CN116003542A - 生产柠檬酸的微生物及其构建方法和应用 - Google Patents
生产柠檬酸的微生物及其构建方法和应用 Download PDFInfo
- Publication number
- CN116003542A CN116003542A CN202111235410.0A CN202111235410A CN116003542A CN 116003542 A CN116003542 A CN 116003542A CN 202111235410 A CN202111235410 A CN 202111235410A CN 116003542 A CN116003542 A CN 116003542A
- Authority
- CN
- China
- Prior art keywords
- leu
- ala
- ser
- val
- gly
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Abstract
本发明公开了生产柠檬酸的微生物及其构建方法和应用。本发明基于对黑曲霉M202菌株的基因组数据挖掘，出乎意料地发现4个蛋白的失活可以提高黑曲霉柠檬酸的产量，进一步分析发现，这4个蛋白的编码基因属于一个功能未知的基因簇，而该基因簇的失活也可促进柠檬酸的生产，显著提高菌株的产酸水平。此外，生产菌株中的柠檬酸转运蛋白活性增强，导致柠檬酸外排强化，进一步提升柠檬酸的生产。本发明可以为本领域技术人员构建柠檬酸生产菌株提供新的思路和靶点，为构建更高产柠檬酸的生产菌株提供借鉴。
Description
技术领域
本发明属于分子生物学和生物工程领域，具体涉及来源于黑曲霉的内源性多肽在生产柠檬酸中的应用、生产柠檬酸的菌株、这种菌株的构建方法和应用。
背景技术
柠檬酸是一种十分重要的有机酸，是天然的防腐剂和食品添加剂，广泛应用于工业、食品和化妆品行业。主要通过微生物发酵法生产，其主要的生产菌株包括酵母和黑曲霉。黑曲霉是柠檬酸产业中最具竞争力的工业菌株，黑曲霉生产的柠檬酸全球年产量达200万吨，产值超过20亿美元，并以每年5％的速度递增。
由于黑曲霉的遗传改造难度较大，早期的研究主要集中在发酵工艺的优化以提高黑曲霉的产量，如碳源、氮源、pH等发酵条件的不断优化。近年来，随着代谢工程的发展以及黑曲霉遗传操作工具的不断开发，通过基因工程改造提高柠檬酸产量的报道也陆续出现，改造的靶点包括增加其底物乙酰辅酶A的供应、增加柠檬酸的外排、增加呼吸链调节等等。这些遗传改造在不同程度上增加了黑曲霉柠檬酸的产量。然而，随着全球市场对于柠檬酸需求的日益增长，如何发现新的改造靶点，构建高效的黑曲霉工程菌株提升柠檬酸的产量，进一步降低生产成本，是本领域需要解决的问题。
发明内容
为克服现有技术中存在的不足，本发明基于对黑曲霉M202菌株的基因组数据挖掘，出乎意料地发现4个蛋白的失活可以提高黑曲霉柠檬酸的产量，进一步分析发现，这4个蛋白的编码基因属于一个功能未知的基因簇，而该基因簇的失活也可促进柠檬酸的生产，显著提高菌株的产酸水平，此外，生产菌株中的柠檬酸转运蛋白CexA活性增强，导致柠檬酸外排强化，进一步提升柠檬酸的生产。因此，本发明的首要目的是提供一种内源活性多肽在生产柠檬酸中的用途。
本发明的又一目的是提供一种柠檬酸的生产菌株。
本发明的另一目的是提供一种生产柠檬酸的方法。
本发明的再一目的是提供一种柠檬酸生产菌株的构建方法。
为了实现上述目的，本发明采用如下技术方案：
在本发明的第一方面，提供一种内源活性多肽在微生物发酵生产柠檬酸中的用途，所述多肽是：
(A)氨基酸序列如SEQ ID NO:1-4任一项所示；或
(B)与(A)所述蛋白氨基酸序列具有98％以上同源性，且来源于黑曲霉与(A)所述蛋白具有相同活性。
优选地，所述内源活性多肽的氨基酸序列如SEQ ID NO:1-4任一项所示。
优选地，上述用途中，(A)或(B)中的一种内源活性多肽的活性减弱或完全去除。
在本发明的第二方面，提供一种基因簇在微生物发酵生产柠檬酸中的用途，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉。
优选地，所述基因簇的核苷酸序列如SEQ ID NO：5所示。
优选地，上述用途中，(A)或(B)中的整个基因簇失活。
在本发明的第三方面，提供一种柠檬酸的生产菌株，所述菌株被修饰：
1)使得内源活性多肽的活性减弱或完全去除，其中所述内源活性多肽选自：
(A)氨基酸序列如SEQ ID NO:1-4任一项所示；或
(B)与(A)所述蛋白氨基酸序列具有98％以上同源性，且来源于黑曲霉与(A)所述蛋白具有相同活性；
或者，
2)使得一种基因簇失活，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉。
优选地，所述内源性多肽的活性减弱或完全去除可以通过以下方法之一或组合实现：部分敲除或完全敲除多肽的编码基因；编码基因突变；改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译弱化；改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定；或其他任何通过修饰基因编码区及其临近的上下游区域使其失活的方式等。
在优选的实施方式中，使得基因编码序列发生移码突变、错义突变、缺失、起始密码子改变等。
进一步优选地，所述菌株中的内源活性多肽相对于未改性的内源活性多肽活性减弱，是指所述内源活性多肽的编码基因的转录、表达降低至少30％，优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失；或者所述内源活性多肽的活性降低至少30％、优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失。
更进一步地，所述生产菌株中的柠檬酸转运蛋白活性增强。
优先地，所述柠檬酸转运蛋白为氨基酸序列如SEQ ID NO:6所示的多肽。
优选地，所述柠檬酸生产菌株包括但不限于黑曲霉(Aspergilus niger)，构巢曲霉(Aspergillus nidulans)，米曲霉(Aspergillus oryzae)，产黄青霉(Penicilliumchrysogenum)，里氏木霉(Trichoderma reesei)，玉蜀黍黑粉菌(Ustilagomaydis)，嗜热毁丝霉(Myceliophthora thermophila)等菌株中的任一种，优选黑曲霉、构巢曲霉、米曲霉、里氏木霉，最优选黑曲霉。
在本发明的具体实施例中，所述内源活性多肽为氨基酸序列如SEQ ID NO:1-4任一项所示，或者所述基因簇的核苷酸序列如SEQ ID NO：5所示。
在本发明的第四方面，提供一种柠檬酸生产菌株的构建方法，所述方法包括通过遗传修饰：
1)使得内源活性多肽的活性减弱或完全去除，其中所述内源活性多肽选自：
(A)氨基酸序列如SEQ ID NO:1-4任一项所示；或
(B)与(A)所述蛋白氨基酸序列具有98％以上同源性，且来源于黑曲霉与(A)所述蛋白具有相同活性；
或者，
2)使得一种基因簇失活，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉。
优选地，所述内源性多肽的活性减弱或完全去除可以通过以下方法之一或组合实现：部分敲除或完全敲除多肽的编码基因；编码基因突变；改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译弱化；改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定；或其他任何通过修饰基因编码区及其临近的上下游区域使其失活的方式等。
在优选的实施方式中，使得基因编码序列发生移码突变、错义突变、缺失、起始密码子改变等。
进一步优选地，上述构建方法中，所述菌株中的内源活性多肽相对于未改性的蛋白活性减弱，是指所述内源活性多肽的编码基因的转录、表达降低至少30％，优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失；或者所述内源活性多肽的活性降低至少30％、优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失。
更进一步地，所述生产菌株中的柠檬酸转运蛋白活性增强。
优先地，所述柠檬酸转运蛋白为氨基酸序列如SEQ ID NO:6所示的多肽。
优选地，所述柠檬酸生产菌株包括但不限于黑曲霉(Aspergilus niger)，构巢曲霉(Aspergillus nidulans)，米曲霉(Aspergillus oryzae)，产黄青霉(Penicilliumchrysogenum)，里氏木霉(Trichoderma reesei)，玉蜀黍黑粉菌(Ustilagomaydis)，嗜热毁丝霉(Myceliophthora thermophila)等菌株中的任一种，优选黑曲霉、构巢曲霉、米曲霉、里氏木霉，最优选黑曲霉。
在本发明的具体实施例中，所述内源活性多肽为氨基酸序列如SEQ ID NO:1-4任一项所示，或者所述基因簇的核苷酸序列如SEQ ID NO：5所示。
在本发明的第五方面，提供一种生产柠檬酸的方法，所述方法包括：(1)培养第三方面的柠檬酸生产菌株，或者第四方面构建的柠檬酸酸生产菌株，使之生产柠檬酸；和
(2)任选地从步骤(1)中分离柠檬酸。
在本发明的第六方面，提供第三方面的柠檬酸生产菌株或第四方面构建的柠檬酸生产菌株在生产柠檬酸中的应用。
本发明的有益效果
1、本发明基于对黑曲霉M202菌株的基因组数据挖掘，出乎意料地发现4个蛋白的失活可以提高黑曲霉柠檬酸的产量，进一步分析发现，这4个蛋白的编码基因属于一个功能未知的基因簇，而该基因簇的失活也可促进柠檬酸的生产，显著提高菌株的产酸水平，与包含内源性多肽的野生型菌株相比，所述柠檬酸生产菌株的柠檬酸产量提高可至30％。此外，生产菌株中的柠檬酸转运蛋白活性增强，导致柠檬酸外排强化，进一步提升柠檬酸的生产，柠檬酸产量提高幅度在2.01-2.30倍之间。因此，在实践上可用于微生物发酵生产柠檬酸，可进一步降低柠檬酸的生产成本，具有较好的工业应用潜能。
2、本发明经过筛选发现功能未知基因簇的失活可提高工程菌株柠檬酸的产量，为黑曲霉的遗传改造提供了新的思路。
具体实施方式
术语定义
本发明所用的“基因簇”，具体地，是指如SEQ ID NO:5所示的来源于黑曲霉的基因簇，其功能未知。该基因簇中的4个蛋白分别为推定的非核糖体肽合酶(NRPS)、脂肪酸合成相关蛋白(FAS)、推定的聚酮合酶(PKS1)、推定的聚酮合酶(PKS2)，其分别具有SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4的氨基酸序列。
本文所用的“柠檬酸转运蛋白”，是指能够将胞内柠檬酸转运至胞外的外排蛋白。示例地，本发明的柠檬酸外排蛋白具有SEQ ID NO:6的氨基酸序列，或者由SEQ ID NO:6所示氨基酸序列经过一个或数个氨基酸残基的取代、缺失或添加而形成的仍然具有柠檬酸外排蛋白功能的衍生蛋白或多肽。
本发明的术语“多肽”、“活性多肽”和“蛋白质”在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的，它可以包含修饰的氨基酸，并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。
本发明的术语“表达”包括涉及多肽产生的任何步骤，包括但不限于：转录、转录后修饰、翻译、翻译后修饰、和分泌。
本发明所用的术语“外源性”是指某体系中包含了原来不存在的物质。例如，通过转化等方式在某菌株中引入该菌株中原本不存在的编码基因，则该基因对于该菌株是“外源性”的。
本文所用的术语“未改性的”、“野生型的”、“内源性的”、“天然存在的”指在自然界中可以找到的对象。例如，一种存在于生物体中，可以从自然界的一个来源中分离出来并且在实验室中没有被人类有意修改的多肽或多核苷酸序列是天然存在的。
本文所用的术语“自然状态”是指微生物中多肽处于未修饰状态的活性，即自然状态下的活性。
在一些实施方式中，本发明的野生型菌株是指包含内源性多肽的菌株，内源性多肽为氨基酸序列如SEQ ID NO:1-SEQ ID NO:4任一所示序列的多肽，或者与SEQ ID NO:1-SEQ ID NO:4任一所示序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的同一性的多肽；或者，本发明的野生型菌株是指包含一种基因簇的菌株，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉。
本发明所述“多核苷酸”，一般是指多核糖核苷酸和多脱氧核糖核苷酸，其可以是非修饰的RNA或DNA，或修饰的RNA或DNA。
本发明所述“序列同源性”和“同一性百分比”指两个或更多个多核苷酸或多肽之间相同(即同一)的核苷酸或氨基酸的百分比。两个或更多个多核苷酸或多肽之间的序列同一性可通过以下方法测定：将多核苷酸或多肽的核苷酸或氨基酸序列对准且对经对准的多核苷酸或多肽中含有相同核苷酸或氨基酸残基的位置数目进行评分，且将其与经对准的多核苷酸或多肽中含有不同核苷酸或氨基酸残基的位置数目进行比较。多核苷酸可例如通过含有不同核苷酸(即取代或突变)或缺失核苷酸(即一个或两个多核苷酸中的核苷酸插入或核苷酸缺失)而在一个位置处不同。多肽可例如通过含有不同氨基酸(即取代或突变)或缺失氨基酸(即一个或两个多肽中的氨基酸插入或氨基酸缺失)而在一个位置处不同。序列同一性可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中氨基酸残基的总数来计算。举例而言，可通过用含有相同核苷酸或氨基酸残基的位置数目除以多核苷酸或多肽中核苷酸或氨基酸残基的总数且乘以100来计算同一性百分比。
本领域技术人员知晓，为提升活性而对野生型多肽进行突变，找到能实现所需目的的位点更为重要。因此，基于本发明的教导，本领域技术人员会对序列1或2所示氨基酸序列进行突变，并检测突变体的相关活性。此外，本领域普通技术人员也不难知晓，在多肽的某些区域，例如非重要区域改变少数氨基酸残基基本上不会改变生物活性，例如，适当替换某些氨基酸得到的序列并不会影响其活性(可参见Watson等，Molecular Biology of TheGene，第四版，1987，The Benjamin/Cummings Pub.Co.P224)。因此，本领域普通技术人员能够实施这种替换并且确保所得分子仍具有所需生物活性。
在具体的实施方式中，野生型菌株为包含内源性多肽的黑曲霉。本发明所述“修饰”是指任何对野生型菌株进行的遗传操作，包括但不限于各种分子生物学手段。
本发明中术语“活性减弱或完全去除”是指通过修饰使得蛋白在微生物中的细胞内活性与自然状态的蛋白质活性相比有所下降、减弱甚至完全消失。不仅包括由于蛋白质自身活性的减弱而带来的比原始功能更低的效果，也包括产生不表达的基因，或尽管表达但表达产物不具有活性或者具有降低的活性。
本发明中术语“内源多肽活性减弱或完全去除”可以通过修饰来实现，也包括但不限于如下方法的修饰：删除部分或全部编码基因、基因阅读框移码突变、弱化转录或翻译强度、或使用编码具有较低活性的相应酶或蛋白质的基因或等位基因，或使对应基因或酶失去活性，及任选地组合使用这些方法。采用合适的培养方法或基因表达的信号结构的遗传修饰(突变)可以实现基因表达的降低，例如基因表达的信号结构是阻遏基因，活性基因，操纵基因，启动子，弱化子，核糖体结合位点，起始密码子和终止子。本发明的术语“减弱或完全去除”、“降低”、“消失”、“失活”可相互替换。
本发明的术语“表达控制序列”，即调控基因表达的核苷酸序列，是指能提高或降低个体中特定基因的表达的片段，包括但不限于启动子、转录因子结合位点、核糖体结合位点、调控中转录和翻译终止的序列等。
本发明所述“柠檬酸生产菌株”具有本领域普通技术人员通常理解的含义，是指当在培养基中培养时可以生产柠檬酸并且能够积累柠檬酸，或者能够将柠檬酸分泌到培养基中，也就是能够得到胞外的游离柠檬酸的菌株。例如，可以是天然存在的柠檬酸生产菌株，也可以是经过遗传改造获得的柠檬酸生产工程菌株。本发明的柠檬酸生产菌株可以是真核细胞，包括但不限于黑曲霉(Aspergilus niger)，构巢曲霉(Aspergillus nidulans)，米曲霉(Aspergillus oryzae)，产黄青霉(Penicillium chrysogenum)，里氏木霉(Trichodermareesei)，玉蜀黍黑粉菌(Ustilago maydis)，嗜热毁丝霉(Myceliophthora thermophila)等菌株中的任一种，优选黑曲霉、构巢曲霉、米曲霉、里氏木霉，最优选黑曲霉，以及由上述菌株制备的产生柠檬酸的突变体或菌株。
本发明的术语“转化”具有本领域技术人员普遍理解的意思，即将外源性的DNA导入宿主的过程。所述转化的方法包括任何将核酸导入细胞的方法，这些方法包括但不限于电穿孔法、磷酸钙(CaPO4)沉淀法、氯化钙(CaCl2)沉淀法、微注射法、聚乙二醇(PEG)法、DEAE-葡聚糖法、阳离子脂质体法以及乙酸锂-DMSO法。
在本发明中，所述菌株的培养可以根据本领域的常规方法进行，包括但不限于孔板培养、摇瓶培养、批次培养、连续培养和分批补料培养等，并可以根据实际情况适当地调整各种培养条件如温度、时间和培养基的pH值等。
本文所用的术语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的，并不排除额外的、未引述的元件或方法步骤。
本文所用的“约”表示：一个值包括测定该值所使用的装置或方法的误差的标准偏差。
本文所用的“或”的定义仅为替代物以及“和/或”，但除非明确表示仅为替代物或替代物之间相互排斥外，权利要求中的术语“或”是指“和/或”。
本文所用的选择/可选/优选的“数值范围”既包括范围两端的数值端点，也包括相对于前述数值端点而言，所述数值端点中间所覆盖的所有自然数。
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。下列实施例中未注明具体条件的实验方法，通常按照常规条件如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,第四版)中所述的条件，或按照制造厂商所建议的条件。
实施例1.功能未知基因sgRNA表达质粒的构建
基于对黑曲霉M202菌株(购买于上海工微所科技有限公司菌种资源库，公开保藏号M202)的基因组数据挖掘，本发明预测了4个未知基因的功能，将SEQ ID NO:1所示的蛋白预测为非核糖体肽合酶(NRPS)，SEQ ID NO:2所示的蛋白预测为脂肪酸合成相关蛋白(FAS)，SEQ ID NO:3所示的蛋白预测为聚酮合酶(PKS1)，SEQ ID NO:4所示的蛋白预测为聚酮合酶(PKS2)，具体氨基酸序列分别如SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3与SEQ IDNO:4所示。
以黑曲霉M202的基因组SEQ ID NO:5为模板，设计sgRNA表达盒构建的上下游引物，以黑曲霉自身的5S rRNA作为启动子，采用GoldenGate的方法来构建sgRNA的表达质粒。将GoldenGate反应产物转化至大肠杆菌Trans-T1，提取相应的重组质粒后，采用酶切与测序验证，获得各蛋白sgRNA的表达质粒pNRPS-sgRNA1,pNRPS-sgRNA2,pFAS-sgRNA1,pFAS-sgRNA,pPKS1-sgRNA1,pPKS1-sgRNA2,pPKS2-sgRNA1,pPKS2-sgRNA2。具体引物序列如表1所示。
表1 sgRNA表达质粒构建的引物
实施例2.各基因失活菌株的构建
将Cas9蛋白表达载体pCas9(参见Zhang L,et al.Disruption or reducedexpression of the orotidine-5'-decarboxylase gene pyrG increases citric acidproduction:a new discovery during recyclable genome editing in Aspergillusniger.Microb Cell Fact.2020Mar24；19(1):76)、sgRNA表达盒片段与供体DNA片段共转化至黑曲霉M202的原生质细胞，构建各基因失活菌株。具体操作如下：
(1)sgRNA表达盒片段的制备
以5S-Fm(SEQ ID NO:23：GGTTGGAGATTCCAGACTCAG)与sgRNA-Rm(SEQ ID NO:24：AAAAAAGCACCGACTCGGTGCCAC)为引物，分别以实施例1中构建的sgRNA表达质粒为模板进行PCR扩增，分别获得各基因敲除使用的sgRNA表达盒片段NRPS-sgRNA1/NRPS-sgRNA2、FAS-sgRNA1/FAS-sgRNA2、PKS1-sgRNA1/PKS1-sgRNA2、PKS2-sgRNA1/PKS2-sgRNA2。
PCR反应体系为5×FastPfu buffer 10μL,10mM dNTPs 1μL,上游/下游引物各2.5μL，DNA模板0.5μL，FastPfu(TransGene)1.5μL，超纯水32μL。
PCR反应条件为：95℃预变性5min；95℃变性30sec，55℃退火30sec，72℃延伸45sec，35个循环；最后72℃延伸10min。PCR产物进行PCR产物纯化后进行PE G-介导的原生质体转化。
(2)供体DNA片段的制备
供体DNA片段的制备采用一步PCR的方法来完成，设计分别含有4个基因同源臂的上下游引物，以质粒pSilent-1(参见Nakayashiki H,Hanada S,Quoc NB,Kadotani N,TosaY,Mayama S.RNA silencing as a tool for exploring gene function in ascom ycetefungi.Fungal Genet Biol.2005；42:275–83)为模板进行PCR扩增，获得用于4个基因敲除使用的供体DNA片段，分别命名为MH-NRPS-sgRNA1/NRPS-sgRNA2、MH-FAS-sgRNA1/FAS-sgRNA2、MH-PKS1-sgRNA1/PKS1-sgRNA2、MH-PKS2-sgRNA1/PK S2-sgRNA2。PCR反应体系和反应条件同上，相关引物序列如表2所示。
表2供体DNA制备的所用引物
(3)PEG-介导的原生质体转化
黑曲霉M202原生质体悬液的制备参照专利CN112250740A。取100μL原生质体悬液、2μg pCas9、2μg sgRNA表达盒片段与2μg供体DNA，向其加入1mL溶液C(Tris-HCl10mM,CaCl25.54g/L,PEG6000 50％(w/v),pH 7.5)冰浴10min，2mL溶液B并混匀。与预热的含有潮霉素的上层培养基MMSH混匀后，再平铺至下层培养基MMSH平板上。将平板在30℃培养箱中培养3-5天直到长出转化子为止。
(4)基因失活菌株的基因型验证
采用天根新型植物基因组提取试剂盒DP350提取已进行二次传代分纯后转化子的基因组DNA，然后以提取的基因组DNA为模板，用各基因的基因组上下验证引物对转化子进行基因PCR验证。引物具体序列如表3所示。
表3各基因失活菌株的验证引物
PCR反应体系和PCR反应条件同上。对PCR扩增产物进行1％琼脂糖凝胶电泳(150V电压，20分钟)，在凝胶成像系统下观察基因扩增条带，并对各PCR产物进行测序验证，最终获得四个预测蛋白的编码基因敲除的菌株，分别命名为AnSM1，AnSM2，AnSM3与AnSM4。
实施例3.基因失活菌株的柠檬酸发酵
将各基因失活菌株AnSM1,AnSM2,AnSM3与AnSM4与对照菌株M202分别接种于PDA培养基上30℃培养5天，然后用0.9％生理盐水收集孢子，并采用血球计数板进行孢子计数。以106/mL的接种量接种于柠檬酸发酵培养基(玉米淀粉培养基，总糖含量12％)，34℃，250r/min培养96h。
通过快速抽滤收集发酵上清，稀释10倍后，加热煮沸10min，用滤膜过滤后用HPLC检测柠檬酸含量。具体检测条件参照专利CN112250740A。结果发现各基因的失活，均能显著促进柠檬酸的生产。与对照菌株相比，AnSM1,AnSM2,AnSM3与AnSM4的柠檬酸发酵水平分别提升15-25％、20-30％、15-30％与15-25％。
实施例4整个基因簇失活菌株的柠檬酸发酵
通过对上述4个基因的进一步分析，发现其均位于一个功能未知的基因簇上，An09g01690是这个基因簇上的第一个基因，An09g01930是该基因簇上的最后一个基因，整个基因簇全长67,885bp，包括24个基因，其核苷酸序列如SEQ ID NO:5所示。
采用实施例1-2的方法构建整个基因簇失活的菌株。其中，以实施例1中构建的pNRPS-sgRNA1和pPKS2-sgRNA2表达质粒为模板，以实施例2中的5S-Fm与sgRNA-Rm为引物，扩增获得整个基因簇的sgRNA表达盒片段NRPS-sgRNA1/PKS2-sgRNA2；以MH-NRPS-sgRNA1-F和MH-PKS2-sgRNA2-R为引物，以质粒pSilent-1为模板，扩增获得整个基因簇的供体DNA片段MH-NRPS-sgRNA1/PKS2-sgRNA2；采用实施例2的方法将2μg pCas9、2μg sgRNA表达盒片段与2μg供体DNA转化至黑曲霉M202中，以NRPS-g-F和PKS2-g-R为引物对转化子进行PCR验证，最终获得整个基因簇失活的工程菌株AnSMc。采用实施例3的方法对工程菌株AnSMc和对照菌株M202进行发酵验证，结果表明整个基因簇的失活，也可以显著提升柠檬酸的产量，提升幅度为15-30％。
实施例5基因(簇)失活与柠檬酸外排强化提高柠檬酸发酵水平
将柠檬酸外排蛋白的过表达质粒pGm-CexA(参见专利CN112250740A)利用实施例2的方法转化至黑曲霉菌株M202中，获得柠檬酸外排蛋白的过表达菌株AnCex。然后采用实施例1-4的方法，分别敲除柠檬酸高产菌株AnCex中的4个基因和整个基因簇，获得失活菌株AnCSM1，AnCSM2，AnCSM3，AnCSM4和AnCSMc。采用实施例3中的方法对上述菌株进行发酵验证，同时以野生型菌株M202作为对照。结果发现，AnCSM1，AnCSM2，AnCSM3，AnCSM4和AnCSMc的柠檬酸发酵水平可提升至对照菌株的2.01-2.30倍，说明在柠檬酸外排强化的基础上，功能未知基因簇或其中相关蛋白的失活，均可进一步提升柠檬酸的生产。
序列表
<110> 中国科学院天津工业生物技术研究所
<120> 生产柠檬酸的微生物及其构建方法和应用
<160> 40
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2793
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 1
Met Ala Pro Ile Ala Leu Ser Thr Pro Gln Pro Ser His Leu Ser Ile
1 5 10 15
Pro Ser Lys Ser Phe Lys Ser Ile Lys Asp Asp Thr Thr Ile Val Thr
20 25 30
Arg Glu Pro Ala Ser Ile Asp Gly Phe Leu Pro Val Glu Val Lys Thr
35 40 45
Gly Asp Thr Ala Glu Val Ser Gly Val Leu Cys Thr Ala Phe Gly Leu
50 55 60
Ala Leu Arg Cys Tyr Asn Thr Glu Gly Val His Phe Glu Tyr His His
65 70 75 80
Glu Thr Ala Thr Gly Ser Ser Ser Thr Asn Leu Leu Phe Glu Leu Asp
85 90 95
Glu Phe Arg Lys Val Glu Asp Leu Leu Gln Ala Val Lys Ala Thr Leu
100 105 110
His Ser Ala Arg Ser Ala Leu Glu Thr Pro Asp Glu Lys Gln Gln Thr
115 120 125
Gln Ser Tyr Gln Ser Phe Arg Ser Ala Val Ile Phe Arg Glu Glu Ser
130 135 140
Leu Asn Ser Lys His Ile Ser Pro Ser Pro Asn Phe Asp Leu Glu Leu
145 150 155 160
His Ala Thr Asn Ser Arg Asn Gly Ile Glu Ile His Met Leu Tyr Thr
165 170 175
Pro Ser Thr Val Ser Glu Asp Leu Ala Val His Leu Ala Asp Thr Leu
180 185 190
His Arg Thr Phe Glu Leu Val Asn Asn His Ser Glu Thr Glu Leu Ser
195 200 205
Ser Ala Ser Tyr Leu Gly Pro Leu Ser His Lys Arg Ile Phe Asn Trp
210 215 220
Asn Ser Asn Ala Pro Asn Ala Val Ile Glu Cys Ala His Glu Leu Val
225 230 235 240
Arg Lys Thr Ala Ser Leu Gln Pro Gln Ala Pro Ala Ile Asp Ser Trp
245 250 255
Asp Ala Gln Met Thr Tyr Ala Glu Leu Asp Ser Leu Ser Asn His Leu
260 265 270
Ala Thr Val Leu Val Glu Leu Gly Ile Lys Ala Glu Val Ile Val Pro
275 280 285
Leu Cys Phe Glu Lys Ser Ala Trp Tyr Val Val Ala Leu Leu Ala Val
290 295 300
Leu Lys Ala Gly Gly Ala Phe Val Pro Leu Asp Pro Thr His Pro Ser
305 310 315 320
Ser Arg Leu Arg Glu Ile Val Ser Gln Val Asp Ala Pro Ile Ile Leu
325 330 335
Thr Thr Ala Lys His His Asp Leu Phe Asp Asp Phe Ser Val Lys Thr
340 345 350
Met Ile Val Asn Glu Asp Thr Val Gly Thr Ile Thr Ser Gly Asp Gly
355 360 365
Cys Leu Thr Pro Val Thr Val Thr Pro Glu Asn Leu Ala Tyr Val Ile
370 375 380
Phe Thr Ser Gly Ser Thr Gly Lys Pro Lys Gly Thr Met Ile Gln His
385 390 395 400
Gln Ser Phe Cys Ser Gly Ser Leu Arg Gln Gly Glu Ala Ala Asn Met
405 410 415
Gly Pro His Ser Arg Ile Phe Gln Phe Ala Ser Tyr Ser Phe Asp Val
420 425 430
Ser Ile Leu Glu Ile Leu Thr Gly Leu Ile Phe Gly Ala Cys Ile Cys
435 440 445
Ile Pro Asn Glu Gln Leu Gly Arg Ala Asp Phe Val Gln Ser Met Asn
450 455 460
Asp Phe Arg Val Asn Trp Ala Phe Leu Thr Pro Ser Val Leu Lys Val
465 470 475 480
Leu Thr Pro Asp Gln Leu Pro Leu Leu Lys Thr Leu Val Leu Gly Gly
485 490 495
Glu Ala Met Ser Glu Ser Asp Met Met Thr Trp Ala Gly Arg Leu Gln
500 505 510
Leu Met Asn Gly Tyr Gly Pro Ser Glu Cys Ser Val Ala Ala Thr Ala
515 520 525
Asn Thr Gln Leu Asn Pro Gln Ser Ser Pro Arg Asn Ile Gly Arg Ala
530 535 540
Ile Gly Gly Val Cys Trp Ile Val Asp Pro Gln Asn His Asn Val Leu
545 550 555 560
Leu Pro Val Gly Ala Thr Gly Glu Leu Ile Ile Gln Gly Pro Ile Val
565 570 575
Ser Arg Gly Tyr Ile Lys Asp Pro Val Lys Thr Ser Ser Ala Phe Phe
580 585 590
Arg Asn Pro Ala Trp Met Val Pro Gly Thr Ser Ser Ile Trp Asn Arg
595 600 605
Phe Tyr Lys Thr Gly Asp Leu Val Arg Tyr Asp Ala Asp Gly Met Ile
610 615 620
His Phe Ile Gly Arg Lys Asp His Gln Val Lys Leu Asn Gly Gln Arg
625 630 635 640
Met Glu Leu Gly Glu Val Glu His His Leu Trp Thr Asp Pro Val Ile
645 650 655
Arg Asn Gly Met Ala Leu Ile Pro Tyr Thr Gly His Cys Lys Ser Arg
660 665 670
Leu Val Gly Ile Val Ser Leu His Lys His Phe Thr Thr Ala Ser Ile
675 680 685
Gln Thr His Thr Ser Ile Arg Leu Val Asn His Ala Glu Ala Ala Ala
690 695 700
Asp Thr Gln Ala Ile Ala Glu Arg Leu Ser His Leu Leu Pro Thr Tyr
705 710 715 720
Met Val Pro Thr Ile Trp Ile Val Leu Glu Ser Leu Pro Leu Met Ser
725 730 735
Ser Gly Lys Met Asp Arg Lys Lys Val Gly Asp Phe Val Ala Asp Met
740 745 750
Ser Thr Glu Ala Phe Gln His Ile Ile Gly Asn Ser Thr Lys Pro Gly
755 760 765
Asp Lys Ala Ser Trp Thr Lys Thr Glu Ile Leu Val Gln Lys Val Trp
770 775 780
Ala Lys Ala Leu Asn Val Pro Glu Glu Gln Ile Gly Lys His Ser Ser
785 790 795 800
Phe Leu Ser Val Gly Gly Asp Ser Ile Lys Ala Met Ala Val Met Ser
805 810 815
Glu Phe Arg Lys Leu Gly Val Thr Val Leu Val Ala Asp Leu Leu Arg
820 825 830
Tyr Asn Val Thr Glu Ile Ala Ser Lys Leu Glu Glu Ser Gly Ser Ala
835 840 845
Asp Val Leu Arg Arg Ser Glu Thr Asp His Leu Pro Ile Ser His Leu
850 855 860
Gln Arg Thr Phe Leu Glu Ala Ser Arg Ser Gly Asn Ser Ala Ile His
865 870 875 880
His Ala Met Leu Leu Glu Leu Asn His Lys Ile Ser Ile Gln Gln Leu
885 890 895
Asn Asp Ala Leu Asp Ser Ile Val Ser Ser Tyr Pro Ala Leu Thr Leu
900 905 910
Gly Phe Asn Glu Gln Asn Gly Thr Trp Leu Gln Thr Phe Arg Asp His
915 920 925
Asp Glu Leu Glu Asn Pro Val Tyr His Leu Asn Leu Asn His Val Ser
930 935 940
Ser Leu Glu Glu Ala Gln Glu Ile Met Gln His Arg Gln Ala Ser Val
945 950 955 960
Gly Val Ser Gly Asn Pro Asn Leu Met Ile Asp Leu Phe Ser Leu Arg
965 970 975
Gly Thr Gln His Ile Ser Leu Val Val His Ala Ala Ile Leu Asn Ser
980 985 990
Gln Ser Leu Ser Thr Val Ile Gly His Leu Asp Tyr Leu Leu Gln Ser
995 1000 1005
Asn Asp Glu Lys Glu Glu Asn Val Trp Glu Ser Lys Leu Leu Ser Arg
1010 1015 1020
Asp Glu Phe Ser His Ala Trp Leu Asp His Leu Arg Ser Asp Thr Gly
1025 1030 1035 1040
Met Glu Pro Met Asn Gly Pro Leu Gln Ser Val Ala Leu Glu Ser Leu
1045 1050 1055
Ser Gly Tyr Arg Asp Tyr Val Thr Glu Thr Leu Glu Leu Gly Ser Glu
1060 1065 1070
Ile Thr Gly Leu Leu Thr Gly Pro Cys Asn Leu Lys Phe Gly Thr Asn
1075 1080 1085
Ala Gly Asp Ile Phe Leu Ala Thr Leu Ile Leu Ser Tyr His Gln Ile
1090 1095 1100
Leu Glu Glu Asn Asp His Ser Thr Leu Ala Ile Val Glu Thr Thr Ala
1105 1110 1115 1120
Thr Glu Ser Thr Leu Ser Lys His Leu Arg Gly His Val Gly Gln Phe
1125 1130 1135
Ser Ser Leu Gln Pro Val His Val Pro Val Glu His Ser Asp Gln Val
1140 1145 1150
Thr Leu Gln Leu Ile Arg Asp Thr Lys Asp Lys Val Arg Gln Arg Gly
1155 1160 1165
Ile Gly Ser Lys Leu Gly Ser Ser Asn Val Ile Phe Asn Tyr Gly Leu
1170 1175 1180
Ser Ser Arg Leu Thr Glu Gly Ile Val Leu Lys Gln Ala Thr Leu Thr
1185 1190 1195 1200
Arg Arg His Gly Leu Gly Ala Ile Val Pro Thr Thr Phe Glu Val Ser
1205 1210 1215
Cys Leu Leu Glu Asp Gly Gln Phe Arg Ile Thr Val Gly Ser Asp Gln
1220 1225 1230
Val Ser Ala Gln Lys Leu Leu Leu Phe Thr Asp Gln Tyr Arg Gln Asn
1235 1240 1245
Ile Thr Gln Ala Val Glu Ile Leu Ala Ser Asn Thr Ala Lys Glu Phe
1250 1255 1260
Thr Leu Ala Asp Phe Pro Leu Leu Lys Leu Asp His Pro Ser Leu Glu
1265 1270 1275 1280
Arg Val Asn Arg Gln His Leu Pro Arg Val Gly Leu Arg Pro Asn Gln
1285 1290 1295
Ile Glu Asp Met Tyr Pro Cys Ser Pro Leu Gln Gln Gly Leu Leu Met
1300 1305 1310
Ser Gln Ala Arg Lys Gln Gly Ser Tyr Asn Val Phe Ile Gly Trp Glu
1315 1320 1325
Val Val Ser Cys Gly Ala Asn Asp Thr Val Asp Ile Gly Arg Leu Leu
1330 1335 1340
Ser Ser Trp Gln Lys Val Val Asp His His Pro Ala Leu Arg Thr Ile
1345 1350 1355 1360
Phe Thr Asp Glu Phe Gly Gly Lys Glu Pro Tyr Ala Gln Ile Val Leu
1365 1370 1375
Arg Arg Pro Arg Ala Glu Val Ser Val Lys Pro Cys Ser Asp Val Asp
1380 1385 1390
Val Ala Ser Val Leu Glu Glu Thr Ile Glu Leu Gly Thr Asn Pro Val
1395 1400 1405
Val Pro Pro His Arg Phe Ser Ile Asn Val Thr Ala Asp Asp Arg Val
1410 1415 1420
Phe Cys Arg Val Asp Ala Ser His Thr Ile Val Asp Gly Val Ser Lys
1425 1430 1435 1440
Ser Leu Ile Leu Arg Asp Leu Lys Arg Ala Tyr Ala Gly Thr Leu Leu
1445 1450 1455
Ser Pro Thr Gly Gly Ala Lys Tyr Arg Asp Phe Val Gln Tyr Val Ala
1460 1465 1470
Ala Asn Asp Met Ser Ala Ser Ile Ser Phe Trp Gln Asn His Leu Ala
1475 1480 1485
Glu Val Glu Pro Cys Tyr Phe Pro Lys Leu Ala Glu Ala Glu Gly Thr
1490 1495 1500
Pro Glu Leu Lys Tyr Val Gln Val Glu Leu Gly Ile Pro Thr Ser Val
1505 1510 1515 1520
Leu Arg Asp Phe Cys Val Ser Gln Ser Ile Thr Ile Ser Asn Leu Leu
1525 1530 1535
Gln Thr Val Trp Ala Leu Val Cys Arg Val Tyr Val Gly Arg Asp Asp
1540 1545 1550
Ile Cys Phe Gly Tyr Leu Ala Ser Gly Arg Asp Ala Pro Val Glu Asp
1555 1560 1565
Leu Val Asn Met Val Gly Pro Thr Ile Ser Val Leu Ile Cys Lys Ala
1570 1575 1580
Leu Leu Glu Asp Ser Thr Ile Ile Lys Asp Ala Leu Lys Ala Cys Gln
1585 1590 1595 1600
Glu Glu Phe Leu Asp Ser Leu Ala His Gln His Cys Ser Leu Ala Glu
1605 1610 1615
Ile Gln His Ser Leu Gly Leu Ala Gly Met Pro Leu Phe Asn Thr Gly
1620 1625 1630
Ile Ser Phe Gln Thr Met Thr Asp Val Asp Ser Asp Asp Val Ile Thr
1635 1640 1645
Asn Ile Asp Phe Glu Gly Leu Ile Val Arg Glu Pro Thr Glu Tyr Asn
1650 1655 1660
Ile Thr Val His Val Val Asp Ser Pro Asn Asn Val Asp Ile Lys Leu
1665 1670 1675 1680
Gly Tyr Trp Thr Asp Cys Ile Ser Glu Ser Arg Ala Gly Ser Val Ser
1685 1690 1695
Gly Thr Phe Ser Ala Ile Leu Lys Ser Val Val Glu His Pro Glu Ala
1700 1705 1710
Arg Val Asp Gln Leu Asn Val Ile Gly Arg Tyr Asp Leu Ser Gln Ile
1715 1720 1725
Val Ser Trp Asn Gln Asp Val Pro Glu Ile Ser Ser Tyr Thr Val Pro
1730 1735 1740
Asp Leu Val Leu Gln Gln Ile Thr Arg Thr Pro His Ala Ile Ala Ile
1745 1750 1755 1760
Asp Thr Met Ala Glu Lys Ile Thr Tyr Gln Asp Phe Gly Arg Met Trp
1765 1770 1775
Leu Ser Leu Ser Arg Tyr Leu Thr Arg Leu Gly Ile Gly Ala Gly Asp
1780 1785 1790
Tyr Val Pro Leu Thr Phe Glu Lys Ser Ala Trp Ala Ile Val Ala Met
1795 1800 1805
Phe Ala Val Leu Gly Thr Gly Ala Ala Phe Val Pro Leu Asp Pro Lys
1810 1815 1820
Thr Pro Val Glu Arg Leu Arg Glu Ala Ser Ile Gln Thr Ala Ala Asn
1825 1830 1835 1840
Val Val Leu Ala Ser Pro Lys Tyr Ala Ser Asn Trp Asn Asp Leu Ile
1845 1850 1855
Pro Thr Ile Val Ser Val Asp Gln Asp Leu Leu Asp Lys Leu Asn Glu
1860 1865 1870
Glu Gln Asp Asp Leu Asp Gln Gly Val Val Arg Ala Arg Pro His His
1875 1880 1885
Asp Ala Tyr Val Ile Phe Thr Ser Gly Ser Thr Gly Lys Pro Lys Gly
1890 1895 1900
Cys Val Val Gln His Ser Ala Phe Cys Ser Gly Ala Leu Ala Gln Gly
1905 1910 1915 1920
Lys Leu Ala Cys Leu Gly Pro Ser Ser Arg Val Leu Gln Phe Ala Ser
1925 1930 1935
Tyr Ser Phe Asp Val Ser Leu Leu Glu Ile Met Thr Ser Leu Met Phe
1940 1945 1950
Gly Ala Cys Ile Cys Val Pro Asp Glu Asp Leu Ser Lys Asp Ile Lys
1955 1960 1965
His Cys Ile Asn Val Leu Asn Ile Asn Trp Thr Phe Leu Thr Pro Ser
1970 1975 1980
Val Leu Lys Leu Leu Gln Pro Ser Asp Val Pro Ser Leu Lys Thr Leu
1985 1990 1995 2000
Val Leu Gly Gly Glu Ala Leu Ser Lys Gly Asp Ile Leu Asn Trp Ala
2005 2010 2015
Asp Arg Val Gln Leu Tyr Asn Gly Tyr Gly Pro Ser Glu Cys Ser Val
2020 2025 2030
Ala Ala Ala Ala Asn Pro Gly Leu Gln Pro Thr Thr Asp Pro Ala Asn
2035 2040 2045
Ile Gly His Ala Val Gly Gly Val Leu Trp Ile Val Asp Ala Lys Gln
2050 2055 2060
Pro Ser Lys Leu Leu Pro Ile Gly Ala Val Gly Glu Leu Leu Ile Ser
2065 2070 2075 2080
Gly Pro Ile Leu Ala Arg Gly Tyr Leu Gly Asp Pro Glu Lys Thr Ala
2085 2090 2095
Ala Ala Phe Val Lys Gln Lys Ser Phe Val Pro Gly Ser His Lys Arg
2100 2105 2110
Glu Arg Tyr Tyr Arg Thr Gly Asp Leu Val Arg Tyr Asn Ala Asp Gly
2115 2120 2125
Thr Ile His Phe Ile Gly Arg Ala Asp Gly Gln Val Lys Ile Arg Gly
2130 2135 2140
Gln Arg Val Glu Leu Gly Glu Ile Glu Tyr Asn Ile Glu Arg Asp Glu
2145 2150 2155 2160
Asn Ile His Gln Ala Leu Thr Leu Leu Pro Ser Gln Gly Pro Phe Lys
2165 2170 2175
Lys Gln Leu Leu Ala Val Val Ser Phe Lys Gly Phe Asp Leu Pro Val
2180 2185 2190
Glu Glu Gly Arg Leu Thr Leu Val Pro Glu Asp Arg Arg Pro Lys Ile
2195 2200 2205
Met Ala Val Val Thr Arg Ile Ala Asn Thr Ile Ser Ala Ala Leu Pro
2210 2215 2220
Ile Tyr Met Val Pro Ala Leu Trp Val Pro Val Asn Ala Ile Pro Leu
2225 2230 2235 2240
Leu Pro Ser Gly Lys Leu Asp Arg Lys Lys Val Arg Arg Trp Val Glu
2245 2250 2255
Thr Leu Asp Asp Ala Thr Tyr Glu Gln Ile Ala Asn Val Gly Ser Lys
2260 2265 2270
Ala Ser Gly Arg Gly Pro Ser Thr Pro Ala Glu Phe Glu Leu Arg Arg
2275 2280 2285
Ile Trp Ala Ser Val Leu Asn Arg Pro Glu Glu Gln Ile Ser Met Glu
2290 2295 2300
Arg Ser Phe Gln Ser Leu Gly Gly Asp Ser Ile Ser Ala Met Gln Val
2305 2310 2315 2320
Ile Ala Gln Cys Arg Asp Ala Lys Leu Ser Leu Ala Val Lys Asp Leu
2325 2330 2335
Ile Gln Cys Pro Thr Ile Ala Glu Leu Ala Pro Arg Val Arg Pro Leu
2340 2345 2350
Asp Glu Asp Ala Glu Pro Met Glu Asp Asp Ile Asp Val Pro Phe Asn
2355 2360 2365
Leu Ser Pro Gln Gln Thr Trp Glu Phe Asp Leu Lys Gln His Cys Asp
2370 2375 2380
Leu Asn Arg Tyr Asn Thr Thr Phe Leu Leu Ser Val His Ser Asp Ser
2385 2390 2395 2400
Asp Leu Lys Pro Val Val Asp Ala Thr Val Glu Arg His Pro Met Leu
2405 2410 2415
Arg Ala Arg Phe Gln Arg Asp Glu Ser Gly Arg Trp Met Gln Tyr Ile
2420 2425 2430
Pro Leu Asp Arg Ser Asn Ser His Leu Tyr Gln Glu Leu Glu Val Pro
2435 2440 2445
Asp Leu Glu Ala Ile Glu Pro Tyr Thr Ala Pro Asn Gln Ser Gly Phe
2450 2455 2460
Asp Leu Gln Arg Gly Pro Leu Met Arg Ala Ile Ile Phe Lys Leu Pro
2465 2470 2475 2480
Asn Gly Asp Arg Tyr Val Gly Leu Thr Val His His Met Val Ile Asp
2485 2490 2495
Thr Val Ser Trp Arg Ile Leu Leu Gln Asp Met Gln Gln Gly Leu Ser
2500 2505 2510
Thr Gly Thr Ile Thr Ser His Arg Ser Asp Ser Phe Ala Leu Trp Cys
2515 2520 2525
Arg Lys Leu Lys Glu His Ala Asp Glu Lys Trp Thr Pro Glu Tyr Val
2530 2535 2540
Leu Pro Phe Ser Ile Pro Ala Pro Leu Tyr Asp Tyr Trp Gln Met Glu
2545 2550 2555 2560
Glu Ser Met Asn Leu Phe Val Thr Glu Thr Ile Gln Glu Phe Ser Leu
2565 2570 2575
Asp Thr Lys Thr Thr Ala Arg Leu Met Gly Asn Cys Asn His Gln Tyr
2580 2585 2590
Gln Thr Lys Pro Leu Asp Ile Phe Leu Ser Ala Ile Phe His Ala Phe
2595 2600 2605
Ser Ala Thr Phe Thr Asp Arg Pro Val Pro Ala Val Phe Ile Tyr Ser
2610 2615 2620
His Gly Arg Asp Glu Phe Gly Gly Asp Leu Asp Val Ser Asn Thr Ile
2625 2630 2635 2640
Gly Trp Phe Thr Ser Ser Thr Pro Val Val Ile Asn Pro Ser Gly Asp
2645 2650 2655
Ile Val Asp Asp Leu Gln Ala Val Arg Asp Ala Arg Ala Ser Val Pro
2660 2665 2670
Gly Asn Gly Val Pro Tyr Trp Ala Thr Arg Trp Leu Thr Asp Asp Gly
2675 2680 2685
His Ala Ala Phe Glu His His Ser Pro Phe Glu Leu Ser Met Asn Tyr
2690 2695 2700
Leu Gly Gln Tyr Gln Gln Leu Glu Arg Asp Asp Ser Val Leu Arg Tyr
2705 2710 2715 2720
Val Asp Val Lys Lys Pro Lys Ala Thr Gly Ser Gly Ser Gly Leu Leu
2725 2730 2735
Arg Thr Ala Leu Val Glu Thr Gln Ala Ile Val Ile Gly Asp Ser Leu
2740 2745 2750
Gln Ile Lys Phe Val Tyr Asn Thr Arg Met Ala Arg Gln Lys Gln Leu
2755 2760 2765
Leu Asp Trp Gln Ala Lys Val Lys Asp Ser Leu Leu Glu Ile Ala Asn
2770 2775 2780
Arg Leu Gly Asp Gly Val Glu Thr Glu
2785 2790
<210> 2
<211> 2108
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 2
Met Met Thr Val Lys Thr Pro Leu Ser Ser Leu Ser Ser Glu Phe Ala
1 5 10 15
Gly Leu Thr Pro Glu Ile Ser Arg Thr Ser Ser Val Thr Gly Asp Lys
20 25 30
Arg Ser Leu Ile Ala Ala Tyr Gly Ser Val Asn Thr Thr Ile Ser Leu
35 40 45
Pro Thr Leu Gln Leu Pro Leu Ala Asn Gln Leu Trp Lys Asp Phe Arg
50 55 60
Ser Asn Leu Pro Ser Ser Ser Pro Glu Asn Asp Leu Thr Ile Lys Glu
65 70 75 80
Gly Leu Val Ala Glu Phe Ile Asn Phe Cys Ile Thr Arg Ser Ser Asp
85 90 95
Asp Thr His Cys Asp Asp Pro Ala Ala Val Leu Gln Phe Leu Glu Ser
100 105 110
Leu Leu Glu Ala Phe Glu Gln Asp Phe Val Gly Gly Asn Asp Val His
115 120 125
Ser Ala Val Ser His Leu Asp Thr Gln His Lys Thr Pro Ile Ile Lys
130 135 140
Ala Tyr Phe Ala Ala Tyr Gly Thr Leu Ser Arg Lys Pro Arg Arg Gln
145 150 155 160
Pro Ser Ile Leu Leu Glu Ser Val Thr Asp Gly Pro Ser Arg Ile Tyr
165 170 175
Ala Ile Phe Gly Gly Gln Gly Asn Asp Glu Lys Tyr Phe Ala Asp Leu
180 185 190
Lys Asp Thr Tyr Ala Thr Tyr Thr Asp Ile Leu Arg Arg Cys Val Phe
195 200 205
Ser Gly Ala Arg Leu Phe Glu Asp Leu Ala Gly Ala Ser Val Phe Lys
210 215 220
Thr His Phe Gln His Gly Phe Ala Leu Met Asp Trp Leu Glu Lys Pro
225 230 235 240
Glu Ser Glu Pro Glu Thr Ser Tyr Leu Leu Ser Ala Pro Val Ser Met
245 250 255
Pro Leu Ile Gly Leu Leu Gln Met Cys His Phe Ile Val Ala Cys Gln
260 265 270
Thr Met Gly Leu Ser Pro Arg Glu Met His Gly Tyr Leu Ser Gly Val
275 280 285
Thr Gly His Ser Gln Gly Ile Val Val Ala Leu Val Val Ser Val Ser
290 295 300
Asp Thr Trp Glu Asp Phe Tyr Arg His Ser Leu Glu Ala Leu Arg Ile
305 310 315 320
Leu Phe Phe Ile Ser Cys Arg Ser His Glu Ile Phe Pro Pro Gln Phe
325 330 335
Ile Pro Glu Ser Ile Arg Gln Asp Ala Glu Asp Asn Gly Glu Gly Thr
340 345 350
Pro Gly Pro Met Leu Arg Val Arg Asn Leu Arg Gln Gly Gln Leu Gln
355 360 365
Lys Ile Val Asp Glu Val Asn Ser Tyr Leu Pro Asp Glu Ser His Val
370 375 380
Ala Ile Ser Leu Phe Asn Gly Pro Gln Asn Leu Val Val Thr Gly Ser
385 390 395 400
Pro Leu Ser Leu Tyr Gly Leu Ser Arg His Leu Arg Gly Leu Lys Pro
405 410 415
Pro Asn Thr Ser Arg Glu Ala Gln Ile Pro Ala Ser Lys Arg Lys Pro
420 425 430
Lys Ile Glu His Arg Phe Leu Pro Ile Thr Ala Pro Phe His Ser Ile
435 440 445
Tyr Leu Asp Arg Val Val Ser Leu Val Met Lys Asp Leu Gly Asp Ile
450 455 460
Asn Ile Thr Gly Asn Gln Leu Arg Ile Pro Leu Tyr Ala Thr Thr Asp
465 470 475 480
Gly His Asp Met Arg Ser His Gly Ala Ser Asn Leu Ile Gly Asp Ile
485 490 495
Val Ser Met Ile Ala Arg Glu Pro Val His Trp Glu Asn Ala Thr Ile
500 505 510
Leu Pro Asn Ala Thr His Ile Ile Asp Phe Gly Pro Gly Gly Ser Ala
515 520 525
Gly Val Gly Ala Val Thr Met His Asn Lys Ala Gly Gln Gly Val Arg
530 535 540
Leu Ile Leu Gly Gly Ser Met Lys His Ser Glu Glu Tyr Gly Asn Lys
545 550 555 560
Ala Glu Leu Phe Asn Arg Asp Cys Gly Tyr Pro Val Val Tyr Ala Glu
565 570 575
Asn Trp Ala Glu Val Tyr Arg Pro Arg Leu Val Arg Asp Ala Ala Gly
580 585 590
Lys Val Ile Val Gln Thr Lys Phe Ser Glu Leu Leu Gly Leu Pro Pro
595 600 605
Ile Met Val Ala Gly Met Thr Pro Thr Thr Val Pro Trp Asp Phe Val
610 615 620
Ser Ala Thr Met Asn Ala Gly Tyr His Ile Glu Leu Ala Gly Gly Gly
625 630 635 640
Tyr Tyr Ser Lys Gly Ala Leu Glu Glu Ala Ile Asn Lys Ile Val Gln
645 650 655
Asn Gly Ile Pro Gly Arg Gly Val Thr Cys Asn Ile Ile Tyr Ala Ser
660 665 670
Pro Asn Ser Val Arg Trp Gln Ile Pro Leu Leu Gln Glu Leu Arg Ala
675 680 685
Lys Gly Val Pro Ile Asp Gly Ile Thr Ile Gly Ala Gly Val Pro Ser
690 695 700
Val Asp Val Ala Asn Glu Tyr Val Glu Thr Leu Gly Leu Lys His Ile
705 710 715 720
Ala Phe Lys Pro Gly Ser Val Glu Ala Ile Gln Gln Val Ile Asp Ile
725 730 735
Ala Arg Glu Asn Lys Ser Phe Pro Ile Ile Leu Gln Trp Thr Gly Gly
740 745 750
Arg Gly Gly Gly His His Ser Tyr Glu Asp Phe His Ala Pro Ile Leu
755 760 765
Glu Met Tyr Gly Arg Ile Arg Ala Cys Lys Asn Ile Val Leu Val Ala
770 775 780
Gly Ser Gly Phe Gly Gly Ala Asp Asp Thr Leu Pro Tyr Leu Thr Gly
785 790 795 800
Glu Trp Ser Lys Ala Phe Asn Ala Pro Ser Leu Met Pro Phe Asp Gly
805 810 815
Ile Leu Leu Gly Ser Arg Val Met Thr Ala Lys Glu Ala His Thr Ser
820 825 830
Ile Glu Ala Lys Gln Leu Ile Val Asp Ile Pro Gly Ile Asp Asp Ala
835 840 845
Val Trp Asp Gln Thr Tyr Thr Arg Pro Thr Gly Gly Ile Ile Thr Val
850 855 860
Arg Ser Glu Met Gly Glu Pro Ile His Lys Ile Ala Thr Arg Gly Val
865 870 875 880
Leu Phe Trp Ala Glu Met Asp Arg Thr Val Phe Ser Ile Ala Asp Lys
885 890 895
Lys Ala Arg Ala Ala Phe Leu Gln Glu Asn Lys Lys Arg Ile Ile Asp
900 905 910
Arg Leu Asn Asn Asp Phe Gln Lys Val Trp Phe Gly Arg Asp Ser Asp
915 920 925
Gly Lys Ala Cys Glu Leu Asn Glu Met Ser Leu Ser Ala Val Ala Trp
930 935 940
Arg Met Val Glu Leu Leu Tyr Val Lys His Gln Ala Arg Trp Ile His
945 950 955 960
His Ser Leu Gln Gln Leu Thr Phe Asp Phe Leu Gln Trp Leu Glu Asp
965 970 975
Val Val Gly Asn Ser Ser Gly Asn Phe Glu Ser Arg Tyr Trp Gln Thr
980 985 990
Val Gly Gln Ile Glu Glu Pro Phe Ser Ala Ile Glu Gln Phe Leu Ala
995 1000 1005
Lys His Ala Arg Ala Thr Ser Glu Leu Met Asp Val Pro Ser Thr Gln
1010 1015 1020
Met Phe Ile His Leu Cys Gln Arg Arg Gly Gln Lys Pro Val Pro Phe
1025 1030 1035 1040
Ile Pro Val Leu Asp Glu Asp Phe Glu Val Trp Phe Lys Lys Asp Ser
1045 1050 1055
Leu Trp Gln Ser Glu Asp Val Gln Ala Val Pro Gly Gln Asp Val Gly
1060 1065 1070
Arg Thr Cys Ile Leu His Gly Pro Val Ala Ala Lys His Thr Thr Val
1075 1080 1085
Pro Asn Glu Pro Ile Lys His Val Leu Asp Gly Ile His Ser Gln Trp
1090 1095 1100
Val Arg Ser Leu Thr Gly Leu Gly His Asp Asn Glu Gln Ala Gln Thr
1105 1110 1115 1120
Pro Leu Thr Glu Glu Gly Met Ser Glu Val Pro Ser Glu Ile Ser Leu
1125 1130 1135
Thr Arg Thr Tyr Asp Pro Met Leu Phe Gly Glu Glu Leu Pro Ser Ile
1140 1145 1150
Glu Pro Trp Met Gln Arg Leu Ala Gly Thr Gln Lys Gly Trp Arg His
1155 1160 1165
Ala Leu Phe Ser Gln Val Ser Ile Val Gln Gly Asn Ser Leu Cys Asp
1170 1175 1180
Asn Pro Ile Arg Arg Leu Phe Ala Pro Arg His Asn Thr Phe Val Glu
1185 1190 1195 1200
Val Glu Glu Cys Thr Ala Thr Glu Gln Ser Val Val Arg Leu Phe Glu
1205 1210 1215
Arg Thr Ala Asp Val Leu Gln Ile Leu Ala Ile Glu Val Arg Ala Val
1220 1225 1230
Ser Glu Asp Val Ile Lys Ala Thr Leu Phe Glu Asn Arg Thr Val Thr
1235 1240 1245
Gly Ala Pro Ala Gly Leu Glu Leu Leu Phe Thr Tyr His Pro Glu Phe
1250 1255 1260
Pro Phe Ala Pro Ile Arg Glu Val Ile Ser Asp Lys Ile Glu Arg Val
1265 1270 1275 1280
Lys Asp Phe Tyr His Gln Leu Trp Phe Gly Glu Pro Leu Ser Ser Ala
1285 1290 1295
Ser Val Leu Ser Gln Glu Asp Glu Phe Tyr Gly Asp Val Val Ala Val
1300 1305 1310
Thr Ala Glu Asp Ile Asn Lys Phe Asn Gln Cys Ile Arg Ser Thr Ala
1315 1320 1325
Thr Ser Arg Ser Arg Ser Ser Ser Gln Gln Thr Leu Asp Ala Pro Met
1330 1335 1340
Asp Tyr Ala Ile Val Val Ala Trp Lys Ser Val Met Lys Pro Leu Phe
1345 1350 1355 1360
Thr Lys Ser Ile Ser Gly Asp Leu Met Lys Leu Val His Leu Ser Asn
1365 1370 1375
Gln Phe Arg Arg Leu Gly Ser Phe Glu Pro Ile Lys Glu Gly Asp Val
1380 1385 1390
Leu Ser Ser Arg Ala His Ile Gln Ala Ile Leu Asn Glu Asp Ser Gly
1395 1400 1405
Lys Val Val Glu Val Ala Ala Leu Ile Ser Arg Asp Gly Val Asp Ile
1410 1415 1420
Val Glu Val Val Ser Arg Phe Met Tyr Arg Gly Ser Tyr Gly Asp Tyr
1425 1430 1435 1440
Asp Ala Ala Phe Gln Gln Thr Lys Glu Pro Lys Val Glu Leu Gln Leu
1445 1450 1455
Thr Ser Leu Lys Asp Val Val Val Leu His Ser Lys Pro Trp Phe Leu
1460 1465 1470
Pro Arg Asp Lys Asp Leu Asp Leu Lys Gly Tyr Lys Leu Thr Phe Glu
1475 1480 1485
Leu Glu Thr His Ala Gln Phe Gln Ser Lys Ser Val Phe Arg Gln Gln
1490 1495 1500
Arg Cys Phe Gly His Val Tyr Gly Arg Ser Pro Ala Gly Glu Thr Leu
1505 1510 1515 1520
Glu Val Ala Tyr Ile Asp His Thr Val Gly Val Ser Thr Ser Asn Pro
1525 1530 1535
Ile Met Asp Tyr Leu Lys Arg His Gly Ser Val Val Glu Gln Pro Val
1540 1545 1550
Leu Phe Gln Ser Pro Ile Thr Leu Gly Gly Arg Glu Ile Gln Phe Lys
1555 1560 1565
Ser Pro Ser Ser Asn Gln Asp Tyr Ala Ser Val Ser Gly Asp Tyr Asn
1570 1575 1580
Pro Ile His Val Ser Lys Thr Phe Ala Ala Tyr Ala Glu Leu Pro Asp
1585 1590 1595 1600
Thr Ile Thr His Gly Met His Met Ser Ala Arg Val Arg Gly Leu Leu
1605 1610 1615
Glu Gln Leu Thr Val Pro Gly Asn Pro Ser Gly Phe Lys Arg Tyr Gln
1620 1625 1630
Cys Asn Phe Val Gly Met Val Leu Pro Glu Asp Asp Val Glu Leu Val
1635 1640 1645
Leu Gln His Val Gly Met Val Asn Gly Arg Lys Leu Val His Ala Glu
1650 1655 1660
Ala His Arg Ala Gly Ser Gly Asp Lys Leu Ile Val Ala Glu Ala Glu
1665 1670 1675 1680
Ile Glu Gln Pro Ser Thr Gly Phe Leu Phe Thr Gly Gln Gly Ser Gln
1685 1690 1695
Glu Lys Gly Met Gly Met Glu Leu Tyr Ala Ser Ser Pro Ala Ala Arg
1700 1705 1710
Lys Val Trp Asp Leu Ala Asp Lys His Phe Leu Glu Asn Phe Gly Glu
1715 1720 1725
Tyr His Leu Phe His Met Ser Ala Phe Leu Thr Leu Pro Gly Phe Arg
1730 1735 1740
Ile Thr Asp Ile Val Arg Asn Asp Pro Arg Glu Leu Lys Ile His Phe
1745 1750 1755 1760
Gly Gly Ala Arg Gly Arg Ala Ile Arg Glu Asn Tyr Met Ala Leu Arg
1765 1770 1775
Cys Glu Met Ile His Ala Asp Gly Arg Thr Thr Ser Glu Lys Ile Phe
1780 1785 1790
Lys Asp Ile Thr His Asp Thr Lys Ser His Thr Phe Arg Ser Ser Thr
1795 1800 1805
Gly Leu Leu Ser Ser Thr Gln Phe Thr Gln Pro Ala Leu Thr Leu Met
1810 1815 1820
Glu Lys Ala Ile Ile Glu Asp Met Lys Asp Lys Gly Leu Leu Ala Asp
1825 1830 1835 1840
His Cys Ser Phe Ala Gly His Ser Leu Gly Glu Tyr Ser Ala Leu Ala
1845 1850 1855
Ala Ile Ala Glu Ile Met Pro Ile Glu Ser Leu Val Ser Val Val Phe
1860 1865 1870
Tyr Arg Gly Leu Thr Met Gln Met Ala Val Glu Arg Asp Glu Gln Gly
1875 1880 1885
Arg Ser Asn Phe Ser Met Cys Ala Val Asp Pro Ser Arg Leu Ser Lys
1890 1895 1900
Ser Phe Asn Glu Lys Arg Leu Gln Tyr Leu Val Ser Leu Ile Ala Arg
1905 1910 1915 1920
Glu Thr Ser Trp Leu Leu Glu Ile Val Asn Tyr Asn Val Ala Asn Ser
1925 1930 1935
Gln Tyr Val Cys Ala Gly Asp Leu Arg Ala Leu Asp Thr Leu Ser Ser
1940 1945 1950
Leu Ile Gly Glu Leu Lys Arg Arg Gln Ser Asp Phe Gln Ala Ile Leu
1955 1960 1965
Gln Asn Thr Gln Pro Glu Glu Glu Ile Arg Ala Ser Val Leu Asp Thr
1970 1975 1980
Leu His Gln Cys Ala Asn Ala Ala Lys Ala Lys Pro Gln Pro Ile Gln
1985 1990 1995 2000
Leu Glu Arg Gly Val Ala Thr Val Pro Leu Arg Gly Ile Asp Val Pro
2005 2010 2015
Phe His Ser Ser Phe Leu Ser Pro Gly Ile Thr Ala Phe Arg Ala Cys
2020 2025 2030
Leu Tyr Lys Tyr Ile Asp Lys Asp Thr Leu Asp Ala Asp Arg Leu Val
2035 2040 2045
Gly Lys Tyr Ile Pro Asn Leu Thr Ala Arg Pro Phe Glu Val Ser Glu
2050 2055 2060
Gly Tyr Phe Arg Gln Val Phe Arg Leu Thr Lys Ser Pro Ile Ile Ala
2065 2070 2075 2080
Lys Val Leu Ala Glu Trp Arg Glu Tyr Met Asp Gly Glu Arg Gly Leu
2085 2090 2095
Glu Lys Val Thr Arg Ser Ser Glu Val Val Val Ala
2100 2105
<210> 3
<211> 2604
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 3
Met Val Thr Thr Thr Arg Ala Thr Asn Pro Thr Asn Thr Leu Leu Leu
1 5 10 15
Phe Gly Pro Gln Ala Leu Ser Phe Ser Thr Ala Thr Phe Ala Asp Ile
20 25 30
His Ala Arg Val Val Gln Thr Ser Glu Asn Ala Trp Ile Lys Gln Thr
35 40 45
Ile Thr Ser Leu Pro Gly Leu Trp Asp Ala Leu Val Lys Glu Phe Pro
50 55 60
Gln Tyr Gly Ala Leu Glu Gly Lys Gln Leu Leu Arg Asp Leu Asp Arg
65 70 75 80
Trp Phe Glu Thr Gly Thr Met Glu His Ala Glu Pro His Leu Pro Asn
85 90 95
Ile Leu Leu Ser Pro Met Val Val Ile Thr Gln Leu Thr Glu Tyr Val
100 105 110
Asp Tyr Leu Lys Thr Met Pro His Ala Ala Asp Gln Gln Thr Glu Thr
115 120 125
Val Gly Phe Cys Thr Gly Leu Leu Thr Ala Leu Ala Ala Ser Leu Ala
130 135 140
Pro Asp Ile Lys Gly Ile Arg Gln Tyr Gly Ala Ile Ala Ile Lys Leu
145 150 155 160
Ala Met Ile Ile Gly Gly Val Val Asp Val Gln Asp Ile Thr Ser Pro
165 170 175
Asn Gly Pro Ser Lys Ser Leu Ala Val Ala Trp Asp Ser Ala Glu Thr
180 185 190
Gln Asp Arg Leu Asn Gln Ile Ile Asp Gln Ser Pro Glu Ala Tyr Ile
195 200 205
Ser Val Glu Tyr Asp His Asn Arg Pro Thr Ile Thr Thr Ala Ala Arg
210 215 220
Ser Ile Ser Ser Leu Gln Gln Arg Leu Arg Asn Ala Gly Leu Ile Ala
225 230 235 240
Ser Gln Ile Gly Leu Arg Gly Arg Phe His Cys Ala Cys Tyr Glu Lys
245 250 255
Asp Ile Glu Ala Leu Ser Lys Phe Cys Asp Ser Val Pro Ser Leu Cys
260 265 270
Leu Pro Asp Ala Ala Val Leu Ala Leu Pro Thr Arg Ser Asn Asp Ala
275 280 285
Gly Ser Phe Ile Leu Ser Gly Lys Leu His His Cys Ala Leu Arg Ser
290 295 300
Ile Leu Leu Asp Thr Ser His Trp Tyr Gln Thr Leu Glu Val Val Arg
305 310 315 320
Gln Ser Phe Leu Lys Ser Pro Ser Ser Met Val Val Ser Phe Gly Pro
325 330 335
Glu Arg Cys Ile Pro Pro Ser Ile Leu Lys Gly Leu Ser Ser Ile Val
340 345 350
Thr Thr Ala Ala Glu Tyr Gln Pro Ser Tyr Leu His Arg Asp Pro Glu
355 360 365
Leu Cys Asn Pro Asn Glu Ile Ala Val Ile Gly Met Ser Cys Lys Val
370 375 380
Ala Gly Ala Asp Asp Val Asp Glu Phe Trp Asp Leu Leu Cys Lys Ala
385 390 395 400
Glu Ser Gln His Gln Glu Val Pro Lys Glu Arg Phe Gly Phe Glu Ser
405 410 415
Ala Phe Arg Glu Val Asp Pro Thr Arg Lys Trp Tyr Gly Asn Phe Ile
420 425 430
Asn Glu His Asp Cys Phe Asp His Lys Phe Phe Lys Lys Ser Ala Arg
435 440 445
Glu Ile Ala Ala Thr Asp Pro Gln Gln Arg Gln Met Leu Gln Val Ala
450 455 460
Tyr Gln Ala Val Glu Gln Ser Gly Tyr Phe Thr Thr Pro Lys Ser Asp
465 470 475 480
Lys Asp Arg Lys Ile Gly Cys Tyr Ile Gly Val Cys Ala Ala Asp Tyr
485 490 495
Glu Tyr Asn Val Ala Cys His Pro Pro Asn Ala Phe Met Ala Thr Gly
500 505 510
Asn Leu Lys Ser Phe Val Ala Gly Lys Ile Ser His Trp Phe Gly Trp
515 520 525
Thr Gly Pro Gly Leu Cys Ile Asp Thr Ala Cys Ser Ser Ser Leu Val
530 535 540
Ala Val His Gln Ala Cys Gln Ala Ile Leu Thr Gly Asp Cys Thr Ala
545 550 555 560
Ala Leu Ala Gly Gly Ala Asn Ile Ile Thr His Pro Leu Trp Tyr Gln
565 570 575
Asn Leu Ala Ala Ala Ser Phe Leu Ser Pro Thr Gly Gln Cys Lys Pro
580 585 590
Phe Asp Ala Ser Ala Asp Gly Tyr Cys Arg Gly Glu Gly Phe Ala Ala
595 600 605
Val Phe Leu Lys Lys Met Ser Ala Ala Ile Ala Asp Gly Asp Met Ile
610 615 620
Ile Gly Ser Ile Lys Ala Thr Ala Val Asn Gln Asn Gln Asn Cys Thr
625 630 635 640
Pro Val Phe Val Pro Asn Ala Pro Thr Leu Ser Asp Leu Phe Arg Asp
645 650 655
Val Leu Asp Arg Ser Gln Leu Thr Ala Asn Gln Ile Thr Val Val Glu
660 665 670
Ala His Gly Thr Gly Thr Gln Val Gly Asp Pro Ala Glu Tyr Glu Ser
675 680 685
Ile Arg Asn Val Leu Gly Gly Pro Ser Arg Ser Thr Pro Leu Leu Phe
690 695 700
Gly Ser Val Lys Gly Leu Val Gly His Thr Glu Cys Thr Ser Gly Ala
705 710 715 720
Val Ser Leu Val Lys Thr Leu Leu Met Gln Gln His Glu Ala Ile Pro
725 730 735
Pro Gln Pro Ser Phe Asp Arg Leu Asn Pro Glu Ile Pro Val Ser Glu
740 745 750
Ser Asp Asn Met Gln Ile Ala Thr Arg Phe Ser Pro Trp Thr Ala Glu
755 760 765
Tyr Arg Ala Ala Leu Ile Asn Asn Tyr Gly Ala Cys Gly Ser Asn Ala
770 775 780
Ser Met Val Val Ala Gln Ala Pro Arg Thr Glu Gln Lys Arg Ser Ala
785 790 795 800
Thr Arg Arg Thr Ser Val Val Leu Asp Tyr Pro Phe Arg Leu Cys Gly
805 810 815
Ser Asp Asp Arg Ala Leu Arg Ala Tyr Ser Glu Arg Leu Val Arg Phe
820 825 830
Ile Ala Ser Gly Ile Lys Asp Gly Ile Ser Val Ala Asp Leu Ala Phe
835 840 845
Asn Val Cys Arg Gln Ser Asn Pro Thr Leu Asp Arg Ser Leu Ala Phe
850 855 860
Ala Cys Arg Thr Thr Gln Glu Val Glu Glu Lys Leu Arg Ala Phe Val
865 870 875 880
Ala Gly Asn Gln Gly Leu Ile Ser Ala Ser Arg Ser Lys Thr Pro Arg
885 890 895
Glu Val Ile Leu Cys Phe Gly Gly Gln Ile Ser Asn Tyr Val Gly Leu
900 905 910
Asp Arg Glu Val Tyr Asp Asn Val Ala Leu Leu Arg Lys His Leu Ala
915 920 925
Ile Cys Asp Ala Ala Cys Arg His Leu Gly Val Asp Ser Ile Phe Pro
930 935 940
Gly Ile Phe Gln Lys Ser Pro Ile Ser Asp Pro Val Lys Leu Gln Thr
945 950 955 960
Ile Leu Phe Ser Thr Gln Tyr Ser Ser Ala Lys Ala Trp Met Asp Ser
965 970 975
Gly Val Arg Pro Val Ala Ala Val Gly His Ser Phe Gly Glu Leu Thr
980 985 990
Ala Leu Cys Val Thr Gly Ile Leu Ser Leu Ala Asp Ala Met Lys Met
995 1000 1005
Ile Val Gly Arg Ala Thr Val Ile Arg Asp Phe Trp Gly Glu Asp Lys
1010 1015 1020
Gly Ser Met Ile Ala Val Glu Ala Asp Glu Asn Arg Val Gln Arg Leu
1025 1030 1035 1040
Leu Ala Glu Ala Ala Lys Gln Cys Glu Leu Ile His Val Arg Ala Pro
1045 1050 1055
Thr Ile Ala Cys Val Asn Gly Pro Thr Ser Tyr Thr Leu Ala Gly Pro
1060 1065 1070
Val Lys Ser Ile Asp Ile Val Thr Glu Val Ile Ser Arg Leu Ser Asp
1075 1080 1085
Ser Gly Pro Ser Ile Arg Ser Lys Arg Leu Lys Val Thr Asn Ala Phe
1090 1095 1100
His Ser Thr Leu Val Glu Pro Leu Met Glu Glu Leu Glu Lys Val Gly
1105 1110 1115 1120
Gln Gln Leu Thr Phe Asn Ala Pro Thr Ile Gln Leu Glu Arg Ala Ile
1125 1130 1135
Glu His His Ser Asp Ala Thr Leu Thr Ser Asp Tyr Val Pro Asp His
1140 1145 1150
Met Arg Asn Pro Val Tyr Phe Asn Gln Ala Val Gln Arg Leu Ala Gln
1155 1160 1165
Gln Tyr Pro Asp Ser Val Trp Leu Glu Ala Gly Ser Asn Ser Thr Ile
1170 1175 1180
Thr Ser Met Ala Ser Arg Ala Leu Gly Ser Pro Lys Ser Leu His Phe
1185 1190 1195 1200
Gln Ala Val Asn Ile Thr Ser Asp Asp Ser Trp Ser Met Leu Ile Thr
1205 1210 1215
Ser Thr Leu Ser Leu Trp Lys Gln Gly Ile Ser Thr Asn Phe Trp Ala
1220 1225 1230
Tyr His Ala Lys Gln Thr Tyr Glu Tyr Asn Pro Val Leu Leu Pro Pro
1235 1240 1245
Tyr Gln Phe Glu Pro Ser Arg His Trp Met Glu Leu Lys Val Pro Ser
1250 1255 1260
Phe Met Ser Asn Gly Asn Val Gln Cys Gly Pro Arg Asp Glu Glu Glu
1265 1270 1275 1280
Pro Pro Lys Thr Leu Trp Ser Leu Ile Glu Ala Ser Asp Lys Val Ala
1285 1290 1295
Arg Phe Gln Ile Asn Thr Ala Ala Pro Lys Tyr Val Glu Leu Val Ser
1300 1305 1310
Gly His Val Ile Ala Asn Thr Ala Pro Ile Cys Pro Ala Thr Val Glu
1315 1320 1325
Val Asp Ile Val Val Glu Ala Leu Arg Ser Leu Arg Pro Asp Phe Met
1330 1335 1340
Asp Ser Asn Leu Gln Pro Gln Val Leu Ala Val Thr Asn Gln Ser Pro
1345 1350 1355 1360
Ile Cys Ile Asp Pro Asn Arg Ser Val Trp Leu Glu Cys Gln Ala Met
1365 1370 1375
Asp Ser Asn Leu Val Trp Glu Trp Arg Ile Val Ser Asp Ser Leu Gln
1380 1385 1390
Glu Pro Gly Thr Ser Ser Ser Ala His Val Leu Gly Lys Leu Ala Phe
1395 1400 1405
Leu Ser Gly Gln Asp Glu Val Lys Gln Gln Glu Ser Glu Phe Met Arg
1410 1415 1420
Leu Glu Arg Leu Ile Gly His Gln Arg Cys Val Asp Leu Leu Asn Thr
1425 1430 1435 1440
Thr Glu Ala Asp Asp Ile Ile Gln Gly Arg Asn Ile Tyr Thr Thr Phe
1445 1450 1455
Ala Gly Val Val Asp Tyr Gly Glu Gln Tyr Arg Gly Leu Lys Lys Ile
1460 1465 1470
Val Gly Lys Gly Leu Glu Ser Ala Gly Arg Val Gln Lys Lys Pro Ser
1475 1480 1485
Glu Glu Ser Trp Leu Asp Ala His Leu Gly Asp Cys Phe Ser Gln Val
1490 1495 1500
Gly Gly Ile Trp Val Asn Cys Met Thr Asp His Asn Pro Asp Asp Met
1505 1510 1515 1520
Phe Ile Ala Thr Gly Phe Glu Lys Trp Val Arg Ser Pro Ala Leu Arg
1525 1530 1535
His Gly Gln Pro Arg Pro Glu Ile Trp Asp Val Leu Ala Cys His His
1540 1545 1550
Arg Ser Ser Glu Gln Thr Tyr Leu Thr Asp Ile Phe Ile Phe Asp Ala
1555 1560 1565
Glu Gln Gly Ala Leu Thr Glu Val Ile Leu Gly Ile Asn Tyr His Lys
1570 1575 1580
Val Ala Lys Ala Ser Met Ser Lys Ile Leu Ser Arg Leu Ser Gly Thr
1585 1590 1595 1600
Glu Ala Ala Pro Ser Ser Ser Thr Arg Ala His Pro Thr Ser Ser Ser
1605 1610 1615
Ser Pro Arg Leu Pro Gly Pro Phe Val Pro Glu Asp Lys Ser Gln Asn
1620 1625 1630
Glu Thr Gln Thr Ala Gly Thr Asn Ala Val Ala Lys Lys Lys Ser Glu
1635 1640 1645
Lys Ser Ala Gln Gln Asn Val Leu Asp Lys Thr Arg Ala Leu Leu Ala
1650 1655 1660
Glu Ile Ser Gly Leu Glu Pro Ser Glu Ile Glu Ala Glu Thr Gly Leu
1665 1670 1675 1680
Ala Asp Ile Gly Ile Asp Ser Leu Met Gly Met Glu Leu Ala Arg Asp
1685 1690 1695
Leu Glu Ala Leu Phe Lys Cys Pro Leu Leu Gly Asp Glu Leu Ala Asn
1700 1705 1710
Val Thr Thr Phe Gln Gly Leu Val Glu Tyr Val Gln Ser Ala Val Gly
1715 1720 1725
Val Pro Ala Asn Gly Asp Glu Pro Asp Asn Thr Asn Ala Asp Glu Val
1730 1735 1740
Phe Glu Glu Asp Asn Leu Ala Ala Ser Pro Ser Ser Ser Ser Ser Ser
1745 1750 1755 1760
Thr Asn Leu Thr Glu Asp Ser Ser Leu Asp Pro Thr Glu Thr Thr Thr
1765 1770 1775
Asn Ile Ser Ser Tyr Pro Gly Gln Thr Lys Thr Glu Lys Pro Ala Met
1780 1785 1790
Pro Pro Ala Ser Ser Lys Thr Leu Glu Leu Ser Pro Ala Trp Val Leu
1795 1800 1805
Glu Ala Phe Glu Glu Ser Lys Gln Leu Thr Asp His Phe Ile Glu Gln
1810 1815 1820
Tyr Arg Cys Ala Asn Tyr Val Asp Thr Ile Leu Pro Lys Gln Thr Gln
1825 1830 1835 1840
Leu Cys Val Ala Leu Thr Val Glu Ala Phe Glu Lys Leu Gly Cys Pro
1845 1850 1855
Ile Arg Ser Ala Val Ala Gly Gln Lys Leu Glu Arg Ile Leu His Ile
1860 1865 1870
Pro Lys His Ala Gln Leu Ala Gln Tyr Leu Tyr Arg Leu Leu Ser Ala
1875 1880 1885
Asp Ala Arg Leu Ile Asn Leu Thr Glu Asp Gly Arg Ile Thr Arg Thr
1890 1895 1900
His Met Ala Leu Pro Lys Pro Ser Asp Gln Ile Leu Gln Asp Leu Leu
1905 1910 1915 1920
Arg Leu Tyr Pro Asp His Glu Trp Ala Asn Arg Leu Ala Ala Phe Thr
1925 1930 1935
Gly Ala Arg Leu Ala Glu Val Leu Lys Gly Glu Thr Asp Gly Leu Gly
1940 1945 1950
Leu Ile Phe Gly Thr Asp Glu Gly Arg Glu Leu Val Ala Gly Leu Tyr
1955 1960 1965
Gly Asp Ser Leu Leu Asn Lys Leu Ser Tyr Arg Gln Met Glu Asp Ile
1970 1975 1980
Ile Thr Arg Leu Ala Ser Arg Ile Pro Arg Asp Ser Gly Pro Leu Lys
1985 1990 1995 2000
Ile Leu Glu Met Gly Ala Gly Thr Gly Gly Thr Thr Arg Gly Met Ala
2005 2010 2015
Pro Leu Leu Ala Arg Leu Gly Ile Pro Val Glu Tyr Thr Phe Thr Asp
2020 2025 2030
Leu Ser Gly Ser Phe Val Ala Ala Ala Arg Lys Lys Tyr Gln Lys Glu
2035 2040 2045
Tyr Pro Phe Met Lys Phe Gln Val His Asp Ile Glu Lys Pro Pro Ser
2050 2055 2060
Asp Gln Leu Arg His Ser Gln His Ile Val Ile Ala Ser Asn Ala Ile
2065 2070 2075 2080
His Ala Thr His Ser Leu Thr Asp Ser Ser Arg His Val Arg Glu Phe
2085 2090 2095
Leu Lys Thr Asp Gly Phe Leu Met Ile Val Glu Met Thr Gln Pro Val
2100 2105 2110
His Trp Val Asp Ile Ile Phe Gly Leu Phe Asp Gly Trp Trp Leu Phe
2115 2120 2125
Ala Asp Gly Arg Asp His Ala Ile Ala Ser Ala Gly Trp Trp Glu Lys
2130 2135 2140
Val Phe Gln Ser Val Gly Tyr Gly Gln Val Asp Trp Thr Asp Gly His
2145 2150 2155 2160
Arg Pro Glu Val Gln Ile Gln Arg Val Ile Ile Ala Leu Ala Ser Gly
2165 2170 2175
Pro Arg Tyr Gly Arg Gln Pro Leu Pro Pro Ala Pro Pro Pro Asn Leu
2180 2185 2190
Val Pro Gly Ser His Ala Ser Arg Gln Ala Ala Val Asn Glu Tyr Leu
2195 2200 2205
Asp Lys Tyr Thr Lys Gly Phe Thr Leu Pro Ala Gln Thr Ser Asn Thr
2210 2215 2220
Asp Ile Ser Asn Ser Thr Ser Tyr Trp Glu Lys Gln Cys Val Leu Ile
2225 2230 2235 2240
Thr Gly Ala Thr Gly Ser Leu Gly Val His Leu Val Ala Ala Val Ala
2245 2250 2255
Ala Leu Asp Asp Val Gln Thr Val Ile Cys Leu Asn Arg Arg Ser Pro
2260 2265 2270
Met Asp Pro Asp Leu Arg Gln Gln Gln Ala Phe Glu Arg Arg Gly Ile
2275 2280 2285
Leu Leu Glu Ala Ala Ser Met Ser Lys Ile Arg Val Leu Gln Thr Asp
2290 2295 2300
Ser Ser Lys Pro Gln Leu Gly Leu Thr Asp Glu Val Tyr Ser Ser Leu
2305 2310 2315 2320
Val Thr Ser Thr Thr His Ile Ile His Asn Ala Trp Pro Met Thr Gly
2325 2330 2335
Lys Arg Pro Leu Ser Gly Leu Glu Gln Gln Phe Leu Val Met Arg Asn
2340 2345 2350
Leu Leu Asp Leu Ala Ala Gln Cys Ser Ser Thr Arg Pro Ala Asn Ala
2355 2360 2365
Pro Arg Ile Val Phe Gln Phe Ile Ser Ser Ile Ala Thr Val Gly Tyr
2370 2375 2380
Tyr Pro Leu Trp Ser Gly Gln Thr Leu Val Pro Glu Ala Arg Met Gly
2385 2390 2395 2400
Ile Glu Ser Val Leu Ala Asn Gly Tyr Gly Glu Ala Lys Tyr Val Cys
2405 2410 2415
Glu Gln Met Leu Asp Arg Thr Leu His Gln Tyr Pro Asp Arg Phe Arg
2420 2425 2430
Ala Met Ala Val Arg Leu Gly Gln Ile Ala Gly Ser Arg Thr Ser Gly
2435 2440 2445
Tyr Trp Asn Pro Met Glu His Leu Ser Phe Leu Phe Lys Ser Ala Gln
2450 2455 2460
Thr Leu Gln Val Phe Pro Asp Phe Thr Gly Asp Phe Asp Leu Leu Leu
2465 2470 2475 2480
Arg Ser Thr His Ser Asn Ser Thr Thr Asp Gln Pro Ile Tyr His Ile
2485 2490 2495
Asp Asn Pro Val Arg Gln Ser Trp Ser Glu Met Val Pro Val Leu Ile
2500 2505 2510
Asp Ala Leu Gly Ile Pro Ala Gln Asn Val Ile Pro Phe Ala Asp Trp
2515 2520 2525
Val Cys Arg Val Arg Ala Phe Pro Gly Gln Val Glu Trp Asp Asn Pro
2530 2535 2540
Ser Ala Leu Leu Ile Asp Phe Leu Asp Asp His Phe Leu Arg Met Ser
2545 2550 2555 2560
Cys Gly Gly Leu Leu Leu Asp Thr Lys Arg Ala Cys Glu His Ser Pro
2565 2570 2575
Thr Leu Ala Ala Val Gly Pro Val Thr Ala Glu Leu Ala Arg Lys Tyr
2580 2585 2590
Ile Gln Ser Trp Lys Glu Met Gly Phe Leu Asn Pro
2595 2600
<210> 4
<211> 2473
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 4
Met Ala Ser Glu Thr Ala Ser Ala Met Asp Pro Gln Tyr Arg Met Glu
1 5 10 15
Leu Glu Val Val Tyr Glu Ala Leu Glu Ser Ala Gly Ile Pro Met Glu
20 25 30
Ser Ile Lys Gly Thr Asn Thr Ser Val Tyr Gly Gly Val Met Phe Arg
35 40 45
Asp Tyr His Asp Thr His Ser Arg Asp Leu Asp Thr Leu Pro Arg Tyr
50 55 60
Phe Met Thr Gly Asn Ala Ala Thr Met Ala Ser Asn Arg Ile Ser His
65 70 75 80
Phe Tyr Asp Leu Arg Gly Pro Ser Met Thr Val Asp Thr Gly Cys Ser
85 90 95
Thr Ser Leu Thr Ala Leu His Leu Ala Cys Gln Asn Leu Arg Ser Gly
100 105 110
Glu Ser Asn Met Ser Ile Val Thr Gly Ala Ser Leu Met Ile Asn Pro
115 120 125
Asp Val Phe Leu Ser Met Ser Asn Ile Gly Phe Leu Ser Pro Asp Gly
130 135 140
Ile Ser Tyr Ala Phe Asp Ser Arg Ala Asn Gly Tyr Gly Arg Gly Glu
145 150 155 160
Gly Val Gly Ala Leu Leu Val Lys Arg Leu Asp Asp Ala Leu Arg Asp
165 170 175
Gly Asp Ser Ile Arg Ala Ile Ile Arg Glu Thr Gly Val Asn Gln Asn
180 185 190
Gly Lys Thr Pro Ser Ile Thr Ala Pro Gln Gln Ala Ala Gln Glu Ala
195 200 205
Leu Ile Arg Gln Cys Tyr Glu Arg Val Asn Leu Asp Pro Ala Gln Thr
210 215 220
Thr Tyr Val Glu Ala His Gly Thr Gly Thr Pro Ala Gly Asp Pro Leu
225 230 235 240
Glu Val Gly Ala Leu Ala Ala Ala Leu Gly Gly Ser Arg Ser Ala Glu
245 250 255
His Pro Leu Tyr Leu Gly Ser Ile Lys Ala Asn Ile Gly His Thr Glu
260 265 270
Ala Ala Ser Gly Val Ala Ser Ile Ile Lys Val Ala Leu Ala Leu Glu
275 280 285
Lys Gly Gln Ile Pro Pro Asn Thr Gln Leu Asn Thr Pro Asn Ser Glu
290 295 300
Leu Arg Leu Asn Asp Arg Asn Met Glu Val Pro Val Ser Thr Gln Arg
305 310 315 320
Trp Pro Val Gly Lys Gly Pro Arg Arg Ala Ser Val Asn Asn Phe Gly
325 330 335
Phe Gly Gly Ser Asn Ala His Ala Ile Leu Glu Ser Pro Pro Val Gly
340 345 350
Ser Thr Asn Gly Thr His Val Asn Gly Thr His Thr Thr Asn Gly Thr
355 360 365
Ala Gly Met Asn Gly Thr Thr Gly Glu Asn Gly Ala Asn Arg Thr Asn
370 375 380
Gly Gln Leu Lys Ser Asn Gly Pro Val Val Asn Gly Asn Lys Thr Asn
385 390 395 400
Ile Thr Lys Arg Glu Thr Pro Trp Val Phe Arg Leu Ser Ala Lys Asp
405 410 415
Ala Gln Thr Cys Gln Gln Met Ala Ala Asp Leu Ser Thr Tyr Ile Glu
420 425 430
Ser His Pro Pro Val Asp Glu Glu Ala Phe Leu Gly Arg Leu Ala Tyr
435 440 445
Thr Leu Gly Ser Arg Arg Ser Val Phe Ser Trp Thr Ala Ala Val Ser
450 455 460
Ala Arg Ser Leu Ala Glu Leu Thr Arg Ala Leu Asp Asp Asp Glu Arg
465 470 475 480
Leu Val Pro Ser Arg Ala Ala Pro Ser Leu Arg Leu Gly Trp Val Phe
485 490 495
Thr Gly Gln Gly Ala Gln Trp Tyr Ala Met Gly Arg Glu Leu Ile Ala
500 505 510
Thr Tyr Pro Val Phe Arg Ser Thr Ile Leu Glu Cys Asp Arg Tyr Met
515 520 525
Thr Glu Met Gly Ser Thr Trp Thr Leu Met Glu Glu Leu His Arg Glu
530 535 540
Glu Ser Thr Ser Gln Val Asn Asn Ile Val Tyr Ser Leu Gly Leu Ala
545 550 555 560
Thr Ala Ile Gln Ile Ala Leu Val Glu Leu Leu Trp Ser Trp Gly Ile
565 570 575
His Pro Thr Ala Val Thr Gly His Ser Ser Gly Glu Ile Ala Ala Ala
580 585 590
Tyr Ala Ser Lys Ala Leu Asp Met Lys Ser Ala Ile Gly Ile Ala Tyr
595 600 605
Leu Arg Gly Val Leu Ala Glu Lys Phe Asp Asp Lys Ile Leu Gly Lys
610 615 620
Gly Gly Met Met Ala Val Gly Leu Gly Arg Lys Pro Val Glu His Tyr
625 630 635 640
Leu Ser Arg Val Thr Ala Gly Tyr Cys Val Val Ala Cys Val Asn Ser
645 650 655
Gln Tyr Ser Val Thr Ile Ser Gly Asp Ile Pro Ala Ile Asp Gln Leu
660 665 670
Glu Gln Leu Leu Gln Glu Asp Gln Val Phe Ala Arg Arg Leu Arg Val
675 680 685
Asn Gly Ala Phe His Cys Glu Gln Met Arg Pro Met Ala Asp Leu Phe
690 695 700
Asp Trp Ser Leu Arg Tyr Leu Leu Thr Pro His Pro Asp Phe Gly Ser
705 710 715 720
Val Leu Phe Ser Ser Pro Lys Thr Gly Ser Arg Ile Gln Asp Gly Thr
725 730 735
Ile Leu Ala Thr Ser Ser His Trp Val Gly Asn Met Leu Gln Ala Val
740 745 750
Glu Phe Glu Ser Ser Phe Arg His Met Cys Phe Gly Asp Pro Ser Pro
755 760 765
Lys Gly Ala Lys Gly Thr Gln Asp Val Asp Leu Val Leu Glu Ile Gly
770 775 780
Pro His Gly Ala Leu Gly Gly Pro Ile Gln Gln Leu Met Thr Leu Pro
785 790 795 800
Glu Phe Glu Gly Ser Gly Ile Ser Tyr Leu Pro Thr Leu Val Arg Lys
805 810 815
Gln Asp Ala Val Phe Ala Met Gln Arg Leu Ala Ile Asp Leu Thr His
820 825 830
Arg Gly Tyr Pro Val Asp Leu Asn Ala Val Asn Phe Pro His Gly Thr
835 840 845
Leu Ser Leu Ser Ile Leu His Asp Leu Pro Ser Tyr Pro Trp Asn His
850 855 860
Ser Thr Arg Tyr Trp Leu Glu Pro Arg Arg Asn Arg Ala Asp Arg Gln
865 870 875 880
Arg Gln Ala Pro Pro Ser Asp Leu Val Gly Tyr Ser Gln Pro Ser Ile
885 890 895
Thr Pro Leu Ala Arg Thr Trp Arg His Ile Ile Arg Leu Ser Asp Leu
900 905 910
Pro Trp Leu Gly Asp His Arg Val Gln Ser Ser Ile Val Phe Pro Gly
915 920 925
Ala Gly Leu Val Ser Met Ala Ile Glu Gly Met Arg Gln Val Ala Ala
930 935 940
Gly Arg Gln Gln Thr Val Ser Ala Tyr Glu Leu Arg Asp Val Asp Ile
945 950 955 960
Ala Lys Ala Leu Thr Val Pro Glu Ala Asp Glu Gly Val Glu Val Gln
965 970 975
Leu Asn Ile Arg Pro Cys Asp Glu Gln Met Leu Gly Thr Lys Asp Trp
980 985 990
Leu Ala Phe Gln Ile Phe Ser Val Ser Gly Asp Ser Arg Trp Thr Glu
995 1000 1005
His Cys Ser Gly Arg Ile Ser Val Ile Thr Thr Ser Asp Ser Thr Pro
1010 1015 1020
Leu Pro Ser Ala Ile Pro Ser Gln Ser Glu Asp Leu Tyr Asn Arg Arg
1025 1030 1035 1040
Ile Asp Pro Arg Tyr Met Trp Ala Ala Met Arg Ser Val Gly Ile Tyr
1045 1050 1055
His Gly Pro Leu Phe Gln Asn Ile His Gln Val Leu Ala Lys Pro Ser
1060 1065 1070
Ala Ser Arg Thr Ile Phe Ala Ile Ala Asp Thr Ala Ala Val Met Pro
1075 1080 1085
Lys Lys Tyr Gln Thr Pro His Val Leu His Pro Thr Thr Leu Asp Ser
1090 1095 1100
Val Phe Gln Ala Ala Tyr Thr Leu Leu Pro Glu Ser Gly Ala Arg Leu
1105 1110 1115 1120
Pro Ser Ala Met Val Pro Arg His Ile Arg Ser Val Arg Val Ser Ala
1125 1130 1135
Gln Ile Ser Asn Ser Pro Ala His Asp Leu Glu Ala Tyr Ala Thr Leu
1140 1145 1150
Asn Arg Asp Tyr Asp Ala Gln Ser Phe Glu Thr Ser Leu Thr Val Val
1155 1160 1165
Asp Ala Lys Asp Gly Asn Ser Pro Val Leu Glu Val Asp Gly Leu Thr
1170 1175 1180
Cys Gln Ser Leu Gly Arg Ala Leu Asp Arg Glu Ala Asp Pro His Glu
1185 1190 1195 1200
Asn Glu Ile Cys Ser Arg Trp Glu Trp Ala Pro Asp Ile Gly Thr Leu
1205 1210 1215
Asp Ala Ala Ala Cys Lys Asp Arg Ile Arg Cys Ala Pro Glu Ala Ala
1220 1225 1230
Glu Ile Glu Thr Met Arg Asp Leu Arg Arg Ala Thr Ile Leu Tyr Ile
1235 1240 1245
Leu Asp Ile Val Ser Ser Leu Thr Val Ala Asp Val Gln Gln Leu Arg
1250 1255 1260
Gly His Leu Lys Lys Phe Tyr Val Trp Met Val Glu Gln Leu Lys Lys
1265 1270 1275 1280
Ala Ser Arg Asn Gln Phe Ala Pro Asp Ser Ala Gln Trp Arg Asp Ile
1285 1290 1295
Ser Ala Ala Asp Lys Ala Ala Leu Tyr Glu Lys Val Gly Arg Thr Ser
1300 1305 1310
Val Asn Gly Glu Met Leu Cys Arg Leu Gly Pro Leu Gly Ala Ser Phe
1315 1320 1325
Leu Arg Gln Glu Met Ala Pro Leu Glu Val Met Leu Glu Asn Arg Leu
1330 1335 1340
Leu Phe Arg Tyr Tyr Leu Glu Ala Leu Lys Trp Asp Arg Ser Thr Arg
1345 1350 1355 1360
Gln Val Ser Glu Leu Val Arg Leu Cys Thr His Lys Asn Pro Arg Ala
1365 1370 1375
Lys Ile Leu Glu Ile Gly Ala Gly Thr Gly Gly Gly Thr Gln Val Ile
1380 1385 1390
Leu Glu Ala Leu Gly Lys Glu Asn Gly Ser Ser Thr Gly Ala Arg Phe
1395 1400 1405
Gly Arg Tyr Asp Phe Thr Asp Ile Ser Ala Gly Phe Phe Glu Ala Ala
1410 1415 1420
Lys Glu Arg Phe Gln Asp Trp Ala Asp Leu Met Asn Phe Gln Lys Leu
1425 1430 1435 1440
Asp Ile Glu His Asp Pro Val Ala Gln Gly Phe Glu Glu Gly Ser Tyr
1445 1450 1455
Asp Val Val Ile Ala Cys Gln Val Leu His Ala Thr Lys Ser Met Asp
1460 1465 1470
Arg Thr Leu Thr His Val Arg Lys Leu Leu Lys Pro Gly Gly Lys Leu
1475 1480 1485
Ile Leu Met Glu Thr Thr Arg Asp Glu Leu Asp Val Phe Phe Ala Phe
1490 1495 1500
Gly Leu Leu Pro Gly Trp Trp Leu Ser Glu Glu Glu Glu Arg Arg Thr
1505 1510 1515 1520
Thr Pro Ser Leu Thr Leu Pro Phe Trp Asn Gln Val Leu Ser Arg Asn
1525 1530 1535
Gly Phe Ala Gly Leu Asp Leu Glu Val His Asp Cys Asp Ser Glu Glu
1540 1545 1550
Phe Tyr Ala Phe Ser Thr Ile Leu Ser Thr Ala Gln Ala Pro Ala Leu
1555 1560 1565
Ser Ile Thr Ser Pro Val Thr Ile Val Thr Gly Thr Ser Pro Pro Pro
1570 1575 1580
Thr Ser Trp Met Ser Glu Leu Gln Thr Ala Val Ala Ala His Ile Gly
1585 1590 1595 1600
Cys Gln Pro Val Ile Ala Thr Leu Glu Thr Val Thr Pro Gln Gly Asn
1605 1610 1615
Ile Cys Ile Phe Leu Gly Glu Ala Asp Glu Pro Leu Leu Asp His Val
1620 1625 1630
Ser Asn Pro Val Glu Phe Asp Arg Ile Ile His Leu Ala Thr Arg Cys
1635 1640 1645
Lys Gly Leu Leu Trp Ile Thr Arg Gly Gly Ser Leu Asp Val Asp Lys
1650 1655 1660
Pro Ala Met Ser Leu Ser Gln Gly Leu Leu Arg Thr Leu Lys Ser Glu
1665 1670 1675 1680
Tyr Gln Gly Lys Ser Phe Val Ser Leu Asp Val Asp Pro Arg Arg Ser
1685 1690 1695
Pro Trp Thr Ala Glu Val Val Gln Ala Ile Ser Gln Ile Phe Pro Ala
1700 1705 1710
Ser Phe Ser Glu Thr Thr Asp Pro Ala Thr Cys Glu Phe Glu Tyr Ala
1715 1720 1725
Glu Arg Asp Gly Val Leu His Ile Pro Arg Thr Val Lys Asp Ile Pro
1730 1735 1740
Met Asn Arg Asn Ile Phe Pro Glu Ser Asp Thr Thr Glu Lys Thr Ile
1745 1750 1755 1760
His Cys Arg Phe Arg Asp Ala Ala Arg Pro Leu Arg Met Lys Ile Gly
1765 1770 1775
Thr Pro Gly Leu Ile Asp Thr Leu Val Phe His Asp Asp Leu Asp Ala
1780 1785 1790
Lys Ser Asp Pro Leu Pro Ala Asp Trp Ile Glu Phe Asp Pro Thr Ala
1795 1800 1805
Phe Gly Leu Asn Phe Arg Asp Val Met Val Ala Met Gly Gln Leu Glu
1810 1815 1820
Ala Asn Ala Ile Met Gly Phe Glu Cys Ala Gly Thr Ile Val Arg Leu
1825 1830 1835 1840
Gly Ala Thr Ala Ala Ala Lys Gly Phe Ala Val Gly Asp Arg Val Cys
1845 1850 1855
Thr Leu Leu Arg Gly His Trp Ala Thr Arg Pro Arg Ala Pro Trp Thr
1860 1865 1870
Ser Val Met Arg Ile Pro Gln His Leu Ser Asp Gln Glu Ala Ala Ser
1875 1880 1885
Phe Pro Thr Val Phe Ala Thr Ala Tyr Ile Ala Leu His Glu Thr Ala
1890 1895 1900
Arg Leu Gln Arg Gly Glu Ser Ile Leu Ile His Ala Ala Thr Gly Gly
1905 1910 1915 1920
Val Gly Gln Ala Ala Ile Gln Leu Ala Gln Leu Ile Gly Ala Glu Ile
1925 1930 1935
Tyr Ala Thr Ala Ser Thr Pro Ala Lys Arg Gln Leu Leu His Glu Thr
1940 1945 1950
Tyr Gly Ile Pro Glu Asn Asn Ile Phe Ser Ser Arg Asp Pro Ser Phe
1955 1960 1965
Ala Thr Asp Val His Leu Arg Thr Asp Gly Arg Gly Val Asp Val Val
1970 1975 1980
Leu Asn Ser Leu Ala Gly Arg Leu Leu Gln Glu Ser Phe Asn Cys Leu
1985 1990 1995 2000
Ala Glu Phe Gly Arg Met Val Glu Ile Gly Lys Arg Asp Leu Glu Gln
2005 2010 2015
His Ser Gly Leu Asp Met Tyr Pro Phe Thr Arg Asn Val Ser Phe Ser
2020 2025 2030
Ser Val Asp Leu Leu Thr Trp Gln Ser Arg Arg Gly Ala Asp Ile Ser
2035 2040 2045
Cys Val Leu Gln Ser Leu Ser Lys Leu Leu Gly Glu Lys Lys Ile Met
2050 2055 2060
Pro Val Tyr Pro Leu Thr Leu Tyr Pro Ile Thr Gln Ile Glu Lys Ala
2065 2070 2075 2080
Phe Arg Thr Met Gln Thr Gly Gln His Met Gly Lys Ile Ile Ile Ser
2085 2090 2095
Val Gly Glu Gln Asp Thr Val Pro Val Val Glu Arg Pro Pro Pro Phe
2100 2105 2110
Ser Leu Arg Ser Asp Ala Ser Tyr Val Ile Val Gly Gly Leu Gly Gly
2115 2120 2125
Ile Gly Arg Val Leu Cys Glu Trp Met Met Ala Arg Gly Ala Arg His
2130 2135 2140
Leu Ile Ile Ile Ser Arg Asn Ala Arg Pro Gly Pro Phe Val Thr Glu
2145 2150 2155 2160
Leu Glu Gln Gln Gly Cys Glu Val Arg Thr Leu Ala Cys Asp Ile Ala
2165 2170 2175
Ala Glu Asp Gln Leu Ala Ala Ala Leu Ala Gln Cys Ala Asp Met Pro
2180 2185 2190
Pro Ile Lys Gly Val Ile Gln Gly Ala Met Val Leu Lys Asp Thr Val
2195 2200 2205
Leu Glu Gln Met Thr Val Gly Asp Phe Glu Ala Ala Val Arg Pro Lys
2210 2215 2220
Ala Gln Gly Ser Trp Asn Leu His Gln Gln Leu Gly Asp Val Asp Phe
2225 2230 2235 2240
Phe Ile Met Leu Ser Ser Leu Met Gly Val Met Gly Ala Ala Ser Gln
2245 2250 2255
Ala Asn Tyr Ala Ala Gly Gly Ala Phe Gln Asp Ala Leu Ala Thr Tyr
2260 2265 2270
Arg Arg Asn Arg Gly Leu Pro Ala Val Ser Leu Asp Leu Gly Ile Val
2275 2280 2285
Arg Ser Val Gly Phe Val Ala Gln Thr Asp Gly Val Gln Glu Arg Leu
2290 2295 2300
Val Gln Met Gly Val Thr Ser Leu Ser Glu Glu Thr Val Leu Arg Ile
2305 2310 2315 2320
Leu Glu Gln Ala Ile Thr His Pro Thr Gly Pro Pro Gln Ile Ile Thr
2325 2330 2335
Gly Ile Asn Thr Ala Pro Gly Lys His Trp Asp Glu Ala Ser Trp Ile
2340 2345 2350
Gln Asp Pro Arg Phe Ala Ala Leu Arg Tyr Arg Asp Ser Thr Gln Ala
2355 2360 2365
Gly Ser Ser Arg Ala Thr Thr Gly Thr Ala Lys Gln Gly Lys Ile Arg
2370 2375 2380
Asp Gln Leu Ala Glu Ile Ala Ser Pro Val Asp Ala Ala Ala Leu Ile
2385 2390 2395 2400
Cys Gln Glu Leu Met Gln Lys Leu Ala Ser Met Phe Gly Leu Val Val
2405 2410 2415
Glu Glu Met Ser Ala Thr Gln Asp Leu Ser Ser Tyr Gly Val Asp Ser
2420 2425 2430
Leu Val Ala Val Glu Leu Arg Asn Trp Leu Val Ala Gln Val Gly Ala
2435 2440 2445
Glu Val Ser Ile Phe Asp Leu Met Gln Ser Pro Ser Leu Glu Asp Leu
2450 2455 2460
Ser Leu Arg Val Ala Thr Lys Arg Thr
2465 2470
<210> 5
<211> 67885
<212> DNA
<213> 黑曲霉(Aspergillus niger)
<400> 5
tcactcagtc tcaacaccat ctccgaggcg attggcaatc tccagtagcg aatccttcac 60
cttggcctgc cagtcgagga gctgcttctg ccgggccatg cgcgtgttat aaacgaactt 120
gatctgcagc gagtcaccga taacaatggc ttgcgtctca accagcgcgg tgcggaggag 180
accggaccca gatccagttg ccttgggctt cttcacatcc acataacgaa gtaccgaatc 240
gtcgcgctcc agctgctgat actggccaag gtagttcata gacaactcga aggggctgtg 300
atgttcaaag gcggcatggc catcgtcggt cagccagcgg gttgcccagt agggaactcc 360
attgcctggc acactggcgc gcgcatctcg cacagcctgc aggtcgtcga caatatctcc 420
agatgggttt atcactaccg gagtggagga ggtaaaccat ccgatagtgt tagatacatc 480
aagatctcca ccaaactcat cccgaccatg actgtagata aagaccgcag gtacgggacg 540
gtcagtgaac gtcgcgctga aggcatggaa gatcgccgac aagaagatgt caagtggctt 600
agtctggtac tgatggttgc aatttcccat caagcgggcg gttgtcttgg tgtccagaga 660
gaactcctgg atagtttcgg tcacgaaaag gttcatggat tcctccatct gccagtaatc 720
gtacagtgga gcaggaatcg agaaaggtag tacatattcg ggagtccatt tctcatcagc 780
gtgctccttg agtttacggc accacaaggc aaatgagtcg gaacggtggg aggtgattgt 840
cccggtggat aagccttgtt gcatatcttg gagaagaatg cgccatgaaa cggtgtcgat 900
aaccatgtgg tgtacagtaa gtccaacgta ccggtctccg ttcggaagct tgaaaataat 960
cgcacgcatc aatggaccac gctgcaaatc gaagccagac tggttgggcg cagtgtatgg 1020
ttcaatagct tccaagtcag gcacttccaa ctcctggtac agatgactat tggaccggtc 1080
aagaggaata tactgcatcc agcggcccga ttcatcgcgc tggaaccgag cacggagcat 1140
gggatgacgc tctactgttg catccacgac cggttttagg tcggaatcag aatgaaccga 1200
tagcaggaac gtagtgttgt aacgattgag atcgcagtgc tgtttcaggt cgaactccca 1260
ggtctgctga ggagaaagat taaacggaac atcaatatcg tcttccattg gctcagcatc 1320
ttcgtccaaa ggtcgcactc tcggtgccag ttcggcgata gtgggacact gaatgagatc 1380
cttaactgcg agcgagagct tggcatcacg gcactgggcg ataacctgca tagcggagat 1440
ggaatcgcca ccaaggcttt ggaaggaccg ctccatactg atttgctctt cgggtcgatt 1500
tagcacggag gcccaaatgc gtctcagctc aaattcggca ggtgtactcg ggccgcggcc 1560
gctagctttg gatcctacgt tggcaatctg ctcatatgtg gcatcgtcca gggtctcgac 1620
ccatcgccgg accttcttcc ggtccagctt tccggaaggc aacaagggga tcgcgttcac 1680
tggtacccac agggccggca ccatgtaaat cggaagggct gcagatatgg tgttagcgat 1740
tctggtcacg accgccataa tcttgggtct cctgtcctcc ggcacgagag ttaatcgtcc 1800
ctcctcaaca gggagatcga agcctttgaa agaaacaacg gccaatagct gcttcttgaa 1860
tgggccctga gatgggagca aagtgagtgc ctggtggata ttctcatcac gctcgatatt 1920
gtactcgatc tcgcccaatt caacccgctg gccgcgaatc ttcacctgcc catcagctct 1980
gccgatgaaa tgaatcgtgc catctgcatt gtatcgcaca agatcaccag tgcggtaata 2040
ccgctcacgc ttgtgagatc ctggaacaaa gctcttctgt ttgacaaagg cggctgctgt 2100
tttctcagga tcacccaggt agcctctcgc cagaattgga cctgaaatca agagctcacc 2160
aacagcacca atgggaagta gcttcgaagg ttgcttggca tcaacaatcc agagcacacc 2220
accaacagca tgtccaatgt tggctggatc ggtagtaggt tgcaatcctg ggttagctgc 2280
tgctgccaca gagcactcac taggtccgta tccgttgtac agctgtaccc tgtcagccca 2340
gttgagaatg tcaccctttg agagagcttc gccgccaaga accaaagtct tcaatgaggg 2400
cacatcggac ggttgtaaaa gcttaaggac agatggcgta aggaatgtcc agttgatatt 2460
gagcacgttg atgcagtgct tgatgtcctt gctcaagtct tcgtccggca cgcaaatgca 2520
tgcaccaaac atgagtgatg tcatgatttc cagcagacta acgtcgaagc tgtaggaagc 2580
aaactgcagt actcgtgatg agggacccag acaggcaagc tttccttgag ccagggctcc 2640
agagcagaat gcagaatgct gcacaacaca tccttttggc ttgcctgtgc tgcccgaagt 2700
gaagataacg taggcatcat gatgggggcg ggctctcacg acaccttggt cgagatcgtc 2760
ttgttcttcg ttcagcttgt cgaggagatc ctggtcaaca ctgacaatgg ttggaataag 2820
gtcgttccag tttgaagcat atttcggaga tgcaagaaca acgttagcag cagtttgaat 2880
agaggcctcg cgtaggcgct ccaccggggt tttaggatcc agagggacga acgcagctcc 2940
agtacccaga acagcaaaca tggcaacgat agcccatgct gacttttcga acgtcaaagg 3000
cacgtaatcc ccggcgccaa taccaagcct ggtcaagtat cgagacaacg acagccacat 3060
ccgtccaaaa tcctgatagg ttatcttttc ggccatggtg tcaatcgcaa tagcatgagg 3120
ggtacgcgta atctgttgga gcacgaggtc cggtactgtg tacgatgaaa tctccggcac 3180
atcctgattc caagatacaa tctgcgacaa atcataccgg ccgataacat tgagctgatc 3240
aactcgggcc tctggatgtt cgaccacgga tttgaggatg gcagagaatg ttcccgatac 3300
ggatccagct cgggattctg agatacaatc agtccaatat ccgagtttga tgtccacgtt 3360
gtttggcgaa tcgacaacgt gaaccgtgat attgtactac ttcaggtcag ttattgcaaa 3420
gtagttcaga gtaaagtcat acctctgtcg gctcccgtac aatgagaccc tcgaagtcaa 3480
tattggtgat aacgtcgtca ctatcaacgt ctgtcattgt ctggaaggaa atgcctgtgt 3540
taaacagagg cattcctgcg agtccaagtg aatgttgaat ctcggcgagc gagcagtgct 3600
ggtgtgcgag agaatcaagg aactcttcct ggcaagcctt caaggcgtcc ttgataatag 3660
tggagtcctc cagcaatgcc ttgcaaatga ggacactgat agttggcccg accatgttga 3720
ctaggtcttc gacaggtgca tctcttccgg atgccaagta accgaaacaa atatcatctc 3780
ggccgacata cacccgacag acgagtgccc agactgtctg tagaaggttc gagattgtga 3840
tactctggga gacacagaag tcacgcagaa cactggtagg tattcctagc tctacctgga 3900
cgtacttcag ctcaggtgta ccttcagcct cagctaattt gggaaagtag cagggctcga 3960
cttcggcaag atggttctgc cagaaagaaa tgctagcgga catgtcgttt gcagcaacat 4020
attggacgaa gtccctgtac ttagcgcctc cagtgggaga aagaagagtc cccgcataag 4080
ctcgcttcag gtcccttaga atcaatgact tggatacacc atcgacaatg gtatgactag 4140
catcgactcg gcagaacact ctatcatccg ctgtgacatt gattgagaac cgatgtggag 4200
ggacaacagg gttagtgcct agttcaatcg tctcctccag cacagatgca acatcgacat 4260
ctgagcaagg tttcaccgaa acctccgccc tgggacgtct cagaacaatt tgagcgtatg 4320
gctccttgcc gccaaattcg tcggtgaata tagtcctcag ggcagggtga tggtcgacta 4380
ccttttgcca tgaggagagc aatctgccaa tatcaactgt gtcgtttgcg ccgcaagaga 4440
ctacctccca tccaatgaat acattgtaac tgccttgctt cctagcctgg ctcataagga 4500
gaccttgctg caacggagag cagggataca tatcttctat ctgatttggt ctaaggccaa 4560
ctcgtggaag gtgctgacgg ttgactctct caagggatgg atgatcgagt ttgagcaatg 4620
gaaagtccgc caaagtgaat tccttagctg tattggacgc gaggatctcg actgcttgcg 4680
tgatgttctg tctgtactgg tcggtaaata gtaatagctt ctgagcgctg acctgatcgg 4740
atccgactgt gattctgaac tgtccatctt cgagcagaca tgaaacctcg aaagttgtgg 4800
gaacgatggc gcccaagccg tgtcgccgag tgagggtggc ttgcttcaac acgatacctt 4860
ctgtaagtcg gctagacaag ccgtagttga agatgacatt ggaagagcca agcttgctac 4920
ctatccccct ctggcgaact ttgtccttag tatcccggat tagctgaaga gtgacttggt 4980
ccgagtgttc cacaggcaca tgcacgggtt gcagcgaaga gaattgaccg acatgacctc 5040
tcaagtgttt gctaagagtc gactcagtag cggttgtttc gacaatagct aaggtagagt 5100
ggtcattttc ttctagaatt tggtggtatg acaggatcaa ggtcgccaga aatatatccc 5160
ccgcgttggt gccaaatttt aggttgcacg gtccagtgag aagcccagta atctcggagc 5220
cgagttctag cgtctctgtg acatagtctc gatacccact aagagattca agggcgacag 5280
attgcaatgg accattcata ggctccatgc cagtatcgct cctgaggtga tcaagccagg 5340
catgagagaa ctcatctcgg gacaaaagct tactttccca gacgttttcc tctttttcat 5400
catttgactg aagcaagtaa tcgagatggc cgatgacagt acttagagac tgtgaattaa 5460
ggatagctgc atgcacgacg agagaaatgt gctgtgtgcc gcgaagagag aacagatcga 5520
tcatgagatt tgggttacca ctcacaccaa ctgacgcctg acgatgctgc ataatttctt 5580
gtgcttcttc caagctagag acatggttca gattcaagtg gtaaacgggg ttttcaagct 5640
cgtcgtggtc tctgaatgtt tgcaaccatg tcccgttctg ctcattgaag ccgagggtga 5700
gtgctgggta ggaagaaacg atactatcca aggcatcatt cagctgctgg atactaatct 5760
tgtgattgag ttccagtaac atggcgtggt gtatggcgct gttgcccgaa cgggacgcct 5820
cgaggaaggt gcgctgtagg tgtgatatgg gaaggtgatc tgtctcgctt ctccggagca 5880
catccgcaga gccagactcc tccagcttag aggcgatctc agtaacattg taccgcaaca 5940
gatcagctac taggacggtg actccgagtt tgcggaactc tgacatgacg gccatcgctt 6000
tgatagagtc accgcccaca gacaggaagc tgctgtgttt gccaatttgc tcctctggta 6060
cgttcagagc tttcgcccaa accttctgca cgagaatctc tgtcttggtc caagacgcct 6120
tgtcgccagg ttttgttgag ttacctatga tgtgttggaa ggcctcggtg ctcatatcag 6180
caacaaaatc cccaaccttc ttccggtcca tcttgcccga cgacatgagt ggaagactct 6240
ccaggacaat ccagatggtt gggaccatat acgtggggag caggtgtgac agcctttctg 6300
caatcgcttg ggtgtctgct gcggcctcgg catgatttac caggcgaata ctcgtgtgtg 6360
tctgtattga agcggttgta aaatgcttgt ggagagacac gataccaacc aagcgggatt 6420
tgcagtgacc agtgtaggga atgagagcca ttccgttacg gatgactgga tctgtccaga 6480
ggtggtgctc tacctcccct aattccatac gctgtccatt gagcttgact tggtggtcct 6540
tacgcccaat gaagtgaatc atgccgtccg catcatatcg taccagatca ccagtcttgt 6600
aaaagcgatt ccagattgag gatgtacccg gcaccatcca agccgggttt ctaaagaagg 6660
ctgatgaagt cttcacagga tccttgatat atccacgcga gacaatagga ccttggatga 6720
tcagctcacc tgtggcgcca acagggagaa gcacgttgtg gttttgcgga tcaacaatcc 6780
agcatacgcc accaattgct ctaccaatat tccgtgggct ggactggggg ttgagctgag 6840
tattggcagt ggctgcgacg gagcattcac ttggcccata gccattcatc aactggagcc 6900
ttcccgccca tgtcatcata tccgactctg acatagcttc gcctcccagt accagagtct 6960
tcaaaagcgg cagttgatca ggagtcagca ccttcaacac cgacggtgtc aagaaggccc 7020
agttgacacg aaagtcattc atggactgga caaagtctgc acgaccaagc tgctcgttcg 7080
ggatacagat gcaggcgccg aatataaggc ccgtgaggat ttccaaaata ctcacgtcaa 7140
agctgtatga agcgaactgg aatatccggg aatgaggtcc catgttggcc gcttcaccct 7200
gtcgcagaga cccactgcag aaggactggt gctggatcat cgtacctttg ggctttccag 7260
tgctcccaga ggtaaagatc acatatgcga gattctcagg cgtgacggtg actggagtaa 7320
gacacccgtc accactagtt attgtcccga ctgtgtcttc attgactatc attgtcttga 7380
cagaaaagtc atcgaagaga tcgtgatgtt tggctgtcgt caaaataatc ggtgcatcaa 7440
cttggctgac gatctctcgt agacgtgatg agggatgcgt cgggtcgagg ggaacaaagg 7500
caccaccggc tttgagtact gcaagcaacg ccacgacata ccatgcagac ttttcaaaac 7560
acagaggcac aatcacctcg gccttgattc ccagttccac caacaccgta gccagatggt 7620
ttgacagaga atccagctct gcatacgtca tctgtgcatc ccatgaatca atagcaggag 7680
cctgtggttg caaggacgct gtctttctaa ctagctcatg agcgcactca atcaccgcat 7740
taggcgcgtt ggaattccag ttgaaaatcc tcttatggct gagtgggccc aagtagcttg 7800
cactcgaaag ctcagtctct gagtggttgt tgaccaattc gaatgtccga tggagagtat 7860
cggccaaatg gactgcaagg tcctcagata ctgtagacgg agtatagagc atgtggatct 7920
caatgccatt tctactattt gtggcatgaa gttcgaggtc aaactatatg cggtaagtct 7980
ctatctttcg gttgctgatg aacaggtggg ttttgggtct tacatttggt gatggcgaga 8040
tgtgtttact atttaaactc tcttcacgaa agattacggc gctgcggaag gactggtagg 8100
attgagtttg ctgtttctcg tctggtgtct ccaaggctga tcgagcacta tgcaatgttg 8160
ccttcactgc ttggagaagg tcctcaactt tgcggaactc gtccaactcg aacaatagat 8220
ctgtggttat cgttagcttg tgctgatttt ggggctctgc atacaatact cactcgtgga 8280
gctggaacca gttgctgtct cgtgatggta ttcaaaatgc acgccttcgg tattatagca 8340
acgaagggca aggccaaagg ctgtgcagag tactccagat acctctgctg tgtctcctgt 8400
cttcacttca acaggcaaaa agccgtctat cgaagcaggt tctcttgtga ctatggtggt 8460
gtcatctttg atgctcttga aagacttgga agggatgcta agatgagatg gttgaggagt 8520
ggataaagcg atcggagcca ttttgcagct gattgatgag tgaaatgtcg gcttgcaaac 8580
tgatagatgc agactgcagg ccaccttaat taaggaaagt tatggaatga aatgaaagct 8640
tctcttccgg gtgatatgtc catgtcgaga aagccttgaa caaagatgtt cggccttcga 8700
aggcttagat cgaacccggg agccaatact ttgctcccct gtcagcttcg tgttgtaggt 8760
ctcaagcaac aggggctgaa ttgatctatc gtgcgactga agttgatacg atccttctgc 8820
atgccctcat cgcgtaattg tcgagacgtc tgtatagacc ttagtctcga taccacgtct 8880
gataccaaac cgtcatgtaa taccatgtaa cgctggtacg gtaatggtga cggtaatgat 8940
actgcaatcg gggggagatg tcagacttga ccggaacggg aggacccacc ggccacatgg 9000
atagcaaaga tccttcctgc attgttcaag ccttcgcacg tcgcgccgca acgccgatcg 9060
tgtggggtcc aaaacatggg agtgcaagag ccgctgcagg atccgacctg caccaacggc 9120
gtggttagat ttacgcaccc gccaatgagc agcacccacg cttaaacagc gtctccacgg 9180
tcgtctgtgc ctatgattat gcccctcctg gacatacacg cgctcatgtt ggcgcattgc 9240
cccgactatg tcgggcacag acaggcagca gctctctgct gggattccca cctcaggatt 9300
tcggatagca ttcgagcacc ccaagcagtg ctatcgactt aggcagtgct agtctacgat 9360
tgccagactg aatccgagca gtgtagggtc taaagcacaa actgtatcgc tttgctccag 9420
atggcacaca tccgcgcttg ctctcagtac tccgcagaac aggcgaagtc gcaatgaact 9480
gcaccatcgc ctctgaagat gctttcgggc ctcaagctcg ggcatgttat ggcggatacg 9540
actttacttt gttgttcgag gagtctgtga tgacgatcgg cccgatcgga cttttactcc 9600
tatgtctttt ccctagagtg gtctacctat acagaaagtc actgagagat ggaagtaaag 9660
atggcgctta tacagccaaa ctcgtaggtc acaattttta tcccactgta cccgagtcgt 9720
cgtcgcatcg ttgacttgtc gcagatcagc tacggatggt tcgctgccat ccaaacggct 9780
attctagtat cctggacatc gggcgatgaa cccacaacac gcacgactgt cccagctgcg 9840
gcgctcagtc tggtcgccgc tgttgcgctc ggtctcgcag cgcacttcga acacacgcgc 9900
tccgtgaaac cgccccttgt tatcgaaatt taccttctat tcactatcgc cttcgatgcg 9960
gttcgactac gcacgctatg gggtatggga tatcatggtc acatattcaa gttatcgact 10020
atcttcgtca tatggaaagt ggtgttgctc gtgcaggagg cctggcctcg ggagcttggt 10080
ttgttggaac agtcgtacag tcctgaagag aaggtcggct ggatcaatcg gcgcatgttt 10140
tggtgggtga acccgttgct gtttctaggt tccaagaaag atttgcaagc ggatgatctc 10200
ttcaccctta accgctccct gcaatctgaa gtatgttcga agagcttctc ggaggtttgg 10260
caaaatggta agcgtgtttc ccctttcctg ttctttgaaa ccattgccaa ctatgaacag 10320
caaagccggc aagaaagggc aaaaaaaatg gacttctttg gattatggct tgggagcaca 10380
aattggtcct tctgactgcg gttcttccgc ggttgtgctt cacagccttc accttcaccc 10440
agccgttcct tatcgacgct gtcgttaaat acctgcagac tgtgtcggag aatcacaacc 10500
gtaaagatgg atacggatta cttggtgcat atataattgt atacgtcggc ctgggagtga 10560
gtttgagtag atctggacta tcttgcagag tgctgatact ggctcagatt tccgaagcat 10620
gctatcagca tatcacgtac cgcactattg tcctcatgag gggttgcctt gtgccgctaa 10680
tctatgaaaa gacgctctct atggacccca aaaaggctga agaatatgcg ccagtaacgt 10740
tgatgagcgc tgacattgag aagatagctt tcggaatgcg ctacatgcat gaggcttggg 10800
gaaatattat tgagatagct ttggctctgt ggctgctata tcgggagctg aactatggag 10860
ggctgagtcc cattcttatt gccgtcggta agactcttca atggagcaga caagatgaag 10920
cttggtactg acattcaact agtttgcggg gctaccgctg ctacaatggc accagctgtc 10980
gggaggcggc aggcgacttg ggttgagggc atacagaaga gaatcgatgt cacaacatac 11040
atgatcaact caatgaagag cttgaagttg gaaggactga ccccttggtt catggacttt 11100
gtacagaatt tgagggtcaa ggaaatagaa tacgctaata gattcagatc ttttctcata 11160
tacgctgtca gcttgtgtta gtacattgtt cccattgttc atgattagct gctcactaac 11220
ctgcgctagc attcggaact tcagtcatat cgcccgtcgt cggcttcggc gtattcatcg 11280
ccttatcgcg atctgacaat ggcccggagc tgacgacctc aagggcattc accacattgt 11340
ctctgttttc agtccttcag aacccaatgt caatgctact gcaatctgtt cccaacctca 11400
tctcagccgt cggatctatc gagcgcgtgc gcgtgttctt gatgaccgag aacaccagag 11460
aggtgggctt ccttagctcc tttacctctg agcaaaccct gttcccagaa atggacaacc 11520
tccgggacat ggagatgaag gataccgaaa tggagaatgc agtcgaggca ttgaactgga 11580
gcgtgggatg ggtatctact cgcgaaccgg tcgtgaaacg aatgacattt aagatccggc 11640
cgtctacact tacgatcatc acgggcccaa ctggttgtgg aaaatcgaca ttacttgctg 11700
gtttgagggg agagacagcc gtggcaaagg ggtacatgag atgccgcttt ccctctgctg 11760
cattttgcag tcaggatcca tggctgcaga atggaaccat actgagtaat attctcggcc 11820
cggctacgta cgaaccgcga tggttccggg aggtcacgca ggcttctgga ttgaggcagg 11880
acttgaaatc gatgccgttg ggtgttcaga cggttgtggg aagtaaggga ttgtcattga 11940
gtgggggtca aaaacatcgg gtggtgggtt attcgtttcg acatagagct catggttgtc 12000
gaatactaac aacaacaatt acaggcactt gcaagggctc tttattcgcg acagcccttg 12060
ttgcttcttg atgacatatt cagtggattc gatgcagaga cagagaagct ggtcatcgca 12120
cggctatttg gccagagtgg cttttgtagg aaacacaacc tgaccactat ccttgcaacc 12180
cattccagta agtggaaaac ctgcgcactc aagaaggata gctactaatt tggctgttga 12240
agctcgcctt tcctcctctg cagactacac catcacactt gatagcaaag tccatttttc 12300
ggaaaaggag ggatcgattg tttctagtct tcctgggcct agcggtggtg actttctctc 12360
agagcccata ttgagcgatg caagattgag ttggattgtg aggcagtcca accccgatgt 12420
accgcacgag cagcttgccg cacagcttga aacatctgat gctagccgcc ggacaggaga 12480
tgtgacgata tacaagtact acctggaaat ggtcggaata ctcaattctg tggttttctt 12540
tatcgcggtt gcgattttca cattcagtct tgcatttccg agtgagtgcc gcaatccgct 12600
atatctgatc aaagctcaca gatgcaggtg tatgggtcca atggtgggct gccgcaaacg 12660
aaaagcaccc atacaaggac ctcggcatgt atttgggtgt atatgctttc ctggcagtta 12720
tggcggagct gtcgttattc ttaggatgct gataagtcta tacgctgtct ctacaaaagg 12780
cgatggctaa cctaaagcac gcatatgatg agcaatatga ttccccgagc gtcaaggaag 12840
ttgcataaga tcctccttcg cacggtgcta aagtaaggtt ttttgaccat accgaagact 12900
tctgctgact cctgcagtgc tcccatatcc ttctttcaca caaccgattc tggtgtgacg 12960
accaacaagt aagatcctat cgctgtatga tgatatgaag ctcaagaggc attaggttca 13020
gtcaagatat gcaactggtg gacatggagc taccccttgc tcttgttgaa acaagcgtcg 13080
gtatgtccaa tatatcgttc tctggtgttg aatagatgac ctttgctaac ctaactaagc 13140
tctcatgtct gcaatagcac agctcctaat agtattcatc actggaaagt accttgcagc 13200
tatcattccg ctatgcctgg cagtattcta cttcctgcaa aagttctacc ttcggacatc 13260
acgacagcta cggttcatgg agcttgaagc caaatctccc ctctattcca acttcatgga 13320
gaccgtgaac ggactggtaa ccattcgtgc tttcggatgg cagtctaact ttcttacgaa 13380
tatatgttgc ctaacagatg catcgcaacg gccttattac ctgctcttcg ttatccagcg 13440
atggctcact ctcgtccttg acatggttgt ggcaggaatg gcgacgctga ttgttggcat 13500
tgctgtcggc gctcccgggt ccattggagc cggttctgca ggactggggt tgctgaatat 13560
catcaacgtg agcgaaagtt tgaagcagct aatttcgaat tggactgtgt tagaaacttc 13620
aattggagca gtgtcacgtg tcaggcagtt tcaggatcag gttcagcctg aagataggcc 13680
ggatgggcac attgaagttc cagcgcgctg gccggagaga ggcgacgtta agttcttcaa 13740
tgtttttgct tcttacaggt aatgcctccg cacggaattc agaagccata ctgactggta 13800
atttgataga cctgatgacc aacccgttct ccatgacgtg acattatcaa tagcagcggg 13860
tgagatggtc gctatttgcg gtcctagtgg caggtacgtt gccttggtct ctcctacaga 13920
tcgctacata atactaaggt cgtgaagtgg caagagctca ctagttcaac tcctcttccg 13980
ccttctcgaa cccgacaagg gtcagattac catcgacgga ctggacatta gctctatctc 14040
ttgccaggac atccggtcat ctctctcgtg catatcccaa gcagtcacca tccttcccgg 14100
tactgtacga caaaacatag accctcttgg gaaggaacct gacgagagta ttatcaatgt 14160
cttaaggaag gtcaagctat gggacattgt atcaacgcaa cttggtggct tggacggcct 14220
ggttcaggaa gaaagcttct cgcagggaca gaagcagctt cttcgcttag ctgctgctat 14280
gttgaggaag agcaaagttg ttgttctaga tgaagcgacg agtaggtatg tttatcatcg 14340
catctcaaat ctagtgatcg ggtatgcctt aaggtcgaat atagctgata ttgtgccgca 14400
cagtgtcgac cccgaaactg acgatctcat gcagcggctg attcgcacag cattcgcaga 14460
ttgcacagtc attgcagttg tgcatcgaat ccatacaatc cttgattttc acaaggtggt 14520
tgtcattgaa agtggacgca ttatcgagtg tggccctccg aaagagttgc tggccgaaaa 14580
tggcctgttc agccaacttt acggaaaagg aaccaccact accaccatag acatggaccc 14640
cgagaagtcc tggctcagat tttgagcgat tagaagctca cgagattttc cctttcgatg 14700
tggccttctc aaggtgctat tatcaggcgt tgtgtttata gacggtgctt ggatgtgatc 14760
ggtgatgttt gacgtcggtg catctactgc cgctcagtgg catacagcac cggcgatata 14820
ctgataatca catttctcat ttgttctgct gcattccata acatccccaa tgcgttttgc 14880
cgccttattc tgcttatgca gcagtgactc attaaacgga attaattcct tcataattcc 14940
aaatacttcc ggatcaacca ttccggctta tcaggccacg tctcccaacg gttgagcata 15000
aaatataaga tttatgtttt ttctatacaa ttctctacat tagatcagca taacacttca 15060
ctttgggtgt atcaagacca tactcagcat ggcgtcccac tatctcagca cgtcgccgga 15120
taccaacatc catttcacca ttactaaagc acagtctcca tcttcaccta cgccccttct 15180
tctctttctc cactactggg gtggttcgag cgcaacatgg cacaagcaaa cctcacctac 15240
ttccccacac accctgagca acatatacaa cactgttaca gttgatcttc gcggctgggg 15300
tcagtccaca ggacctgcag actccggcgc cagctcgaaa gactactcca ttacgcccat 15360
ggcatctgat atcgtctcca tgctatcgca cctccaaagc accacctccc tcctcgacaa 15420
cggcgtgatc ctagtcggtc actccatggg agcaaaggtt acactggcta ctcttgccaa 15480
gctttccgac aatcaattat ccctcgtgaa gggcctcgtg cttgtcgctc cggcgccccc 15540
aactccattg gtactccctg ctgagatgag cgaacagcaa cgcaaagcat atgataacga 15600
aggctccgtt cggtggacgg tcgagaacgt tctatccagc gtacagaata tttcgggtta 15660
cgatatggag ttgatcgtca aggatagtct ggcaggaagt actcttgctc gggacggctg 15720
gattctacat ggtatgcaag aagatattac atctgcttta gatgaggtgt ctagtcggct 15780
tgagggccgg aaggttaagg tgggtgtgct tgctggggcg gatgacattg ttgagaacaa 15840
ggatcgggtc gagaaggagg ttgctggggc gcttaccaaa agaggctttg aggtgcagtt 15900
tgaggtacta gggggcgtaa agcatttgat tccattggag agcccggaga gcgtggctag 15960
ggcgattcgt tcgactgttg aatagtgttt atatttagat gtcttgactg aagaagacga 16020
gatccagtca gtattcttgt aatattcatt ctacccatgt atgtaaagcg aggtggcaat 16080
gcaaatctca ttgactttca cgtctttgag acttgaagct ctttcactaa tttgaaattt 16140
atatgtacga aagccacgtc tgggtgcata taagatgggg ctgtggttta gtggtataat 16200
attcccttag catgggagag gtccggggtt cgattccccg cagctccatt ttttgctcat 16260
attttttagt atcactaaga ggtacatgaa catgatttac acacatgcct taaacccata 16320
acactaaatg atgttttggg cgaagacact cgagactcat actcatgtgt ttctacaccc 16380
tacagtgtca tacaaggtat gttaacattt gtagccatca tcgatcctac tctcatgtta 16440
gggtgtgcca aagtcacccg acaatgaaat gttcccgaat cccagttctc cgcatcattg 16500
acgatactag gtttccgctt gtgatcagct ggtgtctgga tggaagattt tagtcaaggg 16560
actggctagc tcacaagctc ccgccaagaa actcagctgc ggagttgaaa agactgcccc 16620
tggatataca ataatacaat attcgcatta accaataagc tccttcacct tcttatcagc 16680
gactgtaaat gagtagccgc tgctgcacag gtggcaccat aagctcggaa atagagtttc 16740
gcaaagagca gcataagaca cgtgcatgcc acgtttcgaa gccgattagc gttggggctg 16800
gaatgttcca ggataattgc attaatcaat gtactaggta tctgtattaa tctcagtctc 16860
gaaattccac tcacatatga catcatgggc tgtcgccatc ggtcaggggc tgataatatc 16920
atccaaaagg atccgaggag agccggctgc agccgcaatt ggaggatctc ccgcaaggga 16980
ttcagttcct gactcaagag cagcgatcct cccctgatga taggctagga ttaccccgga 17040
gggatgtgtc cattctttgt ctggctccac tgaagcttcg cgttccaaca cttgaggcac 17100
tacatttgat acaccaattt gggacattta gcgccgttgt gtttggtatc catatgttat 17160
acctgtaaca attggtaata ctgttttacc aacaatacgt ttgtgtatgg aacgtcaaga 17220
acacccatca ccgtcacgta tgcagcctcc cattcagctc cgcctcgtcg gatcctgcaa 17280
catgcatttt tacgctgcat attagattat ccttctgttg tacactcggt gccaaatcgc 17340
tcggtcttgc cactgctgac cgagatctcc gatctggccc agtccgttat cgtgagatga 17400
tcgcattgcc aggcttatct ccccgtgcca ccgtgtgtgg tatgatatat cacttcacag 17460
ggcaactgtt ctgagtccag attctggagt gcagttgagt ctcctttcac tttattccct 17520
catgatgacc gtcaagacgc cgttgtcgag tttgagctcg gaatttgcgg gcctcactcc 17580
cgaaatctcc cggacgtctt ctgtcactgg cgacaaacgg tcattgatcg ctgcatatgg 17640
atctgttaat acgacgatct ccctacctac attacagctg ccgttggcaa accaattatg 17700
gaaggacttc agatcgaact tgccttcgtc ctccccagag aacgacttaa ctattaaaga 17760
aggactcgtc gccgagttca tcaacttctg catcacccgg tctagcgacg acactcattg 17820
tgacgacccc gcggcagtac tccagttcct cgaatctctg ttagaagctt ttgagcaaga 17880
ctttgttggt ggaaatgatg ttcactccgc cgtatcacac cttgacactc aacataaaac 17940
ccccataatc aaagcatact tcgcagcata cggaaccctc agtcgcaagc ctaggcgtca 18000
accttctatt ctcctcgagt ccgttacaga tggaccgtca cgtatatacg caatatttgg 18060
tggtcaagga aatgacgaga aatatttcgc ggacctaaaa gacacctacg cgacttacac 18120
cgacatcctc cgtcggtgtg tcttctccgg agccaggttg ttcgaggacc tcgctggggc 18180
atctgtattc aaaacccact ttcaacatgg cttcgcgctc atggactggc tggaaaagcc 18240
cgaatctgag ccagagacct cgtatcttct gtctgcaccc gttagcatgc ccctgatcgg 18300
tctcctccaa atgtgccact tcatcgtggc ctgtcaaaca atgggactct caccgagaga 18360
gatgcatggc tatttgtccg gtgtaacggg tcattcacag ggaattgtcg tagccctcgt 18420
ggtgtctgtt tccgatactt gggaagactt ctaccgacac tctctcgaag ccctccgtat 18480
acttttcttc atctcctgtc gcagccacga aattttccct ccccagttca ttcctgagtc 18540
aataagacag gacgcagaag ataacgggga aggaacacct ggacccatgc tgagagtaag 18600
aaacctccga cagggtcaac tacaaaagat cgtcgatgaa gtcaacagct atctcccaga 18660
tgaatcccat gtcgctatat ctctctttaa tggcccccag aacctcgttg ttaccgggtc 18720
gcctctcagt ttgtacggct tgagccgcca cttgcgtgga ttaaagcccc caaatactag 18780
ccgcgaagca cagatcccgg ctagcaagcg aaagcctaag atcgagcatc gcttccttcc 18840
cattacagca cccttccaca gcatatacct cgatagagtc gtttctctag ttatgaaaga 18900
cctcggggat atcaacatca caggcaacca actacgcatt ccgctttacg ctacaaccga 18960
tggccacgac atgcgttctc acggagccag taacctaatc ggtgatatcg tgagcatgat 19020
tgcgcgcgaa ccagtacact gggagaatgc aaccatcctt ccgaacgcta ctcacataat 19080
cgacttcggt ccgggtggca gtgctggtgt gggcgctgtt actatgcata acaaggcagg 19140
ccagggtgtg cgactgatcc taggtggctc catgaagcac agcgaggagt atgggaataa 19200
agctgaactt ttcaaccgtg actgcggcta tcctgttgtg tatgcggaga actgggcgga 19260
ggtgtacagg ccgcgcctag tcagagatgc agcgggtaag gtcattgttc agaccaagtt 19320
tagcgagttg ttgggtctgc ctcctattat ggttgctggt atgactccca ctactgtgcc 19380
atgggacttt gtctctgcta ccatgaacgc gggctaccat atcgagcttg ctggcggtgg 19440
atactacagt aagggtgcct tggaggaggc aatcaacaag attgtgcaga acggaatccc 19500
agggcgaggg gtaacatgca atatcattta tgccagcccc aactctgttc gctggcaaat 19560
tcccctgctg caggagctcc gtgccaaggg tgttcccatt gatggaatta caattggagc 19620
aggtgtcccc tcagtcgacg tagccaatga gtacgtcgag acacttggac taaagcatat 19680
cgccttcaag cccgggtctg ttgaggctat tcagcaggtc atcgacatcg cccgcgagaa 19740
caagtccttc ccaattattc tccagtggac tggtggcaga ggcggcggcc accactctta 19800
tgaggacttc catgccccaa tcttggagat gtatggtcgc atcagagcgt gcaagaacat 19860
cgttcttgtc gctggtagcg gctttggtgg tgccgacgat actctgcctt atttgactgg 19920
cgagtggagc aaggcattca atgcgccttc cctgatgcca tttgacggta tcctactcgg 19980
tagtagagtt atgacagcca aggaggccca taccagtatt gaggccaagc agctgattgt 20040
ggatatcccc ggtatagacg atgcggtgtg ggaccagact tacacacgac ctactggagg 20100
aatcattacc gttcggtccg aaatgggcga gccaattcat aagatcgcaa ccaggggagt 20160
actcttctgg gccgaaatgg accgaaccgt gttcagcatt gcagacaaaa aagcaagggc 20220
tgccttcttg caagagaaca agaagcgtat tatcgaccgt ctcaacaatg atttccagaa 20280
ggtgtggttt ggacgagact cagatggcaa ggcctgcgag ttgaacgaaa tgtccctatc 20340
cgcggttgca tggcgaatgg tcgagctact ctacgtcaag catcaggccc gctggataca 20400
tcacagcctg cagcagttga cgttcgactt tcttcaatgg ctcgaggacg tcgttggaaa 20460
ctcatccggt aacttcgagt cccgatactg gcagacagtt ggccagatcg aggagccctt 20520
ctcggccatt gaacagttcc tagccaaaca tgctagagca acttcggagc tgatggatgt 20580
accaagcaca cagatgttta ttcacctctg ccagcgtcgc ggccagaagc ctgttccatt 20640
tattcctgtt ctcgacgagg acttcgaggt atggtttaag aaagattcac tgtggcagtc 20700
ggaagacgtt caggcagtgc cgggccagga tgttggcagg acctgcattc tacatggacc 20760
tgtcgccgcc aagcacacca cggtaccaaa cgagccgatc aagcatgtgt tggatggtat 20820
ccactctcag tgggtcaggt cccttacagg gctgggacat gacaatgaac aagctcagac 20880
gccactgacc gaagagggga tgagtgaagt gccaagcgaa atatctctaa cccgaacata 20940
cgacccaatg cttttcggtg aggagctgcc ctcaattgag ccctggatgc aacgactggc 21000
tggcacgcag aagggctgga gacatgctct cttctcgcag gtttctatcg tccaagggaa 21060
cagtctgtgc gataacccca ttagacgact ctttgctcct cgacataata cctttgtcga 21120
agtagaggag tgcactgcta ccgaacagtc tgtcgttcgc cttttcgagc gaacagcaga 21180
tgtgctgcag atcttggcta ttgaggtgcg tgctgttagc gaggacgtaa tcaaggccac 21240
actgttcgaa aatcgcactg tcactggagc accggcgggg ctcgagcttc tatttaccta 21300
tcatcctgag tttccgttcg cgcctatccg cgaggtaatc tccgacaaga ttgagcgcgt 21360
taaggacttt tatcatcaac tttggttcgg tgagccgctg agctctgcta gtgttctctc 21420
ccaggaagat gaattctacg gtgatgttgt tgcagtaacc gctgaggata tcaataaatt 21480
caatcagtgc atccgcagta cagccacatc aagatctcga tcgagctctc agcagacact 21540
ggatgctccc atggattacg cgatcgtcgt tgcctggaaa tcggtcatga agccgctgtt 21600
cactaagagc atcagcggcg atctcatgaa actggtccat ctttccaatc aatttcgcag 21660
acttggaagc tttgaaccca tcaaggaggg cgatgttctt tcgtcaagag cacatatcca 21720
agccatcctg aatgaagact caggcaaagt tgtggaagtt gccgccttga tcagcaggga 21780
tggtgtggat attgttgagg tcgttagccg cttcatgtac cgcggatcat atggcgacta 21840
tgacgcagcc tttcagcaga caaaggagcc caaggtcgag ctccagctca caagtctcaa 21900
ggatgtggtt gttcttcact ccaagccgtg gtttctgcct cgggacaaag atctagatct 21960
caagggttat aagctcacat tcgagctgga aacgcatgcc cagttccaaa gcaagtcggt 22020
tttccgccag cagaggtgct ttggtcacgt ttatggtcgc tccccagcag gcgagactct 22080
cgaggtagct tacattgatc acaccgtggg cgtctcgacc agcaatccca tcatggacta 22140
cctcaagcgt catggaagtg tagtagagca gccggtcctg ttccagtcac caatcacact 22200
cggcggcaga gagatccaat tcaagtcacc ctcatccaat caagactatg cttctgtctc 22260
cggtgactat aacccaatcc atgtgtccaa gacctttgcc gcctacgcag agcttccaga 22320
cacaatcacc cacggcatgc acatgagcgc aagggtccgt ggcttacttg agcagcttac 22380
tgtccctgga aacccaagtg gctttaagcg ctatcagtgt aattttgttg ggatggttct 22440
tccggaggac gatgtggaac tggtactgca gcacgttggc atggtgaacg gacggaagct 22500
tgttcatgca gaggcccatc gcgccggtag cggggacaaa ctgatcgttg cggaggcaga 22560
aattgagcag cccagtactg gtttcttatt taccggccaa ggaagccaag aaaagggaat 22620
gggcatggaa ctttacgcct ccagccctgc agcgcggaag gtgtgggatc tggctgacaa 22680
gcacttcctt gagaatttcg gtgagtatca tctctttcac atgagtgcat ttctaacgtt 22740
accaggcttc cgcattaccg acattgtccg caatgatccc agagaactca aaatccactt 22800
cggtggtgct cgcggacgcg ctatccggga aaactacatg gctcttcgct gtgagatgat 22860
ccatgcggat gggcgcacaa cctcagaaaa gatcttcaaa gacatcacac atgataccaa 22920
gtcacacaca tttcgatcaa gcaccggact tctctcttct actcagttta cccagccagc 22980
actgaccctc atggagaaag ctatcattga agacatgaaa gacaaggggc tgttggcaga 23040
ccactgtagc ttcgctggcc attctctggg agaatactct gcattggcag caattgcaga 23100
gattatgcct attgagagtt tggtatcagt ggttttctac cgcggtttga cgatgcagat 23160
ggctgttgag cgcgacgagc aaggaaggtc gaacttctct atgtgcgccg tcgacccgag 23220
tcggctgtcg aagtgtatgt ccctttactt cctttcgcga ctggttactg actacgtcta 23280
gctttcaacg aaaaacgcct gcagtacttg gtatcgctca ttgctagaga gacaagctgg 23340
ttgcttgaaa ttgtaaacta caatgtcgcc aactctcaat acgtctgcgc gggtgatgtt 23400
agtcaatcaa tttcactcct aaagaaactc ctactaatac atcctagctt cgagccctcg 23460
acaccctctc ctcattgatc ggcgaactca agcgccgtca atctgacttc caggctattc 23520
tccagaatac tcaacccgaa gaggagattc gtgccagcgt actcgataca ctccaccaat 23580
gcgccaacgc cgccaaagca aagccccaac ctatccagct tgagcgggga gtggctaccg 23640
tgccactccg gggaatcgac gtgcccttcc actcatcctt ccttagccca ggcattacag 23700
ccttccgtgc atgtctctac aagtacattg acaaggatac gctggatgcg gaccgcctgg 23760
tgggcaagta tatcccgaac ctgacggcgc ggccatttga agtcagcgaa ggatacttcc 23820
ggcaggtgtt ccggttgaca aagtcaccga tcattgcgaa ggtcttggca gaggtacgtt 23880
gtctatcttg aatctctagg cttttatgtg ctgatttggt tgtttacagt ggcgagagta 23940
tatggatggt gagcgtgggt tggaaaaggt cactcggtct tcggaggttg tcgttgcata 24000
gctgcatcag atttacatag ggttttggcg ttgttgatac cagaatgtct ttcatgataa 24060
gcaattctat gattatatat gacattctgt cctttttcta caacccgtga tctgtcagga 24120
gaattatcaa attgaagtta agtctaaacg gtgccatgct ataaggtatt tctctcaatt 24180
cctgcaacta tctggctaca ccaatgaaga tcacgaggta cattgaacag tccattaacg 24240
ctcagctttt tcacaaattt gaattatgtt cacgaagcat atgctcattt ctccttaagt 24300
tggctatgtc tatatgcctc gagatctaag aatattccac tcatgttctg caaagcccat 24360
gctacttttg actaagacca agctacccac gtctatcagt cacatcgacc tcacttcaga 24420
atacaccaat cgccttcccg tcccccctaa acatcgcatg caatccatcc aacgcaagga 24480
atgactttgc aacttccaaa cccctcttcc tctcctcctc gacctgctcc cacgaccaga 24540
acccctcctt catcttgacc cctttctccg gcaacaatcc agcccgcgag agcactgtct 24600
ccggactcac tgatactctc cccttcttga aaacatacct cgtgacagca gaagccaaaa 24660
tacacttctc atccttgaca cccgtcgcgc tgcccccaaa cactacctcc cctttgtctt 24720
tcttcccgcc ggtcttgaca aaatgactaa cgagatacac ccatttctca tcccaactta 24780
gcactctcgt atggatctca tacgcttggt atggttttat ctctctctta aagacacatg 24840
ctgtggatcc gagtgcgata ttcagctctc cgccggagaa ccccagcgcg agctggtctt 24900
tgaataaact ggagatcaag tgcacgcggt tgatgtctag gtcggtgaag taggttgcgt 24960
tggacttgtg catattgtag tcaatttcga gtagcggggt ggaggatgtc gtaatgaccg 25020
ggtagaagag ggattggggg ccatgggtgg ttatgaccac tttacgggca cggttgagga 25080
tgatgtgttc tttgaggcag ttgaggaggc ggacctgtgt gtttgattgt ttttttagta 25140
tgtcgaggtg ggttgattga gtaattgaat tacgaggcgt ctgaagtgag ggtaatggga 25200
aggggaacga acgtgccaga caaaagggag gcccttcagg tttaggatga tgtaggcccc 25260
tcctgcgaag agcagggtgt tttgccaggt tattgagttg aggagggctg ctgtgtccat 25320
tttgccttga aaatggttgt ggtgtcaatg tgcgctggaa aggatggatg gctttggtcg 25380
tatgatgaac gactaatgaa agagagtgca gtgtcggaac ctatggtagg aaatctgttg 25440
ctcaatcaat gactgtcgga accactgtca gcacggtgcc tcctattgtt acactcggtg 25500
ctcgagtgtt gcttgaccca ttccctgcag aggaaagcct tgctagctct tgctgatacc 25560
atggtccagg tgcagcttca gctccctgca cctgcaggcc ggatccttcg gcgttttgct 25620
ttcttgggtg ggtaatacaa cgtacattat tgtatcgaga tacatgacat gcatacatgc 25680
ttgtttaata cattacatac cggtattaca aacataccaa acatacatac gccaaacctc 25740
caaggatgta tacagcacca tttggacacc cgcactcatc tcatatatac gcagcatacg 25800
atatatatca ttcgctgtta atgcccacgt gggaccaagc ctcccgacac gctgcggcag 25860
gatgcgcaga tccacctatg gagtcgcatg gcctgccatg tgccaggaac catcgtggtg 25920
gagatgggag accatttttc cccgagatgg tgtccgtgac atggttcatt taccacgttt 25980
ctcttagctc ctagctgacc gatgaatcat ttgggatgct catgacgagg ggcgttacac 26040
gtggtggctt tactggtgta tagacatggc ttgggcggga ggccatggtc aggggggatc 26100
atcggtgttc gggtgttgta aaaacaatgg agggtgcctt ttcagtagct taggagcttg 26160
gtgatatttt ggtggattgc gtgtgcatag tgcctagaca ttctggtcgg ctcccataat 26220
cagtattggt ctgaaatgtc ggctgttgat tatgcaatac tgagttcgcg tggcatcatt 26280
ctgaatgggc ggcgactgtg ccgtagggca ttgccatctt tgactcatat gggcatatga 26340
tgtggctcat gtataacact tgtcctattg accaggagac ttcaacttgt aactacgatt 26400
ggataatact catcgtacta atactaccaa gtacgtcgta gatgttactc tcagtaggac 26460
agtatcaaaa tatatatccg aacattgaac attcgatagt attcactcat gatggatatc 26520
caattccacg tgagtgagcg tcagagttac agaaagaaaa tatatagttt attcatgaga 26580
tgcttttaac ataggacaca tcgtaatctt ggcattgtcg atgttggggt cgccatggtg 26640
gtgaggacat cagggtaggt aaatctcgat actgggtgaa aaatgtgtcc cctcattaag 26700
ctatagaacg accgtccata tataaactgg gtcccatagc tcagttggtt agagcgtaac 26760
gctaatagcg ttaaagtcga gggttcgagc ccctctggga ccatcttttt tcctatccgc 26820
cgggaccggc aacttatttt tcgtggccca cctacctaac aaacagcaga ctttagatgt 26880
tgtgagcacg aaagaaagcc cctgcgtttg ttccatttct tgtttctctt tcgtcaatag 26940
gtcaaatagc acgaacgagg aacaccactt atgtctcgta ggtctgggac ggttaatcac 27000
ttattcatct tacatgaatg aagatgacag gcaagacagc cgtgaagacc caccgcggtt 27060
agacaaaaac aaaagaaagt ccttggagca caattaggta gcgaagtgta tgtagcttac 27120
cttccccttc catattgact taacaaacaa tatgggcaaa tgcgtctaaa tatcgcgcat 27180
acagaatagt gtgcagtcaa attcacacaa gcaggaacga ttcagattta tcacaaacag 27240
gttttccaag aataagccaa gaataatttt ttctatgagc aatggttatc agcagaacat 27300
cagactatct gccacaagga gtggcctaaa acaatcatat atgaagggtg aatcagaatc 27360
atcccctaac gatccagaag aagggcgccg cagttagaag tccctccacc ttgtatcgtc 27420
ggaggtacca agcaacccgt cttctgtgca atcttcaaga tctcctccac caccacccga 27480
gcatccttca ttttgatggg aacccgatgg tttccctccc actctattaa agaagctgtc 27540
tcacgattac aatatctctc cagaagttcc cgatgatgtt ccaacccggg atcctgcagc 27600
ccgtgtacat gtatggtggg aagagataga accgccgaag acaccgacgc catagcatcc 27660
atatcaagag gaatcatggg gcctctcccc gcaagcaaga cagcgaagcg gaagttgact 27720
ccaccataca ccccaaccat cccagatccc ccattctgtt cctgaaagag caggctagca 27780
gccagccttg cgccctgact gaaaccaagc actgcaaccc actccccatc tgcccctgat 27840
cggtcatcct cttctattgt atgacggagg gagttgagga ttttctttgc aacgttggta 27900
ggtgagatcg aggggttcgg cggcattggg atccagctct tgaagtcacc gaagtgtcga 27960
tagaccgaga cgacatctgg gcctggctgc gaggggaaag gcgcttcggc gaagcagagc 28020
cggaagtgag ggcttagtag tgtgctcagg actcggcatt gggcgcggaa aatgcgggcg 28080
ttggtgccgc ctccatggag gcagaggatg cgtggtaagc ggagtgttgt gtcgtctggt 28140
agtttggcgg gcatactgtg tactaggcta tgatttatcg agagtggaaa gttaaagaga 28200
tgatggattt ggtgatgaag ggagttatga acccgaggaa tggaggctca tgctacaaaa 28260
gcaggacagg gttctacata caaagacaca tgggtgccac ctgtcaggtt gggacagaag 28320
aactatcgat gtgccagacg tcttagattg atcgccagtg agatcttgct tttctttctg 28380
atacgagatg aagatcgcgt taatagctct tgattggtca tccgctactc tgccacgcgg 28440
agtttcaaat agcccttatt cggagttttg gctagtgcct cttacccaat gtactccacc 28500
atggaagtcc actccgaatc ttgattgaga gcctccatat cggcatcagt cagacaggtc 28560
aacgagtcac attctccctg agggttctgt ggaaagaagt atagcagact gggaaacgga 28620
atgccaggga aattaggtcg ccgaatgaag tccggtcgcc ccagcttccc aaacaccgga 28680
aacagatcta cgcgtagtac cgacgacgac cccacatctg tgtctgggtt gaagcgcccg 28740
ccatatgcaa tcgtggactt gtcaggctcc tgcgcgatga atgtcgcaaa gcttcgcacg 28800
tggtaagcgg tattcgcccg gttcaattcc cggcgcatgt gacgcgcaat ctcaccgagc 28860
ggcagatcaa ccagctctcg gaatgtgagc caagtggtga cattgtgaac tagatctccc 28920
atgtattcgg cagggatccc taggaccttg cggccgtcca tggcccggga gaatcgggac 28980
cgcgcgtccg gggtgttccg tcgacggtgt cgcaccgtta tgtacttctt ccaacaaaaa 29040
gcactgacag catcgtcggt cgtgatgtag ggcacctctg ggtctggggt catggtttga 29100
ctggcgaggt ccttcagctc ggccagcttt gctgcggtga agcgcaagaa atgccaccgg 29160
gcgggatcag ggcgtaccac tggctgcgcg tctgtgatcg ggggtcgaat gtggtgggaa 29220
tgatctagca tgggttcgtc cggggtcaat agcgggatga tgttccgtcg atcgcgattg 29280
acctgttcca ggagagaatc ggagaattgt tcaccccgca tagccatggc gaccaggcgg 29340
gcgaagccga aaagaccgcc tgcatcggtc atgttatgtt gggtgacaaa gtcgagaaag 29400
actcccccgt cgacaaagct ggcctggagg cggatcaccc ggaatgggtc ctcctccgag 29460
tcctcgaagc gctgaggaaa ggcatttgtc ggggctagca gacgcgagtc cagcatcgaa 29520
ttgggtgcct ttagacgttg gatctccttg taaggaggga aggcgtgggt aacatcgcgc 29580
accacgatga gaggatccgg aggcggacag ggaacaacaa tgtatcgccc tgaattgtct 29640
gggcctttgc cgacgttgac gactttgccg gccatccagg gtacatgagc tcggatctgg 29700
cgcactgcag cttctagatc ggcgattatg gtgtcttgtg acaatccttc gggcatggca 29760
aatggaagca tgatgtgact gtagacttgt agcatcggca gctgaccgag gatatcctca 29820
taccgaccga ggtcttcata ctctggtcgg aaaccgttcg ttgccatggc ttttactgat 29880
gagcttggct gctcggtgga agggcttgag ccggagggaa gtacatttag atatagacat 29940
tgatgcttgt tttcatgtat ttgtatgata ttatactata gccctactac ttattgtttt 30000
tactgccgga cattcaattc gatgaagaat aagtgagcat ctgatcgggc acacccggag 30060
tttctcgtgc ggacttgcga tgcccttggg tatccgcacg tgtgcggaga tctctgccag 30120
ctcatccttc tggactaatc agcccacgac gttttgtccg agccgaagat ggacgtttcc 30180
tcaccttgag cacagctctc tatttcaaca ccctggatag ttgatcctgc gctgttagag 30240
taagaaaaaa gaaaacaaga attcgagagc tcacgatggg gtcccttgca aagaaggttc 30300
tgcttacagg tcagtgcaat ccattgttac ccgatacccg cagctgatga actaatgtct 30360
gttgagtaat gtaggcggca caggcttcat tgcctcgcac attttgactg agctactgga 30420
cgccgggtaa gcaatcctcg cgcgagacag cagatccttc taatagaatt tcaggtatca 30480
ggtggtagtg accgtccgca cacacgataa gggccgccag cttctggaat cgttgtcgaa 30540
caccggcgac aaggcggcat cgtacgtggt ggtggaggat atcgccaaag atggggcgta 30600
tgacgaggta aacctcacat ctatgcaggc tttagcagat gtccagagtg ctgactgact 30660
gtgcaaaatt aggctattca atccatctca gacctggacc tcgtagttca cacggcctcg 30720
ccgtatcatt tcaactacac tgatcctaaa acagacttcc ttgacccagc gatcaaaggc 30780
acagccggac tgctcgcctc catcaaggct tacgcgccga cagtcaagcg cgtagtttta 30840
acttcctcat ccgctactat tgtcactcct cccaaccacc cggaggtata cgatgagacc 30900
agctacgggt cggtgacctg ggaagaggcc atggtgccgc aagttacata tcgcgccagt 30960
aaggtatgag ctacttccat ccctgcacta caacagaccc tcattgacac acgcgacccg 31020
gaagatattc gccgagcgcg ccgccttcga cttcatcgag aacgacaagc caaacttcga 31080
ccttgtcaca atcaaccccc cgctagtatt cggaccgaag ccgcgccatg ttactgatct 31140
caaagcacta aacacctcga accacatcat ccgtgacacg atgctcggca agtggaaaga 31200
tgggggcgca cctatcgcca tacctttcac ctgggtggat gtgcgtgatg ttgccttcgc 31260
tcaccgtcaa gcgttggagc tgcccgaagt cagcggccag cgctttttca ccgtggcagg 31320
ccatttctcg aacaagcgaa ttgcggaggc tatccgtgcc acgcacccgg agctggccga 31380
caggctgcct cctagtgatg tgcccgacga tttgccagag ggtatttatg gatttgataa 31440
tagcaagtct cgtaaggtgc tggggatgac tttccgcgac ttgaacactt gcgtgggcga 31500
tgcggtgacc tcaatgctag aacaggcttg agtgtagcta ttcaagataa gttttcgatg 31560
gcatacgggc caccaaatgt caagaagcat gataaggata gcttaggcaa gattgtgaaa 31620
gccggttctc aatgcaagga gatgatgatg tgatcctagg aagacatagc cgcgtagcag 31680
gcaagatatt agattgagac tgtctactgt agatgctcat aattcaactg cccattcatc 31740
tacaacttct caatcctttt gtcctccaac aaccgcttca gaatcttccc actcgcattc 31800
cgcggaatcg catcaataaa acgcactcca ccatccaacc ttttatactt cgccagccgc 31860
agcgccacaa actccttcac ctcctcctcc cgtagtgact gatcctgtct caccacatac 31920
gccctcggat actccgtctt cgcctcagca tcgtacaccc ccacaaccgc gacatcctga 31980
atgcgcggat gattcctcag cacgccctcc acctccgccg gcgcaacttg gaaccctcgc 32040
accttgataa tgtccttttt ccgatcaagc agataccact ttcttgtctt gccgtcgcag 32100
tacccgaggt cgcctgttct atagaacccg tctttatcga aggcgctctc attggcggaa 32160
gggagattga agtatccacg cacgatggta ggtccccgta gacagacttc acccgtagtg 32220
tccggggcag tgatatcgtc tccgtccgtg ttgattatct tcgcttccat acccggaagt 32280
aaccgcccta cacttcctgt gtcgtcggat tcagggtatg gtatcatggt cgctatacag 32340
gaggactctg tcatgcccca gacttgggtg aaggttgcgt cgggggcgag gagagattga 32400
aagcgcgcct gggttgtttt gtctagtggg gcggcgccgc agcggacgat tttgagggat 32460
tgtagtgagt gggagtattt cggggttgtg cagtgggaga ggattgtaag aataattggc 32520
gggacggctg tgcattcggt gatgttgtgt ttggcgacgg tggttagaaa aaggtctggc 32580
tcgaagcggc gaagcacata ggatgggtca ccaagacgga gggtgctgaa gtgtgcagat 32640
ggggctgcgg cggcgtggaa gaaggggaga gagaggatcc gacttgtcta caggacgtca 32700
gctagatgtg gggtactacc tgggcatgca gttctgacct cgaacgggac ggggtgtgca 32760
tcgtacacca gcgtatgttg cgcaataata ttgtagtggg taaagtctac tggtttaggg 32820
agacccgtag tgccggaact gaagagtctc gctactacgg tcgttttggc atggttggga 32880
tcatcgagac ggtgccagtc agcctcgcca tgtttcagca gggcggtcca atgagcgagt 32940
cccgtcgtcg ctacaacctg ggactcgcgc gttgtgaaag cccagatgcg gtctttcgga 33000
atgccgttgc cctcagcggc agggatcatg cgatctagca tctctggatc actgatcacg 33060
aatttggcct gcgctactcg gaggtgatag ttgagttcgt gggatgtata gcccggattg 33120
gtgcccatgt atattcctcc agctgcgatg atgcctagca caaggatgct gtaatagatc 33180
tggtcgatat gtaagcggag atacgtagga aaaataaatt atcttgggtt ggggtgcaaa 33240
catcattgaa agaatgaagg cacacgcaat cgccgtcctt cagaccggct gctcgcaaac 33300
cagcggccaa ctggcgcacc agagtgcggg cttgtcgcca cgagatggaa cggctcgtat 33360
cggaggcatc catatagacc tgaggtgttt tgttgcataa gtgtccagct ctcaattggt 33420
tggggatgat tgactcaccg gcttttcgat ctcatatcgt gcctggtcga atatccagga 33480
aataatatcc tggactggga tgtcaatgct gccctcgccc tggaaaaaca tgattaaaat 33540
atgagaagcg gggaaaataa cgaagaaagg atctgtgtca tgtagtacga gaggggtaga 33600
cttcaaaggg ctcttggctg ggcctataag ccctagtcac ggtcatgtga attagtccga 33660
ggcttgagca tcgagaggcg cggctagcat tgaatgccga tccctctcag gttcaaagtc 33720
cgatcgtaaa ctccgagtga agatcgcccc gcctctacgc agcacaacct gagcagagga 33780
gcctcacaaa aaatatgcct cgttattggt caaggatgga tcgactgtat atacaagcag 33840
tgacactaca gctccaagga ctcctccaat aacggaacag ggttactcca gctgaatcgc 33900
tcttcaggat cccatttttg cttcagccga gtgagatttt ccagcttttc agcggtatac 33960
cacacatctg gcccctcatc gccatgagcg tagttaacat agacggccag attgtcgaat 34020
ccactggact gctgaaagtg ccggcgagac tgtttcagga aggcgttgac cgggtcttca 34080
aggtaggggt cggtatacca gccctcaaga ttgctgcatg ttaagagtta gtgtctacac 34140
aaattttggt ggctgaggag ctccttacat atgcatcttg atctgccggt ggggataggc 34200
cgtattggag tccagcgtgg acagggctcc ttgcgtggga tagcgttgca caaggaagcg 34260
gccatcgtag gtgggattct ggcgatagaa ctgcagtagt tgctccatgt aggactccca 34320
ggcaggtacg tcggtgtggt tcagcccgat gctgtatata ttgaccgctt ggttcttagc 34380
gcaggcccct ccttcctgag ctgctaggcc gaagaaggtg gtgtcgagca gggcattcca 34440
gggcacagtc acactgcggg acatgattgg ggtgagactg gtgaaggggc tcagtagctc 34500
cagcgcctgc tcctcagggc cgtagtatat agcgttgacc agcacaagtg gcttttgaaa 34560
tgatcagtac ccacaaggaa attcagttga tggatgctta cctggtcgat cgtccggtta 34620
taggccaccg ccagggtcaa tgctagctca ggcggaagcg tatcatcgaa cgcgctcagt 34680
gcctgccaaa tggaagcgtg ctcagtcgcc gcgaagatga agtctacatt agtgacaagg 34740
cccccatggg tggcttggtg tgtgcgatag gtagccgagg tgacgactcc aaagttggac 34800
ccagcaccac gcaggcccca gaaaagctct gggtactgtg tctcagaaac ctcgataagg 34860
tctccaatcg gggtgactag gcgaacggac tcgagcgaat ctagcagtaa cccatgcagc 34920
ccctgcagac ttccgatacc gcctcccagg gtcatgccca caactcctac gcaaggagct 34980
gacggtgtca ctatttcccg ttgtgctgag tacagaggat cgatgatgtc cgagaacacc 35040
gtcgctccgg ccacagtcag tcgctcagcg tccacatcga gatgaacatc cttgaactgt 35100
tgcaagtcga tggcaatacc gtggcggaac cgctcaaagg taactgtggc tccgtggcca 35160
gcaccgacta ccaggaaatt gatggcatgc tctcgtgaga ttttgatcta aactccactg 35220
cattatcagt tcatgtcgca caggcttggc gaggcatacc gacaatatgt tggatgtcat 35280
gctcggttgc gggtataatg gcaccgagat agtggggtgc gccatgcaca gtccaacgtt 35340
gtgtggtgac atgcgtgtag ttggcatcag atgctaagac aatttttgca ttaggtgata 35400
ggctggggcc atacaatccc tggaggtcaa gtgtagtctg ggaggcccag agccatgata 35460
gaagccaaga caggatgcct gacagctgca tggcgtaaaa gattctggat tgatatgacc 35520
tagcgcaagt tagagccaat agttcttagt tataatactg tccaatttta tcaaagaaaa 35580
ctacgggtct cctgcgtatc tgatcatcag tgcgataagc agatggcaaa aaccccgagg 35640
ggaaactccg gtagttggaa gggcactaat tcaaccccga atcctgacta tcacagctct 35700
ctcatactat aatgactagt agctgctgat acaacaaaca aaaacagata tctgaagtgt 35760
cttcgcccct gtccttgccg catacaccaa gtcatcagag caaaacctct aaccaggcgg 35820
tagcctctat atccactctc aacgtctaca aataggcaat aaatcaaact ccaatatcta 35880
aatcaattga ttgcatcaaa taaatgctcc cgttatcttg gcagtaagca ctaaaatctt 35940
atgtctgagt aacggccatc ctgcacccca cctcaaaaca atgaagagat agatagaccc 36000
cttccaatcg actcacacct caccaactca caatctacca tcatgagtac agactcgatc 36060
gaagttgcca ttataggcgc cgggatcacg ggaatcaccc tggccctggg cctcctgtct 36120
cgcggcattc ccgtccgcgt ctacgagcga gcccgcgact ttcacgaaat tggagccggt 36180
atcggtttca cccccaacgc cgaatgggcg atgaaagtcg tcgacccgcg cattcaagct 36240
gctttcaaac gcgtcgctac ccccaatgcc tccgactggt tccagtgggt ggacggattc 36300
aacgagtccg gtaccgaccc gcgcgagacc gaggaacagc tactcttcaa gatctacctc 36360
ggcgagcgtg gatttgaggg ctgccaccgt gccgacttcc taggtgagct ggcacgtcta 36420
ctaccggaag gtgtggtgac attccagaag gcgctggata ccgtggagcc tgcagcagat 36480
aatagcctcg gccagcttct tcgattccaa gatggcacga cagctaccgc ccacgcggtg 36540
atcggctgcg atggcattcg gtcgcgcgtt cgtcagatcc tcctaggtga agaccatccg 36600
acagcatcag cccattacag tcataaatat gcagcacgcg gccttattcc catggaccgc 36660
gcccgggagg cgctgggcga agataaagtg gcgacacgct tcatgcatct cggtccggat 36720
gcccatgccc tgaccttccc cgttagccat gggtccttgt tgaacgtcgt cgccttcgtc 36780
acggacccta acccttggcc atatgctgat cgctggacgg cgcaggggcc caagaaagac 36840
gtgacggctg ccttttcccg ctttggtccg accatgcgca ccataattga cctcttgcct 36900
gatcctattg atcaatgggc cgtttttgat acatacgacc atcccccaaa tacgtattcc 36960
cggggagctg tctgtatagc aggggatgct gctcatgccg cggctccgca tcacggtgca 37020
ggtgcaggtt gtggtgtgga agacgcggct gtgctgtgcg ctgtgcttca tatggctgcg 37080
aaaaaagtta acaccgcaaa aactggttct gaggggaaag ccgctcttat cacggccgca 37140
ttcgaaacct atgattcggt ttgtcgcgag cgtgcgcagt ggctggtgga aagtagtcgc 37200
gttatcggta atctgtatga gtggcaggat aaggaggtag ggtcggatgc ttccaggtgc 37260
cacgatgagg tgtattggcg ctctcatcgc atttgggact atgatattga tgcgatgatg 37320
agagagacag ctgaggtgtt tgaggcgcag gtagctgggg tggcgagaaa ttagtatacc 37380
tttgactact tattacaata tgaggtctgt agtgctagga atattctttc gtgtatgtaa 37440
aatttattcc accgcctatt gctgaactca tggggaccga gtctatgtat ctgcaggtga 37500
ctccaacccc cttgcaccat atagctgtta tttcccggct accctcttga gcatgtcggc 37560
tcaggcttcg actagcaagc ggtgtcctct tggagccatg atcaaatact gttcctgagc 37620
cagatcctca tcccgcatgc cgttctggcg cgacatctga acctctggaa accggcgtag 37680
gaatagagtt gtcgtaagtc ggatttccat gagcgcaaga ctattgccga tgcacactaa 37740
taacggtaac aattagtgat atttacccta tgatattgtg attaggctgt ggaagtcaat 37800
cttactcctc gaggcgccac caaagggcat gtaagcgtct ttcatctcct gggtcggaga 37860
ttcccagcga ctggggtcga atctacattc aattagccac acagacagca tcgaaagata 37920
tgaatacttg gggttgggtt tgtacttctc cgggtttgga aagatggcct catttcgatg 37980
taatgtatac gcttgagtcg aaaccgtagt tccaccaggc aggtaatatc cagcaagcgt 38040
agccccgcca gcaggcacca cacgaggcaa agcagacgag acaacagcat atttacgtag 38100
agtttctgtg atgacctgat tgaggtacgg cagatgtcga agctcatcgt ggcggaaatt 38160
ctcaggcaaa gtacggattt ctgccagcag cttctgccgg atctcattgt ccctacagat 38220
cgcatagatg agatataata acgtgacggc agttgtatgg ctccctgcgg taatatatga 38280
gccagccaag tatgtcagct ggaagtcgtc aaacttgtct gccttaaaga gtttagtaaa 38340
taaggtaggc ttcggactct ctggctgttc ctggatatgc tgtctatatt gctgaataga 38400
ccgcctcgcc catttaccaa gaagccctat tgcctgaatg gcatgcttga aaggatgaac 38460
gggcatgaat cgtcccagta cgaagacaaa ggggaaggtc gtccgccagg cgtgcatgat 38520
acccactttg tccaggtagt ccatgaccca ggcggtttcc tgtcaaggcg tgtcaagatg 38580
catagtcaag tctctaatac caaagtccgc cactcacctt gggacgcgcc agcaggccaa 38640
tgggttcgcc aaagctcaat tcgctcacga cgtcggtcgc catatacgtc caccatttgt 38700
agaggtctga taccccctgt gactgcaaat cggaggcaat ccgatcaatg gtagcatgta 38760
catggttggt gaccatgggc tcaactttgc ccaagctggt atcagacaat ggcccggcta 38820
gtaatcgccg ccattcagag tactgagtgg gctcgatgag ggtgagcaag ctagtcaccc 38880
cgggtggtgt caaagacttg taccactcgg acttgagata tccgccgcgc acgcgatgga 38940
tctcacggac tgctggcaga tcacagatgt cgacttctgt aggagtcacg cggacaatag 39000
ggcctaccgc caggagcgcc tcataacatt agctataagc ttagtaatga tgaaagacgg 39060
gtatcgctca ccgtatttac ggtgtagata gtcgacatac tcaacctttg tccccgcaaa 39120
ccagtagtat tgctcgatca atgaactcca tttcgaaatt tgaggaccag ggatatgtga 39180
tagaggattc cttactgccc gatacacgat tgtctatagg ggctgagtca gctctaccaa 39240
ttgtggtgtt tgagatgagg gttgcctact gagagactga gagctaagac acagccagcc 39300
agaggcaaga aaatgcccgg tagctgggca aggctttcca tgtcgcagta acctacagct 39360
atggagatct tctcaaacat aggtgtcaag aaacaggata ctgaatttca agagccgtgg 39420
ctatctgcct gtctggcagc tgtagaactg aaggggcgtt gatatggtgt tttgcagctt 39480
tgatgactgg cagtagaaat tccatacaat aacggaatga tggattagag atagaaacga 39540
ctcgcaaaaa ctgttcattg gccacgaaat tgcaaagata atgagcaaag gcagctgcgg 39600
gagaaactcc attgatataa gtccgagcga cgcggggtca gaaaatcccc tagccacata 39660
ctcggacttc ggtggatcac agaatttcca cccctggaag aggaagcata cgatttgtgc 39720
aagctgatga gtactatcaa ggctttctcg taagagctat cttgaggaaa cgtgaagagt 39780
cctgcccggg cgaaactccg gctcaacagg atgcctcaca tcagcgagat aatgcctgca 39840
gagaataaga aaaagtccga gaggctacca tgtgtttctg ctgcagcatt ttttacgtgg 39900
cagtgaattg ttctggatct atacttgttt tgatattgca ttacaagtca tgcatttatc 39960
tggccatcac agcatcggaa aagcgccggt agggattgat tgattgcttg attaccagtt 40020
cttcgacctt gcaattattc cttacatctc ggcttgccct tcagccttat ccgccatggt 40080
cacaacgaca agggctacta acccgacaaa tacccttctt ctattcggcc cgcaagccct 40140
ttccttctcc accgcgacgt ttgccgatat acatgcacga gtagtgcaaa cctcagagaa 40200
tgcttggatt aagcagacca tcaccagtct accagggctg tgggatgccc ttgtgaagga 40260
atttccacag tatggagcgc tggaaggcaa gcagttgctg cgggatctcg ataggtggtt 40320
cgagacgggg acgatggagc atgctgaacc ccatcttcct aacatccttc tcagccctat 40380
ggtagtcata actcagttga cagaatatgt ggactacttg aagacgatgc cgcatgcagc 40440
agatcagcaa acagaaaccg ttggattttg taccggcctt ttgactgctc ttgccgcatc 40500
acttgcccct gacatcaagg gtatccgcca gtacggcgcc attgcgatca agctggcgat 40560
gattattgga ggggtggtag atgtgcaaga tatcacaagc ccaaatggcc cttcaaaatc 40620
attagccgtg gcttgggatt cggctgaaac acaggaccga ctgaaccaga ttattgacca 40680
gtctccagag gtgagtgcac acagcaagac acaatgaaaa aagacaatct aattctaatc 40740
taaacataaa ggcgtatata tcggtagaat acgaccataa ccgaccgaca ataacaacag 40800
cagccagaag catctcttcc ttgcaacagc ggttacgcaa tgccggcctc attgctagcc 40860
agattggatt gcgcggtcgc ttccactgtg catgctatga gaaagatatt gaggccttga 40920
gcaaattctg cgattctgta cccagcctgt gtctgccaga tgccgctgtg ctggctctgc 40980
cgacacgttc caatgatgcc gggtctttta ttctcagcgg gaagctccat cattgcgcac 41040
ttcgatccat tctcctggat acatcccatt ggtatcagac tctagaggta gtccgacaat 41100
cattcttaaa atcgccttcc tcgatggtgg tttcttttgg acccgagcga tgcattccac 41160
cgtctatact gaaaggcctt agctcgatag ttaccacagc ggctgaatat caaccatcat 41220
acttgcacag agatccagag ctttgcaatc cgaatgagat tgctgtgatt ggcatgtcct 41280
gcaaggtggc tggtgccgac gatgtggatg agttctggga ccttctatgc aaggccgagt 41340
ctcagcatca agaggtgccc aaggagcgat ttggattcga aagtgccttt cgcgaggtcg 41400
accccacacg gaagtggtat ggtaacttca tcaacgaaca tgactgcttc gaccacaaat 41460
tctttaagaa gagcgcccgc gagattgccg caaccgatcc gcagcagagg cagatgctgc 41520
aagttgctta tcaggcagtg gagcagtcgg ggtactttac gacgcccaag tcagacaagg 41580
accgaaagat cgggtgttac attggggtgt gtgcagcgga ttatgagtac aatgtggcat 41640
gccatcctcc gaatgctttc atggccacgg gcaacctgaa gagttttgtg gcaggcaaga 41700
tcagccactg gtttggctgg actggaccag gactctgcat cgacacggca tgctcatctt 41760
ccttggtggc tgttcatcag gcgtgtcaag ctatcttgac cggcgactgc accgcagcat 41820
tagctggcgg tgccaacatc atcacccacc cgttgtggta ccagaacctg gctgctgcct 41880
ccttcctcag cccgacaggt caatgtaaac cgttcgatgc cagcgcggat ggttattgca 41940
gaggcgaggg cttcgccgcg gtattcctca agaagatgtc ggccgccatc gccgatggag 42000
acatgatcat cggcagtatt aaagccactg cagtcaacca gaatcagaac tgcaccccag 42060
tcttcgtacc caatgcgcct acactatcgg atctcttccg tgacgtgttg gaccgatccc 42120
agctgacagc taaccagatc acggtagtgg aggcccatgg cacgggaacc caggtcggcg 42180
atccagccga gtatgaaagc attcggaatg tgttgggcgg tccgtcacgg tcaacgccac 42240
ttcttttcgg atccgtaaag ggcctggtcg gtcatacgga gtgtacatcc ggagctgtct 42300
cgctggtgaa aacactcctg atgcagcagc atgaggcaat cccacctcag cccagctttg 42360
acagattgaa tccggaaatc cctgtgtccg agtcggacaa tatgcagatt gccaccaggt 42420
tttcaccctg gactgcagaa taccgtgctg ctctcatcaa caactacggg gcctgtggat 42480
ctaatgcgtc aatggtcgta gcccaggcgc caagaacgga gcagaagcgg agtgctacaa 42540
gacggacctc cgttgtcctc gactacccct tccgtctctg tggatccgat gatcgggcct 42600
tgcgagccta cagtgagcga ttagtgcgct tcatagctag tggcataaag gacggaattt 42660
ccgtggcaga tctcgctttc aacgtttgtc ggcagtccaa ccctactctg gatcgatcat 42720
tggcgtttgc ctgtcgtaca acacaggagg tggaggaaaa gctgagggct ttcgtcgccg 42780
ggaaccaagg actcatctca gccagtcggt cgaagacacc tcgcgaggtc atcttgtgtt 42840
tcggaggcca gatctctaat tatgtcggcc tggatcgaga agtctacgat aatgttgctc 42900
tactccggaa gcacctcgcc atctgtgacg ctgcatgtcg ccatctcggg gttgacagta 42960
tctttcccgg catcttccaa aaatcaccaa tttccgaccc ggtgaagctt caaacgattc 43020
tcttctccac ccaatactcc tcggcgaaag cgtggatgga ttccggggtc cgccctgttg 43080
ctgcggtggg ccatagcttc ggtgagctaa ccgcactttg cgtcacgggt atactgagcc 43140
ttgccgacgc aatgaagatg attgttggac gtgccaccgt tattcgtgat ttctggggag 43200
aagacaaggg atccatgatt gcagttgagg cagacgaaaa cagagtccag cggctgcttg 43260
ctgaagcagc caaacagtgc gagctgattc acgtaagagc acctaccatt gcatgcgtta 43320
acggaccgac cagttacaca ttagcgggac cggttaaaag cattgacatc gtcacggaag 43380
tcataagtag attatctgac tctggaccct caatccgctc gaagcgatta aaggtgacca 43440
acgcattcca ttccaccctc gtcgagcctc tgatggagga gctcgagaag gtcggacagc 43500
aattgacgtt caacgcacca accatacaac ttgaacgagc tattgagcac cactcggatg 43560
ctacgcttac aagcgactat gttcccgacc atatgcgcaa cccggtatat ttcaaccagg 43620
cagttcagcg gctggctcag cagtacccgg atagcgtgtg gctggaggcc gggtccaact 43680
ccaccatcac ttcgatggct agccgagccc taggatctcc caagtccctt cacttccaag 43740
ccgtgaacat cacctcggac gattcgtggt ccatgctgat tacgagtaca ctgagtcttt 43800
ggaaacaagg catatccact aacttctggg cctaccatgc gaagcagaca tacgagtaca 43860
atcctgtttt gctcccaccg taccagttcg agccatccag acattggatg gagctcaagg 43920
ttccaagctt catgagcaat gggaatgtcc aatgtggccc acgggatgaa gaagagcctc 43980
ctaaaacgct atggagctta atcgaggcaa gcgataaagt ggcccgtttc cagatcaata 44040
cggcggctcc gaagtatgtg gagctcgtct ccggtcatgt catcgccaac acggcgccta 44100
tctgtcctgc aactgtggaa gttgatattg tggtggaagc attgcgtagt ctgcgtccgg 44160
atttcatgga ctctaatcta cagccacagg tgctggcggt cacgaatcaa tcgcccatct 44220
gtatcgatcc gaatcgctcc gtgtggttgg aatgccaagc gatggactcc aacttagtat 44280
gggagtggag gattgtcagt gacagcttgc aagagcccgg aacatcctcc tcagctcacg 44340
tgctcggaaa gttggctttc ctttcagggc aagatgaggt gaaacaacaa gaaagtgagt 44400
tcatgcggtt ggagcgattg atcgggcacc aacggtgtgt ggacctgctc aacactaccg 44460
aggcagacga tatcattcaa ggtcgtaaca tatacacgac cttcgctggt gttgttgatt 44520
acggagagca gtatcgcgga ctgaagaaga ttgttggcaa aggtcttgag tctgccgggc 44580
gcgtacagaa gaaaccatcg gaagagtcat ggctagacgc acacctcgga gattgcttca 44640
gtcaggttgg cggcatctgg gttaactgca tgacggacca caatccagat gacatgttca 44700
ttgcgactgg gtttgagaag tgggtacgtt cccctgcgct gcgacatggc cagccaagac 44760
ctgagatatg ggatgttctg gcttgccatc accggtcgtc ggaacagacc tatttgacgg 44820
acatcttcat attcgacgct gaacaaggtg ctctgacgga agtcattttg ggcatcaact 44880
accacaaggt tgcaaaggcc tcgatgagta agattctctc gcgtctttct ggaactgagg 44940
cagccccttc gtcaagcaca cgtgcccacc caacatcatc ttcgtccccc cgactgcccg 45000
ggccattcgt gccagaggat aagagtcaga atgagacaca aactgcaggc acaaatgccg 45060
tcgcaaagaa gaagtcagag aagtcggccc aacagaacgt gctcgacaag acgcgagccc 45120
tcctggccga aatatcgggg ctagagccta gtgagatcga agctgagact ggtctggctg 45180
acatcgggat cgattcgttg atgggtatgg agctggcacg agatcttgag gcgctcttta 45240
aatgcccctt actcggtgat gagctggcga atgtgaccac cttccagggt ctggttgagt 45300
acgtgcaatc tgcggtcggg gtgccagcca acggcgacga acccgacaat actaatgcgg 45360
acgaagtctt cgaagaagac aacctcgccg caagtccctc gagcagcagc tcttccacca 45420
atcttacgga agacagctcg ctcgacccaa cagagactac aactaacatc tcttcctacc 45480
ctggtcagac aaaaacagaa aagcctgcca tgccgcccgc ttcatccaag actctagagc 45540
tttcccctgc gtgggtcctc gaagccttcg aggagtcgaa gcagctcacc gatcacttca 45600
tcgaacagta tcggtgtgct aactacgtcg ataccatctt gcccaagcaa acccagttgt 45660
gtgttgccct gaccgtggaa gcctttgaga aactggggtg tccaatacgc agcgccgtcg 45720
ccggccagaa gctcgagcga atcctccaca ttccgaagca tgcgcaattg gcacagtatc 45780
tataccgact actaagcgct gacgcacggc tcataaatct gaccgaagac ggtcgcatca 45840
cccgcacgca catggctctt cctaagccaa gtgatcagat cctccaggac ctcctccggc 45900
tatatcccga ccacgaatgg gccaaccgtc tcgccgcttt cacgggcgct cgactcgcag 45960
aagtgctgaa gggagagaca gatggcctgg gattgatctt tggcaccgac gaaggtcgcg 46020
aactcgtagc gggcctatat ggagactctc tgctgaacaa gctttcatac cgacagatgg 46080
aggacattat tacacgactc gcctctagga ttcccagaga cagcggtcca ctgaagattc 46140
ttgaaatggg agcaggcacc ggaggtacca cgaggggaat ggctcctcta ttagctcgac 46200
taggaatccc cgtggagtat accttcaccg acctgtccgg atcgtttgtt gccgcagccc 46260
ggaagaagta ccagaaggaa tatccgttca tgaaattcca ggtgcacgat atcgaaaagc 46320
cccccagcga tcagctccga cactcgcagc acattgtgat cgccagtaac gccatccacg 46380
caacacacag cttgactgac tcctcccgcc atgttcgcga atttctgaag accgacggct 46440
tcctcatgat cgtggagatg acccagccgg ttcactgggt ggacattatc ttcggattgt 46500
ttgatggctg gtggctcttc gccgacggtc gggatcatgc gattgcctcc gcaggctggt 46560
gggagaaggt ctttcagtca gtgggatatg gccaagtaga ctggacagat ggccatcgcc 46620
cagaggtgca gattcaacgc gtgataatcg cccttgcctc agggcctcgc tatggccggc 46680
agcctctccc gccggcacct cccccgaatc tcgttccagg aagccatgct agtcgacagg 46740
ccgcggtaaa tgagtatctc gataagtata cgaaagggtt cacgctgccc gctcaaacct 46800
ctaacacaga catctccaac tcaaccagtt attgggagaa gcaatgtgtg ctaatcaccg 46860
gcgcgaccgg cagcttaggg gtgcatctcg tggccgccgt agcagcattg gacgacgtcc 46920
aaaccgtgat atgcctaaac cggcgcagcc caatggaccc agaccttcgc caacaacagg 46980
cattcgagcg acgcggtatc ctcctcgaag ccgccagcat gtccaagatc cgcgtcctac 47040
aaaccgacag cagcaagccg caactaggac tcaccgatga agtctacagc agcctcgtaa 47100
cctccacaac gcacatcatc cacaacgcat ggcccatgac cggtaaacga cccctatcgg 47160
gcctggaaca acaattccta gtcatgcgaa acctcctcga tctagcagca caatgctcct 47220
ctactcgacc agccaacgcc ccgcggatcg tcttccaatt catctccagc atcgcaacag 47280
tgggatatta ccctctatgg agtggacaga ccctggtgcc tgaggcccgc atggggatcg 47340
agtccgtact ggccaacggc tacggcgaag ccaagtacgt ctgcgagcag atgctcgacc 47400
gcactctgca tcaatatccc gaccggttcc gcgctatggc tgtccgactg ggtcagattg 47460
cgggatcccg gacaagcggg tactggaacc cgatggagca cctgtccttc ttgttcaaat 47520
cggcgcagac attgcaagtg ttcccagact tcacaggtga tttgtgctgg acgcccgtta 47580
atgacgtcgc tgctacgctg agtgaccttc tacttcgatc tacgcattca aatagtacga 47640
ccgaccaacc aatataccac atcgataatc cagtccgaca atcctggagc gagatggtac 47700
cggtgttgat cgatgcgctc ggcatcccgg ctcaaaatgt tattccattt gcggactggg 47760
tctgtcgggt gagggcattc cccggtcaag tggaatggga taacccttct gcattgttga 47820
ttgatttttt ggatgatcat ttcttgcgca tgtcgtgtgg agggctgttg ttggatacga 47880
agcgagcgtg tgagcattcg ccaactttgg cggctgttgg gccggtcact gccgagttag 47940
cgaggaagta tattcagtca tggaaggaga tgggattttt gaatccttga gtctaagatt 48000
tggttgattt gtttggtagt tgctagataa tttggacaat gaactgttgc ttagtgattg 48060
tggactatca cgcagagtct agattacgta ggctagagag aacgataatt tgcagaagtc 48120
gctgctatgg atagctgaat tggcgctcct gatggctgat gatatgtatt gcatttcatt 48180
tagctttagt attcaaattg attagatagg actacacgag ggtaacttgc tagtcttgtg 48240
taccacaagc atggcaatat atatactacc aatctgtcta ggcttaaatg ccgctgagtc 48300
gttgcctaat atactaccat cacaatggat taggagaacc ggacactata aatagaaacc 48360
atagtcaaat atctaaaaag ttacaagcat gttcaactgg catcttatca tctaatctag 48420
tgagcagagg accaaaacgc gcgtacgacc atgacagcct cccgggccgc ctcgagcttc 48480
gagaccagga tcatgaccgg cggatgtcct gagctgctgc ttcctgtcga gccaatcgat 48540
tcacagtcat caaatatttc aaggcgtcct gcacctcccc taacagcttt ggggtatcat 48600
tatgggcaac agggtgctgc aatacctctg cacataggcc tagtagaata ggagtgaagg 48660
gatgcacctt gaagcagctg aaatggcgta gattacgtgc tagaacgaca gtctcgcggg 48720
ccgcctggag cgcgcatgcc tcgtagtcgg gtaggaggct gactgtttcg ggctcccagg 48780
gcgtggactg gaggatgtca tacagggaga ggacacccgc ttgcccgatc atcttggcaa 48840
acaggagcat agggtcgctg gattccgcgg ccgtgggaag cgaaaagctt agctgcaggt 48900
gggctgtgag cgtcgtgtcg atccactgat ggcgagtcca gacatcctga agcggatttc 48960
ctcggaggag ctcagccatg gatagatggc ggtgtgacag agtgcggcca ctgatagtgg 49020
ctagcaggat acatttagca aaggaggata ggggactggt aatatccgta cccgcgagtg 49080
cttcggccaa aaagcccatt gaaatagggt gaccacattg gaattcactc tcgggcatgg 49140
ggagtcgggt cagcgcttgc tcggtcaggg tgagagagcc cttatggcgt aaatttaggg 49200
aacagtccat tatgtaggcc atccacacgg tgcgtcgttt ctcctcggcc accacccatt 49260
tagaatctgg gtctaggtcc aggtcgagca gcgaaagagg cgaagcgtct atctccggca 49320
ggcgcagcag ctgcactagt cggaatacac gaccggcgct catccagccg cgagagtatg 49380
agacctgcat aaactcgtaa atggcgatga ggatccacgc ttgcacctgc tcgaaatccg 49440
gggctgtcaa caaagtgtcg tccgactcga gggactccag tgtgtggcgc gtggtccgat 49500
acagtgctac gcgtacctcg ggcaactggc tactcacgcc agcggccagc gtgcgaatag 49560
cctgctgcag cccccgtcgg tttgccgatt gctgaggttc ccgtgcccag gaaaagtatc 49620
gatggcgttg caatatgggg accatcggct ggacgcgctc gaaaaacacc tggtccctga 49680
gccggtgagt ctgatgactt caatctcgta atggaaggga agataaagca tacaggtctt 49740
ccagaatgtt ttcggatact tcctcagagg ggtccgggga ctgggaagct ggcgaagagg 49800
cggacagcgc tttatccagc agctcatcgt ccgagatggg atcaacgagc atgccatcgc 49860
gctgctccag aagacaccgc tcgagggtgg ctgttcccag gacacataat cagcaacaag 49920
ctcaatcctc aggaaggaaa ttcaaggccc taccgatgcg gcccttcaag gcctttagat 49980
gacccttctt tggaccacgc gccgggcgtg caaggttggt gacacaataa acaccagcat 50040
caacgcagtt ctgacactgc ggctggcggt cgcaccgtag cttccggcga cggcattcct 50100
cacaggccga accagggccc tggcgcgagg tcttgtggtt tgtagtgttg tttttggtgg 50160
ttgtggttcg ggagtctgac atgatcagtc accgtcacag ttacgatcac aatcacgatc 50220
catcaagcgc gataggctga cttcttcagg gaaagaagga cccttatctt gacatccagc 50280
gtacttaacc aactcaagct gctgcctcat ggccacaaaa ggctcgccga tccagtgtgc 50340
aggaggccaa tggcatgcca aacacggcac gtcccacacc aagcgccacc aaatcgtcgt 50400
ttatctcccc ctcatcatga gtctctcatg gctgaccgat aaatttcaca gcccgcaaga 50460
aggattgaag gattgaagga tgaaaggcca taccacatcc acttgcggct tccaaaaggg 50520
cggcgtgttt gccatcggct cacctcatac ttccccatct tttcccttcc cgtgcgtcag 50580
cagcgtgcag gaatggattt gtgtatatac gaggcgaccg gggggagatt cctggtagtg 50640
gctgaccaaa gagaatcccc gagacgaaca taagcccgcc gataagcaga gccttgttgg 50700
ctccccaatc aacatcgatg gccggcagag gaatgctgaa acggccaaca tagtattgcg 50760
aagaatatag gattgtctct tatcagcgct ggaagggtct gatcactcgc tgccatatgg 50820
cagagtgcag tatgcaggat aggtgccgga gatcctgctg ctcgttcagc tcgcctaccc 50880
tgcgttctgt cggcccagac catgtggaag gacgggaagc acggtgcgtg cacagacgcc 50940
ggcgggtttt gctcatagta tggggccaat cgcagggcgc tggaagagag gagcaggtcc 51000
accataggcc gaaatgaagc tttgtggctc ttccgggccg ccaggttgat ttacttcttc 51060
aacaccccat atgagcttgg cctgcgaggc tggaagcgat tggatcatct agtcaactaa 51120
cgaatcctgc tgttgccacc cccatttccc ctcattgatc agtggaacgt gggagaaaac 51180
tccggtggaa agtccgggta attaagatga ctgaccccgc agtatcattt ttttttctga 51240
ctagtactac tacggaatat aatcgaaggc atcgataaag aataatcaca ccaatcaacg 51300
agccctgcac atcatgattc atcactcctg aaagccgatg tctcatagcc ggcttacggc 51360
aacgtcacca ccagcttctt agccgaaacc ccttgtttca aggcattcaa ggccccttga 51420
accgcctcca atccagtgcc caccacctca gcctcgggcg cagcaatgaa gcttccgtgt 51480
gctagagctt caccgaggta tccgtagagc tgatggctga cctcattatc cttgacggtg 51540
ctggcgaaga tgaacttgca ggacactccg tccggcacgt tcgggggtgc aggcatcgac 51600
aaagcgacaa acttgtcacc ctcggactcg gcaaccaggt taagacaagc ctccgccgct 51660
tcattgcggg tctgaggaac cacgccagca atagctaggg ctcctgcgca cgtcttgcct 51720
cgaaaggcct ctcgaagatc gtcacggatt gacggtttct tgtagtcgaa caccgccgta 51780
gcccctagac tcttgaccaa gtcgaaatta tgcggggaac aagtagtgat gacctcgtat 51840
ccagccgcga cagccagctg gatggcgttg cagcccacac tggtggctcc accccaaatc 51900
agtagggtct tccccgtccg ggttggggtc agcgacggga tctgcagacc cagatagtcc 51960
ttttggaata gaccaacgat cgcggtggag aaggccagag ggacaacagc tgcctgttga 52020
tattcgagag cgtggggcag aggagaagcc atgttctcta gcaacaccac atactgttgg 52080
aaggctcctt cagcgctgtt gttgctgttg gttcccactg cgtggccgac cacgcgatca 52140
ccaggcttaa agcgggtgac tcccggacca atcgcaacga cttcacccgc gatatcctct 52200
cccagcacca gtggataagt gacccacgga aagagagtct cgcccatcat ctgagtcgca 52260
tagtcaaccg cattgaccgc cagggcatgg acccgcacga cgatgcggtt ctctcccgct 52320
gttgggtacg gcgcttcctg gacgaccagc gactggttct tgccgggaac gatagcggcg 52380
aagttggtgg gcatattgat ggttgagatt tgaaaagata gacactcaac aaaattgatg 52440
tcagatatgc caagaagtaa ttaataagta agacggaata actgcttatt tacggctgat 52500
ggggcagtgg gggaatcgtc tggcaagagg ggcagtgaac tatatacaaa gcagatcggg 52560
tttggtgcaa tcccgacggc caggagataa gataggctct cttaactccg aaagcttgcg 52620
catatttatg cccttccttt caatttgtcg gatgatcaac cttcttgcgt aatgtttctc 52680
aggggcgttc caaactcccc gtcgggattc aatgccggcg gagtttctgt tattgacggc 52740
catgaatgtg ttggtttatt tgtttcccct gaatggaggg cgatgaagta catctttctg 52800
ttcagatcta cacaggatca gcggccgtct gtaacgatta cgttgaacta ccagaaagga 52860
gagctgtctc tcgatagctt tgcgagcgcg gctagtcatt cgaccatggc ccctctgcga 52920
cccaccgatg ctgacgccag gaatgcatct tccgataccc ccgaaaagga tttaccatca 52980
gcaggtacca gacagtcgtg gtggccgtgg tcgagaacag ccgcgccttc tcgcgcccct 53040
agaggtgtgt gattacggcg atggtctagt cttcctcgct gatttaggca gctgagatgt 53100
attggccagc tgtgttaatt cggaggacga tgagtatcat ctaaaagtcc catcgaaggg 53160
caaaagcaaa gagagatatg aggacttttg gaacgacgtt gtgaattttt tccatccatt 53220
ttctcgttcc aatggtgagt taaccctaca tagcatacag gtagatggag tatatattgc 53280
tatttgagcc acatgctgcc tacgtagaac aagacaaatg tgtttgtcat acaggatgga 53340
aaatatattg tatcatcatt aggaagcatt cgcaccactg tcttcttttt ttccatctag 53400
gaattcccgt atagcagcaa caatgacctc gtggtcggca gcctcctcct gcagcccact 53460
cacagcaatt acccccacga ccccttcgac tccctccacg cgaatcggaa acccacccgc 53520
ctgcaaggca tactcgtatc gttccaccgg tgacagagca aactgctcag ccaacacctc 53580
tatcccctcc ataactgaag gcccgacaca cacggactca tttgcacgag ttggcttcaa 53640
ccggcagtgg aggtaccagg tagacacctg ccagcggagg actgtcttcg tcttgcgctt 53700
aagccaaaga acatggtcca ctcgggttcc cggctctgta gcacagtgga aaactaactg 53760
cggagctgat gagccagaca ggcagatcga gatcacggct ggtcgtctct tgtgggcggg 53820
caaagccagt atgtgggatc gtagcgccac cccaagtgtc catgccgtgg ttgtcgtaaa 53880
gctgggaaag accaaggctt gttcttgggc caaaacaagg gcgggctatc ccatctagct 53940
gattagccag aagttatcat attgtcggcg gggtgtgggc aaggaagatt gattttctca 54000
cgtcagccgg gttatgcgat gaaacaggct cttggggggc catggtagct gccggaatac 54060
ctgggtaatc cgagcgagaa tttgaagcgg tgaaagtcct taaggtcctt caagagcgct 54120
tccttcgact taaagcagag ttcagagata gattgcaaac tgtcagtggg ccatcgtccg 54180
ggatcgtctg gctggagttt ttgatactcc gcattggagc aggtttagcg agaccatgct 54240
aaaagcttat tctgctgctc ctggtcctat cacgaattgc caagatgaac cgtcttaagg 54300
agatcgtgcc ctgccaagct gcctttgtgt caggaagacc acttacacta cccactatct 54360
cgtttcgtca tgactgtaca tcctcccgcc gtggcagacg agacctcccc actactgcca 54420
tcgcaagatg ggcctggaca taatggcatc gttcctgcag caaccaagcc caaagaactt 54480
caatcatcaa tgagtcaggt cgcgttactt tgttgtgctc gagcaattga cccgctggcc 54540
ttcttcacta ttttcccgtt cgtcaaccag atgattgccg acacggcagg tatcgacgaa 54600
gcggacgtcg gcttctacag cggcatcatt gtcagccctc tacctcctct cttagtacca 54660
ttacgctaac aacttccacc ttcaggaatc gttgttttct gtgactcaga tgatgttaat 54720
gattccctgg gcacgagcag cagatcggat gggacgcaag ccggtcctga tcctctccct 54780
ggccggttta tccgtctcat ctgcgctgtt cggattcagt cggactttag gccaaatggt 54840
tttcttcaga tgcttggcgg ggacgttcgg tgggaccgtt gtgtacgtga tatgctgaca 54900
cgttgggacg ggtgagacgc tgatcatgaa ttgtatagta cggtccgagt gatgatttcc 54960
gagaatagca ctccagacac ccaggcccgg gcattcagct acttctcctt ggcgaacacc 55020
attggaacgg tgattggccc tctgcttggt aagctaatgt ttatccctgc cggcagcacg 55080
cctgacacat gctgatgtgg gctccgtagg cggagcactt tgtcgtcctg gtggtgtgtt 55140
ccgtcactac ccgtatgcgt tgcccactgt cgcggccgga gcttttggag taactgtcac 55200
ggttgcctgt ctgatgttcg tcaacgaggt gagaggccat gggtaaggaa caatgacaca 55260
attcgcggaa gctaagatgg gtcctccaga cgcggaaacc cgcagatcat acacctcatg 55320
aaacagcctc gccaacatgg actagtgcca agatcctccg atcccaaggc gtgctaccag 55380
ttctctacat tcacggccat tcgatgatgt tggcattcgc gtacactgcg ggtaagctct 55440
ctttcgagcg tcccaaatat ctacaaaccc taaccagctc tcttattgaa gtttccccgg 55500
tattctactt cacatcacca cgtctaggcg gctacggttt ctctccattc tatatatcac 55560
tcttccttgg cggatcgggg atcgctcaaa ccatctggct agtgctagta tacccgccac 55620
tacacaaacg tctcggcacg ggtaatatct tacgaggctt atgtttcgtt tggattattt 55680
tcttggctgc tactgtagga gccagtgttc tccaccgtca tggtgagatg gtagcgttct 55740
ggattttggc gccgctagct ttggtactag gcagcagtgt tgcaatgcag ctgagtgaga 55800
cacatcccaa atttcctcac tactctatgg aggtggaggg ctgatagcta atggtattaa 55860
gcggctatgc aactcgcgct cgactccgtc tcaccgtcac ccgccgcctt aggcacgctc 55920
aatgccatgt ccttggccat catcagcttt cttcgcgctg tcgccccagc aatgttcact 55980
tccatgtacg caagtaccct taagcttagt agtcctggat tttacacatt ttggctcgtg 56040
ctgggtgggc tagttcttgt cctggcattt acactgcgct ggctgcctga gcaggtggag 56100
aaggctcctc gcaagctggg tcggtcgagc gcctgaggac agaagatatt atgatcgtgt 56160
tgtcgcatca gtgtcgacaa tgctgtccct tactatttat taacttgacg acaaatcact 56220
gtcccgggtt attagacatg ggtgttgaca tcttcgagtt tattgaacat agtaccaagg 56280
atacgcggta attagcggaa ataggtgcaa gccggaaccg tggctgaacc tcagagttca 56340
gaatgctgct agattggaac atactataca agcgaataac gtaccaatca cggtactatt 56400
tatgaacaag tatccaagaa tcaaatttgc gcaaggggga cagtaccact cgaccctaca 56460
tgatacagct caatgcgttt acgcagcgag atttaggatt tgaatcagac atcctacggg 56520
cgttctcagg cgctctacaa gggttttccg gtagtgaaaa gtactatgga atacccttcc 56580
ccctttttga agcaaggtta ttatggcact cgatgcatgg gagataccag agaagggctt 56640
ctgatgcagg tgacgtgttc cattcatagc cctcgtccta aatattgggt gacatcgagc 56700
ttctggcatg ttatgacgat gcttactctg tttcacttgc taggtgggga attccagacc 56760
tcgaatacgg aggacccaag agttatcgta cttgaagaag gatctgtaca tcccgaatgg 56820
ggtcacccat agtcagactt ccggtatata cacactgctt tctcgttctt gttatgtttc 56880
aattcaacga ccaaaagact cgcctgatga aaaaatcaga ttggacgtct gaacatgtaa 56940
gatatagaat gagctccagg aaccagagct tcgattggga ttccatcact ataagtttaa 57000
gcccctggct cgagcatgtg actatggtaa taggttgagt agatctaact gtcgatctgt 57060
caacggcgcc gacgacgact ccgttgcatc gccttgtagt taacctctaa cgtttaataa 57120
atcttgaata agccgaaaca aaacttatat tcattatatg taaataggaa gaatgtgata 57180
gtcgttactc actctgcaaa tctaaacacc cgatgcaaaa gccaattaat gccgcggtat 57240
ggacaccggc acaactgata tatatacaca tgttacgcag caactgatct agggcaacgg 57300
caccggataa ttgaaactga aaagctcatc cggatcccac ttagacttca actgcgagag 57360
cttaggcagg tgttgtggcg agtaccaaac atcagcgcct tcatcgccat gagcgtaatt 57420
caagtagatc tccatctcgc taaagccact agtacgctgg aactgtgagc ggatggaccg 57480
catgaaggaa ttgacggcgt tatcgagggt agaattatca ctggggtagg agttttcgaa 57540
taacctatac ccagtgtgtt agtcacggaa tgaatcgtgt tggatggcag aagcatacag 57600
atgagaattg atgtttcggt agccatagcc gtaggaggtg ctgttggctg gaacggctgc 57660
cgccgctgct gaactgtagc ggtcgacaac gaatattccc gaataatcgg ggtgagcctg 57720
ggagaaggcc tgcagctcat tgaagaaggt tgtgtaggtg tcgatgtttg tctcggccag 57780
tcccagtccc caggtgttga gataggaacc ggtggtgcag gcatcaccgt ccgctccaaa 57840
ggccatcgag cttgtcacgt tgggccaggc taccatcatt agattggttt ctgttgcatt 57900
gagcgctgta aaattggtta ggtaagatac tccctcttct tttggcccgt agtagatcgc 57960
attaacgatc agctccatct gtcaccccgt ttgggttagt cttgaaaatc atgaaagtga 58020
ggctacgtgt gaacgaacct cttgagtggt ttgattgaat cccgagtatg ccgtcaatga 58080
cagctccggg ggaagcgtct cgtcaaaaga ctggaagatc tcccatacgg acctattctc 58140
gctagcgggg aataggtaat tcgcgctcat ggccaggccg ttatttgtgg cattgtagat 58200
cttgtaagtg gctgaggtga tgatgccaaa gttggcaccg gcgccgcgga tggcccagaa 58260
gagatcggca ttttcagagg tagatacagt gaggacatca ccgctgggcg ttaccagctc 58320
gacggacagt agggagtcga gaatcagccc atgcaggcct tgtagatttc ctatcccgcc 58380
gccgatagtc gccccagcga gtccgacaca cggtgcggta ccggtagctg ggtaaatgct 58440
gttagcatag ggctgtagac attttggatg ctggggggga gagcaagcag catacgcagc 58500
tctttacccg cctcatagag aggcggaatg atgtcctcaa agcgtactgc tccaccaacg 58560
gtcatggtat tgttggaagc gtcaatcgaa acagtattga agttgctaat atcaatttcg 58620
atgccattgg tacaattaac ataggtgatg gtcgcgccgt ggccgcctgc tgtggcgaga 58680
aaagggatct tgttggatgc agctagggtc acctagtccg caattttgcg gtgagctgtg 58740
gtacttgttg tctgataatt gaatggaagg cgtcgtctta caatgttttg tatatctttg 58800
accgtagctg gcttgattgc cccgatgtag gtgggggcgt cgtatgtggt ccatctctgt 58860
gttacgtcct ctgtgtagtt tgtgtcggaa ggtagaaaga tctgcgcgcc cggagacagg 58920
ctggaactga agatggatgt gatgttgtgc gtggcagcag acgagagtgt ggttagcccc 58980
agagagcaaa gaagaattgt ccgaaacatg attggccgac gaggactgaa cgagaagaca 59040
aacggcgaga cactttggaa taggtccccc ttatcagtag cgtactatat atttttgagt 59100
cctggatttc caatgatggg gattatgcta ctccgaggga agcgcaataa ctccgtgctt 59160
atgcgtattg tcagaccggc ttgagagtga ctggaaggat gcgggtccct gttggctagc 59220
aaaagtccga agggaaactc catcgcatgg ctgcaagtgt gattatctca gcctaagagc 59280
actctgcttt cacgcgagag gtacgttaag ccagtgtttc agaggcgagg agccacgcga 59340
gaactgcagg gtcgtccgat gcactgcgga gtaattcgcc ggacttgccc taaacaggaa 59400
gtagaaccag gacaatctcc atgtggttgg tacggcagag tatcatatcc gccaagagag 59460
tcaacgagga tcaactgatg gaatataatg aaaataggct tcatggccct atccaaaaat 59520
cagcccagtc cagaacggtg atttcccctc cattcgacgc tacatacccc tcttgaacat 59580
taacgtagat tgtacattct tttctcttca gacctgtgag cgatttaccc acttacaatg 59640
gccggagagt attcaacggc cccgatggcc atcataggca tggcctgccg cttctctggc 59700
ggcgccacga gccccgagaa gttgtgggac atgatcgtac aacggcgatc cggctggtcg 59760
gagataccaa cctcacggtt taacgcaaat ggactttacc atccaaacgg cgagcgtgtg 59820
gggacggtaa gacatgctta ttctgtccct tcaagtgatc aagttgaccc gtgcctttct 59880
ctctagaccc acgtgaaagg tggtcacttt ctcgaagatg atattgcctg cttcgatgcg 59940
gccttcttcg gcatggcctc agaaaccgct agtgtaggat tgttccgtcg aattctccca 60000
gggggcctac caccaaaatg aactgacatg aaggctgcta ggctatggac ccgcaatatc 60060
ggatggaact cgaggttgtc tacgaggctc tggagagcgg tatgcagtca cctttcacgc 60120
ctatcaccgc ccgaggactg acacatttgc ttgcagcggg gattccaatg gagtccatta 60180
aaggaacgaa tacttcagta tatgggggtg tgatgtttcg cgactaccac gacactcata 60240
gccgtgactt ggacacgcta cctcgatact ttatgacagg caatgctgcc acgatggcgt 60300
cgaaccgcat ttcccacttt tatgaccttc gcgggccgag catgaccgtt gatacaggtt 60360
gctccacaag tctgacggct ctgcacctgg cttgccagaa tttgcgcagt ggagaatcca 60420
acatgtctat cgtgacggga gcaagtctca tgataaaccc cgacgttttc ctcagcatgt 60480
ccaatatcgg gtgagtaact tctagctgtc acacgttgaa caaatggcac tgataaatgt 60540
gaccaagctt tttgtcgccc gacggtatat cgtatgcctt tgacagccgt gccaacgggt 60600
acgggagagg tgaaggagtg ggtgccttac tcgttaagcg attagacgat gctctgcgcg 60660
atggcgactc catccgcgcc atcatccgcg aaaccggggt gaaccagaac ggtaagaccc 60720
ccagcatcac agccccacaa caagctgccc aggaagcttt gatccgccag tgttatgaac 60780
gggtcaactt ggatcccgca cagaccacat acgttgaggc tcacggaacc ggtactccag 60840
ccggcgatcc gctggaggtc ggcgcgctgg cagccgcatt gggaggcagt cgatcggcgg 60900
agcacccgtt gtatctcggc tctatcaagg ccaatattgg acacactgag gctgccagtg 60960
gcgtggccag catcatcaag gtggccctag cgctggagaa gggacaaatt ccgccgaata 61020
cgcaactgaa tacaccgaac agtgagctgc ggttaaatga tagaaacatg gaggtaggct 61080
acaatacctt atttggtaga aaactctcat gcgaacggtt atgtttgaac aggttcctgt 61140
atcaacgcag agatggcctg tggggaaagg tccccgacgt gcctccgtca acaattttgg 61200
ttttggcggt agcaatgctc atgccatttt ggaatcgccc ccggtaggtt ccaccaacgg 61260
cacccatgta aatggaacgc atacaacaaa cggaacggct gggatgaacg gcacaactgg 61320
agaaaatggg gcaaatcgaa ctaacgggca gctaaagagc aatggtccgg tggtaaatgg 61380
caacaaaacc aacattacca aaagggaaac cccatgggtg ttccgcttga gtgcaaagga 61440
tgcccagact tgccagcaga tggccgccga cctaagtacc tatatagagt cccacccacc 61500
agtcgacgag gaagccttcc tgggccgcct tgcatacacc ctgggatcgc gccgttctgt 61560
cttctcatgg accgcagctg tgtcggctag gtctctggca gagttaacac gggcactgga 61620
cgatgatgaa cgtttggtgc cgtcacgcgc agccccttct ttacgattgg gatgggtatt 61680
cactggccaa ggggctcagt ggtatgctat gggtcgtgaa ctaattgcca cttacccggt 61740
atttcggtca accattctgg agtgcgaccg ctacatgacg gagatggggt cgacgtggac 61800
attaatgggt gagtattgag cgctctatct tgaagcaacc tttgctaatt atacataaag 61860
aggaactgca tagagaggaa tccacttcac aggtcaacaa catcgtctac agtctgggtc 61920
tggccaccgc catccagata gccctggtgg agctgctatg gtcctggggc atccatccca 61980
cggctgtgac aggtcattcg agcggtgaga ttgcagcagc gtatgcatcg aaagccctgg 62040
acatgaagtc agccattggc attgcttacc ttcgtggagt gttagctgag aagtttgatg 62100
acaagattct cggcaaaggg ggcatgatgg ccgttgggct tggacgtaaa ccggttgaac 62160
attaccttag tcgcgtcact gcggggtact gcgtcgtggc ctgcgtaaac agccagtata 62220
gcgtcacaat atcaggtgat atcccagcaa tcgaccagct tgaacagttg ctgcaggaag 62280
accaagtctt cgctcggcgt ctccgcgtca atggggcgtt tcactgcgag cagatgcgcc 62340
ctatggctga cctattcgat tggagtctac gctacctgct cacccctcat cccgacttcg 62400
gatcagtcct attctcctcc cccaagacag gctcgcgcat ccaagacggc actatactag 62460
cgacctcgtc gcactgggtg ggaaatatgt tgcaggcggt tgagttcgag agctccttcc 62520
gtcatatgtg ttttggtgat ccaagcccaa aaggcgccaa aggcacccaa gatgtcgacc 62580
tagtactcga gatcgggcca catggagcgt tagggggtcc gattcaacag ctcatgaccc 62640
tccccgaatt tgaaggtagt ggcatttctt acctgcccac tctggtacga aagcaggatg 62700
ccgtcttcgc catgcagcgc ctggcgattg acctaactca cagaggctac cccgtcgatc 62760
tcaatgccgt caatttcccc cacggcacgc tctctctttc catcttgcac gaccttccat 62820
catacccatg gaatcatagc acgagatact ggcttgaacc ccggcgaaac cgagccgaca 62880
gacagcggca agcgccgccg tcggatttgg taggttactc tcaaccaagc atcacgccac 62940
ttgcacgcac ttggcgccac attattcgct tatctgatct gccctggctg ggagatcaca 63000
gggtgcagtc gagcattgtc ttccccggtg ctggcctggt gagtatggcc atagagggca 63060
tgcggcaagt ggcggcgggc cgccagcaaa ccgtgtccgc gtacgagctg cgcgacgtag 63120
atattgccaa agccctcacg gtgcctgagg cagatgaggg agtcgaagtg caattaaata 63180
tccgcccgtg tgatgagcaa atgctgggga cgaaggactg gctggctttc cagatcttct 63240
ccgtctctgg cgacagccgc tggacggagc actgcagtgg acgaatttca gttatcacaa 63300
cctctgacag cactcctctc ccatctgcca taccatccca gtcggaggac ctgtataacc 63360
gccgcattga tccccgctat atgtgggcag ccatgcgatc cgtgggcata taccatgggc 63420
ctctgtttca aaacatccac caagttctgg cgaagccgtc cgcatctcgc accatttttg 63480
ctatcgcaga tacggctgcc gtgatgccga agaagtacca gaccccccat gtgttgcatc 63540
ccaccacgct ggactcggtc ttccaggctg cctatacact tttgcccgag tctggcgcgc 63600
gtctgccttc cgccatggtt cctcgccata tccggtctgt ccgagtttca gcgcagatct 63660
ccaatagtcc agcgcatgac ctggaggcat atgccacgct gaatcgggat tatgacgcgc 63720
agtcattcga gacatccctc actgtggtgg acgcaaagga cgggaacagc ccggtgctcg 63780
aagtggatgg cttgacctgt cagtcacttg gacgagccct ggatcgcgag gcagatccgc 63840
acgagaatga aatctgcagc cggtgggaat gggctccaga tattggcaca cttgatgcgg 63900
ccgcctgcaa ggaccggatt cgctgtgcgc cagaggcagc ggagatagag acgatgcgcg 63960
acctccgccg agccactatt ctatacattc ttgacattgt ctccagcctg acggtggctg 64020
atgttcagca gttgcgggga catcttaaga aattctacgt gtggatggtg gagcagctga 64080
aaaaagccag tcgcaatcag ttcgcgcccg acagcgccca gtggcgcgat atcagcgcgg 64140
cggacaaggc agccctgtac gagaaggtcg gtcggaccag tgtgaatggt gagatgctct 64200
gccgattggg tccacttgga gcatcttttc tacggcaaga aatggctcct ttagaggtaa 64260
tgttggaaaa ccggctttta ttccgctact acctcgaggc gctaaaatgg gatcggtcga 64320
cacgccaggt aagtgagctc gtcaggctct gcacgcacaa gaatccccga gccaagattc 64380
tggaaatcgg agccggcaca ggaggcggta cgcaggtgat cttggaggcc ctgggcaaag 64440
agaatggttc ctcgacgggc gctcgatttg gccgatatga cttcacggat atctctgcgg 64500
gcttcttcga ggctgccaaa gagcgcttcc aagactgggc ggacttgatg aacttccaga 64560
agctagatat tgaacacgat cccgtggctc aagggtttga agaaggctcc tatgatgtcg 64620
taattgcctg ccaggtgctt catgccacca agtcgatgga tcgtactctc acacacgttc 64680
gcaagcttct gaagccaggg ggcaaattga ttctaatgga gaccacccgc gacgaactag 64740
atgtcttctt cgcattcggc cttttgccag gatggtggct gagtattacc tccccttcct 64800
ggcacttctc attatatttg gtatatggca ctaattgtgt tctttaacag gcgaggaaga 64860
ggaacgtcga actaccccat cactcaccct gccattctgg aaccaggttc tatcccgtaa 64920
cggcttcgcc gggttggacc ttgaggtcca cgactgcgac agtgaggaat tctacgcttt 64980
cagcacaatt ctctccaccg ctcaagcccc tgctttatct ataacatcac ccgtaactat 65040
cgtcactggc acatcgccac cgccgacatc ttggatgagc gagcttcaaa cagccgtggc 65100
agctcatatc ggatgtcaac cagtaattgc gacgttggag acagttaccc cccagggtaa 65160
tatctgtatc tttctcggcg aggcggacga gcccctgctg gaccatgtca gtaacccagt 65220
cgaatttgac cgtatcatac acctggccac aagatgcaag ggcctcctgt ggatcacccg 65280
cggcgggtct ttggacgtcg acaaaccggc catgagtctg tcccaagggt tactgcgcac 65340
cctgaagtca gaatatcaag gcaaatcctt tgtctccttg gacgtggatc cacgacggtc 65400
tccatggact gcagaagtag tccaagccat atcccagatt tttcccgctt cgttcagtga 65460
gactactgac cctgccacgt gtgagttcga gtatgcagag cgcgatgggg tcctacacat 65520
tccccgtacc gtgaaggata tccccatgaa cagaaacatt ttccctgaga gcgacacgac 65580
tgagaagacc atacattgcc ggttccgaga tgcggcccgt ccccttcgca tgaagatcgg 65640
gaccccaggg ctcatcgaca ccttggtctt ccacgacgat ttagacgcaa aatccgaccc 65700
gttgcctgca gactggattg aattcgaccc cacggctttt ggtctaaact tccgcgatgt 65760
catggtagca atgggccagt tggaggccaa tgccatcatg ggcttcgagt gcgcgggaac 65820
catcgtccgg ctgggagcca ctgcggcagc gaagggcttt gcagttggcg atcgtgtatg 65880
tacgctactc aggggccact gggcgacgcg gcctcgagca ccctggacca gcgtgatgag 65940
gatccctcaa catctaagcg accaggaggc cgcctctttt cctactgtct ttgctaccgc 66000
ctacatcgcc ctgcacgaaa ctgcccgcct ccaacgcggc gagtcgatcc tcattcacgc 66060
tgctaccgga ggagtcggcc aggctgcgat ccagcttgcc caactaatcg gggcagagat 66120
ctacgccacc gcgagtactc cggcaaagcg gcaattgctc catgagacgt atggtattcc 66180
agagaacaat atcttctcca gtcgagatcc atcattcgcc accgatgtac atttgcggac 66240
cgacggccga ggcgttgacg tcgtattgaa ctcccttgca ggccgtctgt tacaggagag 66300
cttcaactgt ttggcggaat tcggtcgcat ggtagagatc ggcaagcggg atctcgagca 66360
acacagcggc ttagacatgt acccgttcac ccggaacgtc tctttctcgt cggtggactt 66420
gctgacatgg cagagccgga gaggcgcaga tatctcttgt gtcctgcaaa gcctctcgaa 66480
gctactgggc gagaagaaga tcatgccggt ctatcccctt acactatatc ctatcacaca 66540
gatcgaaaaa gccttccgaa ctatgcagac tggccaacac atgggcaaga tcattatctc 66600
ggtcggcgaa caggatactg tgccggtaag tcgcactctt cccagttcca acctcacctc 66660
gactgcaggt gaattcgaca agggctaatg tgacctgggc aggtggtgga gcgtccccca 66720
cctttctctt tgcgatccga cgcatcgtat gtcattgtcg gcggccttgg cggtattgga 66780
cgcgtcctgt gtgaatggat gatggcacgc ggggcacggc atctgatcat catatcgcgg 66840
aatgctcgcc ctgggccttt tgtgactgag cttgagcaac aagggtgtga agttcgcacc 66900
ctcgcctgtg acattgcggc agaggatcag ctggcggcag cactggcaca gtgtgctgac 66960
atgcctccta ttaaaggcgt catccagggc gctatggtgc tcaaagtatg ttgcctccct 67020
tcatttcata tcgatctcta gtggatgggt attgcagcat atactaacat ttacatagga 67080
taccgttctc gagcaaatga cagtcggcga cttcgaagcg gcagtacgcc ccaaagcgca 67140
gggaagctgg aacctgcacc agcagctggg cgacgttgac ttcttcataa tgctttcttc 67200
cttgatggga gttatgggag ccgccagcca agcaaactac gccgccgggg gtgccttcca 67260
ggatgccctg gccacgtacc gccgcaaccg gggtctgccc gcagtgagtc tggatctggg 67320
aattgtccga tcagtagggt tcgtagccca gaccgatggc gtgcaagagc gtctcgtcca 67380
gatgggggtt acctctctgt ctgaagaaac agttctccgg atcctcgaac aagccatcac 67440
gcatcccaca ggtccacccc agatcatcac cggtattaac accgccccag gtaaacactg 67500
ggacgaagcc tcctggatcc aagacccgcg ctttgccgct ctgagatacc gcgacagtac 67560
gcaggctgga tcttcccgtg ctactactgg gacggcgaaa cagggaaaga taagggatca 67620
gcttgcagag attgcttccc cagtggatgc tgctgcactt atctgccagg agttgatgca 67680
gaaattagcc agcatgtttg gtctggtggt cgaggagatg tctgctactc aagatctgtc 67740
ttcctatgga gtggactcgc tggtagcggt ggaactgcgc aattggcttg tcgcgcaggt 67800
gggtgctgag gtttccatct ttgatttgat gcagagtccg tctttggagg acttatccct 67860
gcgggtggca acgaagcgga cgtag 67885
<210> 6
<211> 524
<212> PRT
<213> 黑曲霉(Aspergillus niger)
<400> 6
Met Ser Ser Thr Thr Ser Ser Ser Arg Ser Asp Leu Glu Lys Val Pro
1 5 10 15
Val Pro Gln Val Ile Pro Arg Asp Ser Asp Ser Asp Lys Gly Ser Leu
20 25 30
Ser Pro Glu Pro Ser Thr Leu Glu Ala Gln Ser Ser Glu Lys Pro Pro
35 40 45
His His Ile Phe Thr Arg Ser Arg Lys Leu Gln Met Val Cys Ile Val
50 55 60
Ser Leu Ala Ala Ile Phe Ser Pro Leu Ser Ser Asn Ile Tyr Phe Pro
65 70 75 80
Ala Leu Asp Asp Val Ser Lys Ser Leu Asn Ile Ser Met Ser Leu Ala
85 90 95
Thr Leu Thr Ile Thr Val Tyr Met Ile Val Gln Gly Leu Ala Pro Ser
100 105 110
Phe Trp Gly Ser Met Ser Asp Ala Thr Gly Arg Arg Pro Val Phe Ile
115 120 125
Gly Thr Phe Ile Val Tyr Leu Val Ala Asn Ile Ala Leu Ala Glu Ser
130 135 140
Lys Asn Tyr Gly Glu Leu Met Ala Phe Arg Ala Leu Gln Ala Ala Gly
145 150 155 160
Ser Ala Ala Thr Ile Ser Ile Gly Ala Gly Val Ile Gly Asp Ile Thr
165 170 175
Asn Ser Glu Glu Arg Gly Ser Leu Val Gly Ile Phe Gly Gly Val Arg
180 185 190
Met Leu Gly Gln Gly Ile Gly Pro Val Phe Gly Gly Ile Phe Thr Gln
195 200 205
Tyr Leu Gly Tyr Arg Ser Ile Phe Trp Phe Leu Thr Ile Ala Gly Gly
210 215 220
Val Ser Leu Leu Ser Ile Leu Val Leu Leu Pro Glu Thr Leu Arg Pro
225 230 235 240
Ile Ala Gly Asn Gly Thr Val Lys Leu Asn Gly Ile His Lys Pro Phe
245 250 255
Ile Tyr Thr Ile Thr Gly Gln Thr Gly Val Val Glu Gly Ala Gln Pro
260 265 270
Glu Ala Lys Lys Thr Lys Thr Ser Trp Lys Ser Val Phe Ala Pro Leu
275 280 285
Thr Phe Leu Val Glu Lys Asp Val Phe Ile Thr Leu Phe Phe Gly Ser
290 295 300
Ile Val Tyr Thr Val Trp Ser Met Val Thr Ser Ser Thr Thr Asp Leu
305 310 315 320
Phe Ser Glu Val Tyr Gly Leu Ser Ser Leu Asp Ile Gly Leu Thr Phe
325 330 335
Leu Gly Asn Gly Phe Gly Cys Met Ser Gly Ser Tyr Leu Val Gly Tyr
340 345 350
Leu Met Asp Tyr Asn His Arg Leu Thr Glu Arg Glu Tyr Cys Glu Lys
355 360 365
His Gly Tyr Pro Ala Gly Thr Arg Val Asn Leu Lys Ser His Pro Asp
370 375 380
Phe Pro Ile Glu Val Ala Arg Met Arg Asn Thr Trp Trp Val Ile Ala
385 390 395 400
Ile Phe Ile Val Thr Val Ala Leu Tyr Gly Val Ser Leu Arg Thr His
405 410 415
Leu Ala Val Pro Ile Ile Leu Gln Tyr Phe Ile Ala Phe Cys Ser Thr
420 425 430
Gly Leu Phe Thr Ile Asn Ser Ala Leu Val Ile Asp Leu Tyr Pro Gly
435 440 445
Ala Ser Ala Ser Ala Thr Ala Val Asn Asn Leu Met Arg Cys Leu Leu
450 455 460
Gly Ala Gly Gly Val Ala Ile Val Gln Pro Ile Leu Asp Ala Leu Lys
465 470 475 480
Pro Asp Tyr Thr Phe Leu Leu Leu Ala Gly Ile Thr Leu Val Met Thr
485 490 495
Pro Leu Leu Tyr Val Glu Asp Arg Trp Gly Pro Gly Trp Arg His Ala
500 505 510
Arg Glu Arg Arg Leu Lys Ala Lys Ala Asn Gly Asn
515 520
<210> 7
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
caccaggtcc tcagatactg taga 24
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aaactctaca gtatctgagg acct 24
<210> 9
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
caccgagacg caagccattg ttat 24
<210> 10
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
aaacataaca atggcttgcg tctc 24
<210> 11
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
caccacctac attacagctg ccgt 24
<210> 12
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
aaacacggca gctgtaatgt aggt 24
<210> 13
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
caccaagtca gcgaaggata cttc 24
<210> 14
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
aaacgaagta tccttcgctg actt 24
<210> 15
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
caccctcgat aggtggttcg agac 24
<210> 16
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aaacgtctcg aaccacctat cgag 24
<210> 17
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caccttcttg cgcatgtcgt gtgg 24
<210> 18
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
aaacccacac gacatgcgca agaa 24
<210> 19
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 19
caccagtcac ggctatgagt gtcg 24
<210> 20
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 20
aaaccgacac tcatagccgt gact 24
<210> 21
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 21
cacctatctc agagcggcaa agcg 24
<210> 22
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 22
aaaccgcttt gccgctctga gata 24
<210> 23
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 23
ggttggagat tccagactca g 21
<210> 24
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 24
aaaaaagcac cgactcggtg ccac 24
<210> 25
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 25
aaatagtaga aatggcattg agatccacat gctctatact caggaaacag ctatgac 57
<210> 26
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 26
ccatgcgcgt gttataaacg aacttgatct gcagcgagtc tgtaaaacga cggccagt 58
<210> 27
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 27
cattgatcgc tgcatatgga tctgttaata cgacgatctc caggaaacag ctatgac 57
<210> 28
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 28
agaccttcgc aatgatcggt gactttgtca accggaacac tgtaaaacga cggccagt 58
<210> 29
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 29
tccacagtat ggagcgctgg aaggcaagca gttgctgcgg caggaaacag ctatgac 57
<210> 30
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 30
ttggcgaatg ctcacacgct cgcttcgtat ccaacaacag tgtaaaacga cggccagt 58
<210> 31
<211> 57
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 31
cgaatacttc agtatatggg ggtgtgatgt ttcgcgacta caggaaacag ctatgac 57
<210> 32
<211> 58
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 32
cagtagtagc acgggaagat ccagcctgcg tactgtcgcg tgtaaaacga cggccagt 58
<210> 33
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 33
gatgacacca ccatagtcac aag 23
<210> 34
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 34
cgagtccaag tgaatgttga atc 23
<210> 35
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 35
atgatgaccg tcaagacgcc gttg 24
<210> 36
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 36
atggggttat cgcacagact gttc 24
<210> 37
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 37
gtcacaacga caagggctac taac 24
<210> 38
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 38
aacctgactg aagcaatctc cgag 24
<210> 39
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 39
agtgctatgg acccgcaata tc 22
<210> 40
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 40
ggcttggact acttctgcag tc 22
Claims (10)
1.一种内源活性多肽在微生物发酵生产柠檬酸中的用途，其特征在于，所述多肽是：
(A)氨基酸序列如SEQ ID NO:1至SEQ ID NO:4任一项所示；或
(B)与(A)所述蛋白氨基酸序列具有98％以上同源性，且来源于黑曲霉与(A)所述蛋白具有相同活性；
优选地，所述内源活性多肽的氨基酸序列如SEQ ID NO:1至SEQ ID NO:4任一项所示；
优选地，上述用途中，(A)或(B)中的一种内源活性多肽的活性减弱或完全去除。
2.一种基因簇在微生物发酵生产柠檬酸中的用途，其特征在于，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉；
优选地，所述基因簇的核苷酸序列如SEQ ID NO：5所示；
优选地，上述用途中，(A)或(B)中的整个基因簇失活。
3.一种柠檬酸的生产菌株，其特征在于，所述菌株被修饰：
1)使得内源活性多肽的活性减弱或完全去除，其中所述内源活性多肽选自：
(A)氨基酸序列如SEQ ID NO:1至SEQ ID NO:4任一项所示；或
(B)与(A)所述蛋白氨基酸序列具有98％以上同源性，且来源于黑曲霉与(A)所述蛋白具有相同活性；
或者，
2)使得一种基因簇失活，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉；
优选地，所述内源性多肽的活性减弱或完全去除可以通过以下方法之一或组合实现：部分敲除或完全敲除多肽的编码基因；编码基因突变；改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译弱化；改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定；或其他任何通过修饰基因编码区及其临近的上下游区域使其失活的方式等；
进一步优选地，所述菌株中的内源活性多肽相对于未改性的内源活性多肽活性减弱，是指所述内源活性多肽的编码基因的转录、表达降低至少30％，优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失；或者所述内源活性多肽的活性降低至少30％、优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失。
4.根据权利要求3所述的生产菌株，其特征在于，所述生产菌株中的柠檬酸转运蛋白活性增强；
优选地，所述柠檬酸转运蛋白为氨基酸序列如SEQ ID NO:6所示的多肽；
优选地，所述柠檬酸生产菌株包括但不限于黑曲霉(Aspergilus niger)，构巢曲霉(Aspergillus nidulans)，米曲霉(Aspergillus oryzae)，产黄青霉(Penicilliumchrysogenum)，里氏木霉(Trichoderma reesei)，玉蜀黍黑粉菌(Ustilago maydis)，嗜热毁丝霉(Myceliophthora thermophila)等菌株中的任一种，优选黑曲霉、构巢曲霉、米曲霉、里氏木霉，最优选黑曲霉。
5.根据权利要求3或4所述的生产菌株，其特征在于，所述内源活性多肽为氨基酸序列如SEQ ID NO:1至SEQ ID NO:4任一项所示，或者所述基因簇的核苷酸序列如SEQ ID NO：5所示。
6.一种柠檬酸生产菌株的构建方法，其特征在于，所述方法包括通过遗传修饰：
1)使得菌株中内源活性多肽相对于未改性的蛋白活性减弱或完全去除，其中所述内源活性多肽选自：
(A)氨基酸序列如SEQ ID NO:1至SEQ ID NO:4任一项所示；或
(B)与(A)所述蛋白氨基酸序列具有98％以上同源性，且来源于黑曲霉与(A)所述蛋白具有相同活性；
或者，
2)使得一种基因簇失活，所述基因簇是：
(A)核苷酸序列如SEQ ID NO:5所示；或
(B)与(A)所述核苷酸序列具有98％以上同源性，且来源于黑曲霉；
优选地，所述内源性多肽的活性减弱或完全去除可以通过以下方法之一或组合实现：部分敲除或完全敲除多肽的编码基因；编码基因突变；改变编码基因的启动子、翻译调控区或编码区密码子令其转录或翻译弱化；改变编码基因序列使其mRNA稳定性减弱或编码的蛋白的结构不稳定；或其他任何通过修饰基因编码区及其临近的上下游区域使其失活的方式；
进一步优选地，所述构建方法中，所述菌株中的内源活性多肽相对于未改性的蛋白活性减弱，是指所述内源活性多肽的编码基因的转录、表达降低至少30％，优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失；或者所述内源活性多肽的活性降低至少30％、优选至少40％，更优选至少50％，例如至少60％、至少70％、至少80％、至少90％、至少95％、至少96％、至少97％、至少98％、至少99％或完全消失。
7.根据权利要求6所述的构建方法，其特征在于，所述生产菌株中的柠檬酸转运蛋白活性增强；
优选地，所述柠檬酸转运蛋白为氨基酸序列如SEQ ID NO:6所示的多肽；
优选地，所述柠檬酸生产菌株包括但不限于黑曲霉(Aspergilus niger)，构巢曲霉(Aspergillus nidulans)，米曲霉(Aspergillus oryzae)，产黄青霉(Penicilliumchrysogenum)，里氏木霉(Trichoderma reesei)，玉蜀黍黑粉菌(Ustilago maydis)，嗜热毁丝霉(Myceliophthora thermophila)等菌株中的任一种，优选黑曲霉、构巢曲霉、米曲霉、里氏木霉，最优选黑曲霉。
8.根据权利要求6或7所述的构建方法，其特征在于，所述内源活性多肽为氨基酸序列如SEQ ID NO:1至SEQ ID NO:4任一项所示，或者所述基因簇的核苷酸序列如SEQ ID NO：5所示。
9.一种生产柠檬酸的方法，其特征在于，所述方法包括：(1)培养权利要求3-5任一项所述的柠檬酸生产菌株，或者权利要求6-8任一项的构建方法获得的柠檬酸酸生产菌株，使之生产柠檬酸；和
(2)任选地从步骤(1)中分离柠檬酸。
10.权利要求3-5任一项所述的柠檬酸生产菌株或权利要求6-8任一项的构建方法获得的柠檬酸生产菌株在生产柠檬酸中的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111235410.0A CN116003542A (zh) | 2021-10-22 | 2021-10-22 | 生产柠檬酸的微生物及其构建方法和应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111235410.0A CN116003542A (zh) | 2021-10-22 | 2021-10-22 | 生产柠檬酸的微生物及其构建方法和应用 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116003542A true CN116003542A (zh) | 2023-04-25 |
Family
ID=86018031
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111235410.0A Pending CN116003542A (zh) | 2021-10-22 | 2021-10-22 | 生产柠檬酸的微生物及其构建方法和应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116003542A (zh) |
-
2021
- 2021-10-22 CN CN202111235410.0A patent/CN116003542A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6169174B1 (en) | Cotton plant gene | |
| AU2023204276A1 (en) | Novel CRISPR-associated transposases and uses thereof | |
| Moore et al. | The α-mating type locus of Cryptococcus neoformans contains a peptide pheromone gene | |
| Mazel et al. | Green light induces transcription of the phycoerythrin operon in the cyanobacterium Calothrix 7601 | |
| Viotti et al. | Multiplicity and diversity of cloned zein cDNA sequences and their chromosomal localisation | |
| CN108822194B (zh) | 一个植物淀粉合成相关蛋白OsFLO10及其编码基因与应用 | |
| CN109988231B (zh) | 水稻基因OsGRF4在提高植物耐冷性中的应用 | |
| CN112852650B (zh) | 一种高产檀香烯和檀香醇的酿酒酵母工程菌及其构建方法与应用 | |
| CN114133438B (zh) | 一种紫心甘薯花色素苷合成调控因子IbEIN3-2及其应用 | |
| CN107759676B (zh) | 一种植物直链淀粉合成相关蛋白Du15与其编码基因及应用 | |
| CN111073898B (zh) | 小麦条锈病抗性基因YrU1及其克隆与应用 | |
| CN110343158B (zh) | 水稻半卷叶基因srl10及其应用 | |
| CN109134633B (zh) | 抗稻瘟病蛋白和基因、分离的核酸及其应用 | |
| CN113667682B (zh) | Yh66-rs11190基因突变体及其在制备l-缬氨酸中的应用 | |
| Mosrin et al. | The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail | |
| CN107446031B (zh) | 一种植物谷蛋白转运储藏相关蛋白OsVHA-E1及其编码基因与应用 | |
| CN114456244B (zh) | 基因OsR498G1018986900.01及其编码的蛋白在调控水稻垩白中的应用 | |
| CN116003542A (zh) | 生产柠檬酸的微生物及其构建方法和应用 | |
| CN108866075A (zh) | 影响番茄果色形成调控基因yft2的可变剪切子及应用 | |
| CN109295071A (zh) | 一种水稻花器官发育调控基因peh1及其编码的蛋白质和应用 | |
| CN108586595B (zh) | 水稻mis2基因及其编码蛋白与应用 | |
| CN113999828A (zh) | 草莓甲基转移酶mta和mtb基因在控制草莓成熟中的应用 | |
| CN112708628A (zh) | 玉米百粒重主效QTL位点qKW4a及其候选基因和应用 | |
| JP2001503988A (ja) | Ａｐｐのエンドサイトーシスを阻害し得るペプチド及び対応するヌクレオチド配列 | |
| CN113929756A (zh) | Gl11蛋白和编码gl11蛋白的基因在调控水稻粒形和千粒重中的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |