcctgacgga tggccttttt gcgtttctac 240
aaactctttt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa 300
ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt 360
gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg 420
ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg 480
gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg 540
agcacttttg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg 600
gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga 660
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg 720
gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag 780
aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc 840
gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg 900
ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc tcagttcggt gtaggtcgtt 960
cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc 1020
ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc 1080
actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg 1140
tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca 1200
gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc 1260
ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat 1320
cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt 1380
ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttggggtg ggcgaagaac 1440
tccagcatga gatccccgcg ctggaggatc atccagccat tcggggtcgt tcactggttc 1500
ccctttctga tttctggcat agaagaaccc ccgtgaactg tgtggttccg ggggttgctg 1560
atttttgcga gacttctcgc gcaattccct agcttaggtg aaaacaccat gaaacactag 1620
ggaaacaccc atgaaacacc cattagggca gtagggcggc ttcttcgtct agggcttgca 1680
tttgggcggt gatctggtct ttagcgtgtg aaagtgtgtc gtaggtggcg tgctcaatgc 1740
actcgaacgt cacgtcattt accgggtcac ggtgggcaaa gagaactagt gggttagaca 1800
ttgttttcct cgttgtcggt ggtggtgagc ttttctagcc gctcggtaaa cgcggcgatc 1860
atgaactctt ggaggttttc accgttctgc atgcctgcgc gcttcatgtc ctcacgtagt 1920
gccaaaggaa cgcgtgcggt gaccacgacg ggcttagcct ttgcctgcgc ttctagtgct 1980
tcgatggtgg cttgtgcctg cgcttgctgc gcctgtagtg cctgttgagc ttcttgtagt 2040
tgctgttcta gctgtgcctt ggttgccatg ctttaagact ctagtagctt tcctgcgata 2100
tgtcatgcgc atgcgtagca aacattgtcc tgcaactcat tcattatgtg cagtgctcct 2160
gttactagtc gtacatactc atatttacct agtctgcatg cagtgcatgc acatgcagtc 2220
atgtcgtgct aatgtgtaaa acatgtacat gcagattgct gggggtgcag ggggcggagc 2280
caccctgtcc atgcggggtg tggggcttgc cccgccggta cagacagtga gcaccggggc 2340
acctagtcgc ggataccccc cctaggtatc ggacacgtaa ccctcccatg tcgatgcaaa 2400
tctttaacat tgagtacggg taagctggca cgcatagcca agctaggcgg ccaccaaaca 2460
ccactaaaaa ttaatagtcc ctagacaaga caaacccccg tgcgagctac caactcatat 2520
gcacgggggc cacataaccc gaaggggttt caattgacaa ccatagcact agctaagaca 2580
acgggcacaa cacccgcaca aactcgcact gcgcaacccc gcacaacatc gggtctaggt 2640
aacactgagt aacactgaaa tagaagtgaa cacctctaag gaaccgcagg tcaatgaggg 2700
ttctaaggtc actcgcgcta gggcgtggcg taggcaaaac gtcatgtaca agatcaccaa 2760
tagtaaggct ctggcggggt gccataggtg gcgcagggac gaagctgttg cggtgtcctg 2820
gtcgtctaac ggtgcttcgc agtttgaggg tctgcaaaac tctcactctc gctgggggtc 2880
acctctggct gaattggaag tcatgggcga acgccgcatt gagctggcta ttgctactaa 2940
gaatcacttg gcggcgggtg gcgcgctcat gatgtttgtg ggcactgttc gacacaaccg 3000
ctcacagtca tttgcgcagg ttgaagcggg tattaagact gcgtactctt cgatggtgaa 3060
aacatctcag tggaagaaag aacgtgcacg gtacggggtg gagcacacct atagtgacta 3120
tgaggtcaca gactcttggg cgaacggttg gcacttgcac cgcaacatgc tgttgttctt 3180
ggatcgtcca ctgtctgacg atgaactcaa ggcgtttgag gattccatgt tttcccgctg 3240
gtctgctggt gtggttaagg ccggtatgga cgcgccactg cgtgagcacg gggtcaaact 3300
tgatcaggtg tctacctggg gtggagacgc tgcgaaaatg gcaacctacc tcgctaaggg 3360
catgtctcag gaactgactg gctccgctac taaaaccgcg tctaaggggt cgtacacgcc 3420
gtttcagatg ttggatatgt tggccgatca aagcgacgcc ggcgaggata tggacgctgt 3480
tttggtggct cggtggcgtg agtatgaggt tggttctaaa aacctgcgtt cgtcctggtc 3540
acgtggggct aagcgtgctt tgggcattga ttacatagac gctgatgtac gtcgtgaaat 3600
ggaagaagaa ctgtacaagc tcgccggtct ggaagcaccg gaacgggtcg aatcaacccg 3660
cgttgctgtt gctttggtga agcccgatga ttggaaactg attcagtctg atttcgcggt 3720
taggcagtac gttctcgatt gcgtggataa ggctaaggac gtggccgctg cgcaacgtgt 3780
cgctaatgag gtgctggcaa gtctgggtgt ggattccacc ccgtgcatga tcgttatgga 3840
tgatgtggac ttggacgcgg ttctgcctac tcatggggac gctactaagc gtgatctgaa 3900
tgcggcggtg ttcgcgggta atgagcagac tattcttcgc acccactaaa agcggcataa 3960
accccgttcg atattttgtg cgatgaattt atggtcaatg tcgcgggggc aaactatgat 4020
gggtcttgtt gttggcgtcc cggaaaacga ttccgaagcc caacctttca tagaaggcgg 4080
cggtggaatc gaaatctcgt gatggcaggt tgggcgtcgc ttggtcggtc atttcgaagg 4140
gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc agtactgttg 4200
taattcatta agcattctgc cgacatggaa gccatcacag acggcatgat gaacctgaat 4260
cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg 4320
ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg 4380
attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc 4440
accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta 4500
ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg 4560
aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaact ccggatgagc 4620
attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt 4680
tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc 4740
aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt 4800
atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgtcgaagc 4860
tcggcggatt tgtcctactc aagctgatcc gacaaaatcc acacattatc ccaggtgtcc 4920
ggatcggtca aatacgctgc cagctcatag accgtatcca aagcatccgg ggctgatccc 4980
cggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac 5040
cgcctggccc tgagagagtt gcagcaagcg gtcggtgtca aattcgccag cgtgttgtaa 5100
aagcgggcag tgagcgcaac gcaattaatg tgagttagcg cgaattgatc tggtttgaca 5160
gcttatcatc gactgcacgg tgcaccaatg cttctggcgt caggcagcca tcggaagctg 5220
tggtatggct gtgcaggtcg taaatcactg cataattcgt gtcgctcaag gcgcactccc 5280
gttctggata atgttttttg cgccgacatc ataacggttc tggcaaatat tctgaaatga 5340
gctgttgaca attaatcatc cggctcgtat aatgtgtgga attgtgagcg gataacaatt 5400
tcacacagga aacagaccat gggtaccgag ctcgaattca cttacctgca gccatgccat 5460
aacaggaatg ttcctttcga aaattgagga agccttatgc cctacaaccc tacttagctg 5520
ccaattattc cgggcttgtg acccgctacc caataaatag gtgggctgaa aaatttcgtt 5580
gcaatatcaa caaaaaggcc tatcattggg aagtgtcgca ccaagtactt ttgcgaagcg 5640
ccatctgacg gattttcaaa agatgtatat gctcggtgcg gaaacctacg aaaggatttt 5700
ttaccctaat ttctactctt gtagatacca cagaaggtat tccatcaaat aaaacgaaag 5760
gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg 5820
agtaggacaa atctcgagcg atctttgtag tcgtaacgag ggccgtgaaa aggcactcat 5880
aagcgttaga ctgcaaaagt tcggggtagg ttccctaaaa ttcaaattat ttttttgaac 5940
ttacgccaag ctgttcaaat tgcctgagta gatgacgttt ccgatctgga attcagcatt 6000
cgacaatgag ccgtttgagg tgttgaacgc gtattgcagg ttcgttgtcg cgccggcacc 6060
cagtggtagg tcgagtggga tgttgtcgtc gccggagagg gtttcgatgt tggttttgtt 6120
gccgttgtag tcttcgatgt ccagggtggg tggatcgatg gcctcggctg gcagggctac 6180
gtcgtttagg tttttcaccg cgatgtgaat aattgagcca ccgttggtgc cgtagcgggt 6240
gcctcggaag ataaattcaa cattgagacc tgggtcttca accctgatat cgccgcttgg 6300
gattgtgatt gggcttacct ctttggcttc gaacaccggg gttgaggtag tggttacttc 6360
catggtttcc tcagcggaaa cggcttggct atcagcactt tcacccgaac agcctgcaag 6420
aagtgcgacg gctaacaggg ctgggattgt cctcaacttc acttcgggct ccttcttagt 6480
aataggttcg tagaaaagtt tactagccta gagagtatgc gatttcctga actcgaagaa 6540
ttgaagaatc gccggacctt gaaatggacc cggtttccag aagacgtgct tcctttgtgg 6600
gttgcggaaa gtgattttgg cacctgcccg ccgttgaagg aagctatggc agatgccgtt 6660
gagcgcgagg tcttcggata cccaccagat gctactgggt tgaatgatgc gttgactgga 6720
ttctacgagc gccgctatgg gtttggccca aatccggaaa gtgttttcgc cattccggat 6780
gtggttcgtg gcctgaagct tgccattgag caaaaagctt ggcgtgaagg tctacgcacc 6840
ggactctact tatttgatgt ggttggactt cgctggcacc aagattgaag aggcgccttc 6900
taaaattctt cgtgaggagg gtaaggtcat gctgaatgat ggcgcagctt ttggtggttt 6960
cacaacctgc gctcgtctta attttgcgtg ttccagagag acccttgagg aggggctgcg 7020
ccgtatcgcc agcgtgttgt aaataatgag taaaaagtct gtcctgatta cttctttgat 7080
gctgttttcc atgttcttcg gagctggaaa cctcatcttc ccgccgatgc ttggattgtc 7140
ggcaggaacc aactatctac cagctatctt aggatttcta gcaacgagtg ttctgctccc 7200
ggtgctggcg attatcgcgg tggtgttgtc gggagaaaat gtcaaggaca tggcttctcg 7260
tggcggtaag atctttggcc tggtgtttcc tattgctgcc tatttgtcca tcggcgcgtt 7320
ttacgcgctg ccgaggactg gggcggtgag ctattcgacg gcggttggcg ttgataatgc 7380
gctttattcg ggcttgttta actttgtgtt ttttgcggtg gcactggcgt tgtcgtggaa 7440
tccgaatggc attgcagaca aattgggtaa gtggctcacc ccggctctac tcacgttgat 7500
tgtggtgctg gtggtgttgt cggtagccaa gttggatggc acgccaggtg agccaagtag 7560
tgcgtatgcg cagcaacctg cgggggcggg tttgcttgag ggctacatga cgatggatgc 7620
gattgctgcg ttggcgtttg gcatcgtggt gatttctgcg ttcaagtacc aaaaggttaa 7680
caaggtccgc acggcaactg tcgtgtcggc gttcattgcc ggaattttgt tggcgctggt 7740
ttatcttggt ttgggctcgc cacagggtag ctggtagttt gaaaatcaac gccgttgccc 7800
ttaggattca gtaactggca cattttgtaa tgcgctagat ctgtgtgctc agtcttccag 7860
gctgcttatc acagtgaaag caaaaccaat tcgtggctgc gaaagtcgta gccaccacga 7920
agtccaggag gacatacata atttctactc ttgtagat 7958
<210> 7
<211> 33
<212> DNA
<213> Corynebacterium glutamicum
<400> 7
catcgaatgg tgcaccacag ggtagctggt agt 33
<210> 8
<211> 31
<212> DNA
<213> Corynebacterium glutamicum
<400> 8
tagatggaca ttgtatgtcc tcctggactt c 31
<210> 9
<211> 33
<212> DNA
<213> Corynebacterium glutamicum
<400> 9
caggaggaca tacaatgtcc atctaccaag agt 33
<210> 10
<211> 34
<212> DNA
<213> Corynebacterium glutamicum
<400> 10
aaaacagcca aggttagtta ttgcggttct ggac 34
<210> 11
<211> 32
<212> DNA
<213> Corynebacterium glutamicum
<400> 11
gcaataacta accttggctg ttttggcgga tg 32
<210> 12
<211> 33
<212> DNA
<213> Corynebacterium glutamicum
<400> 12
ctaccctgtg gtgcaccatt cgatggtgtc aac 33
<210> 13
<211> 24
<212> DNA
<213> Corynebacterium glutamicum
<400> 13
tttatgaagg cacccatgtg gttg 24
<210> 14
<211> 309
<212> DNA
<213> Corynebacterium glutamicum
<400> 14
ccacagggta gctggtagtt tgaaaatcaa cgccgttgcc cttaggattc agtaactggc 60
acattttgta atgcgctaga tctgtgtgct cagtcttcca ggctgcttat cacagtgaaa 120
gcaaaaccaa ttcgtggctg cgaaagtcgt agccaccacg aagtccagga ggacatacat 180
aatttctact cttgtagatt gaaggcaccc atgtggttgc atcaaataaa acgaaaggct 240
cagtcgaaag actgggcctt tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt 300
aggacaaat 309
<210> 15
<211> 56
<212> DNA
<213> Corynebacterium glutamicum
<400> 15
gagctcgaat tcacttacct gcagtctaga ctcgagcgat ctttgtagtc gtaacg 56
<210> 16
<211> 40
<212> DNA
<213> Corynebacterium glutamicum
<400> 16
gaccttcacg ccaagctttt tgctcaatgg caagcttcag 40
<210> 17
<211> 40
<212> DNA
<213> Corynebacterium glutamicum
<400> 17
ctgaagcttg ccattgagca aaaagcttgg cgtgaaggtc 40
<210> 18
<211> 40
<212> DNA
<213> Corynebacterium glutamicum
<400> 18
actaccagct accctgtggc gagcccaaac caagataaac 40
<210> 19
<211> 41
<212> DNA
<213> Corynebacterium glutamicum
<400> 19
ctctcctgag taggacaaat ccgccgggag cggatttgaa c 41
<210> 20
<211> 50
<212> DNA
<213> Corynebacterium glutamicum
<400> 20
ctgcaggtaa gtgaattcga gctcggtacc catggtctgt ttcctgtgtg 50
<210> 21
<211> 20
<212> DNA
<213> Corynebacterium glutamicum
<400> 21
tttcaccaca gaaggtattc 20
<210> 22
<211> 381
<212> DNA
<213> Corynebacterium glutamicum
<400> 22
ccatgccata acaggaatgt tcctttcgaa aattgaggaa gccttatgcc ctacaaccct 60
acttagctgc caattattcc gggcttgtga cccgctaccc aataaatagg tgggctgaaa 120
aatttcgttg caatatcaac aaaaaggcct atcattggga agtgtcgcac caagtacttt 180
tgcgaagcgc catctgacgg attttcaaaa gatgtatatg ctcggtgcgg aaacctacga 240
aaggattttt taccctaatt tctactcttg tagataccac agaaggtatt ccatcaaata 300
aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta tctgttgttt gtcggtgaac 360
gctctcctga gtaggacaaa t 381
<210> 23
<211> 31
<212> DNA
<213> Corynebacterium glutamicum
<400> 23
aactgcagcc atgccataac aggaatgttc c 31
<210> 24
<211> 29
<212> DNA
<213> Corynebacterium glutamicum
<400> 24
ccgctcgaga tttgtcctac tcaggagag 29
<210> 25
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 25
atgtccatct accaagagtt 20
<210> 26
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 26
ttagttattg cggttctgga 20
<210> 27
<211> 20
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 27
caacttcact tcgggctcct 20
<210> 28
<211> 19
<212> DNA
<213> Corynebacterium glutamicum
<400> 28
taagtccact tgagacgga 19
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