具体实施方式

下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。

下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。pK18mobsacB质粒：Addgene公司；pK18mobsacB质粒中具有卡那霉素抗性基因作为筛选标记。pXMJ19质粒：BioVector质粒载体菌种细胞基因保藏中心；pXMJ19质粒中具有氯霉素抗性基因作为筛选标记。NEBuilder酶：NEB公司。如无特殊说明，实施例中的培养基为配方为表1的培养基(余量为水，pH为7.0)。不含卡那霉素培养基即表1所示的培养基。含卡那霉素的培养基由表1所示的培养基和卡那霉素组成，卡那霉素的含量为50μg/ml。如无特殊说明，实施例中的培养指的是32℃静置培养。实施例中的单链构象多态性聚丙烯酰胺凝胶电泳(sscp-PAGE)：采用的胶浓度为8％，电泳胶的组成见表2；电泳条件为：使用1×TBE缓冲液，120V电压，电泳时间10h。

如无特殊说明，以下实施例中的定量试验，均设置三次重复实验，结果取平均值。

表1

表2

实施例1、谷氨酸棒杆菌CGMCC21260的获得

谷氨酸棒杆菌ATCC15168：ATCC中编号为15168的谷氨酸棒杆菌(Corynebacteriumglutamicum)。

将谷氨酸棒杆菌ATCC15168进行诱变，获得谷氨酸棒杆菌(Corynebacteriumglutamicum)YPFV1。

谷氨酸棒杆菌(Corynebacterium glutamicum)YPFV1，已于2020年11月30日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址为：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所)，保藏登记号为CGMCC No.21260。谷氨酸棒杆菌(Corynebacterium glutamicum)YPFV1，又称为谷氨酸棒杆菌CGMCC21260。

实施例2、构建重组菌YPV-031

P1：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGGCTTCGTTTGCTTGAGCGTG-3'；

P2：5'-CGCTGCAGGTTGCATTGAACCACGAAGGTCAGGT-3'；

P3：5'-ACCTGACCTTCGTGGTTCAATGCAACCTGCAGCG-3'；

P4：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTAGCCACCGAGCGCTGCGCG-3'。

P5：5'-ACGTTTCAACCGCTACCTCG-3'；

P6：5'-ATCCCACTCGCGACCCCAAAC-3'。

一、构建重组质粒

1、以谷氨酸棒杆菌ATCC15168为模板，采用引物P1和引物P2组成的引物对进行PCR扩增，回收扩增产物(720bp)。

2、以谷氨酸棒杆菌ATCC15168为模板，采用引物P3和引物P4组成的引物对进行PCR扩增，回收扩增产物(678bp)。

3、同时将步骤1回收的扩增产物和步骤2回收的扩增产物作为模板，采用引物P1和引物P4组成的引物对进行PCR扩增(Overlap PCR)，回收扩增产物(1364bp)。经测序，扩增产物如序列表的序列5所示。

4、取pK18mobsacB质粒，采用限制性内切酶Xba I进行单酶切，回收线性化质粒。

5、将步骤3回收的扩增产物与步骤4回收的线性化质粒共孵育(采用NEBuilder酶，50℃孵育30min)，得到重组质粒pK18-YH66-RS10865^A844C,C846A。经测序验证，重组质粒pK18-YH66-RS10865^A844C,C846A中具有序列表的序列5所示的DNA分子。

二、构建重组菌YPV-031

1、采用重组质粒pK18-YH66-RS10865^A844C,C846A对谷氨酸棒杆菌CGMCC21260进行电击转化，然后培养。

2、挑取步骤1中的菌株，采用含15％蔗糖的培养基培养，然后挑取单菌落，分别采用含卡那霉素的培养基和不含卡那霉素的培养基进行培养，筛选在含卡那霉素的培养基上不能生长且在不含卡那霉素的培养基上可以生长的菌株。

3、取步骤2筛选的菌株，采用引物P5和引物P6组成的引物对进行PCR扩增，然后回收扩增产物(283bp)。

4、取步骤3的扩增产物，先95℃变性10min再冰浴5min，然后进行sscp-PAGE。电泳时，采用重组质粒pK18-YH66-RS10865^A844C,C846A的扩增片段(即以重组质粒pK18-YH66-RS10865^A844C,C846A为模板，采用引物P5和引物P6组成的引物对进行PCR扩增的扩增产物)为阳性对照，采用谷氨酸棒杆菌CGMCC21260的扩增片段(即以谷氨酸棒杆菌CGMCC21260为模板，采用引物P5和引物P6组成的引物对进行PCR扩增的扩增产物)为阴性对照，水作为空白对照。由于片段结构不同，电泳位置不同，电泳位置与阴性对照不一致且与阳性对照一致的菌株为筛选的目的菌株(等位替换成功的重组菌株)。

5、根据步骤4的结果，将筛选得到的菌株的步骤3的扩增产物进行测序验证，得到重组菌YPV-031。与谷氨酸棒杆菌CGMCC21260相比，重组菌YPV-031的差异仅在于：将谷氨酸棒杆菌CGMCC21260基因组中的序列表的序列4所示的YH66-RS10865基因取代为了序列表的序列2所示的YH66-RS10865^A844C,C846A基因。序列2和序列4仅存在两个核苷酸差异，位于第844位和第846位。重组菌YPV-031即将谷氨酸棒杆菌CGMCC21260中的YH66-RS10865基因进行突变(两个位点突变)得到的工程菌株。

实施例2、构建重组菌YPV-032和重组菌YPV-033

P7：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGCATGACGGCTGACTGGACTC-3'；

P8：5'-ACTGGGATTTCAGGTATCCAAATCGGACTCCTTAAATGGG-3'；

P9：5'-CCCATTTAAGGAGTCCGATTTGGATACCTGAAATCCCAGT-3'；

P10：5'-CTATGTGAGTAGTCGATTTATTATTCCTCAGGAGCGTTTG-3'；

P11：5'-CAAACGCTCCTGAGGAATAATAAATCGACTACTCACATAG-3'；

P12：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCTGCATAAGAAACAACCACTT-3'。

P13：5'-GTCCGCTCTGTTGGTGTTCA-3'；

P14：5'-TGTTACCGCGGACAGGTCCG-3'。

P15：5'-GCAAGCAGCAGATCGCTACC-3'；

P16：5'-TGGAGGAATATTCGGCCCAG-3'。

一、构建重组菌YPV-033

1、以重组菌YPV-031为模板，采用引物P7和引物P8组成的引物对进行PCR扩增，回收扩增产物(806bp)。

2、以重组菌YPV-031为模板，采用引物P9和引物P10组成的引物对进行PCR扩增，回收扩增产物(3014bp)。

3、以重组菌YPV-031为模板，采用引物P11和引物P12组成的引物对进行PCR扩增，回收扩增产物(783bp)。

4、取pK18mobsacB质粒，采用限制性内切酶Xba I进行单酶切，回收线性化质粒。

5、将步骤1回收的扩增产物、步骤2回收的扩增产物、步骤3回收的扩增产物与步骤4回收的线性化质粒共孵育(采用NEBuilder酶，50℃孵育30min)，得到重组质粒033。经测序验证，重组质粒033中具有序列表的序列6所示的DNA分子。

6、采用重组质粒033对谷氨酸棒杆菌CGMCC21260进行电击转化，然后培养，然后对各个单菌落分别进行PCR鉴定(采用引物P13和引物P14组成的引物对)，能扩增出1921bp条带的菌株为阳性菌株。

7、挑取步骤6中的阳性菌株，采用含15％蔗糖的培养基培养，然后挑取单菌落，分别采用含卡那霉素的培养基和不含卡那霉素的培养基进行培养，筛选在含卡那霉素的培养基上不能生长且在不含卡那霉素的培养基上可以生长的菌株。

8、取步骤7筛选的菌株，采用引物P15和引物P16组成的引物对进行PCR扩增，扩增出2013bp条带的菌株为YH66-RS10865^A844C,C846A基因整合到谷氨酸棒杆菌CGMCC21260基因组上的阳性菌株，将其命名为重组菌YPV-033。重组菌YPV-033为基因组上过表达YH66-RS10865^A844C,C846A基因的工程菌株。

二、构建重组菌YPV-032

将模板均由“重组菌YPV-031”替换为“谷氨酸棒杆菌ATCC15168”，其他同步骤一。

得到YH66-RS10865基因整合到谷氨酸棒杆菌CGMCC21260基因组上的阳性菌株，将其命名为重组菌YPV-032。重组菌YPV-032为基因组上过表达YH66-RS10865基因的工程菌株。与重组菌YPV-033相比，重组菌YPV-032的差异仅在于：整合到谷氨酸棒杆菌CGMCC21260基因组的外源DNA的序列中，序列4取代了序列2。

实施例3、构建重组菌YPV-035和重组菌YPV-034

一、构建重组菌YPV-035

1、以重组菌YPV-031为模板，采用引物P17和引物P18组成的引物对进行PCR扩增，回收扩增产物(3044bp)。经测序，扩增产物如序列表的序列7所示。

P17：5'-GCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCTGGATACCTGAAATCCCAGT-3'；

P18：5'-ATCAGGCTGAAAATCTTCTCTCATCCGCCAAAACTTATTCCTCAGGAGCGTTTG-3'。

2、取pXMJ19质粒，采用限制性内切酶EcoR I酶切进行单酶切，回收线性化质粒。

3、将步骤1回收的扩增产物与步骤2回收的线性化质粒共孵育(采用NEBuilder酶，50℃孵育30min)，得到重组质粒pXMJ19-YH66-RS10865^A844C,C846A。经测序验证，重组质粒pXMJ19-YH66-RS10865^A844C,C846A中具有序列表的序列7所示的DNA分子。

4、将重组质粒pXMJ19-YH66-RS10865^A844C,C846A电转导入谷氨酸棒杆菌CGMCC21260，得到重组菌YPV-035。重组菌YPV-035为通过质粒过表达pXMJ19-YH66-RS10865^A844C,C846A基因的工程菌株。

二、构建重组菌YPV-034

将模板由“重组菌YPV-031”替换为“谷氨酸棒杆菌ATCC15168”，其他同步骤一。

得到重组菌YPV-034。重组菌YPV-034为通过质粒过表达YH66-RS10865基因的工程菌株。与重组菌YPV-035相比，重组菌YPV-034的差异仅在于：通过质粒过表达的外源DNA的序列中，序列4取代了序列2。

实施例4、构建基因组上缺失YH66-RS10865基因的工程菌株

P19：5'-CAGTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGTCGGCCACTTTAATAATCCT-3'；

P20：5'-TATCTGTCCCTTGAGGTGATTTCCACACCTCCTGTTGGAA-3'；

P21：5'-TTCCAACAGGAGGTGTGGAAATCACCTCAAGGGACAGATA-3'；

P22：5'-CAGCTATGACCATGATTACGAATTCGAGCTCGGTACCCAACAATGTAAACAAGATGTC-3'。

P23：5'-TCGGCCACTTTAATAATCCT-3'；

P24：5'-AACAATGTAAACAAGATGTC-3'。

一、构建重组质粒

1、以谷氨酸棒杆菌ATCC15168为模板，采用引物P19和引物P20组成的引物对进行PCR扩增，回收扩增产物(上游同源臂片段，730bp)。

2、以谷氨酸棒杆菌ATCC15168为模板，采用引物P21和引物P22组成的引物对进行PCR扩增，回收扩增产物(下游同源臂片段，725bp)。

3、同时将步骤1回收的扩增产物和步骤2回收的扩增产物作为模板，采用引物P19和引物P22组成的引物对进行PCR扩增(Overlap PCR)，回收扩增产物。经测序，扩增产物如序列表的序列8所示。

4、取pK18mobsacB质粒，采用限制性内切酶Xba I进行单酶切，回收线性化质粒。

5、将步骤3回收的扩增产物与步骤4回收的线性化质粒共孵育(采用NEBuilder酶，50℃孵育30min)，得到重组质粒pK18-ΔYH66-RS10865。经测序，重组质粒pK18-ΔYH66-RS10865中具有序列表的序列8所示的DNA分子。

二、构建重组菌YPV-036

1、采用重组质粒pK18-ΔYH66-RS10865对谷氨酸棒杆菌CGMCC21260进行电击转化，然后培养，然后对各个单菌落分别进行PCR鉴定(采用引物P23和引物P24组成的引物对)。能同时扩增出1341bp及4110bp条带的菌株为阳性菌株。只扩增出4110bp条带的菌株为转化失败的出发菌，其中的4110bp片段如序列表的序列9所示。

2、挑取步骤1中的阳性菌株，采用含15％蔗糖的培养基培养，然后挑取单菌落，分别采用含卡那霉素的培养基和不含卡那霉素的培养基进行培养，筛选在含卡那霉素的培养基上不能生长且在不含卡那霉素的培养基上可以生长的菌株。

3、取步骤2筛选的菌株，采用引物P23和引物P24组成的引物对进行PCR扩增，只显示单一扩增产物且大小为1341bp的菌株为YH66-RS10865基因编码区被敲除的阳性菌株。

4、取步骤3筛选得到的菌株，再次采用引物P23和引物P24组成的引物对进行PCR扩增并测序，将测序正确的菌株命名为重组菌YPV-036。与谷氨酸棒杆菌CGMCC21260的基因组DNA相比，重组菌YPV-036的差异仅在于缺失了序列表的序列4所示的DNA分子。

实施例5、发酵制备L-缬氨酸

供试菌株分别为：谷氨酸棒杆菌CGMCC21260、重组菌YPV-031、重组菌YPV-032、重组菌YPV-033、重组菌YPV-034、重组菌YPV-035和重组菌YPV-036。

发酵罐：BLBIO-5GC-4-H型号的发酵罐(上海百仑生物科技有限公司)。

发酵培养基的配方见表3，余量为水。

表3发酵培养基配方

发酵控制工艺见表4。

完成接种的初始时刻，体系OD值为0.3-0.5。

发酵过程中：用于调pH的为氨水；发酵体系中有泡沫时，加入适量消泡剂antifoam(CB-442)；通过补加70％葡萄糖水溶液控制体系含糖量(残糖)。

表4发酵控制工艺

完成发酵后，收集上清，采用HPLC检测上清中的L-缬氨酸产量。

结果见表5。重组菌YPV-031和重组菌YPV-036的L-缬氨酸产量显著高于谷氨酸棒杆菌CGMCC21260。结果表明，抑制YH66-RS10865基因表达可以提高L-缬氨酸产量，过表达YH66-RS10865基因降低L-缬氨酸产量。

表5 L-缬氨酸发酵实验结果

以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。

序列表

<110> 宁夏伊品生物科技股份有限公司

<120> YH66-RS10865基因改造得到的工程菌及其在制备缬氨酸中的应用

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660 665 670

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

675 680 685

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

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

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

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

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

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

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

885 890 895

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

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 2

<211> 2769

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 2

atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60

cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120

gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180

cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240

tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300

aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360

ccaggcatcg gcgtcggcgg gcacatttcc acttacgcag gcgcagcccc tctgtacgaa 420

gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480

ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540

gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg tattccgtcc 600

taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660

ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720

aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780

gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840

gttcaatgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900

gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960

gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020

acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080

ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140

tggaagctgc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200

cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260

ggccacaatt tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320

ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380

ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440

gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500

caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560

gctaccacca tggcgactgt tcgcaccttc aaggaactga tgcgcgataa gggcttggct 1620

gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680

ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740

atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800

ggttccatgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860

attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920

gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980

ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040

gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100

ggcatcgacc gcatgtacgg cccaggcaag ggcgaagatg ttatctacta catcaccatc 2160

tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220

aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280

tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340

gttcgtgcaa acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400

cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460

cagctgaagc agacctctgg cccatacgtc gcagtgtctg acttctccac tgatctgcca 2520

aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580

ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640

gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700

gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760

gaggaataa 2769

<210> 3

<211> 922

<212> PRT

<213> Corynebacterium glutamicum

<400> 3

Met Ala Asp Gln Ala Lys Leu Gly Gly Lys Pro Ser Asp Asp Ser Asn

1 5 10 15

Phe Ala Met Ile Arg Asp Gly Val Ala Ser Tyr Leu Asn Asp Ser Asp

20 25 30

Pro Glu Glu Thr Asn Glu Trp Met Asp Ser Leu Asp Gly Leu Leu Gln

35 40 45

Glu Ser Ser Pro Glu Arg Ala Arg Tyr Leu Met Leu Arg Leu Leu Glu

50 55 60

Arg Ala Ser Ala Lys Arg Val Ser Leu Pro Pro Met Thr Ser Thr Asp

65 70 75 80

Tyr Val Asn Thr Ile Pro Thr Ser Met Glu Pro Glu Phe Pro Gly Asp

85 90 95

Glu Glu Met Glu Lys Arg Tyr Arg Arg Trp Ile Arg Trp Asn Ala Ala

100 105 110

Ile Met Val His Arg Ala Gln Arg Pro Gly Ile Gly Val Gly Gly His

115 120 125

Ile Ser Thr Tyr Ala Gly Ala Ala Pro Leu Tyr Glu Val Gly Phe Asn

130 135 140

His Phe Phe Arg Gly Lys Asp His Pro Gly Gly Gly Asp Gln Ile Phe

145 150 155 160

Phe Gln Gly His Ala Ser Pro Gly Met Tyr Ala Arg Ala Phe Met Glu

165 170 175

Gly Arg Leu Ser Glu Asp Asp Leu Asp Gly Phe Arg Gln Glu Val Ser

180 185 190

Arg Glu Gln Gly Gly Ile Pro Ser Tyr Pro His Pro His Gly Met Lys

195 200 205

Asp Phe Trp Glu Phe Pro Thr Val Ser Met Gly Leu Gly Pro Met Asp

210 215 220

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

225 230 235 240

Lys Asp Thr Ser Asp Gln His Val Trp Ala Phe Leu Gly Asp Gly Glu

245 250 255

Met Asp Glu Pro Glu Ser Arg Gly Leu Ile Gln Gln Ala Ala Leu Asn

260 265 270

Asn Leu Asp Asn Leu Thr Phe Val Val Asn Cys Asn Leu Gln Arg Leu

275 280 285

Asp Gly Pro Val Arg Gly Asn Thr Lys Ile Ile Gln Glu Leu Glu Ser

290 295 300

Phe Phe Arg Gly Ala Gly Trp Ser Val Ile Lys Val Val Trp Gly Arg

305 310 315 320

Glu Trp Asp Glu Leu Leu Glu Lys Asp Gln Asp Gly Ala Leu Val Glu

325 330 335

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

340 345 350

Asp Gly Ala Tyr Val Arg Glu His Phe Phe Gly Arg Asp Pro Arg Thr

355 360 365

Ala Lys Leu Val Glu Asn Met Thr Asp Glu Glu Ile Trp Lys Leu Pro

370 375 380

Arg Gly Gly His Asp Tyr Arg Lys Val Tyr Ala Ala Tyr Lys Arg Ala

385 390 395 400

Leu Glu Thr Lys Asp Arg Pro Thr Val Ile Leu Ala His Thr Ile Lys

405 410 415

Gly Tyr Gly Leu Gly His Asn Phe Glu Gly Arg Asn Ala Thr His Gln

420 425 430

Met Lys Lys Leu Thr Leu Asp Asp Leu Lys Leu Phe Arg Asp Lys Gln

435 440 445

Gly Ile Pro Ile Thr Asp Glu Gln Leu Glu Lys Asp Pro Tyr Leu Pro

450 455 460

Pro Tyr Tyr His Pro Gly Glu Asp Ala Pro Glu Ile Lys Tyr Met Lys

465 470 475 480

Glu Arg Arg Ala Ala Leu Gly Gly Tyr Leu Pro Glu Arg Arg Glu Asn

485 490 495

Tyr Asp Pro Ile Gln Val Pro Pro Leu Asp Lys Leu Arg Ser Val Arg

500 505 510

Lys Gly Ser Gly Lys Gln Gln Ile Ala Thr Thr Met Ala Thr Val Arg

515 520 525

Thr Phe Lys Glu Leu Met Arg Asp Lys Gly Leu Ala Asp Arg Leu Val

530 535 540

Pro Ile Ile Pro Asp Glu Ala Arg Thr Phe Gly Leu Asp Ser Trp Phe

545 550 555 560

Pro Thr Leu Lys Ile Tyr Asn Pro His Gly Gln Asn Tyr Val Pro Val

565 570 575

Asp His Asp Leu Met Leu Ser Tyr Arg Glu Ala Pro Glu Gly Gln Ile

580 585 590

Leu His Glu Gly Ile Asn Glu Ala Gly Ser Met Ala Ser Phe Ile Ala

595 600 605

Ala Gly Thr Ser Tyr Ala Thr His Gly Lys Ala Met Ile Pro Leu Tyr

610 615 620

Ile Phe Tyr Ser Met Phe Gly Phe Gln Arg Thr Gly Asp Ser Ile Trp

625 630 635 640

Ala Ala Ala Asp Gln Met Ala Arg Gly Phe Leu Leu Gly Ala Thr Ala

645 650 655

Gly Arg Thr Thr Leu Thr Gly Glu Gly Leu Gln His Met Asp Gly His

660 665 670

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

675 680 685

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

690 695 700

Met Tyr Gly Pro Gly Lys Gly Glu Asp Val Ile Tyr Tyr Ile Thr Ile

705 710 715 720

Tyr Asn Glu Pro Thr Pro Gln Pro Ala Glu Pro Glu Gly Leu Asp Val

725 730 735

Glu Gly Leu His Lys Gly Ile Tyr Leu Tyr Ser Arg Gly Glu Gly Thr

740 745 750

Gly His Glu Ala Asn Ile Leu Ala Ser Gly Val Gly Met Gln Trp Ala

755 760 765

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

770 775 780

Ile Tyr Ser Ala Thr Ser Trp Val Asn Leu Ala Arg Asp Gly Ala Ala

785 790 795 800

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

805 810 815

Phe Val Thr Thr Gln Leu Lys Gln Thr Ser Gly Pro Tyr Val Ala Val

820 825 830

Ser Asp Phe Ser Thr Asp Leu Pro Asn Gln Ile Arg Glu Trp Val Pro

835 840 845

Gly Asp Tyr Thr Val Leu Gly Ala Asp Gly Phe Gly Phe Ser Asp Thr

850 855 860

Arg Pro Ala Ala Arg Arg Phe Phe Asn Ile Asp Ala Glu Ser Ile Val

865 870 875 880

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

885 890 895

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

900 905 910

Val Ser Val Asp Pro Asn Ala Pro Glu Glu

915 920

<210> 4

<211> 2769

<212> DNA

<213> Corynebacterium glutamicum

<400> 4

atggccgatc aagcaaaact tggtggcaag ccctcggatg actctaactt cgcgatgatc 60

cgcgatggcg tggcatctta tttgaacgac tcagatccgg aggagaccaa cgagtggatg 120

gattcactcg acggattact ccaggagtct tctccagaac gtgctcgtta cctcatgctt 180

cgtttgcttg agcgtgcatc tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac 240

tacgtcaaca ccattccaac ctctatggaa cctgaattcc caggcgatga ggaaatggag 300

aagcgttacc gtcgttggat tcgctggaac gcagccatca tggttcaccg cgctcagcga 360

ccaggcatcg gcgtcggcgg gcacatttcc acttacgcag gcgcagcccc tctgtacgaa 420

gttggcttca accacttctt ccgcggcaag gatcacccag gcggcggcga ccagatcttc 480

ttccagggcc acgcatcacc aggtatgtac gcacgtgcat tcatggaggg tcgcctttct 540

gaagacgatc tcgatggctt ccgtcaggaa gtttcccgtg agcagggtgg tattccgtcc 600

taccctcacc cacacggtat gaaggacttc tgggagttcc caactgtgtc catgggtctt 660

ggcccaatgg atgccattta ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc 720

aaggacacct ctgaccagca cgtctgggcc ttccttggcg acggcgaaat ggacgagcca 780

gaatcacgtg gtctcatcca gcaggctgca ctgaacaacc tggacaacct gaccttcgtg 840

gttaactgca acctgcagcg tctcgacgga cctgtccgcg gtaacaccaa gatcatccag 900

gaactcgagt ccttcttccg tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc 960

gagtgggatg aacttctgga gaaggaccag gatggtgcac ttgttgagat catgaacaac 1020

acctccgatg gtgactacca gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac 1080

ttcttcggac gtgacccacg caccgcaaag ctcgttgaga acatgaccga cgaagaaatc 1140

tggaagctgc cacgtggcgg ccacgattac cgcaaggttt acgcagccta caagcgagct 1200

cttgagacca aggatcgccc aaccgtcatc cttgctcaca ccattaaggg ctacggactc 1260

ggccacaatt tcgaaggccg taacgcaacc caccagatga agaagctgac gcttgatgat 1320

ctgaagttgt tccgcgacaa gcagggcatc ccaatcaccg atgagcagct ggagaaggat 1380

ccttaccttc ctccttacta ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag 1440

gaacgtcgcg cagcgctcgg tggctacctg ccagagcgtc gtgagaacta cgatccaatt 1500

caggttccac cactggataa gcttcgctct gtccgtaagg gctccggcaa gcagcagatc 1560

gctaccacca tggcgactgt tcgcaccttc aaggaactga tgcgcgataa gggcttggct 1620

gatcgccttg tcccaatcat tcctgatgag gcacgtacct tcggtcttga ctcttggttc 1680

ccaaccttga agatctacaa cccgcacggt cagaactacg tgcctgttga ccacgacctg 1740

atgctctcct accgtgaggc acctgaagga cagatcctgc acgaaggcat caacgaggct 1800

ggttccatgg catcgttcat cgctgcgggt acctcctacg ccacccacgg caaggccatg 1860

attccgctgt acatcttcta ctcgatgttc ggattccagc gcaccggtga ctccatctgg 1920

gcagcagccg atcagatggc acgtggcttc ctcttgggcg ctaccgcagg tcgcaccacc 1980

ctgaccggtg aaggcctcca gcacatggat ggacactccc ctgtcttggc ttccaccaac 2040

gagggtgtcg agacctacga cccatccttt gcgtacgaga tcgcacacct ggttcaccgt 2100

ggcatcgacc gcatgtacgg cccaggcaag ggcgaagatg ttatctacta catcaccatc 2160

tacaacgagc caaccccaca gccagctgag ccagaaggac tggacgtaga aggcctgcac 2220

aagggcatct acctctactc ccgcggtgaa ggcaccggcc atgaggcaaa catcttggct 2280

tccggtgttg gtatgcagtg ggctctcaag gctgcatcca tccttgaggc tgactacgga 2340

gttcgtgcaa acatttactc cgctacttct tgggttaact tggctcgcga tggcgctgct 2400

cgtaacaagg cacagctgcg caacccaggt gcagatgctg gcgaggcatt cgtaaccacc 2460

cagctgaagc agacctctgg cccatacgtc gcagtgtctg acttctccac tgatctgcca 2520

aaccagatcc gtgaatgggt cccaggcgac tacaccgttc tcggtgcaga tggcttcggt 2580

ttctctgata cccgcccagc tgctcgtcgc ttcttcaaca tcgacgctga gtccattgtt 2640

gttgcagtgc tgaactccct ggcacgcgaa ggcaagatcg acgtctccgt tgctgctcag 2700

gctgctgaga agttcaagtt ggatgatcct acgagtgttt ccgtagatcc aaacgctcct 2760

gaggaataa 2769

<210> 5

<211> 1364

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 5

cagtgccaag cttgcatgcc tgcaggtcga ctctaggctt cgtttgcttg agcgtgcatc 60

tgcaaagcgc gtatctcttc ccccaatgac gtcaaccgac tacgtcaaca ccattccaac 120

ctctatggaa cctgaattcc caggcgatga ggaaatggag aagcgttacc gtcgttggat 180

tcgctggaac gcagccatca tggttcaccg cgctcagcga ccaggcatcg gcgtcggcgg 240

gcacatttcc acttacgcag gcgcagcccc tctgtacgaa gttggcttca accacttctt 300

ccgcggcaag gatcacccag gcggcggcga ccagatcttc ttccagggcc acgcatcacc 360

aggtatgtac gcacgtgcat tcatggaggg tcgcctttct gaagacgatc tcgatggctt 420

ccgtcaggaa gtttcccgtg agcagggtgg tattccgtcc taccctcacc cacacggtat 480

gaaggacttc tgggagttcc caactgtgtc catgggtctt ggcccaatgg atgccattta 540

ccaggcacgt ttcaaccgct acctcgaaaa ccgtggcatc aaggacacct ctgaccagca 600

cgtctgggcc ttccttggcg acggcgaaat ggacgagcca gaatcacgtg gtctcatcca 660

gcaggctgca ctgaacaacc tggacaacct gaccttcgtg gttcaatgca acctgcagcg 720

tctcgacgga cctgtccgcg gtaacaccaa gatcatccag gaactcgagt ccttcttccg 780

tggcgcaggc tggtctgtga tcaaggttgt ttggggtcgc gagtgggatg aacttctgga 840

gaaggaccag gatggtgcac ttgttgagat catgaacaac acctccgatg gtgactacca 900

gaccttcaag gctaacgacg gcgcatatgt tcgtgagcac ttcttcggac gtgacccacg 960

caccgcaaag ctcgttgaga acatgaccga cgaagaaatc tggaagctgc cacgtggcgg 1020

ccacgattac cgcaaggttt acgcagccta caagcgagct cttgagacca aggatcgccc 1080

aaccgtcatc cttgctcaca ccattaaggg ctacggactc ggccacaatt tcgaaggccg 1140

taacgcaacc caccagatga agaagctgac gcttgatgat ctgaagttgt tccgcgacaa 1200

gcagggcatc ccaatcaccg atgagcagct ggagaaggat ccttaccttc ctccttacta 1260

ccacccaggt gaagacgctc ctgaaatcaa gtacatgaag gaacgtcgcg cagcgctcgg 1320

tggctagggt accgagctcg aattcgtaat catggtcata gctg 1364

<210> 6

<211> 4523

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 6

cagtgccaag cttgcatgcc tgcaggtcga ctctagcatg acggctgact ggactcgact 60

tccatacgag gttctggaga agatctccac ccgcatcacc aacgaagttc cagatgtgaa 120

ccgcgtggtt ttggacgtaa cctccaagcc accaggaacc atcgaatggg agtaggcctt 180

aaatgagcct tcgttaagcg gcaatcacct tattggagat tgtcgctttt cccatttctc 240

cgggttttct ggaacttttt gggcgtatgc tgggaatgat tctattattg ccaaatcaga 300

aagcaggaga gacccgatga gcgaaatcct agaaacctat tgggcacccc actttggaaa 360

aaccgaagaa gccacagcac tcgtttcata cctggcacaa gcttccggcg atcccattga 420

ggttcacacc ctgttcgggg atttaggttt agacggactc tcgggaaact acaccgacac 480

tgagattgac ggctacggcg acgcattcct gctggttgca gcgctatccg tgttgatggc 540

tgaaaacaaa gcaacaggtg gcgtgaatct gggtgagctt gggggagctg ataaatcgat 600

ccggctgcat gttgaatcca aggagaacac ccaaatcaac accgcattga agtattttgc 660

gctctcccca gaagaccacg cagcagcaga tcgcttcgat gaggatgacc tgtctgagct 720

tgccaacttg agtgaagagc tgcgcggaca gctggactaa ttgtctccca tttaaggagt 780

ccgatttgga tacctgaaat cccagtgagc gcaccactcc ccttacgtca cagtctgtaa 840

aacaaatctt cggtgttgcg tatccttgtt aataacttat gcgttgactc attcgtgcac 900

ttcggcgtgt cacaattagg tacgaccaag aatgggaccg ggaaaccggg acgtataaac 960

gaaataaaac attccaacag gaggtgtgga aatggccgat caagcaaaac ttggtggcaa 1020

gccctcggat gactctaact tcgcgatgat ccgcgatggc gtggcatctt atttgaacga 1080

ctcagatccg gaggagacca acgagtggat ggattcactc gacggattac tccaggagtc 1140

ttctccagaa cgtgctcgtt acctcatgct tcgtttgctt gagcgtgcat ctgcaaagcg 1200

cgtatctctt cccccaatga cgtcaaccga ctacgtcaac accattccaa cctctatgga 1260

acctgaattc ccaggcgatg aggaaatgga gaagcgttac cgtcgttgga ttcgctggaa 1320

cgcagccatc atggttcacc gcgctcagcg accaggcatc ggcgtcggcg ggcacatttc 1380

cacttacgca ggcgcagccc ctctgtacga agttggcttc aaccacttct tccgcggcaa 1440

ggatcaccca ggcggcggcg accagatctt cttccagggc cacgcatcac caggtatgta 1500

cgcacgtgca ttcatggagg gtcgcctttc tgaagacgat ctcgatggct tccgtcagga 1560

agtttcccgt gagcagggtg gtattccgtc ctaccctcac ccacacggta tgaaggactt 1620

ctgggagttc ccaactgtgt ccatgggtct tggcccaatg gatgccattt accaggcacg 1680

tttcaaccgc tacctcgaaa accgtggcat caaggacacc tctgaccagc acgtctgggc 1740

cttccttggc gacggcgaaa tggacgagcc agaatcacgt ggtctcatcc agcaggctgc 1800

actgaacaac ctggacaacc tgaccttcgt ggttcaatgc aacctgcagc gtctcgacgg 1860

acctgtccgc ggtaacacca agatcatcca ggaactcgag tccttcttcc gtggcgcagg 1920

ctggtctgtg atcaaggttg tttggggtcg cgagtgggat gaacttctgg agaaggacca 1980

ggatggtgca cttgttgaga tcatgaacaa cacctccgat ggtgactacc agaccttcaa 2040

ggctaacgac ggcgcatatg ttcgtgagca cttcttcgga cgtgacccac gcaccgcaaa 2100

gctcgttgag aacatgaccg acgaagaaat ctggaagctg ccacgtggcg gccacgatta 2160

ccgcaaggtt tacgcagcct acaagcgagc tcttgagacc aaggatcgcc caaccgtcat 2220

ccttgctcac accattaagg gctacggact cggccacaat ttcgaaggcc gtaacgcaac 2280

ccaccagatg aagaagctga cgcttgatga tctgaagttg ttccgcgaca agcagggcat 2340

cccaatcacc gatgagcagc tggagaagga tccttacctt cctccttact accacccagg 2400

tgaagacgct cctgaaatca agtacatgaa ggaacgtcgc gcagcgctcg gtggctacct 2460

gccagagcgt cgtgagaact acgatccaat tcaggttcca ccactggata agcttcgctc 2520

tgtccgtaag ggctccggca agcagcagat cgctaccacc atggcgactg ttcgcacctt 2580

caaggaactg atgcgcgata agggcttggc tgatcgcctt gtcccaatca ttcctgatga 2640

ggcacgtacc ttcggtcttg actcttggtt cccaaccttg aagatctaca acccgcacgg 2700

tcagaactac gtgcctgttg accacgacct gatgctctcc taccgtgagg cacctgaagg 2760

acagatcctg cacgaaggca tcaacgaggc tggttccatg gcatcgttca tcgctgcggg 2820

tacctcctac gccacccacg gcaaggccat gattccgctg tacatcttct actcgatgtt 2880

cggattccag cgcaccggtg actccatctg ggcagcagcc gatcagatgg cacgtggctt 2940

cctcttgggc gctaccgcag gtcgcaccac cctgaccggt gaaggcctcc agcacatgga 3000

tggacactcc cctgtcttgg cttccaccaa cgagggtgtc gagacctacg acccatcctt 3060

tgcgtacgag atcgcacacc tggttcaccg tggcatcgac cgcatgtacg gcccaggcaa 3120

gggcgaagat gttatctact acatcaccat ctacaacgag ccaaccccac agccagctga 3180

gccagaagga ctggacgtag aaggcctgca caagggcatc tacctctact cccgcggtga 3240

aggcaccggc catgaggcaa acatcttggc ttccggtgtt ggtatgcagt gggctctcaa 3300

ggctgcatcc atccttgagg ctgactacgg agttcgtgca aacatttact ccgctacttc 3360

ttgggttaac ttggctcgcg atggcgctgc tcgtaacaag gcacagctgc gcaacccagg 3420

tgcagatgct ggcgaggcat tcgtaaccac ccagctgaag cagacctctg gcccatacgt 3480

cgcagtgtct gacttctcca ctgatctgcc aaaccagatc cgtgaatggg tcccaggcga 3540

ctacaccgtt ctcggtgcag atggcttcgg tttctctgat acccgcccag ctgctcgtcg 3600

cttcttcaac atcgacgctg agtccattgt tgttgcagtg ctgaactccc tggcacgcga 3660

aggcaagatc gacgtctccg ttgctgctca ggctgctgag aagttcaagt tggatgatcc 3720

tacgagtgtt tccgtagatc caaacgctcc tgaggaataa taaatcgact actcacatag 3780

ggtcgggcta gtcattctga tcagcgaatt ccacgttcac atcgccaatt ccagagttca 3840

caaccagatt cagcattgga ccttctagat cagcattgtg ggcggtgaga tctccaacat 3900

cacagcgcgc tgtgcccaca ccggcggtac aacttaggct cacgggcaca tcatcgggca 3960

gggtgaccat gacttcgccg atccctgagg tgatttggat gttttgttcc tgatccaatt 4020

gggtgaggtg gctgaaatcg aggttcattt cacccacgcc agaggtgtag ctgctgagga 4080

gttcatcgtt ggtggggatg agattgacat cgccgattcc agggtcgtct tcaaagtaga 4140

tgggatcgat atttgaaata aacaggcctg cgagggcgct catgacaact ccggtaccaa 4200

ctacaccgcc gacaatccat ggccacacat ggcgcttttt ctgaggcttt tgtggaggga 4260

cttgtacatc ccaggtgttg tattggtttt gggcaagtgg atcccaatga ggcgcttcgg 4320

gggtttgttg cgcgaagggt gcatagtagc cctcaacggg ggtgatagtg cttagatctg 4380

gttggggttg tgggtagaga tcttcgtttt tcatggtggc atcctcagaa acagtgaatt 4440

cagtggtgag tagtccgcgg ggtggaagtg gttgtttctt atgcagggta ccgagctcga 4500

attcgtaatc atggtcatag ctg 4523

<210> 7

<211> 3044

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 7

gcttgcatgc ctgcaggtcg actctagagg atcccctgga tacctgaaat cccagtgagc 60

gcaccactcc ccttacgtca cagtctgtaa aacaaatctt cggtgttgcg tatccttgtt 120

aataacttat gcgttgactc attcgtgcac ttcggcgtgt cacaattagg tacgaccaag 180

aatgggaccg ggaaaccggg acgtataaac gaaataaaac attccaacag gaggtgtgga 240

aatggccgat caagcaaaac ttggtggcaa gccctcggat gactctaact tcgcgatgat 300

ccgcgatggc gtggcatctt atttgaacga ctcagatccg gaggagacca acgagtggat 360

ggattcactc gacggattac tccaggagtc ttctccagaa cgtgctcgtt acctcatgct 420

tcgtttgctt gagcgtgcat ctgcaaagcg cgtatctctt cccccaatga cgtcaaccga 480

ctacgtcaac accattccaa cctctatgga acctgaattc ccaggcgatg aggaaatgga 540

gaagcgttac cgtcgttgga ttcgctggaa cgcagccatc atggttcacc gcgctcagcg 600

accaggcatc ggcgtcggcg ggcacatttc cacttacgca ggcgcagccc ctctgtacga 660

agttggcttc aaccacttct tccgcggcaa ggatcaccca ggcggcggcg accagatctt 720

cttccagggc cacgcatcac caggtatgta cgcacgtgca ttcatggagg gtcgcctttc 780

tgaagacgat ctcgatggct tccgtcagga agtttcccgt gagcagggtg gtattccgtc 840

ctaccctcac ccacacggta tgaaggactt ctgggagttc ccaactgtgt ccatgggtct 900

tggcccaatg gatgccattt accaggcacg tttcaaccgc tacctcgaaa accgtggcat 960

caaggacacc tctgaccagc acgtctgggc cttccttggc gacggcgaaa tggacgagcc 1020

agaatcacgt ggtctcatcc agcaggctgc actgaacaac ctggacaacc tgaccttcgt 1080

ggttcaatgc aacctgcagc gtctcgacgg acctgtccgc ggtaacacca agatcatcca 1140

ggaactcgag tccttcttcc gtggcgcagg ctggtctgtg atcaaggttg tttggggtcg 1200

cgagtgggat gaacttctgg agaaggacca ggatggtgca cttgttgaga tcatgaacaa 1260

cacctccgat ggtgactacc agaccttcaa ggctaacgac ggcgcatatg ttcgtgagca 1320

cttcttcgga cgtgacccac gcaccgcaaa gctcgttgag aacatgaccg acgaagaaat 1380

ctggaagctg ccacgtggcg gccacgatta ccgcaaggtt tacgcagcct acaagcgagc 1440

tcttgagacc aaggatcgcc caaccgtcat ccttgctcac accattaagg gctacggact 1500

cggccacaat ttcgaaggcc gtaacgcaac ccaccagatg aagaagctga cgcttgatga 1560

tctgaagttg ttccgcgaca agcagggcat cccaatcacc gatgagcagc tggagaagga 1620

tccttacctt cctccttact accacccagg tgaagacgct cctgaaatca agtacatgaa 1680

ggaacgtcgc gcagcgctcg gtggctacct gccagagcgt cgtgagaact acgatccaat 1740

tcaggttcca ccactggata agcttcgctc tgtccgtaag ggctccggca agcagcagat 1800

cgctaccacc atggcgactg ttcgcacctt caaggaactg atgcgcgata agggcttggc 1860

tgatcgcctt gtcccaatca ttcctgatga ggcacgtacc ttcggtcttg actcttggtt 1920

cccaaccttg aagatctaca acccgcacgg tcagaactac gtgcctgttg accacgacct 1980

gatgctctcc taccgtgagg cacctgaagg acagatcctg cacgaaggca tcaacgaggc 2040

tggttccatg gcatcgttca tcgctgcggg tacctcctac gccacccacg gcaaggccat 2100

gattccgctg tacatcttct actcgatgtt cggattccag cgcaccggtg actccatctg 2160

ggcagcagcc gatcagatgg cacgtggctt cctcttgggc gctaccgcag gtcgcaccac 2220

cctgaccggt gaaggcctcc agcacatgga tggacactcc cctgtcttgg cttccaccaa 2280

cgagggtgtc gagacctacg acccatcctt tgcgtacgag atcgcacacc tggttcaccg 2340

tggcatcgac cgcatgtacg gcccaggcaa gggcgaagat gttatctact acatcaccat 2400

ctacaacgag ccaaccccac agccagctga gccagaagga ctggacgtag aaggcctgca 2460

caagggcatc tacctctact cccgcggtga aggcaccggc catgaggcaa acatcttggc 2520

ttccggtgtt ggtatgcagt gggctctcaa ggctgcatcc atccttgagg ctgactacgg 2580

agttcgtgca aacatttact ccgctacttc ttgggttaac ttggctcgcg atggcgctgc 2640

tcgtaacaag gcacagctgc gcaacccagg tgcagatgct ggcgaggcat tcgtaaccac 2700

ccagctgaag cagacctctg gcccatacgt cgcagtgtct gacttctcca ctgatctgcc 2760

aaaccagatc cgtgaatggg tcccaggcga ctacaccgtt ctcggtgcag atggcttcgg 2820

tttctctgat acccgcccag ctgctcgtcg cttcttcaac atcgacgctg agtccattgt 2880

tgttgcagtg ctgaactccc tggcacgcga aggcaagatc gacgtctccg ttgctgctca 2940

ggctgctgag aagttcaagt tggatgatcc tacgagtgtt tccgtagatc caaacgctcc 3000

tgaggaataa gttttggcgg atgagagaag attttcagcc tgat 3044

<210> 8

<211> 1415

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 8

cagtgccaag cttgcatgcc tgcaggtcga ctctagtcgg ccactttaat aatcctcctc 60

gtgtgggccc cgatgtgttt ttcgattaca tggattcaac atgaaaccgc ggggctattg 120

atatatccga attgcacatt accgtccaac cggtactttg aaccaccttt ccctggaatt 180

ttttcctttt cctccccctt tacgctcaag aatcaatgaa ttcaatcact ggccagcgat 240

taacttttcg agttttcagt cttggatttc cacaattctc ttcaaaataa tggtggctag 300

atttttcatc aaaccttcac caaaaggaca tcagacctgt agttttatgc gattcgcgtc 360

aaacgtgaga gaaacatcac atctcgcggg aaactacccg ataattcttt gcaaaacttt 420

gcaaagcgga atgaacatgc agctagtttc cgtagaaatg ttctttaaaa aatccacaac 480

aattgccagg aagcacaccg attgatggat acctgaaatc ccagtgagcg caccactccc 540

cttacgtcac agtctgtaaa acaaatcttc ggtgttgcgt atccttgtta ataacttatg 600

cgttgactca ttcgtgcact tcggcgtgtc acaattaggt acgaccaaga atgggaccgg 660

gaaaccggga cgtataaacg aaataaaaca ttccaacagg aggtgtggaa atcacctcaa 720

gggacagata aatcccgccg ccagacgtta gtctggcggc gggattcgtc gtaaagcaag 780

ctctttttag ccgaggaacg ccttgtcaga caatgttgcg cccttgatgt tggcgaactc 840

ctgcagcaaa tcgcgcacag tcaacttcga cttggtagcc tgatctgcct ggtagacaat 900

ctggccttca tgcatcatga tcaggcgatt gcccaggcga attgcctgtt ccatgttgtg 960

cgtgaccata agcgtagtca gagttccatc tgccacgatc ttttcggtca aggtggtcac 1020

aagctctgca cgctgtggat caagtgctgc ggtgtgctca tccaacagca tgattttagg 1080

ttgagtaaaa ccagccatca gcagggacaa tgcctgacgc tgaccgccag agagcaaacc 1140

aactttggca gtgagcctgt tttccagacc cagctcgagg cgctcaagtt cctgcttgaa 1200

ttgctcacgg cgcttcgagg tcagtgcaaa gcccaatcca cggcgcttgc cgcgcagcaa 1260

cgcgatggcc agattctctt caatggtgag attcggcgcg gtgccggcca gaggatcctg 1320

gaaaacgcgg ccgatgtagc gggcacgctt gtgctctgac atcttgttta cattgttggg 1380

taccgagctc gaattcgtaa tcatggtcat agctg 1415

<210> 9

<211> 4110

<212> DNA

<213> 人工序列(Artificial Sequence)

<400> 9

tcggccactt taataatcct cctcgtgtgg gccccgatgt gtttttcgat tacatggatt 60

caacatgaaa ccgcggggct attgatatat ccgaattgca cattaccgtc caaccggtac 120

tttgaaccac ctttccctgg aattttttcc ttttcctccc cctttacgct caagaatcaa 180

tgaattcaat cactggccag cgattaactt ttcgagtttt cagtcttgga tttccacaat 240

tctcttcaaa ataatggtgg ctagattttt catcaaacct tcaccaaaag gacatcagac 300

ctgtagtttt atgcgattcg cgtcaaacgt gagagaaaca tcacatctcg cgggaaacta 360

cccgataatt ctttgcaaaa ctttgcaaag cggaatgaac atgcagctag tttccgtaga 420

aatgttcttt aaaaaatcca caacaattgc caggaagcac accgattgat ggatacctga 480

aatcccagtg agcgcaccac tccccttacg tcacagtctg taaaacaaat cttcggtgtt 540

gcgtatcctt gttaataact tatgcgttga ctcattcgtg cacttcggcg tgtcacaatt 600

aggtacgacc aagaatggga ccgggaaacc gggacgtata aacgaaataa aacattccaa 660

caggaggtgt ggaaatggcc gatcaagcaa aacttggtgg caagccctcg gatgactcta 720

acttcgcgat gatccgcgat ggcgtggcat cttatttgaa cgactcagat ccggaggaga 780

ccaacgagtg gatggattca ctcgacggat tactccagga gtcttctcca gaacgtgctc 840

gttacctcat gcttcgtttg cttgagcgtg catctgcaaa gcgcgtatct cttcccccaa 900

tgacgtcaac cgactacgtc aacaccattc caacctctat ggaacctgaa ttcccaggcg 960

atgaggaaat ggagaagcgt taccgtcgtt ggattcgctg gaacgcagcc atcatggttc 1020

accgcgctca gcgaccaggc atcggcgtcg gcgggcacat ttccacttac gcaggcgcag 1080

cccctctgta cgaagttggc ttcaaccact tcttccgcgg caaggatcac ccaggcggcg 1140

gcgaccagat cttcttccag ggccacgcat caccaggtat gtacgcacgt gcattcatgg 1200

agggtcgcct ttctgaagac gatctcgatg gcttccgtca ggaagtttcc cgtgagcagg 1260

gtggtattcc gtcctaccct cacccacacg gtatgaagga cttctgggag ttcccaactg 1320

tgtccatggg tcttggccca atggatgcca tttaccaggc acgtttcaac cgctacctcg 1380

aaaaccgtgg catcaaggac acctctgacc agcacgtctg ggccttcctt ggcgacggcg 1440

aaatggacga gccagaatca cgtggtctca tccagcaggc tgcactgaac aacctggaca 1500

acctgacctt cgtggttaac tgcaacctgc agcgtctcga cggacctgtc cgcggtaaca 1560

ccaagatcat ccaggaactc gagtccttct tccgtggcgc aggctggtct gtgatcaagg 1620

ttgtttgggg tcgcgagtgg gatgaacttc tggagaagga ccaggatggt gcacttgttg 1680

agatcatgaa caacacctcc gatggtgact accagacctt caaggctaac gacggcgcat 1740

atgttcgtga gcacttcttc ggacgtgacc cacgcaccgc aaagctcgtt gagaacatga 1800

ccgacgaaga aatctggaag ctgccacgtg gcggccacga ttaccgcaag gtttacgcag 1860

cctacaagcg agctcttgag accaaggatc gcccaaccgt catccttgct cacaccatta 1920

agggctacgg actcggccac aatttcgaag gccgtaacgc aacccaccag atgaagaagc 1980

tgacgcttga tgatctgaag ttgttccgcg acaagcaggg catcccaatc accgatgagc 2040

agctggagaa ggatccttac cttcctcctt actaccaccc aggtgaagac gctcctgaaa 2100

tcaagtacat gaaggaacgt cgcgcagcgc tcggtggcta cctgccagag cgtcgtgaga 2160

actacgatcc aattcaggtt ccaccactgg ataagcttcg ctctgtccgt aagggctccg 2220

gcaagcagca gatcgctacc accatggcga ctgttcgcac cttcaaggaa ctgatgcgcg 2280

ataagggctt ggctgatcgc cttgtcccaa tcattcctga tgaggcacgt accttcggtc 2340

ttgactcttg gttcccaacc ttgaagatct acaacccgca cggtcagaac tacgtgcctg 2400

ttgaccacga cctgatgctc tcctaccgtg aggcacctga aggacagatc ctgcacgaag 2460

gcatcaacga ggctggttcc atggcatcgt tcatcgctgc gggtacctcc tacgccaccc 2520

acggcaaggc catgattccg ctgtacatct tctactcgat gttcggattc cagcgcaccg 2580

gtgactccat ctgggcagca gccgatcaga tggcacgtgg cttcctcttg ggcgctaccg 2640

caggtcgcac caccctgacc ggtgaaggcc tccagcacat ggatggacac tcccctgtct 2700

tggcttccac caacgagggt gtcgagacct acgacccatc ctttgcgtac gagatcgcac 2760

acctggttca ccgtggcatc gaccgcatgt acggcccagg caagggcgaa gatgttatct 2820

actacatcac catctacaac gagccaaccc cacagccagc tgagccagaa ggactggacg 2880

tagaaggcct gcacaagggc atctacctct actcccgcgg tgaaggcacc ggccatgagg 2940

caaacatctt ggcttccggt gttggtatgc agtgggctct caaggctgca tccatccttg 3000

aggctgacta cggagttcgt gcaaacattt actccgctac ttcttgggtt aacttggctc 3060

gcgatggcgc tgctcgtaac aaggcacagc tgcgcaaccc aggtgcagat gctggcgagg 3120

cattcgtaac cacccagctg aagcagacct ctggcccata cgtcgcagtg tctgacttct 3180

ccactgatct gccaaaccag atccgtgaat gggtcccagg cgactacacc gttctcggtg 3240

cagatggctt cggtttctct gatacccgcc cagctgctcg tcgcttcttc aacatcgacg 3300

ctgagtccat tgttgttgca gtgctgaact ccctggcacg cgaaggcaag atcgacgtct 3360

ccgttgctgc tcaggctgct gagaagttca agttggatga tcctacgagt gtttccgtag 3420

atccaaacgc tcctgaggaa taaatcacct caagggacag ataaatcccg ccgccagacg 3480

ttagtctggc ggcgggattc gtcgtaaagc aagctctttt tagccgagga acgccttgtc 3540

agacaatgtt gcgcccttga tgttggcgaa ctcctgcagc aaatcgcgca cagtcaactt 3600

cgacttggta gcctgatctg cctggtagac aatctggcct tcatgcatca tgatcaggcg 3660

attgcccagg cgaattgcct gttccatgtt gtgcgtgacc ataagcgtag tcagagttcc 3720

atctgccacg atcttttcgg tcaaggtggt cacaagctct gcacgctgtg gatcaagtgc 3780

tgcggtgtgc tcatccaaca gcatgatttt aggttgagta aaaccagcca tcagcaggga 3840

caatgcctga cgctgaccgc cagagagcaa accaactttg gcagtgagcc tgttttccag 3900

acccagctcg aggcgctcaa gttcctgctt gaattgctca cggcgcttcg aggtcagtgc 3960

aaagcccaat ccacggcgct tgccgcgcag caacgcgatg gccagattct cttcaatggt 4020

gagattcggc gcggtgccgg ccagaggatc ctggaaaacg cggccgatgt agcgggcacg 4080

cttgtgctct gacatcttgt ttacattgtt 4110