CN106148380B - 一种高效穿梭表达载体及其构建方法与应用 - Google Patents

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

一种高效穿梭表达载体及其构建方法与应用

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(Em^r)、质粒pC194的头孢菌素抗性基因(Cm^r)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 IDNO: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EcoRI/HindIII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，1：1kb DNA Ladder Marker；2：质粒pHTL的EcoR I单酶切；3：质粒pHTL的NotI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，1，5，6：1kb DNA Ladder Marker；2：质粒pHTL-gfp双酶切；3：100bp DNALadder 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，1，3，8：1kb 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，1，5：1kb DNA Ladder Marker；4：100bp 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℃，200rpm培养12h；按2％接种量接种到20mL的LB培养基中37℃，200rpm过夜培养；将菌液收集在1.5mL离心管中，12000rpm离心1min，收集所有菌体，用TE洗一次；加入200μL TE缓冲液震荡混匀；加入200μL 2％的SDS缓冲液混匀；加入400μL异硫氰酸胍已酸钠混合液；12000rpm离心5min，将上清转移到一个新的离心管中，加入600μL异丙醇混匀；12000rpm离心10min去上清，加入500μL 70％乙醇混匀；12000rpm离心10min，倒去上清，室温晾干至无乙醇气味；加入50μL灭菌的去离子水，加入5μLRNA酶溶液，37℃孵育30min，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PrimeSTARMax 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 I和Hind III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(如图1)。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 IDNO: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1842bp的lipa基因。分别用限制性内切酶EcoR I和Hind III、HindIII和XhoI、SalI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390nm，发射波长为507nm，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5mL含有红霉素(10μg/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8h后收获。取1mL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450μLGUS基液重悬菌体，加入50μL的X-Gluc母液，混匀。37℃保温，5min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8h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(100mg/mL)，37℃保温30min，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410nm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±0.07U/mg)，同时，无木糖诱导的工程菌WB800-lipa的菌体酶活力为0.34±0.05U/mg.，诱导后的脂肪酶活力为72.73±0.25U/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12h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12h按1％接种量转接一次为一代工转接10代。每一代取样1mL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7)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12h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12h按1％接种量转接一次为一代工转接10代。每一代取样1mL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20mL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200μL TE缓冲液震荡混匀；加入200μL 2％的SDS缓冲液混匀；加入400μL异硫氰酸胍已酸钠混合液；12000rpm离心5min，将上清转移到一个新的离心管中，加入600μL异丙醇混匀；12000rpm离心10min去上清，加入500μL 70％乙醇混匀；12000rpm离心10min，倒去上清，室温晾干至无乙醇气味；加入50μL灭菌的去离子水，加入5μLRNA酶溶液，37℃孵育30min，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(如图8)。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9。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10，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1842bp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11)，证明构建正确，序列见(SEQID 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5mL含有红霉素(10μg/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24h后收获。取1mL培养基，离心收集上清。加入450μLGUS基液和50μL的X-Gluc母液，混匀。37℃保温，肉眼观察染色结果(图12)。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410nm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±0.07U/mg)，同时，无木糖诱导的工程菌B8-pHTZlipa的菌体酶活力为0.41±0.09U/mg，诱导后的脂肪酶活力为181.2±1.75U/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410nm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±0.08U/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±0.11U/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。