CN106148380A - 一种高效穿梭表达载体及其构建方法与应用 - Google Patents
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Abstract
本发明公开了属于生物技术领域的一种高效穿梭表达载体及其构建方法与应用。本发明是在E. coli-B. subtilis穿梭载体pHT315的基础上构建的,通过将枯草芽孢杆菌Bs168的木糖启动子序列与来自E. coli表达载体pET21b的T7表达区序列融合,构建了一个高效穿梭表达载体pHTL,以gfp,gus和lipa为报告基因,检测载体的表达能力和遗传稳定性;同时,将来自纳豆芽孢杆菌的碱性蛋白酶信号肽序列代替载体pHTL上的T7肽编码区,构建成高效分泌型穿梭表达载体pHTZ,以gus,tgl1和lipa为报告基因,检测载体的表达能力。两种载体均可以正确表达相应蛋白并具有生物学活性,具有广泛的应用前景。
Description
技术领域
本发明属于生物技术领域,具体涉及一种大肠杆菌-枯草芽孢杆菌高效穿梭表达载体及其构建方法与应用。
背景技术
枯草芽孢杆菌(Bacillus subtilis)是一种极具潜力的外源蛋白表达宿主菌,不论是用来高效表达外源蛋白还是当做口服疫苗和全细胞酶催化剂的载体,都有着许多其他细菌无可比拟的优势,是研究较多、应用广泛的生物安全菌。B. subtilis特有的生物安全性、遗传操作简单、可廉价大规模培养及芽孢具有的较强的抗逆性,使得B. subtilis的基因工程菌产品,特别是一些工业酶制剂如α-淀粉酶、蛋白酶、碱性磷酸酶等,已占据了世界工业酶制剂产量的一半以上;还有一部分抗生素、维生素、肌苷、核黄素等也已进入商业化生产。从转录模式上分类,B. subtilis的启动子可分为组成型启动子和诱导型启动子。常见的组成型启动子有P43启动子,诱导型启动子有sacB启动子、麦芽糖启动子Pglv、Pspac启动子、PxylR启动子。PxylR启动子中编码木糖异构酶(XylA)的基因序列和木酮糖激酶(XylB)的基因序列构成了B. subtilis木糖代谢途径的重要调操纵子-木糖操纵子(xyl操纵子)。一般木糖操纵子片段包括整个xylR编码区序列,PxylA、PxylB、xylO、XylA前的CRE(catabolite-responsive
element)区和SD序列区。将外源基因的编码序列替代XylA,利用木糖代谢途径中木糖与xylR的相互作用,诱导控制PxylA启动子表达外源蛋白。
外源基因转入B. subtilis菌体内需要有载体的参与,主要是质粒载体。B. subtilis使用的第一代质粒载体主要来自其他革兰氏阳性细菌,如:pUB110、pHTZ21和pLS11等,第二代使用于B. subtilis的质粒载体是穿梭质粒,由第一代非B. subtilis的革兰氏阳性菌天然质粒与E. coli质粒(一般为pBR322或其衍生的质粒)构成的载体,第三代质粒载体是由B. subtilis的隐性质粒与E. coli质粒构成的嵌合载体,如:pHP3、pMTLBS72、pHT01和pHT43。pHP3由质粒pTA1060的复制子片段、质粒pE194的红霉素抗性基因(Emr)、质粒pC194的头孢菌素抗性基因(Cmr)和E. coli质粒pUC9的复制子片段组成,在E. coli和B. subtilis中都能表现所有的抗生素抗性,而且具有较高的B. subtilis转化效率和稳定的遗传性。质粒pMTLBS72由来自野生B. subtilis的质粒的复制子片段和E. coli质粒pMTL21C组合构建,具有较好遗传稳定性,以此为基础添加强启动子构建成功的质粒pHT01,pHT43是已经用于生产的商品质粒,在连续传代和发酵罐培养中都比较稳定。
随着基因工程的飞速发展,作为引导蛋白质分泌的信号肽也倍受关注。外源蛋白质在宿主菌,如大肠杆菌中的表达形式多为细胞内不溶性表达(包涵体),少数为细胞外分泌表达。利用信号肽的特性来引导外源蛋白质定位分泌到细胞特定区间,提高可溶性,可避免因包涵体复性带来的困难。目前研究采用的信号肽基本来自表达系统自身的信号序列或外源信号序列,或两者兼而有之。研究表明,多种外源基因连上信号肽后,在原核表达系统,如大肠杆菌、L型细菌、乳酸杆菌和芽孢杆菌等中都得到了分泌表达。信号肽因其特有的功能在外源基因的分泌表达上被广泛应用。
B. subtilis表达系统是继E. coli表达系统之后研究最为成熟的原核表达系统。B. subtilis作为外源蛋白分泌表达的宿主菌具有很大发展潜力。利用诱导方便,表达效率高的启动子构建稳定遗传的穿梭表达载体是实现外源蛋白高效表达的有效途径。因此,构建一个便于诱导、表达效率高和遗传操作简便的E. coli-B. subtilis穿梭表达载体,为今后研究更高效的穿梭表达载体奠定基础,对研制枯草芽孢杆菌工程菌的实验研究和工业生产的日益迫切的需求提供了更为广阔的前景。
发明内容
在本发明的目的是构建一个高效的含有木糖启动子的E. coli-B. subtilis穿梭表达载体和一个带有信号肽的分泌型穿梭表达载体并对其效率进行评估,以满足实验研究和工业生产的日益迫切的需求。
一种高效穿梭表达载体,所述表达载体为pHTL,其核苷酸序列如序列表SEQ ID NO:1所示。
所述高效穿梭表达载体的构建方法为:
(1)获得木糖启动子序列PxylR和大肠杆菌表达载体pET-21b中T7表达区序列的融合片段PxylR-T7;
所述表达区序列包括T7短肽,多克隆位点区,T7终止子;
(2)融合片段PxylR-T7插入经酶切修饰后的骨架载体pHT315的多克隆位点之间。
一种重组表达载体,其在上述高效穿梭表达载体的多克隆位点之间插入目的基因。
所述重组表达载体为pHTL-gfp,pHTL-gus和pHTL-lipa,其序列如SEQ ID NO:14、SEQ ID NO:15和SEQ ID NO:16所示。
包含上述重组表达载体的工程菌。
所述工程菌为B8-pHTLgfp,B8-pHTLgus和B8-pHTLlipa,含有上述重组表达载体pHTL-gfp,pHTL-gus,pHTL-lipa。
所述表达载体在基因gfp,gus和lipa表达中的应用,表达蛋白为GFP,GUS和LipA。
进一步的,所述步骤(1)的方法为:以B. subtilis 168菌株基因组DNA为模板,用引物对xyl-5/xyl-3扩增木糖启动子PxylR序列,大小为1473bp;以大肠杆菌表达载体pET-21b质粒为模板,用引物对T7-5/T7-3扩增得到无启动子的pET-21b中T7表达区片段,大小为213bp,其中包括T7短肽、多克隆位点区(MCS)、T7终止子;回收两个PCR产物,不添加引物,利用PrimeSTAR Max DNA
Polymerase进行互补延伸,形成融合片段的中间产物,以此为模板,用引物对xyl-5/T7-3进行融合PCR的扩增,获得木糖启动子PxylR片段和载体pET-21b的T7表达区的融合片段PxylR-T7,大小为1686bp,所述骨架载体的pHT315经EcoR /Hind 双酶切后,用Klenow酶和Fastap酶先后进行黏末端补平反应以及对补平的片段进行去磷酸化处理;
所述xyl-5序列如序列表SEQ ID NO:2所示,所述xyl-3序列如SEQ ID NO:3所示,
所述T7-5序列如SEQ ID NO:4所示,所述T7-3序列如SEQ ID NO:5所示。
一种分泌型穿梭表达载体,将来自纳豆芽孢杆菌碱性蛋白酶信号肽序列取代T7表达区序列插入上述载体pHTL的木糖启动子PxylR和多克隆位点之间。
所述分泌型穿梭表达载体的序列如序列表SEQ ID NO:17所示。
所述分泌型穿梭表达载体的构建方法为:
1)获得木糖启动子PxylR部分序列和纳豆芽孢杆菌碱性蛋白酶信号肽序列Apre的融合片段PxylRstuI-Apre;
2)插入经酶切后的骨架载体pHTL的PxylR中Stu I位点和多克隆位点的BamH I之间。
进一步的,所述步骤1)的方法为:以质粒pHTL为模板,用引物对xyls-5/xyls-3扩增木糖启动子PxylR部分序列PxylRstuI,大小为1167bp;以纳豆芽孢杆菌基因组为模板,用引物对apre-5/apre-3扩增得到纳豆芽孢杆菌中编码信号肽的DNA区段Apre,大小为96bp;回收两个PCR产物,不添加引物,利用PrimeSTAR Max DNA
Polymerase进行互补延伸,形成融合片段的中间产物,以此为模板,用引物对xyls-5/apre-3进行融合PCR的扩增,获得木糖启动子PxylRstuI和纳豆芽孢杆菌信号肽表达区的融合片段PxylRstuI-Apre,大小为1263bp,所述骨架载体的pHTL经Stu I/BamH I双酶切;
所述xyls-5序列如SEQ ID NO:18所示,所述xyls-3序列如SEQ ID NO:19所示,
所述apre-5序列如SEQ ID NO:20所示,所述apre-3序列如SEQ ID NO:21所示。
信号肽插入PxylR和多克隆位点之间的方法和限制性内切酶的选择:在PxylR和Apre相接处通过引物设计引入限制性内切酶PmlI的酶切位点CAC|GTG,在Apre和多克隆位点之间通过PCR引物设计引入了限制性内切酶Ngo MIV酶切位点序列G|CCGGC;同时在信号肽末端和Ngo MIV之间引入了有利于信号肽酶酶切的氨基酸序列AlaGluAla,其编码序列为:“GCGCAGGCT”。
所述分泌表达载体上替换Apre信号肽序列的策略:通过扩增信号肽的引物引入上述酶切位点,即5’端引物引入Pml I酶切位点、3’端引物引入Ngo MIV酶切,通过上述一对引物扩增得到的任何信号肽的DNA编码区可以通过这两个酶切位点插入pHTL的PxylR和多克隆位点之间,以替换Apre信号肽并提高多克隆位点处插入外源蛋白表达效率和信号肽切除效率。
一种重组表达载体,在上述分泌型穿梭表达载体的多克隆位点之间插入目的基因。
所述重组表达载体分别为pHTZ-gus,pHTZ-tgl1,pHTZ-lipa,其序列如SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24所示。
包含上述重组表达载体的工程菌。所述工程菌为B8-pHTZgus,B8-pHTZtgl1和B8-pHTZlipa,含有重组表达载体pHTZ-gus,pHTZ-tgl1,pHTZ-lipa。
所述表达载体在基因gus,tgl1和lipa蛋白中的应用,表达蛋白为GUS,Tgl1和LipA。
本发明的有益效果:本发明是在E. coli-B. subtilis穿梭载体pHT315的基础上构建,通过将枯草芽孢杆菌Bs168的木糖启动子序列与来自E. coli表达载体pET21b的T7表达区序列融合,构建了一个高效穿梭表达载体pHTL,以gfp,gus和lipa为报告基因,检测载体的表达能力和遗传稳定性;同时,将来自纳豆芽孢杆菌的碱性蛋白酶信号肽序列代替载体pHTL上的T7肽编码区,构建成高效分泌型穿梭表达载体pHTZ,以gus,tgl1和lipa为报告基因,检测载体的表达能力,两种载体均可以正确表达相应蛋白并具有生物学活性,具有广泛的应用前景。
附图说明
图1 为pHTL载体构建示意图。
图2 为pHTL载体诱导表达区构建示意图。
图3 为pHTL载体PCR和酶切鉴定;
图中,1:1 kb DNA Ladder Marker;2:质粒pHTL的EcoR I单酶切;3:质粒pHTL 的Not I单酶切;4:质粒pHTL的Xho I单酶切;5:质粒pHTL的SmaI单酶切;6:质粒pHTL的Kpn I单酶切;7:质粒pHTL的PCR产物。
图4:重组质粒pHTL-gfp,pHTL-gus和pHTL-lipa酶切鉴定;
图中,1,5,6:1 kb DNA Ladder Marker; 2:质粒pHTL-gfp双酶切;3:100 bp DNA Ladder Marker;4:质粒:pHTL-gus双酶切;7:质粒:pHTL-lipa双酶切。
图5 为菌株B8-pHTLgfp的荧光显微镜观察结果;
A:WB800可见光下观察;B:WB800紫外光下观察;C:B8-pHTLgfp可见光下观察;D:B8-pHTLgfp紫外光下观察。
图6 为菌株B8-pHTLgus的颜色变化观察结果;
图中,A:WB800;B:B8-pHTLgus。
图7 为菌株B8-pHTLgfp的质粒稳定性分析。
图8 为pHTZ载体构建示意图。
图9 为pHTZ载体表达区构建示意图。
图10 为pHTZ载体PCR和酶切鉴定;
图中,1,3,8:1 kb DNA Ladder Marker;2:质粒pHTZ 的Stu I/BamH I双酶切;4:质粒pHTZ的PCR产物;5:质粒pHTZ的Xho I单酶切;6:质粒pHTZ的Sal I单酶切;7:质粒pHTZ的Hind III单酶切。
图11 为重组质粒pHTZ-gus,pHTZ-tgl1,pHTZ-lipa酶切鉴定;
图中,1,5:1 kb DNA Ladder Marker;4:100 bp DNA Ladder Marker;2:质粒pHTZ-gus双酶切产物;3:质粒pHTZ-lipa双酶切产物;6:质粒pHTZ-tgl1双酶切产物。
图12 为菌株B8-pHTZgus的颜色变化观察结果;
图中,A:B8-pHTZgus;B:WB800。
图13 为菌株B8-pHTZtgl1产酶蛋白的量与BSA交联程度的SDS-PAGE分析;
图中,A:WB800;B:B8-pHTZtgl1。
具体实施方式
下面结合附图和具体实施例对本发明做进一步说明。
实施例1
(1)B. subtilis168基因组提取
无菌条件下,挑取B. subtilis 168单菌落,接种于LB培养基中,37 ℃,200 rpm培养12 h;按2%接种量接种到20 mL的LB培养基中37 ℃,200 rpm过夜培养;将菌液收集在1.5 mL离心管中,12000 rpm离心1 min,收集所有菌体,用TE洗一次;加入200 µL TE缓冲液震荡混匀;加入200 µL 2%的SDS缓冲液混匀;加入400 µL异硫氰酸胍已酸钠混合液;12000 rpm离心5 min,将上清转移到一个新的离心管中,加入600 µL异丙醇混匀;12000 rpm离心10 min去上清,加入500 µL 70%乙醇混匀;12000 rpm离心10 min,倒去上清,室温晾干至无乙醇气味;加入50 µL灭菌的去离子水,加入5 µLRNA酶溶液,37 ℃孵育30 min,用1%的琼脂糖凝胶进行电泳,检测提取的的基因组DNA。
(2)融合片段扩增
分别以B. subtilis 168基因组DNA为模板用引物对xylr-5/xylr-3(SEQ ID NO:2/ SEQ ID NO:3)扩增木糖1473bp的启动子PxylR序列;以大肠杆菌表达载体pET-21b质粒为模板,用引物对T7-5/T7-3(SEQ ID NO:4/ SEQ ID NO:5)扩增得到213bp的无启动子的pET-21b中T7表达区片段,其中包括多克隆位点区;回收两个PCR产物,不添加引物,利用PrimeSTAR Max DNA
Polymerase进行互补延伸,形成融合片段的中间产物,以此为模板,用引物对xylr-5/T7-3进行融合PCR的扩增,获得木糖启动子序列PxylR和载体pET-21b表达区的融合片段PxylR-T7,大小为1686bp。
(3) 质粒pHT315平端化
质粒pHT315使用限制性内切酶EcoR 和Hind 双酶切,将回收的片段用klenow酶进行黏末端补平反应,利用Fastap酶对补平的片段进行去磷酸化处理,电泳回收去磷酸化的质粒pHT315酶切补平片段。
(4)穿梭表达载体pHTL的构建
将融合片段PxylR-T7用限制性内切酶Sam I进行单酶切,回收后的片段与质粒pHT315酶切补平去磷酸化得到的片段连接,得到重组质粒pHTL。该质粒含有PxylR序列和T7肽(不含启动子)、多克隆位点区、T7终止子,物理图谱如图2。重组质粒经多种限制性内切酶单酶切、PCR及测序鉴定(SEQ ID NO:6/ SEQ ID NO:7),证明构建正确图3,序列见(SEQ ID NO:1)。
(5) 新型高效穿梭表达载体的效率评估
以质粒pMD-19T-gfp为模板,引物对5F-H/3R-H(SEQ ID NO:8/ SEQ ID NO:9)进行PCR反应扩增大小为756bp的gfp基因;以质粒pBI121为模板,引物对gusp-5/gusp-3(SEQ ID NO:10/ SEQ ID NO:11)进行PCR反应扩增大小为1812bp的gus基因;以质粒pMD-19lipa为模板,引物对LSal-5/Lhind-3(SEQ ID NO:12/ SEQ ID NO:13)进行PCR反应扩增大小为1842 bp的lipa基因。分别用限制性内切酶EcoR 和Hind 、Hind和Xho、Sal和Sph I双酶切回收的gfp、gus和lipa基因,导入pHTL的相应酶切位点间,转化E. coli JM109,得到重组质粒pHTL-gfp,pHTL-gus和pHTL-lipa,经酶切测序鉴定正确(图4),证明构建正确,序列见(SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16)。再转入E. coli SCS110去甲基化后,采用热击转化的方法将重组质粒导入B.subtilis WB800感受态细胞中,获得工程菌B8-pHTLgfp,B8-pHTLgus和B8-pHTLlipa。
工程菌B8-pHTLgfp中GFP的表达:挑选重组工程菌单菌落接种于LB液体培养基中培养过夜,离心收集细菌,弃去上清,用灭菌的生理盐水洗涤2次。挑去菌体涂在载玻片上在荧光显微镜下观察。用灭菌生理盐水稀释菌体至OD为1,用荧光分光光度计扫描适合检测gfp基因表达的发射波长和激发波长。结果如图5,荧光分光光度计扫描菌体的最佳激发波长为390 nm,发射波长为507 nm,说明pHTL载体可以正确表达gfp基因。
工程菌B8-pHTLgus中GUS表达检测:挑取工程菌单菌落接种于5 mL含有红霉素(10 μg /mL) LB液体培养基中,活化后转接新的液体培养基中震荡培养至菌体OD为0.1时,加入终浓度为1%的D-Xylose,诱导8 h后收获。取1 mL培养基,离心去掉上清收集菌体,灭菌的生理盐水洗涤2次,保留菌体。用450 μLGUS基液重悬菌体,加入50 μL的X-Gluc母液,混匀。37 ℃保温,5 min后肉眼观察染色结果(图6)。证明载体pHTL能成功的高效表达GUS蛋白。
工程菌B8-pHTLlipa中脂肪酶的表达:建立对硝基苯酚(p-NP)的浓度标准曲线。将活化的工程菌B8-pHTLlipa转接LB液体培养基中震荡培养,当菌体OD为0.1时,加入终浓度为1%的D-Xylose,诱导8 h后收获。离心收集菌体,用无菌生理盐水洗2遍。甩干水分,测菌体干重。同时,收获以同样接种培养同时期收获但不加D-Xylose的工程菌WB800-lipa和以同样接种培养同时期收获的B. subtilis WB800菌体,作为脂肪酶表达的参照。用细胞破碎液重悬待测的菌体,加入溶菌酶溶液(100 mg/mL),37 ℃保温30 min,用细胞破碎仪进行菌体破碎,收集上清液进行酶活测定。根据建立p-NP浓度与410 nm下吸光值的标准曲线,计算破碎液中的p-NP量,根据脂肪酶活力定义计算收获菌体的脂肪酶的酶活(U/mg),作为对照的WB800几乎无脂肪酶活性(0.25±0.07 U/mg),同时,无木糖诱导的工程菌WB800-lipa的菌体酶活力为0.34±0.05 U/mg.,诱导后的脂肪酶活力为72.73±0.25 U/mg,提高了近214倍,说明载体pHTL成功并高效表达了LipA蛋白。
质粒稳定性检测:接种重组工程菌B8-pHTLgfp于含有红霉素的LB液体培养基中培养活化转接至LB液体培养基中摇瓶培养 12 h 后,以1%的接种量转入新的LB液体性培养基中(不含红霉素),开始连续摇瓶培养试验。每12 h按1%接种量转接一次为一代工转接10代。 每一代取样1 mL进行平板稀释涂布检测质粒的缺失程度。检测时,取菌液用无菌水逐级稀释,取三个合适的梯度,分别涂在含有和不含有红霉素的固体LB培养基平板上,每个处理三个重复。 倒置在 37℃培养箱中培养24h,计算各皿中的菌落数。计算出质粒的稳定率(稳定率=含有抗生素平板的菌落数/不含有抗生素平板的菌落数)。结果(图8)表明在无抗生素选择压力下质粒pHTL传10代后质粒仍有98.5%的存在。
对比试验:接种重组工程菌B8- pHT315gfp(构建过程中gfp插入pHT315的多克隆位点处,构建过程与pHTLgfp相同,省略构建过程)于含有红霉素的LB液体培养基中培养活化转接至LB液体培养基中摇瓶培养 12 h 后,以1%的接种量转入新的LB液体性培养基中(不含红霉素),开始连续摇瓶培养试验。每12 h按1%接种量转接一次为一代工转接10代。 每一代取样1 mL进行平板稀释涂布检测质粒的缺失程度。检测时,取菌液用无菌水逐级稀释,取三个合适的梯度,分别涂在含有和不含有红霉素的固体LB培养基平板上,每个处理三个重复。 倒置在 37℃培养箱中培养24h,计算各皿中的菌落数。计算出质粒的稳定率(稳定率=含有抗生素平板的菌落数/不含有抗生素平板的菌落数)。结果表明在无抗生素选择压力下质粒pHT315传10代后质粒只有48.9%的存在。
实施例2:
(1)纳豆芽孢杆菌基因组提取
无菌条件下,挑取纳豆芽孢杆菌单菌落,接种于LB培养基中,震荡培养过夜,转接到20 mL的LB培养基中继续震荡培养过夜;将菌液收集在离心管中,离心收集所有菌体,用TE洗一次;加入200 µL TE缓冲液震荡混匀;加入200 µL 2%的SDS缓冲液混匀;加入400 µL异硫氰酸胍已酸钠混合液;12000 rpm离心5 min,将上清转移到一个新的离心管中,加入600 µL异丙醇混匀;12000 rpm离心10 min去上清,加入500 µL 70%乙醇混匀;12000 rpm离心10 min,倒去上清,室温晾干至无乙醇气味;加入50 µL灭菌的去离子水,加入5 µLRNA酶溶液,37 ℃孵育30 min,用1%的琼脂糖凝胶进行电泳,检测提取的的基因组DNA。
(2)融合片段的扩增
分别以质粒pHTL为模板用引物对xyls-5/xyls-3(SEQ ID NO:18/ SEQ ID NO:19)扩增1167bp的木糖启动子部分序列PxylRstuI;以纳豆芽孢杆菌基因组为模板,用引物对apre-5/apre-3(SEQ ID NO:20/ SEQ ID NO:21)扩增得到纳豆芽孢杆菌中96bp的信号肽Apre表达区片段;回收两个PCR产物,不添加引物,利用PrimeSTAR Max DNA
Polymerase进行互补延伸,形成融合片段的中间产物,以此为模板,用引物对xyls-5/apre-3进行融合PCR的扩增,获得木糖启动子部分序列PxylRstuI和纳豆芽孢杆菌信号肽表达区的融合片段PxylRstuI-Apre,大小为1263bp,经测序验证正确性。
(3) 分泌型穿梭表达载体pHTZ的构建
融合片段PxylRstuI-Apre用限制性内切酶Stu I/BamH I双酶切并进行胶回收后,连接到经同种酶酶切后的载体pHTL上,获得重组质粒pHTZ。该质粒含有PxylR序列、Apre序列、多克隆位点区、T7终止子,物理图谱如图10。重组质粒经多种限制性内切酶单酶切、PCR及测序鉴定(SEQ ID NO:6/ SEQ ID NO:7),证明构建正确图11,序列见(SEQ ID NO:17)。
(4) 高效分泌型穿梭表达载体的效率评估
质粒pHTL-gus经限制性内切酶Xho I/Hind III双酶切后得到大小为1812bp的gus基因;质粒pHTL-tgl1经限制性内切酶Sal I/Hind III双酶切后得到大小为738bp的tgl1基因;以质粒pHTL-lipa经限制性内切酶Sal I/Hind III双酶切后得到大小为1842 bp的lipa基因。回收后的基因片段导入pHTZ的相应酶切位点间,转化E. coli JM109,得到重组质粒pHTZ-gus,pHTZ-tgl1,pHTZ-lipa,经酶切测序鉴定正确(图12),证明构建正确,序列见(SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25)。再转入E. coli SCS110去甲基化后,采用热击转化的方法将重组质粒导入B.subtilis WB800感受态细胞中,获得B8-pHTZgus,B8-pHTZtgl1和B8-pHTZlipa工程菌。
工程菌B8-pHTZgus中GUS表达检测:挑取工程菌单菌落,接种于5 mL含有红霉素(10 μg /mL )LB液体培养基中,活化后转接新的液体培养基中震荡培养至菌体OD为0.5时,加入终浓度为2%的D-Xylose,诱导24 h后收获。取1 mL培养基,离心收集上清。加入450 μLGUS基液和50 μL的X-Gluc母液,混匀。37 ℃保温,肉眼观察染色结果(图13)。证明分泌型穿梭表达载体pHTZ能成功的高效表达GUS蛋白。
工程菌B8-pHTZtgl1中谷氨酰胺转氨酶的表达:将活化的工程菌转接LB液体培养基中震荡培养,当菌体OD为0.5时,加入终浓度为2%的D-Xylose,诱导24h后离心收获上清,与BSA(1mg/mL)混合后于70℃温育3h,利用SDS-PAGE电泳检测tgl1蛋白含量的表达情况。SDS-PAGE电泳检测方法参见分子克隆实验指南[J.萨姆布鲁克。分子克隆实验指南(第二版,金冬雁等).北京:科学出版社,1992]。结果证明工程菌表达的Tgl1催化BSA发生交联(图13),而B.subtilis WB800菌液与BSA混合后未检测出BSA的聚合条带,说明分泌型穿梭表达载体pHTZ可以成功地表达Tgl1蛋白。
工程菌B8-pHTZlipa中脂肪酶的表达:建立对硝基苯酚(p-NP)的浓度标准曲线。将活化的工程菌B8-pHTZlipa转接LB液体培养基中震荡培养,当菌体OD为0.5时,加入终浓度为2%的D-Xylose,诱导24h后收获。离心收获上清液进行酶活测定。同时,收获以同样接种培养同时期收获但不加D-Xylose的工程菌B8-pHTZlipa和以同样接种培养同时期收获的WB800菌体,作为脂肪酶表达的参照。根据建立p-NP浓度与410 nm下吸光值的标准曲线,计算破碎液中的p-NP量,根据脂肪酶活力定义计算收获的上清中脂肪酶的酶活(U/mg),作为对照的WB800几乎没有脂肪酶活性(0.25±0.07 U/mg),同时,无木糖诱导的工程菌B8-pHTZlipa的菌体酶活力为0.41±0.09 U/mg,诱导后的脂肪酶活力为181.2±1.75 U/mg,提高了441倍,说明载体pHTZ成功并高效表达了LipA蛋白。
对比试验:建立对硝基苯酚(p-NP)的浓度标准曲线。将活化的工程菌B8-pHTLlipa(构建过程lipa的插入位点与pHTZlipa相同,省略构建过程)转接LB液体培养基中震荡培养,当菌体OD为0.5时,加入终浓度为2%的D-Xylose,诱导24h后收获。离心收获上清液进行酶活测定。同时,收获以同样接种培养同时期收获但不加D-Xylose的工程菌B8-pHTLlipa和以同样接种培养同时期收获的WB800菌体,作为脂肪酶表达的参照。根据建立p-NP浓度与410 nm下吸光值的标准曲线,计算破碎液中的p-NP量,根据脂肪酶活力定义计算收获的上清中脂肪酶的酶活(U/mg),作为对照的WB800无论有无木糖诱导都只有微弱的脂肪酶活性(0.22±0.08 U/mg),无木糖诱导的工程菌B8-pHTLlipa同样也只有微弱的脂肪酶活性(0.39±0.11 U/mg),证明在没有木糖诱导的时候,木糖启动子几乎没有启动目的基因的转录,而当有木糖诱导时,木糖启动子则会表现出相当强大的启动目的基因转录的功能,上述事实表明pHTZ是一个典型的诱导型表达载体。
SEQUENCE LISTING
<110> 河北农业大学
<120> 一种高效穿梭表达载体及其构建方法与应用
<130> 1111
<160> 24
<170> Patent In version 3.5
<210> 1
<211> 8170
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 1
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat attaatgttt
ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa aattagcagt
aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta ccaaccttta
gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga ggcgccaacc tcaaataagt
240
tatttgcctt gttttcgcga acaaggctta ttagatacac ctattgtacc
gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat aataaaacgg
atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg tttagatata
gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa gatggtctag
tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca actgtctgac
gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg taaatcctaa
taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa taagacatga
ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg attcacggct
attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga atcacccgaa
agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc caataagcta
tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg caaccaatct
caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa cctcgtaaat
tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa ctcgccagta
tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg attagcaaaa
aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga ggaagaatcc
agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga aagatagggg
agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt attgatctat
tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag gatgaaagta
ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca tatttaactc
ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt tggaattatt
gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg atgttataat
attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga aaagtaacca
ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc ttaccgtaca
taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag ccaatcaaat gcttgagaac
1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa agatcctata
tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta gtaaactccc
cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg tctttggatt
ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc gcttgggtcc
aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac gctattaggt
tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat gtgcgatgaa
tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc gggaaacatt
tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg tggaagtttt
gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt tctccctcat
ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg cgcgggacag
gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc atgcttggaa
agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt aaaatttatg
ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta ataggtaaaa tattaattta
2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat ttatataatc
agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact gatgtcgtaa
aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt ttgaacacca
cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg atagtgcagt atcttaaaat
2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa aatttgtaat
taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata aaacatctac
tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt agattaattc
ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa acaaatcgtt
taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg aacaaaaata
taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata ataaaacaat
tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg catttaacga
cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca
acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac caagatattc
tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt
taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac atctatctga
ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta gggttgctct
tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc tttcatccta
aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata
aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa tatcgtcaac
tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac aatttaagta
ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt aacgggagga
aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa agggaatgga
gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc cctagcgcct
acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt attcaacatt
tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg ggttacatcg
aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa aatcccttaa
cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat actcctagaa
taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca ttgtaatcat
gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat tctgtcctaa
ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg atgatacttc
acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc ttaaaattac ggcttgtggg
6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat agaatccaaa
attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta tgatatcttg ataggacagt
6300
tttttctctt tggtctgaag agattttaat aaagccttct ctgaagcata
caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt ctattgtcat
atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt tgataataac
accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat tttttgcagc
tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa aaacaggcac
attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg agttcggagt
gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta taccaataag
cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa tcaaaatgtc
tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa cgattgttcc
ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc caacggagta
tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg attgtccttg
acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg agacagttga
tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat ttttaaggat
ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag cgatatccac
ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca tagcgaatct
tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa aatactgacg
aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata ggtgatgtac
ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag ggggaaatca
catggctagc 7380
atgactggtg gacagcaaat gggtcgggat ccgaattcga gctccgtcga
caagcttgcg 7440
gccgcactcg agcaccacca ccaccaccac tgagatccgg ctgctaacaa
agcccgaaag 7500
gaagctgagt tggctgctgc caccgctgag caataactag cataacccct
tggggcctct 7560
aaacgggtct tgaggggttt tttgcccaat tcactggccg tcgttttaca acgtcgtgac
7620
tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc
tttcgccagc 7680
tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg
cagcctgaat 7740
ggcgaatggc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
ttcacaccgc 7800
atatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
gccccgacac 7860
ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
cgcttacaga 7920
caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
atcaccgaaa 7980
cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
catgataata 8040
atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
ccctatttgt 8100
ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
ctgataaatg 8160
cttcaataat
8170
<210> 2
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 2
ggcccgggaa atactcctag
aata
24
<210> 3
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 3
ccaccagtca tgctagccat gtgatttccc
ccttaa
36
<210> 4
<211> 36
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 4
ttaaggggga aatcacatgg ctagcatgac tggtgg
36
<210> 5
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 5
aaatcccggg caaaaaaccc
ctcaagac
28
<210> 6
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 6
caggaaacag
ctatgacc
18
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 7
tgtaaaacga
cggccagt
18
<210> 8
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 8
ccggaattcg atggtagatc
tgac
24
<210> 9
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 9
cccaagcttt cacacgtggt
ggtg
24
<210> 10
<211> 32
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 10
cggaagctta tgttacgtcc tgtagaaacc
cc
32
<210> 11
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 11
taactcgagt cattgtttgc
ctccctgctg
30
<210> 12
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 12
aacgtcgaca tgggcatctt
tagc
24
<210> 13
<211> 24
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 13
ggaaagcttt taggccaaca
ccac
24
<210> 14
<211> 8914
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 14
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat
attaatgttt ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca
actgtctgac gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg
attcacggct attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc caataagcta
tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga
ggaagaatcc agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt
tggaattatt gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc
ttaccgtaca taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac
gctattaggt tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg
tggaagtttt gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta
ataggtaaaa tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat
ttatataatc agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt
ttgaacacca cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg
aacaaaaata taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt
catctattca acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt ccttaccatt
taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat
gttccagata aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat
actcctagaa taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca
ttgtaatcat gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg
atgatacttc acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt
tgataataac accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg
agttcggagt gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa tcaaaatgtc
tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg
agacagttga tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat
ttttaaggat ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag
ggggaaatca catggctagc 7380
atgactggtg gacagcaaat gggtcgggat ccgaattcga
tggtagatct gactagtaaa 7440
ggagaagaac ttttcactgg agttgtccca attcttgttg
aattagatgg tgatgttaat 7500
gggcacaaat tttctgtcag tggagagggt gaaggtgatg
caacatacgg aaaacttacc 7560
cttaaattta tttgcactac tggaaaacta cctgttccgt
ggccaacact tgtcactact 7620
ttctcttatg gtgttcaatg cttttcaaga tacccagatc
atatgaagcg gcacgacttc 7680
ttcaagagcg ccatgcctga gggatacgtg caggagagga
ccatcttctt caaggacgac 7740
gggaactaca agacacgtgc tgaagtcaag tttgagggag acaccctcgt
caacaggatc 7800
gagcttaagg gaatcgattt caaggaggac ggaaacatcc
tcggccacaa gttggaatac 7860
aactacaact cccacaacgt atacatcatg gccgacaagc
aaaagaacgg catcaaagcc 7920
aacttcaaga cccgccacaa catcgaagac ggcggcgtgc
aactcgctga tcattatcaa 7980
caaaatactc caattggcga tggccctgtc cttttaccag
acaaccatta cctgtccaca 8040
caatctgccc tttcgaaaga tcccaacgaa aagagagacc
acatggtcct tcttgagttt 8100
gtaacagctg ctgggattac acatggcatg gatgaactat
acaaagctag ccaccaccac 8160
caccaccacg tgtgaaagct tgcggccgca ctcgagcacc
accaccacca ccactgagat 8220
ccggctgcta acaaagcccg aaaggaagct gagttggctg
ctgccaccgc tgagcaataa 8280
ctagcataac cccttggggc ctctaaacgg gtcttgaggg
gttttttgcc caattcactg 8340
gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
ttacccaact taatcgcctt 8400
gcagcacatc cccctttcgc cagctggcgt aatagcgaag
aggcccgcac cgatcgccct 8460
tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
tgcggtattt tctccttacg 8520
catctgtgcg gtatttcaca ccgcatatgg tgcactctca
gtacaatctg ctctgatgcc 8580
gcatagttaa gccagccccg acacccgcca acacccgctg
acgcgccctg acgggcttgt 8640
ctgctcccgg catccgctta cagacaagct gtgaccgtct
ccgggagctg catgtgtcag 8700
aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
gcctcgtgat acgcctattt 8760
ttataggtta atgtcatgat aataatggtt tcttagacgt
caggtggcac ttttcgggga 8820
aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
attcaaatat gtatccgctc 8880
atgagacaat aaccctgata aatgcttcaa
taat
8914
<210> 15
<211> 9973
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 15
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat
attaatgttt ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca
actgtctgac gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg
attcacggct attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc
caataagcta tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga
ggaagaatcc agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt tggaattatt
gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc
ttaccgtaca taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac
gctattaggt tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg
tggaagtttt gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta ataggtaaaa
tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat
ttatataatc agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt
ttgaacacca cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg
aacaaaaata taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt
catctattca acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt
ccttaccatt taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat
gttccagata aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat actcctagaa
taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca
ttgtaatcat gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg
atgatacttc acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt
tgataataac accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg
agttcggagt gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa
tcaaaatgtc tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg
agacagttga tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat
ttttaaggat ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag ggggaaatca
catggctagc 7380
atgactggtg gacagcaaat gggtcgggat ccgaattcga
gctccgtcga caagcttatg 7440
ttacgtcctg tagaaacccc aacccgtgaa atcaaaaaac
tcgacggcct gtgggcattc 7500
agtctggatc gcgaaaactg tggaattgat cagcgttggt
gggaaagcgc gttacaagaa 7560
agccgggcaa ttgctgtgcc aggcagtttt aacgatcagt
tcgccgatgc agatattcgt 7620
aattatgcgg gcaacgtctg gtatcagcgc gaagtcttta
taccgaaagg ttgggcaggc 7680
cagcgtatcg tgctgcgttt cgatgcggtc actcattacg
gcaaagtgtg ggtcaataat 7740
caggaagtga tggagcatca gggcggctat acgccatttg
aagccgatgt cacgccgtat 7800
gttattgccg ggaaaagtgt acgtatcacc gtttgtgtga
acaacgaact gaactggcag 7860
actatcccgc cgggaatggt gattaccgac gaaaacggca
agaaaaagca gtcttacttc 7920
catgatttct ttaactatgc cggaatccat cgcagcgtaa
tgctctacac cacgccgaac 7980
acctgggtgg acgatatcac cgtggtgacg catgtcgcgc
aagactgtaa ccacgcgtct 8040
gttgactggc aggtggtggc caatggtgat gtcagcgttg
aactgcgtga tgcggatcaa 8100
caggtggttg caactggaca aggcactagc gggactttgc
aagtggtgaa tccgcacctc 8160
tggcaaccgg gtgaaggtta tctctatgaa ctgtgcgtca
cagccaaaag ccagacagag 8220
tgtgatatct acccgcttcg cgtcggcatc cggtcagtgg
cagtgaaggg cgaacagttc 8280
ctgattaacc acaaaccgtt ctactttact ggctttggtc
gtcatgaaga tgcggacttg 8340
cgtggcaaag gattcgataa cgtgctgatg gtgcacgacc acgcattaat
ggactggatt 8400
ggggccaact cctaccgtac ctcgcattac ccttacgctg
aagagatgct cgactgggca 8460
gatgaacatg gcatcgtggt gattgatgaa actgctgctg
tcggctttaa cctctcttta 8520
ggcattggtt tcgaagcggg caacaagccg aaagaactgt
acagcgaaga ggcagtcaac 8580
ggggaaactc agcaagcgca cttacaggcg attaaagagc
tgatagcgcg tgacaaaaac 8640
cacccaagcg tggtgatgtg gagtattgcc aacgaaccgg
atacccgtcc gcaaggtgca 8700
cgggaatatt tcgcgccact ggcggaagca acgcgtaaac
tcgacccgac gcgtccgatc 8760
acctgcgtca atgtaatgtt ctgcgacgct cacaccgata
ccatcagcga tctctttgat 8820
gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa
gcggcgattt ggaaacggca 8880
gagaaggtac tggaaaaaga acttctggcc tggcaggaga
aactgcatca gccgattatc 8940
atcaccgaat acggcgtgga tacgttagcc gggctgcact
caatgtacac cgacatgtgg 9000
agtgaagagt atcagtgtgc atggctggat atgtatcacc
gcgtctttga tcgcgtcagc 9060
gccgtcgtcg gtgaacaggt atggaatttc gccgattttg
cgacctcgca aggcatattg 9120
cgcgttggcg gtaacaagaa agggatcttc actcgcgacc
gcaaaccgaa gtcggcggct 9180
tttctgctgc aaaaacgctg gactggcatg aacttcggtg
aaaaaccgca gcagggaggc 9240
aaacaatgac tcgagcacca ccaccaccac cactgagatc
cggctgctaa caaagcccga 9300
aaggaagctg agttggctgc tgccaccgct gagcaataac
tagcataacc ccttggggcc 9360
tctaaacggg tcttgagggg ttttttgccc aattcactgg
ccgtcgtttt acaacgtcgt 9420
gactgggaaa accctggcgt tacccaactt aatcgccttg
cagcacatcc ccctttcgcc 9480
agctggcgta atagcgaaga ggcccgcacc gatcgccctt
cccaacagtt gcgcagcctg 9540
aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
atctgtgcgg tatttcacac 9600
cgcatatggt gcactctcag tacaatctgc tctgatgccg
catagttaag ccagccccga 9660
cacccgccaa cacccgctga cgcgccctga cgggcttgtc
tgctcccggc atccgcttac 9720
agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
ggttttcacc gtcatcaccg 9780
aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
tataggttaa tgtcatgata 9840
ataatggttt cttagacgtc aggtggcact tttcggggaa
atgtgcgcgg aacccctatt 9900
tgtttatttt tctaaataca ttcaaatatg tatccgctca
tgagacaata accctgataa 9960
atgcttcaat
aat
9973
<210> 16
<211> 10015
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 16
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat
attaatgttt ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca actgtctgac
gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg
attcacggct attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc
caataagcta tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga
ggaagaatcc agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt
tggaattatt gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc
ttaccgtaca taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac gctattaggt
tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg
tggaagtttt gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta
ataggtaaaa tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat
ttatataatc agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt
ttgaacacca cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg aacaaaaata
taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt
catctattca acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt
ccttaccatt taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat
gttccagata aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat
actcctagaa taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca
ttgtaatcat gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg
atgatacttc acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt tgataataac
accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg
agttcggagt gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa
tcaaaatgtc tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg
agacagttga tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat
ttttaaggat ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag
ggggaaatca catggctagc 7380
atgactggtg gacagcaaat gggtcgggat ccgaattcga
gctccgtcga catgggcatc 7440
tttagctata aggatttgga cgaaaacgcg tcgaaagcgc
tgttttccga cgccttggcc 7500
atctccacct acgcttacca caatatcgat aacggcttcg
acgaaggcta ccaccagacc 7560
ggtttcggtc ttggcctgcc gctgacgctg atcaccgcgc
tgatcggcag cacccaatcg 7620
cagggcggcc tgccccgcat tccctggaac cccgactccg
aacaggccgc gcaggagacg 7680
gtgaacaatg ccggctggtc ggtcatcagc gccgcgcagc
tgggttacgc cggcaaaacc 7740
gatgcacgcg gcacctatta cggcgagacc gccggttaca
ccaccgcgca ggccgaggtg 7800
ctgggcaaat atgacagcga aggcaatctc accgccattg
gtatctcatt tcgcggtacc 7860
agcggcccgc gcgagtcgct gatcggcgat accatcggcg atgtgattaa
cgatctgctg 7920
gccggtttcg ggccgaaagg ctacgctgac ggctacacgc
tgaacgcctt cggcaatctg 7980
ctgggcgacg tggcgaaatt cgcgcaggcg cacgggctga
gcggcgagga cgtagtggtc 8040
agcggccaca gcctcggcgg gctggcggtc aacagcatgg
cggcgcagag cgacgccaac 8100
tggggcggct tctacgcgca gtccaactat gtcgccttcg
cctcgccgac ccagtacgaa 8160
gccggcggca aggtgatcaa catcggctac gagaacgacc
cggtgttccg cgcgctcgac 8220
ggcacctcgc taaccctgcc gtcactgggc gtacacgatg
cgccgcacgc ctccgccacc 8280
aacaatatcg tcaacttcaa cgaccactac gcgtcggacg
cctggaacct gctgccgttt 8340
tccattctca acattccgac ctggctgtcg cacctgccgt
tcttctatca ggacgggctg 8400
atgcgggtgc tgaactccga gttttattcg ctgaccgaca
aggactcgac catcatcgtc 8460
tccaacctgt cgaacgtgac gcgcggcaat acctgggtgg
aagacctgaa ccgcaacgcg 8520
gaaacgcaca gcggaccgac gtttatcatc ggcagcgacg
gcaatgattt gatcaagggc 8580
ggcaaaggca acgactatct cgagggccgc gacggcgacg
atatcttccg cgacgccggc 8640
ggctataacc tgatcgccgg cggcaaaggc cacaatatct
tcgataccca acaggcgttg 8700
aaaaacaccg aggtcgccta cgacggcaat acgctttacc
tgcgcgacgc caaaggcggt 8760
attacgctgg cagacgacat cagcaccctg cgcagcaaag
aaacctcctg gctgattttc 8820
agcaaagagg tggatcatca ggtgaccgct gcgggattga
aatcggactc gggcctcaaa 8880
gcctatgccg ccgccaccac cggcggcgac ggcgatgacg tcctgcaggc
tcgcagccac 8940
gacgcctggc tgttcggcaa cgccggcaac gacacgctga
tcggccatgc cggcggcaac 9000
ctgaccttcg tcggcggcag cggcgatgac atcctgaagg
gcgccggcaa cggtaatacc 9060
ttcctgttca gcggcgattt cggccgcgac cagctgtatg
gtttcaacgc caccgataaa 9120
ctggtgttta tcggtaccga aggcgccagc gggaatatcc
gcgactatgc cacacagcaa 9180
aacgacgatc tggtgctggc cttcggccac ggccaggtca
cgctgatcgg cgtctcgctc 9240
gatcacttca acaccgatcg ggtggtgttg gcctaaaagc
ttgcggccgc actcgagcac 9300
caccaccacc accactgaga tccggctgct aacaaagccc
gaaaggaagc tgagttggct 9360
gctgccaccg ctgagcaata actagcataa ccccttgggg
cctctaaacg ggtcttgagg 9420
ggttttttgc ccaattcact ggccgtcgtt ttacaacgtc
gtgactggga aaaccctggc 9480
gttacccaac ttaatcgcct tgcagcacat ccccctttcg
ccagctggcg taatagcgaa 9540
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
tgaatggcga atggcgcctg 9600
atgcggtatt ttctccttac gcatctgtgc ggtatttcac
accgcatatg gtgcactctc 9660
agtacaatct gctctgatgc cgcatagtta agccagcccc
gacacccgcc aacacccgct 9720
gacgcgccct gacgggcttg tctgctcccg gcatccgctt
acagacaagc tgtgaccgtc 9780
tccgggagct gcatgtgtca gaggttttca ccgtcatcac
cgaaacgcgc gagacgaaag 9840
ggcctcgtga tacgcctatt tttataggtt aatgtcatga
taataatggt ttcttagacg 9900
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
tttgtttatt tttctaaata 9960
cattcaaata tgtatccgct catgagacaa taaccctgat
aaatgcttca ataat 10015
<210> 17
<211> 8230
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 17
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat attaatgttt
ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca
actgtctgac gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg
attcacggct attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc
caataagcta tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga
ggaagaatcc agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt
tggaattatt gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc
ttaccgtaca taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac
gctattaggt tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg
tggaagtttt gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta
ataggtaaaa tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat ttatataatc
agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt
ttgaacacca cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg
aacaaaaata taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt
catctattca acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt
ccttaccatt taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat gttccagata
aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat
actcctagaa taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca ttgtaatcat
gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg
atgatacttc acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt
tgataataac accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg
agttcggagt gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa
tcaaaatgtc tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg agacagttga
tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat
ttttaaggat ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag
ggggaaatca cgtgagaagc 7380
aaaaaattgt ggatcagctt gttgtttgcg ttaacgttaa
tctttacgat ggcgttcagc 7440
aacatgtctg cgcaggctgc cggcaaggat ccgaattcga
gctccgtcga caagcttgcg 7500
gccgcactcg agcaccacca ccaccaccac tgagatccgg
ctgctaacaa agcccgaaag 7560
gaagctgagt tggctgctgc caccgctgag caataactag
cataacccct tggggcctct 7620
aaacgggtct tgaggggttt tttgcccaat tcactggccg
tcgttttaca acgtcgtgac 7680
tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
cacatccccc tttcgccagc 7740
tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
aacagttgcg cagcctgaat 7800
ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
tgtgcggtat ttcacaccgc 7860
atatggtgca ctctcagtac aatctgctct gatgccgcat
agttaagcca gccccgacac 7920
ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
tcccggcatc cgcttacaga 7980
caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
tttcaccgtc atcaccgaaa 8040
cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
aggttaatgt catgataata 8100
atggtttctt agacgtcagg tggcactttt cggggaaatg
tgcgcggaac ccctatttgt 8160
ttatttttct aaatacattc aaatatgtat ccgctcatga
gacaataacc ctgataaatg 8220
cttcaataat
8230
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 18
ggaggcctat tcctaaatag
aatcc
25
<210> 19
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 19
ccacaatttt ttgcttctca cgtgatttcc
ccc
33
<210> 20
<211> 33
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 20
gggggaaatc acgtgagaag caaaaaattg
tgg
33
<210> 21
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 21
ttaattaatt ggatccttgc
cggcagcctg
30
<210> 22
<211> 10033
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 22
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat
attaatgttt ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca
actgtctgac gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg
attcacggct attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc
caataagcta tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga ggaagaatcc
agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt
tggaattatt gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc
ttaccgtaca taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac
gctattaggt tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg tggaagtttt
gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta
ataggtaaaa tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat
ttatataatc agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt
ttgaacacca cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg
aacaaaaata taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt
catctattca acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt
ccttaccatt taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat
gttccagata aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat
actcctagaa taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca
ttgtaatcat gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg
atgatacttc acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt
tgataataac accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg
agttcggagt gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa
tcaaaatgtc tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg
agacagttga tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat ttttaaggat
ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag
ggggaaatca cgtgagaagc 7380
aaaaaattgt ggatcagctt gttgtttgcg ttaacgttaa
tctttacgat ggcgttcagc 7440
aacatgtctg cgcaggctgc cggcaaggat ccgaattcga
gctccgtcga caagcttatg 7500
ttacgtcctg tagaaacccc aacccgtgaa atcaaaaaac
tcgacggcct gtgggcattc 7560
agtctggatc gcgaaaactg tggaattgat cagcgttggt
gggaaagcgc gttacaagaa 7620
agccgggcaa ttgctgtgcc aggcagtttt aacgatcagt
tcgccgatgc agatattcgt 7680
aattatgcgg gcaacgtctg gtatcagcgc gaagtcttta
taccgaaagg ttgggcaggc 7740
cagcgtatcg tgctgcgttt cgatgcggtc actcattacg
gcaaagtgtg ggtcaataat 7800
caggaagtga tggagcatca gggcggctat acgccatttg
aagccgatgt cacgccgtat 7860
gttattgccg ggaaaagtgt acgtatcacc gtttgtgtga
acaacgaact gaactggcag 7920
actatcccgc cgggaatggt gattaccgac gaaaacggca
agaaaaagca gtcttacttc 7980
catgatttct ttaactatgc cggaatccat cgcagcgtaa
tgctctacac cacgccgaac 8040
acctgggtgg acgatatcac cgtggtgacg catgtcgcgc aagactgtaa
ccacgcgtct 8100
gttgactggc aggtggtggc caatggtgat gtcagcgttg
aactgcgtga tgcggatcaa 8160
caggtggttg caactggaca aggcactagc gggactttgc
aagtggtgaa tccgcacctc 8220
tggcaaccgg gtgaaggtta tctctatgaa ctgtgcgtca
cagccaaaag ccagacagag 8280
tgtgatatct acccgcttcg cgtcggcatc cggtcagtgg
cagtgaaggg cgaacagttc 8340
ctgattaacc acaaaccgtt ctactttact ggctttggtc
gtcatgaaga tgcggacttg 8400
cgtggcaaag gattcgataa cgtgctgatg gtgcacgacc
acgcattaat ggactggatt 8460
ggggccaact cctaccgtac ctcgcattac ccttacgctg
aagagatgct cgactgggca 8520
gatgaacatg gcatcgtggt gattgatgaa actgctgctg
tcggctttaa cctctcttta 8580
ggcattggtt tcgaagcggg caacaagccg aaagaactgt
acagcgaaga ggcagtcaac 8640
ggggaaactc agcaagcgca cttacaggcg attaaagagc
tgatagcgcg tgacaaaaac 8700
cacccaagcg tggtgatgtg gagtattgcc aacgaaccgg
atacccgtcc gcaaggtgca 8760
cgggaatatt tcgcgccact ggcggaagca acgcgtaaac
tcgacccgac gcgtccgatc 8820
acctgcgtca atgtaatgtt ctgcgacgct cacaccgata
ccatcagcga tctctttgat 8880
gtgctgtgcc tgaaccgtta ttacggatgg tatgtccaaa
gcggcgattt ggaaacggca 8940
gagaaggtac tggaaaaaga acttctggcc tggcaggaga
aactgcatca gccgattatc 9000
atcaccgaat acggcgtgga tacgttagcc gggctgcact
caatgtacac cgacatgtgg 9060
agtgaagagt atcagtgtgc atggctggat atgtatcacc
gcgtctttga tcgcgtcagc 9120
gccgtcgtcg gtgaacaggt atggaatttc gccgattttg
cgacctcgca aggcatattg 9180
cgcgttggcg gtaacaagaa agggatcttc actcgcgacc
gcaaaccgaa gtcggcggct 9240
tttctgctgc aaaaacgctg gactggcatg aacttcggtg
aaaaaccgca gcagggaggc 9300
aaacaatgac tcgagcacca ccaccaccac cactgagatc
cggctgctaa caaagcccga 9360
aaggaagctg agttggctgc tgccaccgct gagcaataac
tagcataacc ccttggggcc 9420
tctaaacggg tcttgagggg ttttttgccc aattcactgg
ccgtcgtttt acaacgtcgt 9480
gactgggaaa accctggcgt tacccaactt aatcgccttg
cagcacatcc ccctttcgcc 9540
agctggcgta atagcgaaga ggcccgcacc gatcgccctt
cccaacagtt gcgcagcctg 9600
aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
atctgtgcgg tatttcacac 9660
cgcatatggt gcactctcag tacaatctgc tctgatgccg
catagttaag ccagccccga 9720
cacccgccaa cacccgctga cgcgccctga cgggcttgtc
tgctcccggc atccgcttac 9780
agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
ggttttcacc gtcatcaccg 9840
aaacgcgcga gacgaaaggg cctcgtgata cgcctatttt
tataggttaa tgtcatgata 9900
ataatggttt cttagacgtc aggtggcact tttcggggaa
atgtgcgcgg aacccctatt 9960
tgtttatttt tctaaataca ttcaaatatg tatccgctca
tgagacaata accctgataa 10020
atgcttcaat
aat
10033
<210> 23
<211> 8968
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 23
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat
attaatgttt ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca
actgtctgac gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg
attcacggct attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc
caataagcta tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga
ggaagaatcc agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt
tggaattatt gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc ttaccgtaca
taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac
gctattaggt tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg
tggaagtttt gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta
ataggtaaaa tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat
ttatataatc agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt ttgaacacca
cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg
aacaaaaata taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt
catctattca acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt
ccttaccatt taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat
gttccagata aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag
tactcaccag tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat
actcctagaa taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca
ttgtaatcat gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg atgatacttc
acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt
tgataataac accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg
agttcggagt gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa
tcaaaatgtc tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg
agacagttga tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat
ttttaaggat ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag
ggggaaatca cgtgagaagc 7380
aaaaaattgt ggatcagctt gttgtttgcg ttaacgttaa
tctttacgat ggcgttcagc 7440
aacatgtctg cgcaggctgc cggcaaggat ccgaattcga
gctccgtcga catgattatt 7500
gtatcaggac aattgctccg tccccaggat attgaaaatt ggcagattga
tcaagatctg 7560
aatccgctct taaaagagat gattgagacg cctgttcagt
ttgattatca ttcaattgct 7620
gaactgatgt ttgagcttaa actgcggatg aatattgtag
cagcggcaaa gacgctgcac 7680
aaaagcgggg cgaagtttgc cactttttta aaaacatacg
ggaatacaac gtattggagg 7740
gtttcaccgg agggcgcctt ggagctgaaa tacagaatgc
cgccttcaaa agcgattcgg 7800
gacattgcag agaacggccc gttttatgcg tttgaatgcg
caaccgcaat cgttatcatt 7860
tattacttgg ccttaatcga tacaatcgga gaagataaat
tcaatgccag ctttgacaga 7920
attattttat atgactggca ttatgagaaa ttgccgatat
atacggaaac aggacaccac 7980
tttttccttg gagattgttt gtattttaag aatcctgaat
ttgatccgca aaaggcgcaa 8040
tggagaggcg aaaatgtgat actactgggg gaagataaat
attttgccca tggtcttgga 8100
atcttaaacg gaaagcaaat tattgataag ctgaattctt
ttaggaaaaa aggagcctta 8160
cagtcagcct accttctgtc tcaggcgacc agactggatg
ttccgtctct tttccgcatc 8220
gtccgctaaa agcttgcggc cgcactcgag caccaccacc
accaccactg agatccggct 8280
gctaacaaag cccgaaagga agctgagttg gctgctgcca
ccgctgagca ataactagca 8340
taaccccttg gggcctctaa acgggtcttg aggggttttt
tgcccaattc actggccgtc 8400
gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
aacttaatcg ccttgcagca 8460
catccccctt tcgccagctg gcgtaatagc gaagaggccc
gcaccgatcg cccttcccaa 8520
cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
tacgcatctg 8580
tgcggtattt cacaccgcat atggtgcact ctcagtacaa
tctgctctga tgccgcatag 8640
ttaagccagc cccgacaccc gccaacaccc gctgacgcgc
cctgacgggc ttgtctgctc 8700
ccggcatccg cttacagaca agctgtgacc gtctccggga
gctgcatgtg tcagaggttt 8760
tcaccgtcat caccgaaacg cgcgagacga aagggcctcg
tgatacgcct atttttatag 8820
gttaatgtca tgataataat ggtttcttag acgtcaggtg
gcacttttcg gggaaatgtg 8880
cgcggaaccc ctatttgttt atttttctaa atacattcaa
atatgtatcc gctcatgaga 8940
caataaccct gataaatgct
tcaataat
8968
<210> 24
<211> 10075
<212> DNA
<213> Artificial Sequence
<220>
<223> Artificial Sequence
<400> 24
ccatcctcca aagttggaga gtgagtttta tgtcgcaaat
attaatgttt ctggtgaacc 60
ttatcaaatt ttcgttgatt taatagaaac atagcggtaa
aattagcagt aacttaatag 120
aacggaaatg aaaaaagcca ctctcatatg ctattggcta
ccaaccttta gcgagaatga 180
cttaatcctg tacagccata caggacttcg acttataaga
ggcgccaacc tcaaataagt 240
tatttgcctt gttttcgcga acaaggctta ttagatacac
ctattgtacc gttactctac 300
gaatatttca actagtaatt actagcattg tcatatacat
aataaaacgg atataaaagg 360
gcgttttcta tacctagaag tcttgtaaat gtacagggcg
tttagatata gagaacgccc 420
tttttgtgtt ccgttccagt ggaagctacc actttaaaaa
gatggtctag tgtagccaat 480
gcaggagagt acactcggat atcagttgtc gttgcattca
actgtctgac gtaagcgagg 540
taaaggacac aagccttgca taaaacaagc ctacgggatg
taaatcctaa taatgatgat 600
aaccaagacg ttagcggcaa aaagtgttgg gggttcaaaa
taagacatga ttgtgcgact 660
ggagttaaac agttactcgt aagcggcgat catgacactg attcacggct
attcttgtac 720
aagctagctt tattacaagg atatgcgggt tatatagcga
atcacccgaa agggaacggt 780
gttgggcgtg agaaacgcac cgtacggcgc aatacaatgc
caataagcta tatacggacg 840
gtatagtagt tttgtaagct ataaccgttt gtcgtcaatg
caaccaatct caattcgaga 900
cctcggcatc taagccagta cgaatgagtg ggcgttttaa
cctcgtaaat tttcaacagg 960
ggttactatg cccaaaacta cattcagatt tcctaacaaa
ctcgccagta tgaaaacctt 1020
aagaccttaa agtcaaggga tttgaaggat tttaacctcg
attagcaaaa aatgtagagt 1080
actgaagcaa ctaccattaa ctaagatagt gggggattga
ggaagaatcc agagctgttt 1140
aaatcaagtg aaagacaaga tgaaattaaa agaatagtga
aagatagggg agtggttctc 1200
tatgagaaag gaaatggcta gagaacaaag gcagcggttt
attgatctat tgttagactt 1260
tatggtaaag aatcctcatt tatttgttaa tggtacagag
gatgaaagta ataatgttgt 1320
tacaaaatgt aatagtgata ttaaagaggt tgcggagtca
tatttaactc ttttatagtg 1380
agagggttaa aactaattaa tatgtattaa ggcccaatgt
tggaattatt gtatttcact 1440
aggcaaccta cttactaaaa gtaagattat ccattagtgg
atgttataat attgggtttt 1500
ttaacacaat aatcatcgcc tttcggtgtc gtttgataga
aaagtaacca ttagcgatga 1560
aaaagtcaat ataaaaagcc atccgtaaaa aacggatggc
ttaccgtaca taggatcgtt 1620
ggtagggcgg cgtatcctac atctctggta acttacctag
ccaatcaaat gcttgagaac 1680
ggcggttaga taagcgcgtg gggaaccttt cccacctcaa
agatcctata tcattattat 1740
gttactttct acaggtagta taccatgttc ttatatttta
gtaaactccc cgttagctta 1800
acaggtcttt gtaagcaatt aaacgtccac tattcaatcg
tctttggatt ttcgcaggac 1860
cgttttttag atcgaacata gttgataaga acaaataacc
gcttgggtcc aactttatag 1920
caattagtat atggtcattt aaaatcttta ccaattcaac
gctattaggt tctttaggat 1980
tttgcccgac atagtcgggg tgttcaacga tatcttttat
gtgcgatgaa tatttttcat 2040
aaataccagg atgttgtttc tttacgtgct ttataaatcc
gggaaacatt tttacatcgt 2100
tagaagtgca agtcaagtta tatgtatcta taatgatttg
tggaagtttt gccacaacag 2160
ttggtttatt tacaatcttt tttttattag ccgtcaaatt
tctccctcat ctcgtctctt 2220
tatatcttta ttttatcata aaggagtatt tgaaccgtcg
cgcgggacag gtttatgata 2280
gggatatttt attgaataat tgatggtata agggactttc
atgcttggaa agtggggatt 2340
atgaattaga tgcttgtcca caatatgttc caatgtaatt
aaaatttatg ttcccacctt 2400
gaccaaacat cacgtccata cttaaatcgt ccctccttta
ataggtaaaa tattaattta 2460
ccttaataaa aaaataatgg ataatagtat tcgtctgaat
ttatataatc agggggaact 2520
attgatgctg gggatactat ttacagcggc gccatctact
gatgtcgtaa aggatttgca 2580
agataaagtt atatcattgc aggatcatga ggtagcgttt
ttgaacacca cgatatctaa 2640
tatgttgatc ccccttagaa gcaaacttaa gagtgtgttg
atagtgcagt atcttaaaat 2700
tttgtgtata ataggaattg aagttaaatt agatgctaaa
aatttgtaat taagaaggag 2760
ggattcgtca tgttggtatt ccaaatgcgt aatgtagata
aaacatctac tgttttgaaa 2820
cagactaaaa acagtgatta cgcagataaa taaatacgtt
agattaattc ctaccagtga 2880
ctaatcttat gactttttaa acagataact aaaattacaa
acaaatcgtt taacttctgt 2940
atttatttac agatgtaatc acttcaggag taattacatg
aacaaaaata taaaatattc 3000
tcaaaacttt ttaacgagtg aaaaagtact caaccaaata
ataaaacaat tgaatttaaa 3060
agaaaccgat accgtttacg aaattggaac aggtaaaggg
catttaacga cgaaactggc 3120
taaaataagt aaacaggtaa cgtctattga attagacagt catctattca
acttatcgtc 3180
agaaaaatta aaactgaaca ttcgtgtcac tttaattcac
caagatattc tacagtttca 3240
attccctaac aaacagaggt ataaaattgt tgggagtatt
ccttaccatt taagcacaca 3300
aattattaaa aaagtggttt ttgaaagcca tgcgtctgac
atctatctga ttgttgaaga 3360
aggattctac aagcgtacct tggatattca ccgaacacta
gggttgctct tgcacactca 3420
agtctcgatt cagcaattgc ttaagctgcc agcggaatgc
tttcatccta aaccaaaagt 3480
aaacagtgtc ttaataaaac ttacccgcca taccacagat
gttccagata aatattggaa 3540
gctatatacg tactttgttt caaaatgggt caatcgagaa
tatcgtcaac tgtttactaa 3600
aaatcagttt catcaagcaa tgaaacacgc caaagtaaac
aatttaagta ccattactta 3660
tgagcaagta ttgtctattt ttaatagtta tctattattt
aacgggagga aataattcta 3720
tgagtcgctt ttttaaattt ggaaagttac acgttactaa
agggaatgga gataaattat 3780
tagatatact actgacagct tccaagaagc taaagaggtc
cctagcgcct acggggaatt 3840
tgtatcgcga tgggtacatt gaaaaaggaa gagtatgagt
attcaacatt tccgtgtcgc 3900
ccttattccc ttttttgcgg cattttgcct tcctgttttt
gctcacccag aaacgctggt 3960
gaaagtaaaa gatgctgaag atctgttggg tgcacgagtg
ggttacatcg aactggatct 4020
caacagcggt aagatccttg agagttttcg ccccgaagaa
cgttttccaa tgatgagcac 4080
ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
gacgccgggc aagagcaact 4140
cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
tcacagaaaa 4200
gcatcttacg gatggcatga cagtaagaga attatgcagt
gctgccataa ccatgagtga 4260
taacactgcg gccaacttac ttctgacaac gatcggagga
ccgaaggagc taaccgcttt 4320
tttgcacaac atgggggatc atgtaactcg ccttgatcgt
tgggaaccgg agctgaatga 4380
agccatacca aacgacgagc gtgacaccac gatgcctgta
gcaatggcaa caacgttgcg 4440
caaactatta actggcgaac tacttactct agcttcccgg
caacaattaa tagactggat 4500
ggaggcggat aaagttgcag gaccacttct gcgctcggcc
cttccggctg gctggtttat 4560
tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
atcattgcag cactggggcc 4620
agatggtaag ccctcccgta tcgtagttat ctacacgacg
gggagtcagg caactatgga 4680
tgaacgaaat agacagatcg ctgagatagg tgcctcactg
attaagcatt ggtaactgtc 4740
agaccaagtt tactcatata tactttagat tgatttaaaa
cttcattttt aatttaaaag 4800
gatctaggtg aagatccttt ttgataatct tcatgaccaa
aatcccttaa cgtgagtttt 4860
cgttccactg agcgtcagac cccgtagaaa agatcaaagg
atcttcttga gatccttttt 4920
ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
gctaccagcg gtggtttgtt 4980
tgccgatcaa gagctaccaa ctctttttcc gaaggtaact
ggcttcagca gagcgcagat 5040
accaaatact gtccttctag tgtagccgta gttaggccac
cacttcaaga actctgtagc 5100
accgcctaca tacctcgctc tgctaatcct gttaccagtg
gctgctgcca gtggcgataa 5160
gtcgtgtctt accgggttgg actcaagacg atagttaccg
gataaggcgc agcggtcggg 5220
ctgaacgggg ggttcgtgca cacagcccag cttggagcga
acgacctaca ccgaactgag 5280
atacctacag cgtgagctat gagaaagcgc cacgcttccc
gaagggagaa aggcggacag 5340
gtatccggta agcggcaggg tcggaacagg agagcgcacg
agggagcttc cagggggaaa 5400
cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
tgacttgagc gtcgattttt 5460
gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
agcaacgcgg cctttttacg 5520
gttcctggcc ttttgctggc cttttgctca catgttcttt
cctgcgttat cccctgattc 5580
tgtggataac cgtattaccg cctttgagtg agctgatacc
gctcgccgca gccgaacgac 5640
cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
ccaatacgca aaccgcctct 5700
ccccgcgcgt tggccgattc attaatgcag ctggcacgac
aggtttcccg actggaaagc 5760
gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact
cattaggcac cccaggcttt 5820
acactttatg cttccggctc gtatgttgtg tggaattgtg
agcggataac aatttcacac 5880
aggaaacagc tatgaccatg attacgccaa gctgggaaat
actcctagaa taaaaaaact 5940
catctttaaa gatgagctgt ccattccata aaaaattaca
ttgtaatcat gtccagaaaa 6000
tgatcaatca caatggagga cattcctaat gccggtgcat
tctgtcctaa ggaagatggc 6060
aataattcat agctattgcc taattgggaa taaacccttg
atgatacttc acttctcatt 6120
gaatttaaaa ccataggatg cgattcaatt atgctatttc
ttaaaattac ggcttgtggg 6180
ttgaaagtat ttagaatatt ggtaaggcct attcctaaat
agaatccaaa attttgtaat 6240
gcatttaagg ttccgatatc attcagatgg gcgaggttta
tgatatcttg ataggacagt 6300
tttttctctt tggtctgaag agattttaat aaagccttct
ctgaagcata caattcccag 6360
catcctcggt ttccgcaact gcatttagga ccattaaagt
ctattgtcat atgtcccatt 6420
tctccagaga agccgcttac tcctctatat aaatgattgt
tgataataac accgatccct 6480
attcctgtgc tgatacttac gtaaataatg ttatcgtgat
tttttgcagc tccaaatact 6540
ttttctccat atgcgccagc atttgcctca ttttcaataa
aaacaggcac attgtacttc 6600
tcttgtatcg aagattttaa gtcaatatct ctccagttgg agttcggagt
gaaaacaatt 6660
ttttgatctt tatcaatgag tccaggcacg caaataccta
taccaataag cccgtacgga 6720
gattggggca tttgcgtaat aaagtgatga atcatatcaa
tcaaaatgtc tttcgttatt 6780
tctggagaat tggattccaa atggcggtat tgatcaagaa
cgattgttcc ttcaaggtct 6840
gttaaaatgc cattaatata atccacacca acatctattc
caacggagta tcctgccttt 6900
ttattaaaaa caagcatgac aggtcttctt ccgccacttg
attgtccttg acctatttca 6960
aataccatac tttctttcat taacgtgttt acctgtgatg
agacagttga tttatttaat 7020
ccagtcattt cagataattt tgctcttgaa ataggtgaat
ttttaaggat ttcttttaat 7080
aataactttt gatttacttt tttgacaaag gtttgatcag
cgatatccac ttcatccact 7140
ccatttgttt aatctttaaa ttaagtatca acatagtaca
tagcgaatct tccctttatt 7200
atatctaatg tgttcataaa aaactaaaaa aaatattgaa
aatactgacg aggttatata 7260
agatgaaaat aagttagttt gtttaaacaa caaactaata
ggtgatgtac ttactatatg 7320
aaataaaatg catctgtatt tgaatgaatt tatttttaag
ggggaaatca cgtgagaagc 7380
aaaaaattgt ggatcagctt gttgtttgcg ttaacgttaa
tctttacgat ggcgttcagc 7440
aacatgtctg cgcaggctgc cggcaaggat ccgaattcga
gctccgtcga catgggcatc 7500
tttagctata aggatctgga cgaaaatgcg tcgaaggcgc
tgttttccga cgccttggcc 7560
atctctacct acgcttacca taatatcgat aacggcttcg
atgaaggcta tcaccagacc 7620
ggtttcggcc tcggtctgcc actgacgctg gtcactgcgc tgatcggcag
tacccagtcg 7680
cagggtggcc tgcctggcct cccttggaat cccgactccg
aacaggccgc gcaggaggcg 7740
gtaaacaatg ccggctggtc ggtgatcagc gccgcgcagc
tcggttacgc cggcaaaacc 7800
gatgcgcgcg gcacctacta cggcgagaca gccggttaca
ccaccgcgca ggccgaagta 7860
ctgggcaaat atgatagcga aggcaatctc accgccattg
gcatctcatt tcgcggcacc 7920
agcggcccgc gcgagtcgtt gatcggcgat accatcggcg
atgtgattaa cgatctgctg 7980
gccgggttcg ggccgaaagg ctatgccgaa ggctatacgc
tgaaggcctt cggcaatttg 8040
ctgggcgacg tggcgaaatt cgctaaggcc cacgggctga
gcggtgagga cgtggtggtc 8100
agcggccaca gcctcggcgg gctggcggtc aatagcatgg
cggcgcagag cgacgccaac 8160
tggggcggct tctacgcgca gtccaactat gtcgccttcg
cctcgccgac ccagtacgaa 8220
gccggcggta aggtgatcaa catcggctac gagaacgacc
cggtgttccg cgcgctcgac 8280
ggcacctcac tgaccctgcc gtcactgggc gtacatgatg
cgccgcacgc ctccgccacc 8340
aacaatatcg tcaacttcaa cgaccactac gcgtcggacg
cctggaacct gctgccgttt 8400
tccattctca acattccgac ctggctgtcg cacctgccgt
tcttttatca ggacggcctg 8460
atgcgggtgc tgaactccga gttttattca ctgaccgaca
aggactcgac catcatcgtc 8520
tccaacctgt cgaacgtgac gcgcggcaat acctgggtgg
aagacctgaa ccgcaacgcg 8580
gaaacgcaca gcggaccgac gtttatcatc ggcagcgacg
gcaatgattt gatcaggggc 8640
ggcaaaggca acgactatct cgagggccgc gacggtgacg
atatctttcg cgacgccggc 8700
ggctataacc tgatcgccgg cggaaaaggc cacaatatct
tcgataccca gcaggcgttg 8760
aaaaacaccg aggtcgccta cgacggcaac acgctttacc
tgcgcgatgc caagggcggc 8820
attacgctgg cggacgacat cagcaccctg cgcagcaaag
aaacctcctg gctgattttc 8880
agcaaagagg tggatcacca ggtgacagca accggattga
aatcggatgc gggcctcaaa 8940
gcctatgccg ccactaccac cggcggcgac ggcgatgacg
tcctgcaggc tcgcggccac 9000
gacgcctggc tgttcggcaa cgccggcaac gacacgctga
tcggccacgc cggcggcaat 9060
ctgaccttcg tcggcggcag cggcgatgac atcctgaagg gcgtcggcaa
cggcaatacc 9120
ttcctgttca gcggggattt tggccgcgac cagctgtatg
gcttcaacgc caccgataaa 9180
ctggtgttta tcggtaccga aggcgccagc gggaatattc
gcgactacgc cacgcagcaa 9240
aacgacgatc tggtgctggc cttcgggcac agccaggtca
cgctgatcgg cgtctcgctc 9300
gatcacttca ataccgatca ggtggtgttg gcctaaaagc
ttgcggccgc actcgagcac 9360
caccaccacc accactgaga tccggctgct aacaaagccc
gaaaggaagc tgagttggct 9420
gctgccaccg ctgagcaata actagcataa ccccttgggg
cctctaaacg ggtcttgagg 9480
ggttttttgc ccaattcact ggccgtcgtt ttacaacgtc
gtgactggga aaaccctggc 9540
gttacccaac ttaatcgcct tgcagcacat ccccctttcg
ccagctggcg taatagcgaa 9600
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
tgaatggcga atggcgcctg 9660
atgcggtatt ttctccttac gcatctgtgc ggtatttcac
accgcatatg gtgcactctc 9720
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc
aacacccgct 9780
gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc
tgtgaccgtc 9840
tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc
gagacgaaag 9900
ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt
ttcttagacg 9960
tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
tttctaaata 10020
cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
ataat 10075
Claims (9)
1.一种高效穿梭表达载体,其特征在于,所述表达载体为pHTL,其核苷酸序列如序列表SEQ ID NO:1所示。
2.根据权利要求1所述一种高效穿梭表达载体,其特征在于,所述高效穿梭表达载体的构建方法为:
(1)获得木糖启动子序列PxylR和大肠杆菌表达载体pET-21b中T7表达区序列的融合片段PxylR-T7;
所述表达区序列包括T7短肽,多克隆位点区,T7终止子;
(2)融合片段PxylR-T7插入经酶切修饰后的骨架载体pHT315的多克隆位点之间。
3.一种重组表达载体,其特征在于,其在权利要求1或2所述高效穿梭表达载体的多克隆位点之间插入目的基因。
4.包含权利要求3所述重组表达载体的工程菌。
5.一种分泌型穿梭表达载体,其特征在于,将来自纳豆芽孢杆菌碱性蛋白酶信号肽序列取代T7表达区序列插入权利要求1或权利要求2所述载体pHTL的木糖启动子PxylR和多克隆位点之间。
6.根据权利要求5所述一种分泌型穿梭表达载体,其特征在于,所述分泌型穿梭表达载体的序列如序列表SEQ ID NO:17所示。
7.根据权利要求5或6所述一种分泌型穿梭表达载体,其特征在于,所述分泌型穿梭表达载体的构建方法为:
1)获得木糖启动子PxylR部分序列和纳豆芽孢杆菌碱性蛋白酶信号肽序列Apre的融合片段PxylRstuI-Apre;
2)插入经酶切后的骨架载体pHTL的PxylR中Stu I位点和多克隆位点的BamH I之间。
8.一种重组表达载体,其特征在于,在权利要求5或6所述分泌型穿梭表达载体的多克隆位点之间插入目的基因。
9.包含权利要求8所述重组表达载体的工程菌。
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CN102002509A (zh) * | 2010-05-25 | 2011-04-06 | 江南大学 | 一种大肠杆菌-枯草芽孢杆菌穿梭表达载体及其应用 |
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CN102002509A (zh) * | 2010-05-25 | 2011-04-06 | 江南大学 | 一种大肠杆菌-枯草芽孢杆菌穿梭表达载体及其应用 |
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