CN100413887C - 一种p53负调控分子及其编码基因 - Google Patents
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Abstract
本发明公开了KRAB型锌指蛋白KRANA及其活性片段在制备抑制p53转录功能的负调控分子中的应用;其中,所述KRAB型锌指蛋白KRANA的氨基酸残基序列如SEQ ID NO:1所示。本发明的p53负调控分子对p53的转录抑制作用受ATM的负调控,提示在ATM的调控网络中存在一条新的将信号从ATM向p53传递的支路。本发明不仅扩充了KRAB型锌指蛋白家族的成员,增加了对此类分子作用机制的了解;还为白血病及放射病的防治提供新的靶标和思路。
Description
技术领域
本发明涉及生物技术及医学领域中一种p53负调控分子及其编码基因。
背景技术
真核细胞在受到诸如电离辐射、肿瘤化疗药物等基因毒应激(genotoxin stress)损伤时,细胞作出最重要的细胞学反应有:一是起始DNA损伤检查点机制使细胞阻滞在G1/S期,赢得时间修复损伤,避免损伤的DNA进行复制;或/和阻滞在G2/M期,避免缺陷的染色体进一步分裂;二是启动细胞死亡的程序,清除带有病变倾向的细胞,避免危害机体。ATM(ataxia telangiectasia mutant,毛细血管扩张与共济失调综合征突变致病基因蛋白)是一种丝/苏氨酸激酶,可以感受DNA损伤等应激(stress)信号而激活,启动DNA损伤修复机制,通过细胞周期阻滞、细胞凋亡等过程维护机体基因组的稳定性,降低疾病及肿瘤的风险。ATM位于这种基因毒应激损伤反应网络的起始位置,能够对G1/S、G2/M检查点和Intra-S期进行调控,并参与DNA损伤引起的修复过程和凋亡过程。在过去几年中,许多实验室对ATM在DNA损伤与修复中的功能进行了深入而广泛的研究,已鉴定了近20个ATM的磷酸化底物,如参与G1/S期检查点调控的p53、MDM2、CHK2、RAD9、RAD17;参与G2/M期检查点调控的BRCA1、CHK2、CtIP;参与Intra-S期调控的NBS1、SMC1、FANCD2、CHK1和CHK2;参与DNA修复的ABL以及参与凋亡过程的TRF1等。ATM的这些磷酸化底物均在细胞周期的调控与DNA损伤修复中发挥了必不可少的作用,几乎每个成员的发现都在肿瘤和放射病的研究领域引发了研究热潮,并随后成为肿瘤和放射病治疗的靶点。
p53作为转录因子和抑癌基因,具有极其重要的生理、病理功能,同时,p53又是ATM的重要底物分子。当ATM激活以后,一方面它可以通过对p53的直接磷酸化激活p53,同时又可以通过对p53的一种抑制分子HDM2磷酸化使其解除对p53的抑制功能,增强p53的转录活性,从而促进下游的一些重要基因,如Gadd45、p21及PML等重要的周期调控分子和凋亡调控分子的表达,从而发挥周期抑制及促凋亡的功能。ATM和p53共同介导了DNA损伤修复网络的一条重要途径。
含KRAB(Krueppel-associated box)结构域的锌指蛋白也称KRAB型锌指蛋白,占人类基因组中所有锌指蛋白(799种)的三分之一(290种),是最大的转录因子家族,其中超过220种在胚胎发育、细胞分化、细胞转化及细胞周期的调控中发挥重要功能,并随时间和空间的不同进行特异表达。其结构特征是N端含有一个KRAB结构域,C端含有多个C2H2型锌指结构。KRAB结构域由75个氨基酸组成,为一蛋白质-蛋白质相互作用区,即可以与多种协同转录抑制因子,如:KAP-1、HP1α,MOD1和MOD2等结合,也可以与多种转录因子如c-myc和BRCA1结合,作为转录因子和/或转录调控因子发挥依赖于DNA结合的转录抑制功能。C端锌指的数目不一,一般在3到40之间,参与特定DNA序列的识别,使KRAB结构域具有靶向性地发挥功能。KRAB型锌指蛋白尤其是功能相近的一些成员在基因组上成簇分布,且主要分布在3号、19号及X染色体上。近年来,随着对KRAB结构域转录抑制功能的阐明,此类蛋白引起了研究者越来越多的关注,多种具有重要功能的成员相继被克隆并进行了功能鉴定,如与BRCA1相互作用通过调节GADD45的表达调控细胞周期的ZBRK1;能够抑制c-myc活性的KRIM-1;通过与ZAK相互作用促进细胞增殖的ZZaPK等。但是,目前,对KRAB型锌指蛋白家族成员如何调控它们参与的多种生理功能了解的并不深入,因此对新型KRAB型锌指蛋白的研究不仅能丰富已有的KRAB型锌指蛋白家族,更有助于深入了解它们参与的各种重要功能。
发明内容
本发明的目的是提供一种p53负调控分子及其编码基因。
本发明所提供的p53负调控分子,名称为KRANA(含KRAB结构域和19个锌指的ATM结合蛋白,Krab domain and nineteen zinc fingers-containing ATM-associatedprotein),来源于人,是具有下述氨基酸残基序列之一的蛋白质:
1)序列表中的SEQ ID №:1;
2)将序列表中SEQ ID №:1的氨基酸残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有抑制p53转录功能的蛋白质。
序列表中的序列1由688个氨基酸残基组成。
KRANA为一新型的KRAB型锌指蛋白,含有典型的位于N端的KRAB结构域(自序列1的氨基端第4位至67位氨基酸残基)和19个C2H2型的锌指结构(自序列1的氨基端第138位至665位氨基酸残基),18个SQ和12个TQ基序。自序列1的氨基端第16位丝氨酸残基、第68位丝氨酸残基和第264位丝氨酸残基为潜在的ATM底物位点。
KRANA的编码基因也属于本发明的保护范围。
KRANA的cDNA基因,可具有下述核苷酸序列之一:
1)序列表中SEQ ID №:2的DNA序列;
2)编码序列表中SEQ ID №:1蛋白质序列的多核苷酸;
3)在高严谨条件下可与序列表中SEQ ID №:2限定的DNA序列杂交的核苷酸序列;
4)与序列表中SEQ ID №:2限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
序列表中的序列2由2999个碱基组成,其开放阅读框架(ORF)为自5′端第225位-2291位碱基。
KRANA的基因组基因,可具有下述核苷酸序列之一:
1)序列表中SEQ ID №:3的DNA序列;
2)编码序列表中SEQ ID №:1蛋白质序列的多核苷酸;
3)在高严谨条件下可与序列表中SEQ ID №:3限定的DNA序列杂交的核苷酸序列;
4)与序列表中SEQ ID №:3限定的DNA序列具有90%以上同源性,且编码相同功能蛋白质的DNA序列。
序列表中的序列3由51280个碱基组成,其开放阅读框架(ORF)为自5′端第10641位-50587位碱基,含有5个外显子,4个内含子。KRANA的基因组基因染色体定位于19q13.12。
所述高严谨条件可为在0.1×SSPE(或0.1×SSC),0.1%SDS的溶液中,在65℃下杂交并洗膜。
含有KRANA编码基因的表达载体和细胞系和宿主菌均属于本发明的保护范围。
上述p53负调控分子的活性片段,具有下述氨基酸残基序列之一:
1)自序列1的氨基端第1位至70位氨基酸残基(N1);
2)自序列1的氨基端第1位至244氨基酸残基(N2);
3)自序列1的氨基端第1位至514位氨基酸残基(N3);
4)自序列1的氨基端第240位至688位氨基酸残基组成(C2);
5)自序列1的氨基端第72位至688位氨基酸残基组成(C3)。
上述p53负调控分子活性片段的编码基因也属于本发明的保护范围。
含有上述p53负调控分子活性片段的编码基因的表达载体,细胞系和宿主菌也属于本发明的保护范围。
免疫共沉淀(co-immunoprecipitation,CO-IP)实验显示,在人胚肾HEK293细胞中内源的ATM可以共沉淀外源表达的KRANA,表明二者在体内能够结合;N端的三种截短体均能与ATM结合,而C端三种截短体均不能与ATM结合,表明N端的KRAB区是KRANA与ATM结合所必须的,而锌指区不介导二者的结合。Western blot和CO-IP结果表明,随着ATM活性的增加,其与KRANA的结合程度降低。这提示KRANA很可能特异地与低活性状态的ATM结合。
报告基因实验结果表明KRANA可以显著地抑制p53、雌激素受体(ER)、CREB和ELK1的转录活性,且这种抑制功能具有量效关系,但对c-jun和NF-κB的活性没有显著影响。表明KRANA可以作为转录调控因子选择性的抑制某些重要转录因子的功能。KRANA对带有p21调控区的报告质粒具有明显的抑制功能。HEK293细胞中KRANA的过表达能够降低p21的蛋白表达量;在p53缺陷的肺小细胞癌细胞系H1299细胞中过表达KRANA对p21的表达水平没有影响,而再次过表达p53后,KRANA对p21表达的抑制作用恢复,表明KRANA不是作为转录因子直接调控p21的表达,而是通过影响p53的转录功能而调控p21。这些结果再次证实KRANA具有对p53的转录抑制功能。
为了揭示KRANA分子中发挥抑制p53活性的功能区,将KRANA的六个截短体分别与p53的报告基因质粒共转染HEK293细胞,结果表明N端的三个截短体和C端较长的两个截短体C2和C3均具有转录抑制功能,提示KRANA的N端和C端有独立的转录抑制功能区域。
KRANA作为ATM潜在的磷酸化底物,并且与ATM的结合程度与ATM的活性成反比,这提示ATM的磷酸化可能影响KRANA的转录抑制活性。ATM激活后能够部分抑制KRANA对p53的转录抑制功能,因此激活p53的功能。
RT-PCR和Northern-blot分析显示KRANA只有一个转录本,且在多种血液病来源的细胞系中(如Jurkat,MOLT4,SKO-007)特异表达。
本发明提供了一种新的p53负调控分子,它对p53的转录抑制作用受ATM的负调控,提示在ATM的调控网络中存在一条新的将信号从ATM向p53传递的支路。此支路的存在不仅是对ATM调控网络的补充和完善,而且可能在KRANA特异表达的细胞和组织中对造血调控和DNA损伤信号的传导发挥重要的功能。本发明不仅扩充了KRAB型锌指蛋白家族的成员,增加了对此类分子作用机制的了解;更为重要的是通过对ATM底物知识的扩展,对p53调控机制的完善,增进了对ATM参与的多种生理功能与疾病如白血病、放射病等机制的了解。同时通过揭示KRANA在调控细胞周期自稳状态与DNA损伤修复过程中发挥的功能,为白血病及放射病的防治提供新的靶标和思路。
附图说明
图1为KRANA的19个锌指的对比图
图2为KRANA分子中DNA损伤激酶磷酸化位点的示意图
图3为免疫共沉淀的方法检测KRANA与内源ATM的结合
图4a-图4b为免疫共沉淀的方法确定KRANA与ATM结合的区域
图5为HS、MMS、IR及wortmannin处理后KRANA与ATM结合的变化
图6A为KRANA对转录因子p53转录活性的影响
图6B为KRANA对转录因子ER转录活性的影响
图6C为KRANA对转录因子ELK1转录活性的影响
图6D为KRANA对转录因子CREB转录活性的影响
图6E为KRANA对转录因子c-jun转录活性的影响
图6F为KRANA对转录因子NF-κ转录活性的影响
图7为KRANA对p21表达的抑制功能
图8为在p53缺陷的细胞系肺小细胞肺癌细胞H1299中,KRANA依赖p53对p21表达水平的影响
图9为KRANA具有对p53转录抑制功能区域的确定
图10为HS、MMS、IR及wortmannin处理后KRANA对p53转录抑制功能的变化
图11为RT-PCR技术对KRANA细胞和组织表达谱的确定
图12KRANA的亚细胞定位
具体实施方式
下述实施例中的实验方法,如无特别说明,均为常规方法。
pCMV-Myc真核表达质粒购自Clontech公司。RNA提取试剂TRIZOL、SuperScriptTMIII Reverse Transcriptase及转染试剂LipofecamineTM 2000为Invitrogen产品;T4 DNA连接酶购自Promega公司;限制性内切酶Not I,Sal I,KpnI及PyrobestTM DNA聚合酶购自TaKaRa公司。寡核苷酸由北京奥科生物技术公司合成,DNA序列测定由上海博亚公司完成。c-Myc单克隆抗体购自Clontech公司,ATM多克隆抗体抗体购自CALBIOCHEM公司,HRP标记的羊抗鼠IgG购自北京中山公司。western发光试剂盒购自PIERCE公司,双报告基因荧光素酶检测试剂盒、pRL-CMV水木荧光素酶报告基因质粒购自Promega公司。p21、β-actin抗体购自Santa Cruze公司。随机引物标记试剂盒购自Promega公司。α-32P-dCTP购自亚辉公司。
实施例1、KRANA及其编码基因的获得
(1)将1086种转录因子中各特征性的domain、motif或sequence pattern进行归纳,获得其调和序列。在EST数据库中搜索调和序列的特定程序,在已拼接的HFL22w EST数据库的未知基因序列中搜索新基因对应的EST序列。其中搜索到的一条拼接号为4553的序列,全长2999bp(序列2)。利用NCBI的ORF finder程序进行开放阅读框(ORF)分析,为自序列1的5′端第225位-2291位碱基。
(2)RT-PCR扩增
使用TRIZOL一步法提取Jurkat细胞总RNA,取2μg总RNA采用SuperScriptTMfirst-strand synthesis system来制备cDNA。整个操作均按照试剂盒推荐的条件进行。
5’端正向引物为引物1:5’-t ctg tcg acc atg gct cgg aaatta gtg-3’;3’端反向引物为引物2:5’-aatgcggccgctcacatgtaaacttgtgagc-3’(其中带下划线部分的碱基为两端的Sal I与Not I识别位点)。
以上述引物1和引物2为引物,按照常规方法进行PCR扩增,扩增条件为先94℃5min,1个循环;接着94℃ 1min,55℃ 1min,72℃ 2min,5个循环;然后94℃ 1min,60℃ 1min,72℃ 2min,25个循环;最后72℃ 7min,1个循环。将PCR产物与pCMV-Myc(均经Sal I与Not I酶切)连接后,转入E. coli JM109感受态细胞中,利用氨苄抗性筛选阳性克隆(即pCMV-Myc-KRANA质粒),提取质粒进行酶切鉴定,挑选有插入片段的克隆测序鉴定,结果表明插入的cDNA片段由2999bp组成,具有序列表中序列2的核苷酸序列,将其命名为KRANA。它的开放阅读框架(ORF)为自序列2的5′端第225位-2291位碱基,编码含688个氨基酸的蛋白(KRANA,序列1)。KRANA定位于19q13.12,其基因组序列含有5个外显子,4个内含子,其外显子和内含子的剪接形式如表1所示,具有序列表中序列3的核苷酸序列。表1中,交界区的小写字母表示外显子,大写字母表示内含子。
表1.KRANA的基因组基因中外显子-内含子的交界区
生物信息学分析显示,KRANA为一新型的KRAB型锌指蛋白,含有典型的位于N端的KRAB结构域(自序列1的氨基端第4位至67位氨基酸残基)和19个C2H2型的锌指结构(自序列1的氨基端第138位至161位氨基酸残基序列;自序列1的氨基端第166位至189位氨基酸残基序列;自序列1的氨基端第194位至217位氨基酸残基序列;自序列1的氨基端第222位至245位氨基酸残基序列;自序列1的氨基端第250位至273位氨基酸残基序列;自序列1的氨基端第278位至301位氨基酸残基序列;自序列1的氨基端第306位至329位氨基酸残基序列;自序列1的氨基端第334位至357位氨基酸残基序列;自序列1的氨基端第362位至385位氨基酸残基序列;自序列1的氨基端第390位至413位氨基酸残基序列;自序列1的氨基端第418位至441位氨基酸残基序列;自序列1的氨基端第446位至469位氨基酸残基序列;自序列1的氨基端第474位至497位氨基酸残基序列;自序列1的氨基端第502位至525位氨基酸残基序列;自序列1的氨基端第530位至553位氨基酸残基序列;自序列1的氨基端第558位至581位氨基酸残基序列;自序列1的氨基端第586位至609位氨基酸残基序列;自序列1的氨基端第614位至637位氨基酸残基序列;自序列1的氨基端第642位至665位氨基酸残基序列)(图1)。19个锌指之间极为保守,其一致序列是P Y E C K E C G K A F XR G S Q L S/T Q H Q R I H。与其它KRAB型锌指蛋白不同的是,KRANA含有18个SQ和12个TQ基序,SQ/TQ是ATM与Rad3相关蛋白(ATM-and Rad3-relatedprotein,ATR)/DNA依赖的蛋白激酶(DNA-dependent protein kinase,DNA-PK)的磷酸化位点。经MotifScanner数据库进行严谨性分析,其中的S16、S68和S264为潜在的DNA损伤激酶底物位点(图2中,DNA_dam_kin为DNA损伤激酶)。
实施例2、KRANA与ATM的结合
(1)KRANA与ATM在体内结合的确证
通过免疫共沉淀(IP)实验检测KRANA与ATM的体内结合,具体方法如下:
将pCMV-Myc-KRANA利用转染试剂LipofecamineTM 2000(Invitrogen)转染人胚肾HEK293细胞24h后,25cm2培养瓶以400ul ATM裂解缓冲液(100mM Tris-Cl(pH 7.5),300mM NaCl,2%(V/V)Tween 20,0.4%NP-40,20%甘油;补加1mM Na3VO4(1∶100),1mM DTT(1∶100)和蛋白酶抑制剂(1∶20))裂解,冰浴中超声破碎细胞。12000rpm,离心10min,去沉淀,收集上清(总蛋白提取液)。取出50ul总蛋白提取液备western(IB)分析用。其余总蛋白提取液加入5ul ATM多克隆抗体抗体(购自CALBIOCHEM公司),4℃3h混合器上混匀。加protein A/G-agarose 40ul,4℃放置8h,3000rpm离心5min,弃上清液,沉淀再加ATM裂解缓冲液洗涤,3000rpm离心5min,弃上清,重复三次,得到免疫沉淀(IP)。免疫沉淀悬于50ul 1×SDS-PAGE上样缓冲液中,100℃水浴5min,3000rmp离心5min,吸上清上样,进行10%SDS-PAGE电泳。同时取备western分析(IB)用的总蛋白提取液在同一张胶上的其它泳道进行10%SDS-PAGE电泳。电泳完毕,经电转移(Bio-Rad,80mA,3h)至硝酸纤维素膜上,用3%BSA封闭2小时后,按1∶1000稀释的c-Myc单克隆抗体(购自Clontech公司)室温孵育2小时,TBST洗3次后,加入HRP标记的羊抗鼠IgG(购自北京中山公司),室温50min,加入western发光试剂盒(购自PIERCE公司)中的发光底物进行显色。结果如图3所示,表明人胚肾HEK293细胞内源的ATM可以共沉淀外源表达的KRANA,表明二者在体内能够结合。图3中,泳道1为未转染pCMV-Myc-KRANA的细胞总蛋白提取液,泳道2为转染pCMV-Myc-KRANA的细胞总蛋白提取液,泳道3为未转染pCMV-Myc-KRANA的细胞的ATM的免疫沉淀,泳道4为转染pCMV-Myc-KRANA的细胞的ATM的免疫沉淀,泳道2中的箭头示pCMV-Myc-KRANA表达的融合蛋白Myc-KRANA,泳道4中的箭头示pCMV-Myc-KRANA表达的融合蛋白Myc-KRANA和ATM的免疫沉淀。
(2)KRANA与ATM结合区域的确定
为了确定KRANA与ATM结合的区域,根据KRAB结合域的有无及锌指数目的多少构建了KRANA的六种截短体N1(由自序列1的氨基端第1位至70位氨基酸残基组成,只含有KRAB结构域)、N2(由自序列1的氨基端第1位至244位氨基酸残基组成,含KRAB结构域与N端3个锌指)、N3(由自序列1的氨基端第1位至514位氨基酸残基组成,含KRAB结构域与N端13个锌指)、C1(由自序列1的氨基端第507位至688位氨基酸残基组成,只保留C端5个锌指)、C2(由自序列1的氨基端第240位至688位氨基酸残基组成,只保留C端15个锌指)和C3(由自序列1的氨基端第72位至688位氨基酸残基组成,含有全部19个锌指)。将六种截短体的编码基因分别单转染HEK293细胞,利用CO-IP的方法检测其与ATM的结合。具体方法如下:
以pCMV-Myc-KRANA为模板,在如下引物的引导下,扩增N1,N2,N3,C1,C2和C3的编码基因。
引物1:5’-cgcgtcgaccatggctcggaaattagt-3’(带下划线碱基为Sal I识别位点)
引物2:5’-ctcggtaccggatccatgtaaacttgtga-3’(带下划线碱基为KpnI识别位点)
引物3:5’-ctcggtaccggatccattgggataattctgt-3’(带下划线碱基为KpnI识别位点)
引物4:5’-ctcggtaccggatcctgaactttttgatgtcg-3’(带下划线碱基为KpnI识别位点)
引物5:5’-ctcggtaccggatcctgagtaaaggccattct-3’(带下划线碱基为KpnI识别位点)
引物6:5’-cgcgtcgaccatgagtgacagactt-3’ (带下划线碱基为Sal I识别位点)
引物7:5’-cgcgtcgaccatgcatcaaaaagtttcatac-3’(带下划线碱基为Sal I识别位点)
引物8:5’-cgcgtcgaccatggaatgtagaatggcc-3’(带下划线碱基为Sal I识别位点)
其中引物1和3为N1编码基因的上下游引物;引物1和4为N2编码基因的上下游引物;引物1和5为N3编码基因的上下游引物;引物8和2为C1编码基因的上下游引物;引物7和2为C2编码基因的上下游引物;引物6和2为C3编码基因的上下游引物。
将PCR产物与pCMV-Myc(均经Sal I与KpnI酶切后)连接后,转入E.coli JM109感受态细胞中,利用氨苄抗性筛选阳性克隆,提取质粒进行酶切鉴定,挑选有插入片段的克隆,经测序鉴定,得到六种KRANA截短体的阳性克隆pCMV-Myc-N1,pCMV-Myc-N2,pCMV-Myc-N3,pCMV-Myc-C1,pCMV-Myc-C2和pCMV-Myc-C3。
将pCMV-Myc-N1,pCMV-Myc-N2,pCMV-Myc-N3,pCMV-Myc-C1,pCMV-Myc-C2和pCMV-Myc-C3分别转染HEK 293细胞,参照步骤(1)的方法进行免疫共沉淀结合Western分析,结果如图4a和图4b所示,表明N端的三种截短体均能与ATM结合,而C端三种截短体均不能与ATM结合,说明了N端的KRAB区决定了KRANA与ATM的结合,N端的KRAB区是KRANA与ATM结合所必需的,而锌指区不介导二者的结合。图4a和图4b中,泳道Ly表示细胞总蛋白提取液,泳道IP表示免疫沉淀,箭头示Myc与六种KRANA截短体和ATM的免疫沉淀;图4a中,Lsectin为一种膜蛋白,作为不与ATM结合的阴性对照;图4b中,FL表示KRANA的全长氨基酸序列。
(3)检测HS、MMS、IR及wortmannin处理后KRANA与ATM结合的变化
为分析KRANA与ATM的结合是否与其激活状态相关,选择了影响ATM活性的因素:作为ATM激活条件的电离辐射(ionizing radiation,IR)(20Gy)、热休克(heat shcok,HS)(42℃孵育2h)和DNA烷化剂MMS(0.05%孵育2h)以及作为ATM激酶抑制剂的wortmannin(100nmol/L预先处理1h)。将KRANA转染HEK293细胞后,分别给予如上处理,再进行免疫共沉淀。通过ATM底物p53的Ser15位点的磷酸化水平反映ATM的激活程度。具体方法如下:
A.IR、HS、MMS、IR及wortmannin处理的条件:
IR处理为细胞经20Gy照射2h收细胞;HS处理为将细胞42℃孵育2h后再在37℃孵育3h后收细胞;MMS处理为含0.05%(质量百分含量)MMS的培养基处理细胞2h后收细胞;W处理条件为含100nmol/L wortmannin的培养基处理细胞1h后收细胞;HW处理为含100nmol/L wortmannin的培养基处理细胞1h后再将细胞42℃孵育2h后再在37℃孵育3h后收细胞,IW处理为含100nmol/L wortmannin的培养基处理细胞1h后再经20Gy照射2h收细胞,MW处理为含100nmol/L wortmannin的培养基处理细胞1h后再经含0.05%MMS的培养基处理细胞2h后收细胞。
B.检测HS、MMS、IR及wortmannin处理后KRANA与ATM结合的变化
将pCMV-Myc-KRANA转染HEK293细胞24h后,分别使用HS、MMS、IR及联合wortmannin处理细胞,参照步骤(1)的方法进行免疫共沉淀结合Western分析。Western blot结果显示HS和MMS处理后可以导致p53 S15位磷酸化水平的显著上升,wortmannin可以降低p53 S15位的本底磷酸化水平,而HEK293细胞对于IR不敏感,ATM没有明显的激活。几种处理没有影响p53的蛋白水平。免疫共沉淀结果如图5所示,表明Wortmannin处理细胞,增强ATM与KRANA的结合;HS和MMS处理细胞后ATM与KRANA相互作用消失;加入Wortmannin逆转HS对ATM的激活,与KRANA的相互作用恢复,但对MMS的逆转失败;而由于HEK293细胞对IR不敏感,所以IR对HEK 293细胞中ATM与KRANA的作用不变。图5中,泳道Ly表示细胞总蛋白提取液,泳道IP表示免疫沉淀;C表示空白对照,未经处理的转染pCMV-Myc-KRANA的HEK293细胞。这些结果清楚地表明,随着ATM活性的增加,其与KRANA的结合程度降低。这提示KRANA很可能特异地与低活性状态的ATM结合。
实施例3、KRANA的转录抑制功能
已有研究表明,KRAB型锌指蛋白既可以与转录抑制因子如KAP-1、HP1α、MOD1和MOD2等结合,也可以与转录因子如c-myc和BRCA1结合,其本身作为转录因子和/或转录调控因子发挥依赖于DNA的转录抑制功能。为了研究KRANA的转录抑制功能,选择了几种在周期调控、生长及代谢方面比较具有重要功能的转录因子,检测KRANA是否对其活性有调控作用。具体方法如下:
(1)KRANA对多种转录因子p53、ER、ELK1、CREB、c-jun和NF-κB转录活性的影响
将不同浓度的pCMV-Myc-KRANA(实施例1)与含各种反应元件的荧光素酶表达质粒p21-luc(含有p21启动子区,见Cell,1993;75:817-25),p53的报告基因质粒pG13L(具有13个串连排布的p53的反应元件,见Nature Genetics,1992;1:45-49),AP-1-luc、CREB-luc、ELK1-luc(购自Stratagene公司),pERE-Luc(见MolEndocrinol.1997;11:353-65)或NF-KB-luc(见EMBO J.1990;9(12):3933-44.)共转染HEK293细胞,36h后,收细胞,按照双报告基因荧光素酶检测试剂盒(Promega)推荐的方法检测荧光素酶的活性。HEK293细胞中无雌激素受体的表达,同时转染ER的表达质粒,pER(见Mol Endocrinol.1997;11:353-65)。一般情况下,每组做3复孔,每次数据重复3次,以测定的萤火虫荧光素酶对水母荧光素酶(RL,pRL-CMV水母荧光素酶报告基因质粒购自Promega公司)的比值为准。
p53、ELK1、CREB、c-jun和NF-κB转录因子组的转染量如表2和表3。
表2.KRANA对p53-Luc转录抑制功能的实验的反应体系:
pRL-CMV | pG13L | pCMV-Myc | pCMV-KRANA |
10ng | 5ng | 0.4μg | - |
10ng | 5ng | 0.35μg | 0.05μg |
10ng | 5ng | 0.3μg | 0.1μg |
10ng | 5ng | 0.2μg | 0.2μg |
10ng | 5ng | - | 0.4μg |
表3.KRANA对ELK1、CREB、c-jun和NF-κB转录抑制功能的实验反应体系:
pRL-CMV | 报告基因的转染量 | pCMV-Myc | pCMV-KRANA |
10ng | 0.1μg | 0.4μg | - |
10ng | 0.1μg | 0.35μg | 0.05μg |
10ng | 0.1μg | 0.3μg | 0.1μg |
10ng | 0.1μg | 0.2μg | 0.2μg |
10ng | 0.1μg | - | 0.4μg |
ER转录因子组的反应体系为在表3的反应体系中再加入0.05μg pER。
结果如图6A-图6F所示,表明KRANA可以明显的抑制p53、雌激素受体(ER)、CREB和ELK1的转录活性,且具有剂量效应,但对c-jun和NF-κB的活性无影响。说明KRANA作为转录调控因子具有选择性的转录抑制功能。
(2)KRANA对含有p21启动区报告质粒及p21表达的抑制功能
A.将不同浓度的pCMV-Myc-KRANA与p21-luc(含有p21启动子区,见Cell,1993;75:817-25)共转染HEK293细胞,36h后,收细胞,按照双报告基因荧光素酶检测试剂盒(Promega)推荐的方法检测荧光素酶的活性。质粒转染量如表4:
表4、质粒转染量
pRL-CMV | p21-Luc | pCMV-Myc | pCMV-KRANA |
10ng | 0.1μg | 0.4μg | - |
10ng | 0.1μg | 0.35μg | 0.05μg |
10ng | 0.1μg | 0.3μg | 0.1μg |
10ng | 0.1μg | 0.2μg | 0.2μg |
10ng | 0.1μg | - | 0.4μg |
结果表明KRANA能抑制p21-luc中荧光素酶的表达,且具有剂量效应。
B.KRANA对HEK293细胞(HEK293细胞具有p21、p53的内源表达)中p21表达水平的影响
将不同浓度的pCMV-Myc-KRANA转染HEK293细胞,24h后收细胞,利用Western分析p21、p53、KRANA自身及β-actin的表达量。结果如图7所示,表明随KRANA转染量的增加,p53与β-actin的量无变化,但p21的表达量逐渐降低,从转录水平间接反映KRANA对p21活性的影响。
(3)在H1299细胞中KRANA依赖p53对p21表达水平的影响
在H1299细胞(p53缺陷的肺小细胞肺癌细胞)中分别转染pCMV-Myc-KRANA、含有p53的表达质粒pCMV-p53(见Science,1990;249:912-5))及将两种质粒同时转染,36 h后,收集细胞检测p21、p53、KRANA以及β-actin的表达量。结果如图8所示,表明KRANA依赖p53发挥对p21的抑制功能,说明KRANA不是作为转录因子直接调控p21的表达,而是通过影响p53的转录功能而调控p21。图8中,泳道1为未转染质粒的H1299细胞,泳道2为转染pCMV-Myc-KRANA的H1299细胞,泳道3为转染pCMV-p53的H1299细胞,泳道4为转染pCMV-p53和pCMV-Myc-KRANA的H1299细胞。
(4)KRANA具有对p53转录抑制功能区域的确定
将KRANA的六种截短体pCMV-Myc-N1,pCMV-Myc-N2,pCMV-Myc-N3,pCMV-Myc-C1,pCMV-Myc-C2和pCMV-Myc-C3分别与pG13L(见Nature Genetics,1992;1:45-49)共同转染HEK293细胞,按照双报告基因荧光素酶检测试剂盒(Promega)推荐的方法检测荧光素酶的活性,检测它们对p53-luc的抑制功能,确定KRANA的转录抑制功能区域。结果如图9所示,表明N端的三个截短体和C端较长的两个截短体C2和C3均具有转录抑制功能,说明KRANA的N端和C端有独立的转录抑制功能区域。
(5)HS、MMS、IR及wortmannin处理后KRANA对p53转录抑制功能的变化
将pCMV-Myc-KRANA转染HEK293细胞,收集细胞前,分别使用HS、MMS、wortmannin及联合wortmannin处理细胞,按照双报告基因荧光素酶检测试剂盒(Promega)推荐的方法检测荧光素酶的活性,检测它们对p53-luc的抑制功能,确定KRANA的转录抑制功能的变化。其中,HS处理(H)为将细胞42℃孵育2h后再在37℃孵育3h后收细胞;W处理条件为含100nmol/L wortmannin的培养基处理细胞1h后收细胞;HW处理为含100nmol/L wortmannin的培养基处理细胞1h后再将细胞42℃孵育2h后再在37℃孵育3h后收细胞,MW处理为含100nmol/L wortmannin的培养基处理细胞1h后再经0.05%的MMS的培养基处理细胞2 h后收细胞。结果如图10所示,表明HS处理后,KRANA的转录抑制活性降低了约20%,wortmannin的预先处理阻断了这种作用;MMS处理后p53的活性几乎完全丧失,而wortmannin部分逆转MMS的活性后使得KRANA的转录抑制活性降低了近30%。说明ATM激活后能够部分抑制KRANA对p53的转录抑制功能,因此激活p53的功能。图10中,C表示空白对照,未经处理的转染pCMV-Myc-KRANA的HEK293细胞。
实施例4、KRANA在细胞和组织中的表达谱
(1)RNA的提取:
使用TRIZOL一步法分别提取Jurkat,MOLT4,HL-60,U-937,AHH-1,K-562,Daudi,Raji,Mo7-e(原巨核细胞来源的肿瘤细胞系,购自协和细胞库),sko-007,SMMC-7721,HEK-293,HeLa,HepG2,MCF-7这15种细胞系和均取自22周孕龄的胎儿的胎儿心脏,胎儿肺脏,胎儿脾,胎儿肾脏,胎儿肠,胎儿胃的总RNA。
(2)RT-PCR对KRANA在多种细胞系及组织中的鉴定
A.cDNA的制备:取2μg RNA采用SuperScriptTMfirst-strand synthesis system来制备cDNA。整个操作均按照试剂盒推荐的条件进行。
B.PCR使用引物与条件同实施例1。
结果如图11所示,表明KRANA在Jurkat,MOLT4,SKO-007这三种血液病来源的细胞系中特异表达。图11中,1:Jurkat;2:MOLT4;3:HL-60;4:U-937;5:AHH-1;6:K-562;7:Daudi;8:Raji;9:Mo7-e;10:sko-007;11:SMMC-7721;12:HEK-293;13:HeLa;14:HepG2;15:MCF-7;16:胎儿心脏;17:胎儿肺脏;18:胎儿脾;19:胎儿肾脏;20:胎儿肠;21:胎儿胃;22为Marker(DL2000,从100bp到2000bp)。
实施例5、KRANA的亚细胞定位
(1)带有绿色荧光标签的KRANA表达质粒的构建:
以5’-t ctg tcg acc atg gct cgg aaa tta gtg-3’(带下划线的碱基为Sal I识别位点)和5’-ca ggg atc cca cat gta aac ttg tga gc-3(带下划线的碱基为BamHI识别位点)为上下游引物,以pCMV-Myc-KRANA为模板,按照常规PCR方法进行扩增、回收。回收后经Sal I和BamH I酶切与经同样酶切后的pEGFP-C3质粒(购自Clontech公司)连接,转入E. coli JM109感受态细胞中,利用卡那霉素抗性筛选阳性克隆,提取质粒进行酶切鉴定,挑选有插入片段的克隆,经测序鉴定,得到含有KRANA编码基因的阳性克隆pEGFP-C3-KRANA。
(2)KRANA亚细胞定位的观察:
利用LipofecamineTM 2000(Invitrogen)将pEGFP-C3-KRANA转染MCF-7细胞,24h后,在激光共聚焦显微镜下观察KRANA的定位,并拍照。同时使用DAPI(购自Roche公司)染核,以明确定位。结果如图13所示,表明KRANA多以均匀的形式分布在细胞核内,偶见点状的大颗粒分布。左侧图片为GFP-KRANA融合蛋白的绿色荧光,中间图片为DAPI染核的结果,右侧图片为二者叠加的结果。
序列表
<160>3
<210>1
<211>688
<212>PRT
<213>人属人(Homo sapiens)
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Met Ala Arg Lys Leu Val Met Phe Arg Asp Val Ala Ile Asp Phe Ser
1 5 10 15
Gln Glu Glu Trp Glu Cys Leu Asp Ser Ala Gln Arg Asp Leu Tyr Arg
20 25 30
Asp Val Met Leu Glu Asn Tyr Ser Asn Leu Val Ser Leu Asp Leu Pro
35 40 45
Ser Arg Cys Ala Ser Lys Asp Leu Ser Pro Glu Lys Asn Thr Tyr Glu
50 55 60
Thr Glu Leu Ser Gln Trp Glu Met Ser Asp Arg Leu Glu Asn Cys Asp
65 70 75 80
Leu Glu Glu Ser Asn Ser Arg Asp Tyr Leu Glu Ala Lys Gly Lys Met
85 90 95
Glu Lys Gln Gln Glu Asn Gln Lys Glu Tyr Phe Arg Gln Gly Met Ile
100 105 110
Ile Tyr Asp Lys Met Ser Ile Phe Asn Gln His Thr Tyr Leu Ser Gln
115 120 125
His Ser Arg Cys His Ser Thr Glu Lys Pro Tyr Lys Cys Lys Glu Cys
130 135 140
Gly Lys Ala Phe Arg Arg Ala Ser His Leu Thr Gln His Gln Ser Ile
145 150 155 160
His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Gln Cys Gly Lys Ala Phe
165 170 175
Ser Arg Asp Ser Gln Leu Ser Leu His Gln Arg Leu His Thr Gly Glu
180 185 190
Lys Pro Tyr Ala Cys Lys Glu Cys Gly Lys Ala Phe Thr Gln Ser Ser
195 200 205
Gln Leu Ile Leu His His Arg Ile His Thr Gly Glu Lys Pro Tyr Lys
210 215 220
Cys Glu Glu Cys Gly Lys Ala Phe Ile Arg Ser Ser Gln Leu Thr Arg
225 230 235 240
His Gln Lys Val His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys
245 250 255
Gly Lys Ala Phe Thr Gln Asn Ser Gln Leu Thr Leu His Gln Arg Leu
260 265 270
His Thr Gly Glu Lys Leu Tyr Glu Cys Lys Glu Cys Arg Lys Val Phe
275 280 285
Thr Gln Leu Ser Gln Leu Ile Leu His Lys Arg Ile His Thr Gly Glu
290 295 300
Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Ala Phe Ile Cys Gly Ser
305 310 315 320
Gln Leu Ser Gln His Gln Lys Ile His Asn Gly Glu Lys Pro Tyr Glu
325 330 335
Cys Lys Glu Cys Gly Arg Ala Phe Ile Arg Gly Ser Leu Leu Met Gln
340 345 350
His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Lys Cys Glu Glu Cys
355 360 365
Gly Lys Ala Phe Ile Arg Gly Ser Gln Leu Thr Gln His Gln Arg Ile
370 375 380
His Thr Asn Glu Lys Pro Tyr Glu Cys Lys Glu Cys Gly Lys Met Phe
385 390 395 400
Ser His Gly Ser Gln Leu Thr Gln His Gln Arg Ile His Thr Gly Glu
405 410 415
Lys Pro Tyr Gln Cys Lys Glu Cys Gly Lys Ala Phe Asn Arg Gly Ser
420 425 430
Leu Leu Thr Arg His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu
435 440 445
Cys Lys Glu Cys Gly Lys Thr Phe Ser Arg Gly Ser Glu Leu Thr Gln
450 455 460
His Glu Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys
465 470 475 480
Gly Lys Ser Phe Ile Arg Gly Ser Gln Leu Thr Gln His Gln Arg Ile
485 490 495
His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys Arg Met Ala Phe
500 505 510
Thr Gln Ser Ser His Leu Ser Gln His Gln Arg Leu His Thr Gly Glu
515 520 525
Lys Pro Tyr Val Cys Asn Glu Cys Gly Lys Ala Phe Ala Arg Gly Leu
530 535 540
Leu Leu Ile Gln His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Gln
545 550 555 560
Cys Lys Glu Cys Gly Lys Ala Phe Ile Arg Gly Ser Gln Leu Thr Gln
565 570 575
His Gln Arg Ile His Thr Gly Glu Lys Pro Tyr Glu Cys Lys Glu Cys
580 585 590
Gly Lys Ala Phe Ser His Gly Ser Gln Leu Thr Leu His Gln Arg Ile
595 600 605
His Thr Gly Glu Lys Pro Tyr Glu Cys Arg Glu Cys Arg Lys Ala Phe
610 615 620
Thr Gln Ser Ser His Leu Ser Arg His Gln Arg Ile His Thr Gly Glu
625 630 635 640
Lys Pro Tyr Gln Cys Lys Glu Cys Gly Lys Ala Phe Thr Arg Gly Ser
645 650 655
Gln Leu Thr Gln His Gln Arg Ile His Ile Ser Glu Lys Ser Phe Glu
660 665 670
Tyr Lys Glu Cys Gly Ile Asp Phe Ser His Gly Ser Gln Val Tyr Met
675 680 685
<210>2
<211>2999
<212>DNA
<213>人属人(Homo sapiens)
<400>2
ggcacgaggc ttggctccgc acctgctggg ctggcgaacc cgaaattggg cttggaaccc 60
gggagccaga tcttggaccc tgaactgcac ctctgtcaag agcccagagg agaaagaatg 120
gctgtttcag tagcaatgtc caagctctgc attctccaga ctctgtgctt tcctaagata 180
ggaacccaga agaggactga tcatttcttg cagctctaaa aaccatggct cggaaattag 240
tgatgttcag ggatgttgcc attgacttct ctcaggaaga gtgggaatgc ctggactctg 300
ctcagagaga tttgtataga gatgtgatgt tggagaacta tagcaacttg gtatcactag 360
acttgccttc aaggtgtgca agtaaggact tatctccaga aaagaacact tatgaaacag 420
aattatccca atgggaaatg agtgacagac ttgaaaactg tgatcttgaa gagtccaatt 480
ccagggatta tttggaagcc aaaggcaaga tggagaagca acaagaaaat cagaaggaat 540
atttcaggca agggatgatc atatatgaca aaatgtccat tttcaaccag catacttact 600
tatctcaaca ttcaagatgt cattctactg agaaacccta taaatgtaag gaatgtggga 660
aagccttcag acgagcctca cacctaacac aacatcaaag tattcatact ggtgaaaaac 720
cctatgaatg taagcaatgc gggaaggcct ttagtcgtga ttcacaactc agtcttcatc 780
agagacttca tactggtgag aaaccctatg catgtaagga atgtgggaag gcctttactc 840
aaagctcaca acttatttta catcatagaa ttcatactgg tgaaaaacca tataaatgtg 900
aagaatgtgg gaaagccttt attcgtagct cacaacttac ccgacatcaa aaagttcata 960
ctggtgagaa accttatgaa tgtaaagaat gtgggaaggc ctttactcag aattcacaac 1020
ttacactaca ccagagactt catactggtg aaaagctcta tgaatgtaaa gaatgtagga 1080
aggtctttac tcagctctca caacttattc tgcataagag aattcatacc ggtgagaaac 1140
cctatgaatg taaggaatgt ggaaaagctt ttatttgtgg ctcacagctt tctcaacatc 1200
agaaaattca taatggggaa aaaccatatg aatgtaagga atgtggaagg gcctttattc 1260
ggggctcact actgatgcaa catcagagga ttcatactgg tgaaaaaccc tataaatgtg 1320
aagaatgtgg gaaggccttt atccgtggct cacaacttac tcaacaccag agaattcaca 1380
ccaatgaaaa gccctatgaa tgtaaggaat gtggaaagat gtttagtcat ggctcacaac 1440
ttactcaaca tcagagaata cacactggtg agaaacccta tcaatgtaag gaatgtggaa 1500
aagcgtttaa tcgtggctca ctccttacac gacaccagag gattcatact ggtgagaaac 1560
cctatgaatg taaagaatgt ggaaaaacct ttagtcgtgg ctcagaactt actcaacatg 1620
agcgaattca cacaggtgag aaaccctatg aatgtaagga atgtgggaaa tcttttattc 1680
gtggttccca gcttactcaa catcagagaa tccatactgg tgagaaacct tatgaatgta 1740
aagaatgtag aatggccttt actcagagtt cacatctttc ccaacatcaa agacttcaca 1800
ctggtgagaa accctatgtg tgtaatgaat gtggaaaggc ctttgcgcgt ggcttactac 1860
ttatacaaca tcagagaatt catactggtg agaaaccata tcaatgtaag gaatgtggga 1920
aagcctttat tcgtggttca cagttgactc aacatcagcg aattcacact ggagaaaaac 1980
cctatgaatg caaggagtgt ggcaaggcct ttagtcatgg ctctcagctt actctacatc 2040
agagaatcca tactggtgag aagccctatg aatgcagaga atgtagaaag gcctttactc 2100
agagttcaca tctttctcgg catcagagaa ttcatactgg tgagaaacca tatcaatgta 2160
aggaatgtgg gaaggccttt actcgtggtt cacagctaac tcaacatcag agaattcata 2220
tcagtgagaa atcttttgaa tataaggaat gtgggattga ctttagtcat ggctcacaag 2280
tttacatgtg aattgtctga ttatttgaga tcactatgaa gaggttctct ggttgttagc 2340
agcaaagaat tctcacaaat gtgaatatgg gcgcacattt gcctcataaa gcacagcatc 2400
agataattta tgtgagagaa aatggtagtg tcattcatat agaaaaacat cattactgga 2460
aacctattaa acattagcaa attggagaat agttttaata tagtaaatgt aggaagccct 2520
ttagccatat tgaaaacaaa tatctttttc aacgttatct tagctctact agttgatctt 2580
tttgttatat gtatcatgat acttaacctc taccttggtt taatcatttt aagatagacc 2640
taagtatatt acctttattg taagattctt ggaagtatta tgtaagttat tacatgtaaa 2700
agctcttaga atggtgcctt gaacgtagca taccacaaat attagctacc attttcacta 2760
gtgttatttt agagaatttg catgagagga gagtacttat gagtataatg aatattgaga 2820
aatcttttat caacacatcc aagatgactt ggttgggggg cactgtatgc cataatgaat 2880
gtgagaaagc tgtcatttaa atctcatcca ttattgctat aagaggaaat tcatactgtt 2940
aaaaaaaaaa aaacccagtg gatataatca gtgtattatt aaaaaaaaaa aaaaaaaaa 2999
<210>3
<211>51280
<212>DNA
<213>人属人(Homo sapiens)
<400>3
gcttggctcc gcacctgctg ggctggcgaa cccgaaattg ggcttggaac ccgggagcca 60
gatcttggac cctgaactgc acctctgtca aggtaggagt gtccgggatg ccggagcgga 120
cacgcgcgga gaaagcgtcg ctggggttac tcgtgtggcc gtgtctccgg ttttgtgtgc 180
aggctacagc ctgcctggta ttggcgactg cctgtgcata cacgactgga cgtgtgtggc 240
tgtggttttg tgtatgagca tttgtatgag tgtgagttgt gactctgagg gtgtttgtga 300
ctgtttgctt acctggctgg gatatactta tgtgactgtg attttatgtg tgccgcgtgt 360
ataattgggg tttgcctgtg actatgaagt gtgtgacagt tgtgacaaga gtgtttggat 420
gttcttgtgt cactttgagg ttgtgtgtaa acctgatatc gtgggatgga tttgccagta 480
aatttaaggt gtgtgacagt gttgctgtga cacggtgttt attaccgcgt gtttccctga 540
tttcgtactg ggttgggtga ctgtctgatt gtgtgtgact gtggggttgt gtgtgctgtt 600
ttcatgactg tgtaaagaga ctaaaattgc agctttgaaa gggtgttttt ggcggtatgt 660
gtctctgaca aggtgcttgg atgtaattat atgccttgta cttaatggga ttttgtttga 720
gacaaggtgg tctaacaatg tagcataatt gggtgattgt gcctctagct gtgtaactgt 780
ggaaatgtga ttatgtgtga ctttatgtgg ccatttgtat attctgtgac cttatggggg 840
tttagtccag tacagaagta aatttctttt tggtatttct ggggttcatt tggggctgag 900
gtctgcctat ggattgtgag tcccatgtgg ctctgtgctc ctcccatgtg gctctgagga 960
cttaaggact ttttgatttg accacagtgg ctgccctttc atccatggcc tgtattgctt 1020
catttctgta tccatcctcg gttcaccttg ggaaatgtag ttacttacga gttcacgact 1080
tcttccctgc gtcctcttgc cttatagcct taaggggatt gggtgcaggg aagtggcaag 1140
aagtaaggaa gtcagacatt tggacacctg agtgatgaat aggagttatt tgcaagttac 1200
agacctggtc ttaggatgag aattttaggg ctgataatac cttcaagaca gccactggat 1260
gctgtgcgcg gtggctcaca tctgtaatcc cagcgctttg ggaggcagag gcgggtggat 1320
tacctgaggt catggtttca ccatgacctc tactaaaaat acaaaattag ctgggcgtgg 1380
tggcgcatgc ctgtaatccc agctacttag gaggctgagg caggagaatt gcttgaaccc 1440
gggaggcgga ggttgcagtg agccaagatc aagccactgc actccagcct gggcaacaag 1500
agtgaaactc attctctaaa taaataaata aataggccac tggaaatata atgggactcc 1560
aggagtggga atagtttcag agggagtgct ttaggagaca gccacccagt tctgaacctg 1620
aagcacccca ctgcaggact gtctccagtg tacagctttg aatacagaaa taaattcaag 1680
cccaagacag ctgtgacctg gcccttcttt ctgatacaga aggtagaaga cttattctag 1740
tctcctcctg acttttcttt atgttaaatg agattaagag gatggaggaa gagggtatgt 1800
tataatttag catattcccc aagaaaggcc taatatctca ttccattttt ctttttctga 1860
gagtagagcc cagaggagaa agaatggctg tttcagtagc aatgtccaag gtgagtattt 1920
cctctttgtt tcctgaaata cctttgttat tacaggacat agacattttt gttttctttt 1980
tgaaatttct tgccttctca aatgatagag gggcaaggca atttttggta gattgagagc 2040
aaagaaaatg tgccattaat cgggatagat gcttccccaa tagtggttaa atttacgaag 2100
aatggatttg agactcatgg atttaaaaag gggaagagag aaatagctct attgtgttat 2160
ttaaaaaggg aaagagctct attgtgttat ttctgataca gcaccatttt gattttttaa 2220
ctttttttag cttgttatgg aaagtttcaa atgtacacaa aagtataatg aatatgccta 2280
gcttaaaaac gatcacggcc aggtgtggtg gctcatgcct ataatcccag cactttggga 2340
ggccgaggcg gatggaacga ggttaggaga tcgaaaccat cctggaaaaa ggtgaaaccc 2400
cgtctgtact aaaaattaaa aaaaaaaaat tagttgggcg tggtggtgga tgcacatagt 2460
cccagctact cgggagactg aggcaggaga atggcgtgaa cctgggaggc agagcttgca 2520
gtgagccgag attgcgccac tgcactccag cctgggcaac agagcgagac tctgtctcaa 2580
aaaaaaaaaa aaaaaagcga ttactttcca atcttgtttc atttattcta ccttcagttc 2640
aaaatccttt ctaatttgcc cctttgattt cttgacttat aggttatttt gatgtgtcat 2700
gttttcaaat attttgagga tcttccagag atcttttgtt actgatttct aatttcatta 2760
cattgtgttc acagaatata cttcatatga ttttaatcct tttaaattta ttaagatttg 2820
ttttatgacc cagaatttga tgcatcttag taaatgttcc atgtgcactt gaaaagacta 2880
tattctgtgg ttgttgggtg gagtgttcta taaatgtcaa ctaggtcagg taggttgata 2940
gtatttttta aatcttccat atctttactg agtttttatt tgttggattt ttctccttgt 3000
agtttctttc agtttctgct tcatgtattt tttttttttt tgaggcagag tcttgctttg 3060
tcacccaggc tggagtgcag tggcccaatc ttggctcact gcaacctcca cctcccaggt 3120
tcaagcaacc tccacctccc aggttcaagt gattctccta ccttagcctc ccgagtagct 3180
gggaatacag gcgggcacga ccatgcccag ctaatttttg tatttttttt tttttttttt 3240
tttttttagc agagatgggg tttcaccata ttggccaggc tggtctcaaa cttctgacct 3300
caggtgatct gcccacctcg gcctcccaaa gtgcagggat tacaggtgtg agccactgtg 3360
cctggcctgc ttcatgtatt ttgatgctct gttattaggt atagaaaggt tttagatttt 3420
tacatcctac tggtgaattg acccctttat cattaggaaa tggtcttcct tgtccctcat 3480
aatattcttt gctccaaaca tctactttgc ttgatattta atgtagccat tccagctttc 3540
ttttcattaa tattagcatt gtgtgtcttt ttctatcctt tttcttttaa cctgtttgtg 3600
tttggtattt caagtgcatt tcttacaggc agcatgtaat tgggtttgtg tttttattca 3660
atttggcaat ctccattttt tgttgttgtt gttactgttg ttgagacaga gtctcactct 3720
gtcacccagg ctggagtgca gtggtgtgat ctcagctcac tgcaacctct gcctcctgtg 3780
ttcaagtgat tctcctgcct cagcctccca agtagctggg attacaggca cacaccacca 3840
cacccaccta atgtttttgt acttttagta gagacggggt tttgccatgt tggccaggct 3900
ggtctcaaac tcctgaactc aggtgatctg cctgcctcgg cctcccaaat ttctgggatt 3960
acaggtgtga acaaccacac ccaaccagca atctctatct tttaattgga gtatttagtc 4020
catttatatt tagtgtgatt attgatatgg tttggttcaa atccatcatt ttgccatttg 4080
ttttatattt atcccatctg ttctttattt cctgttctcc cttttctgcc ttcttttgga 4140
ttgagacaga gtttcaaaat aacaatacca atattttaac caaccatatg aatatagaaa 4200
aacaacataa agtcctgttg gagtttgaca ggatatagcc cactagggat ctataatcaa 4260
attactgtgt ttaaaagtcg tctacaataa cacttctcgt atggttaagc tgccagctgg 4320
aaatgcagtt atgctcattt gttttatatt gcttttcatg tttagggatt gcttttctcc 4380
cactatgatt taattttgtt ttaaaattac ataacatata tctaaaatca aaagtacaga 4440
ataagatgaa tttgaagaag tctagtgtcc atttctgtcc cctctacctg ctcctttctt 4500
ccctctatag tagctggggc tacaggcacg cgccaccatg cccagctaat tttttgtatt 4560
ttagtagaga cagggtttca ccatgttggc caggatggtc tcgatctcct gaccttgtga 4620
tccgcccacc tctgcctccc aaagtgctgg gattacaggc gtgagccacc atgcctggcc 4680
ccgtttttgc tttttttttt tttttatttt agtaatccat catacatagt gatgtgcact 4740
tctctgaagt agggcatagt atctggttgt tttcctttat gtgatgttaa cagccagcta 4800
ctgacatctt tatgtgaata tgttatttta tcagtggtta cagaattata ttctaattct 4860
tctgcattta ttagctggac tatatctata aagggagcca tcctttctca atgtcttggt 4920
tacctgaaga atagtttatg caaaaagaaa aactggaatg aaagattggt ttttctctta 4980
ttatttttca gcatctttca aagcttattc tgtgtttttc ttttaaacta ttattattaa 5040
cttgtgtatt aaaacatatc tgttatgttt cgatctgtga caataactgt tcttattaat 5100
gctcacattg ttccatgtat ggcaagtgga aggtttaccc tctctgattt gtctgctttt 5160
ggattccttt ttattctttt tgacatgacc ccagtagtct ttggcagatt ccagactttc 5220
cgtgctgaca gagtattcca ggctcatttt gtacaatttc ctgtcccaaa cataaaatca 5280
gccacttctt tatggagccc tggttccttt tataagaagt gcttttaaga agtcacaaca 5340
aggatagtag gaactctcat tgctcctagt tgtcattgct tatagcctta acagagctgg 5400
tgaaatacaa attttttaga aggagaaaaa aaatcctgag tgtatactga tatttccaat 5460
tgaaataaaa gattatagag tttttcttac tatctttgat tttatatttg tgttttataa 5520
tctgaaaaaa tcttaattcg taatgattaa tgatattaag attctcactg atatatgtgt 5580
gtgtataatc atttcaggtt atatacatat attactttta gactaacaat gccaatatta 5640
ttaataacag tataactact gaaaaactct aagacataac ttcataaagt tataaactct 5700
taagacaact ttgaaaaaca gtttatagtt attttgtttg taggttatat tgggtctaat 5760
gtcaaatgac tgtattttaa agttcccgaa aataatcact tcctttatat gtttaacctt 5820
ccagctttat gcacagataa gtttgtttca ttttgctttt atttcttaga gatttttttt 5880
cattttgatt taattttgtg attatagtta tagaaaacat acttgtggtt ccaaagtcaa 5940
atagatttga ttggtaatac tttataactt tgttgtatgt ctgtcttcct agaagctatt 6000
gagctgtaac ttttttcatg taatatcttt gtgaggtttt agtattatgg ttatttggcc 6060
tcaaaatgta gaacagactg ctgtctcctt tttctgtttt ctgaaaacgt ttgtgaaaga 6120
acagtattat atctgcctta aatattggta gaatttacca gtaaagccat ctgggccttc 6180
tctgtggaca gtttatttgt ttgtttctca cattcaattt ctttaatatc catgggatga 6240
tttgtatttc tgttttttct tgtgtgaatt ttggcaattt ttgttttaag gaatttgtct 6300
atattgttga aattgccaaa attattggca tagaattgta ttatcttttt aattaaagcc 6360
ttaaaaaatc tgtggtccag tctccacttt ggtaatattt ttttttgaaa acttgatagg 6420
ggtttgtaaa ctgtattaat cttttgaaag aattgatttt tgagtttgtt gatattctct 6480
attgtaatct actttatatt gtggattttg tgcttatttt tattctttct tttcttctta 6540
ctttcagctt cacttgctct tctttttcca gcttcctaag gtgggagcct aaatcatttt 6600
tctaacataa gcatttaaca ctattaattt tcctctaaat actgcttttg ttgcagctca 6660
cagaatctga taagtgtgtt tccattgtca ctcagttcgg catatttttc aatgtctttt 6720
tgcatgggtt atttagaagt atgtttaatt tccaaatatt tggagattct tgttactagt 6780
ttgtaattta ataatatgtg gacactgacc acagtatatt ctctgtataa ttcagttttt 6840
aaaatattta ctgttacttg ttttatgacc cagcatctgg tttattttga tgacctttcc 6900
atacgcagtt gttaatgaag agtctgactc ttgctaaaat cctatgtgtg ggttctcagg 6960
ctccaccagg tcattcctgg caggccacac aggggaaagg tggcctcccc gcagcagact 7020
tggtgcactt ctagtgcatt acagtctcta aagggagttg cagtgcagga ctcaggcctc 7080
ccagaccagc catgctgtta tgcgtgttta cattcctaac ccaggcacct ccattaggag 7140
gcctctttat caggtgcctc tgccaagagg acatcctaat ctaggggaga ggggaaactt 7200
tgaagacact ttctttcccc actcttagta tccggtagtt ttaatttttt tctttatttc 7260
ttttttcttt cttctctttt cttttttttt aacatatttt tggagacagg atattgctct 7320
gtcgcccaga ctggagtgta gtggcatgat catagctcat tgcagccttg aactcctgga 7380
cagttcaagc aatcctcctg cctcagcctc ccaagtagct aggactacag gtttgcatca 7440
caatgcctgg ctgatgtttt attattatta ttattattat tattattatt attattatta 7500
ttttgtagag acagattctt gctatgttgc ccaggctggt ctcaaacttc tggcctcaag 7560
taatcttcct gcctcggcct ccccaagtgc tggggttgct tacaggcatg agccaccgtg 7620
cccagccagg cctcactact ttttcactct tagcccttcc cttgtcttct tgtcccctgg 7680
tcccagtcca taaaatgaat gataggagct ttttgttctg cattccctta gcagtgagat 7740
gactcacttc tgcactgatc cacatttgca ctgatctgcc tgaccctctc tgaccctctc 7800
caggtgctat ttcatggttg gggttggggg agtgaaacag tggagtactc agcgctttct 7860
ctgggttagc ctcttaaatt aagtgactac taagtgatta aaggctggtt gtgttaatca 7920
cttattccaa caccttgcag ctcagttctc ttcagcccag cttggctttt ggtaagtggg 7980
tgtggtattc tattaatata tatttatcat cttggccaag gtacttgata tagtggtatg 8040
taaatcctct atattgttat cgattttatt gtctacttgt tctgtcagtt actgagcaaa 8100
agattttaca attcccaact ctgtgggttt gtctctttct tcctttagta ctgtcaattt 8160
ttgcttcaca cattttgaag ctttgttgtt aaatctctac atttggattg ctgtcttctt 8220
gatgagttga tcctccttat tgctagtatt tctctttatc ttgaagtcta ttttgtctta 8280
tactaatata ggcatacaag ctttcttgtg attagagttt gatggtgtat cttcccattt 8340
tacttttagc ccatctttat atttaaaatg tgcttcttgt aaagagcatg tagttggaac 8400
atgcattttt atcttgtctt gttaccattt ttttattgga atggttgaac tattattaat 8460
acaatttagt ttaagctctg ccatagatgg gagcatccat atgtggctct taagaagagc 8520
ttagggccac atgtggtggc ttatacctat aatcccatca ctttgggagg ctgaggcagg 8580
aggattgctt gaggccagga gttcaagacc agcctgggca atgtagcaaa acctcatccc 8640
aacaaaaaaa gtaaaaatta ggtgagtatg atggcacatg cctgcagtcc tagctacttc 8700
gaaggctgag gagggaggat tacttgagcc taggagttca aggttacagt aagctatgat 8760
cataccactg cactccagct tggttgagag tgagaccctg tctcaaaaaa aaaaataaat 8820
aaataaataa ataaataaat aaataaataa ataaataaaa attaagagct tggcactttt 8880
ttggagttta ggtcttttaa atttgtgccc tcagctctct gatgaattta aaagaaatta 8940
tgacttttca tcttatctga attattttta ggttgttagg atgggcccaa gtatctcttg 9000
cagaaatcta tatgttgaag aagtcaaatt ttcagttaaa aatgtattga gcacctgtat 9060
caatgattct ctgcaggggc agttattctc cctagagggt gttttggaaa tatatctagg 9120
tactgttgaa tgttaaagta agtgggttga gggtaaagac ttctagcttt tttgtttgtt 9180
tgtttgagac agagtctcac tctgtcgccc aggctggagt gcagtggcgc gatctcggct 9240
cgctgcaagc tccgcctccc gggttcacac cattctcctg cctcagcctc ctgagtagct 9300
gggactacag gcgctcgcca ccacacccgg ctaatttttt ttatttttag tagagacggg 9360
gtttcaccat gttagccagg atggtctaga tctcctgacc tcgtgatccg cccgcttcgg 9420
cctcccaaag tgctgggatt acaggcgtga gccactgcac ccggccgatt tctagctttt 9480
gaaggagtga ggtctcggat ctaggttaga gattacccaa ctttttctgt aaagggctag 9540
acatttcata tggctttgcg ggccacatgg tctctgctgc aactcttcag ctctgctgct 9600
gtagtgtgaa agtagccaaa ggcactatat aactgaatga gtatggacgt attccaataa 9660
aaccttattt acaaaattag ttggtagaca gtagtttgcc aagttatgat ttagttgtcc 9720
atcagtgcat aacaaaaaat tgtctgtgat ttgcaaaatt gagaaccata cctggaaaaa 9780
atgtgtttat gattttccag gtctggaatc tcttacataa tgcattttgt gcatgacttt 9840
ttcaatttgc tgaatttatc tactttccta ttattggatg gttttactat tagctgtatc 9900
ttccaggaat gcaaccatga tttgattgag aaaaaggctc tacttcgctt aagaaattta 9960
ctgagttttt tttaaccatt tcaaaaagtt atagtattga tggcaatgct aattgtgtta 10020
tttgtcttgc ctatagcaaa tatgtataga aacctccatt tatagataat gcatttctat 10080
ttattctata cgtacacatg aacacacata tatttagcct ttatttcaaa atgtcaaata 10140
taaaaggtgt caacaatact tagttacata ttgtcatttt ataagtctaa atttcttcat 10200
tataagtaat tgtaggcata agcatttaat tatttgtgcc ttctaatgta gttgtgtatg 10260
atcataaata tattgaaata ttaagttgtt cattatctcc atttttgaca tctatagatg 10320
tgtcgttatt acctatgagt ttcatttcag aagtagtaga gacattgtta tattttcttt 10380
ttgtatatag aaagcattga gtttgatcaa gttaagaacc acaacatatg tctgtccccc 10440
ttagtgtgtg ttttgggggt gggagaaaca ggaagaaaag aacaacaaag ataattccaa 10500
ctgaatcatt tgttactttg caaaacacct ggtctcatgg ttctgctttc tcctccgtag 10560
ctctgcattc tccagactct gtgctttcct aagataggaa cccagaagag gactgatcat 10620
ttcttgcagc tctaaaaacc atggctcggg taaattggag tttccttgtg ctcttttcac 10680
ttactttttt acagagcatg tgtgtgtttg tattccttac tcctgaaact tcattcctaa 10740
ctgacctcac tcaggaatgt gctcctcagt ttgtcccatt ctgataagtg atagtgtctt 10800
ccctaagtgt gtctttcgaa tctaaccttt agtcacatac ttactcaaca atatgtatat 10860
tgttcagtaa tctttgacta ggaacactga tctaacaaaa gtagagttcc ctgatttcat 10920
aaggtagagg aggatgttaa atggcaccac acatgaaaac cttttgaact tctctttaga 10980
tctaagttcc caggtagaca agacaggagt tggtcttgac tgggctcact ctcagtatgt 11040
ggcaagcgct catgctttat gagtttttaa gattaggaag atgattaggg actgaataca 11100
gagagatcag ggatcttccc caggaacata taatttaata tggatcaatt tgaaattcga 11160
cattatgtgc tggtcttgaa tgtataaatt tggattatgt ttttcacctt tctattgttt 11220
agaatgatga aaagttacat ttacggatga atggtaaata tgatcatagt gaagatgatc 11280
attcaccagc agaagttgtt gagaattata aactctgaga accaagaaat acttacaaga 11340
tttttacaac ctaaaatgtt caaatatgag cacttattaa gtcagacata ttcattatat 11400
ggagctattc tgtagcctat aaaatattta ctaaattatt gatgacaagg aaaatgttta 11460
tgatatagta ctctgatcta atggtgaatc tggagcttca ataaattctt taattttata 11520
caaatcttat tttataaggt tttacagaaa aattttacca ttttgtttat tccttaaata 11580
atgaagagag aaacttacat ttattgagaa tgtatgatac acaatataca tattgtataa 11640
aatgcttgtg tatgctactt cttttaattg ggacattgca tttattttat tcccagactt 11700
atctgtaaag taagatgttc tgtatgcaga aaatatgtgt agcaggccag gtgcagtggc 11760
tcacacctat aatcccagca ctttgggagg cctaagcctt aaggccagga attcaagact 11820
agcctgggca acataacaag acccctgtct ctacaaaaaa attggaaaat tatcagggca 11880
cagtggcatg tactgtgagt ggcacgtact gtggtctcaa ctactcagga gaatgagaca 11940
ggatcactta agcctaggag ttggaggctg cagtgagcta tgatggtgcc actgcactcc 12000
agcctggatg acagagcaag accctgtctc caaaacaaca acaacaaaac tatagcaata 12060
ggaataatca ggaacatatt tgtacttttc agcccatgtg ctctgttgct tcctttgtgg 12120
agggatcttc atctccctct ccattgaaaa agtatttttt tggccgggca cagtggctca 12180
cacctgtaat cccagcactt tgggaggcct agtcgggtga atcacaaggt caggagttcg 12240
agactagcct ggccaagatg atgaaacctt atctctacta aaaatacaaa aattaactgg 12300
gcgcagtggc aggtgcctgt aatcccagct actcaggagg ctgaggcagg agaattgctt 12360
gaacctagga ggcggaggtt gcagcgtgcc gagatcgcgc cactgcactc cagcctgagc 12420
gacagagcaa gactccatct caaaaaaaaa aaaagaaaga aaaaaagaaa agcatttttt 12480
aaatttccaa aattaacatt ttgtttgttg ttagaaatta gtgatgttca gggatgttgc 12540
cattgacttc tctcaggaag agtgggaatg cctggactct gctcagagag atttgtatag 12600
agatgtgatg ttggagaact atagcaactt ggtatcacta ggtaaggaat ctagccttca 12660
tatttcagga tctacctttg gattggctgc tttctctgct gttatttcaa atttcctttt 12720
taagtaatta actgaatttc ttattctttt caaaggagga gtttgaaatt actggaagta 12780
gacatggttt ctcagggcac ctttatcttt tccatccttc aaaaagcctt accactgcct 12840
tctaatacca tcttccctga tgaacaaagg gctagatttg aagtttggga tacttggaat 12900
attggcagat aaaacagcct atacattgtt acactttaga tctgctaaag gagctctaaa 12960
taattcacac gcccagtcac agatttaaaa aatagatgca gagatgtatg ttttttctga 13020
taaaagagtt tacatcattt gtatagtgat agagtagatc aatgcatttc accaacaact 13080
caaatgaaga caacccatta attagaacaa acacattgtc ttatatgtgc ttggtagttg 13140
aatattacat attaagaact ttgggctggg ctcggtggct cacgcctata atcccggcac 13200
gttgggaggc caaggtgggt agatcacctg aggtcaggag ttcaagacta gcctggccaa 13260
catggcgaaa cccatctcc actaaaaata caaaaattga gctgggcatg gtggtgtgtg 13320
cctgtaatcc cagttactca ggagattgag ggaggagaat tgcttgaacc catgaggtgg 13380
aggttgcagt gagctgagat cacatcactg tactcctcca gcctggatga cagagcgaga 13440
ctccatctca aaaaacaaaa aaaaaagaac ttgcattgga cattgaatga atatgctaga 13500
atcatctttt attgtaaaat agttgagggg gaacctgtga ttgtctaaga aactttattg 13560
ttttcttctg tagtattttt atatgaaaca aaatccgtat caattacaac tttaataagt 13620
gattttccaa cctacatgat aactttattc cttgtgctgg aataaactgt ctcttgcctc 13680
ttccttcctt tctcctctga agcctttagg aagtcagtga ttaacacatg tccctagagg 13740
acccaagtgg tgtggaaaaa aacctatgac ttgaggagaa aagcaagggt ttttcaccat 13800
gatcttccta aagactctta cctaatttct actgcgtcct ttccctgttg gtttttatta 13860
tcattcttcc cactttcaaa agagggaatg gttggccggg cgcggtcttg tatgtgcttg 13920
gtagttgaat attacatatt aagaactttg ggctcggctc ggtggctcac acctgtaatc 13980
ccaacacttt gggaggccga ggagggcgga tcacctgagg tcgggagttc gagaccagcc 14040
tgaccaacat ggagaaaccc cgtctctact aaaaatacaa aaaattagcc gagcatggtg 14100
gcgcatgcca gtaatcccag ctactctgga ggttgaggca ggagaatcgc ttgaacctag 14160
gaggcagagg ttgcagtgag ccgagattgt gccactgcac tccagcctgg gcgacagagg 14220
gagactctgt ctcaaaaaaa aaaaaaaagt gcaaattaaa accacaatga tatagttatt 14280
tcacatgcta taaattggca gcttgacagt accaggcatt ctcaagattt gaaggcagaa 14340
gtgcactata tcttgatatt cacaatagcc aaaaagtggg aacaacccaa atgtccatca 14400
actaatgaat ggataaaatt tggtattttc ttaaaacgaa atattattca gcaataaaaa 14460
ggaatgaagt tctgatacat gctttaacat ggatgaacct tggaaacatg ctataaagaa 14520
atacccgagg ctgggtaatt tataataaaa agagatttac ttagctcatg cttctggagg 14580
ctgtacgagc atagcactag tgtctgcttg gcttctggtg aggtccagga agcttttagt 14640
catggtggaa gggaggggga gtcggtgtgt cactggcaag agagggggca agagagagag 14700
gaggaaattc aggctctttt aaacaaccac atctcgcgtg aactcatagc gtaagaactc 14760
acgacagagt cttgctctgt cgcccaggct agagtgcaat ggcccaatct tggctcactg 14820
caacctcctt ctcctgggct caagcgattc tccttcctca gcctccccag cagctgggat 14880
tacaggcacc tgccaccaca cccagctaat ttttgtattt ttagtagaga tggtatttca 14940
ccttgttggc cagactggtc tcaaactcct gacctcgtga tccgcccacc tccctcccaa 15000
agtgctgaga ctacaggcgt gagccagtgc acccggccaa gaactcactc tttaccacaa 15060
agatggcacg aagctattca tgagggatcc acccccatga tccaaacacc tcccaccaag 15120
ccccacctct gacactggag attacattca atgttagatt acatcaacat gagatttgga 15180
agggacaaac atccaaacta tatcccatgc caagtgaaag aagccaaata caaacactgc 15240
atattatatg attgcattta tatgaaacgt cctcaatagg gaaatctata ctgacagaaa 15300
gtaggttact ggttacccag cactggggat ggggaagagg tagaacagat ggagaatggg 15360
gagtggggag atgagaagtg actgctaatg aacacagaca gcatttctct tgggggtgaa 15420
aaaatgttta aaaattagat tgttgtgatt gttgcacatc tcagtgggta tactaaaagc 15480
cactgaattg tgcagaaata atatggaaat ggttgccaaa cttgtgtgta ctttagaaat 15540
agctacggtc ttttaaaaaa ttccacagcc caggccacac cccagaccaa ttaaataata 15600
atttctaggc atggtacatg agtatcatat tttttgaaat tctctaggtt tttcaaatgt 15660
ttagccatgt ttgataacca ctgcactgtg gtattatctg agaatttttt ttaattgaat 15720
gttcactcca tttaatttat taccaatata cttggattta tttatcccct attttatgct 15780
ttttgtttcc tctttttatc ttacgggctt aaaaaacttt ctccctcctt aatgagggtg 15840
gatacaatcg tgttttattt tttgcaactc cattttcccc cttgttttgt ttggaaaacg 15900
tacagtattt atttattttt aaaagttatc tttaaatttt tatcatgtgt acctaattcc 15960
ttttaatgtc aatggtattt tatagttttt attgattgta attgtgtctg aaaattatat 16020
atttaatgga cattatatat tttgatggat actaaaacat atttattcag tccacataca 16080
atatttggtt tgcatattgc tttgcaataa acttgctcta tctccacccc taggatttca 16140
aggccagcaa gttgaaaaac agacatataa ataatagtat ctactgtgaa ataaattggt 16200
aacctttgtg gcaattagtt gaggttctaa ggagagataa gaaattgaca tctctatgtc 16260
aaatatatat tttattgtca cagaattctt atcagaagta taccagcaat gtatatctat 16320
tatatacttt atcaacagtg gctattagca cgtagaaata tctttgttaa ggccaggcgc 16380
ggtggctcac gcctgtaatc ccagcacttt gggaggctga ggcgggcaga tcacgaggtc 16440
aagagatcaa gaccatcctg gccaacatga tgaaatccca tctctactaa atatacaaaa 16500
attcggtagg tgtggtggca catgcctgta atcccagcta ctcaggaggc tgaggcagga 16560
gaatcgcttg aacctgggag gcggaggttg cagtgagcta agatcgcgcc acagcactcc 16620
agcctagtga tagagagaga ctctgtctca aaaaaaaaaa aaaaaaagaa atatctttgt 16680
tgatttgtag atgaaaaaca gaagaacatt tctgccattg ccggtgaatt taaatacttt 16740
gggggtattt ctgcctgttt tggtacttta gtaattatca ttattgtttg tgtcacttga 16800
caaatacatg cttaatttag tgaagtctaa aattcatgtc taactttcct taagataatt 16860
aaaggatatt gaaatgcttt aactttgatc tgtttctcca atctggtatt gttgtctggt 16920
attttagttc tgttcttttt ttaacctaca aaatgggcat tatttttgtt tcataatggc 16980
aatgtttgct tgtggatttg ttgatgtaat tatcttttat ttgtatgcta ttcctctttc 17040
attatacctt ccttctggga tattttattt atcatgaaat acattaggga aggcttattg 17100
gttttatttt tttcttttct tttcttttct tttttgagat ggggtctcat tcttgttgcc 17160
caggctgtag tgcagtggcg caatttcggc tcactgcaac ctccgcttcc caggttcaag 17220
cgattctcct acttcagcct ccccagtagc tgggattaca ggcgcccgca gccaagccca 17280
gcttattttt taatttttag tagagacggg gtttcgccat gttggccagg ctggtctcga 17340
actcctgacc tcaggtgatc cacccacctc cacctcccag agtgctggga ttacaagtgt 17400
gagctaccac ccccggcctg tcttattgtt aataaatgct ctaagtgttt gtttttgtca 17460
ttgttgcaaa ataaccttgt ttcactgtca ttcttgcctg atgatttaac taggtataaa 17520
attctatact gacaattatt tttttctcag cactctgaag atattattcc actgtcctct 17580
ggcttctatt gttgctcttg agaagtctgc catctgtttc attgttattc cttttgtaag 17640
taatctgtat tttctctggc tgcttttaag atttttttct ttaagattta aatgtaattg 17700
attatattgt ttcacttcta gatgtttgat gttctccccc tctcccagat cttttttttt 17760
taagctcatc cctttctcat ggtttttatc ttatactttc atatatttcc agcacttatg 17820
tgtcatacaa tcccaatttc tgggaaacag gtctaatttt gctgtttctt gtatccatgg 17880
ggactcttgt gtaatgatga attatccata gcactttttt tctccttttc tttgagagtc 17940
caggccaaga ctgaaaagtc ccttgtcatt ttcctttaag gtttgctgga tttgtttttc 18000
tttctctgaa ctttatgtct tgaatatttc caattttgta aagaggtttg tttgtaatcc 18060
ctgctctttt gttgaggaaa ggtcttgtct gttcagtttt ttcagaccat taatatacaa 18120
acatctaggt aattaaaatc aacaaatatc ccaagggtaa ctgtaacttc agagcatatt 18180
tactccatct actttctggt tcctacttca ttgtagcacc cctggatctt gtttactttc 18240
ttttatgctt cacatacttt taaaaggaca tctgtcatat ttttccagta tttgttagtg 18300
ttttttaaga aatcggatgc accatttaca tattaacaat attgaatcct tttattgttc 18360
agcatgctaa atctgctttt ctttttatgt tctctgtaaa gtttcatggt tttttcttat 18420
tctttttttt ttttttgaga cagagtctcg ctgtgtcacc caggctggag tgcagtggcg 18480
tgatctcggc tcactgcaac ctccgactcc ccagttcaag ggattctcct gcctcagcct 18540
cccaagtagc tgggactaca ggcacctgcc accacaccca gataattctt gtatttttag 18600
tagagacagg gtttcactat gttggccagg ctggtcttga attcttgacc ttgtgatctg 18660
cctgcctcgg cctcccaaag tgctgggatt agtttcatgg ttttttctta aaagatcttg 18720
cacatttctt aagcaatttc tcagtacatt ttgtttataa tatgaatagg aatattttgt 18780
tctgtaacat tttctaaatg gctaatactg atatatatta tatatgagaa agtattgatt 18840
tctataaatt gaaaatctta tggaaatata gtttttcagt ggattatagt ggatttctag 18900
gtggacagtt ttgctctcta catacaatat gtggtctctt ccttttctgt ttgtaccttt 18960
aaaaaaaggc aactaaattc agtaggaccc tgtagcggaa tttgaaaaca aatcattctt 19020
atcttttctc aaactttatt ggcagtgttt ctaatgttta actattaaat gtatttctat 19080
agacatctgg tagatactct taactaggtt tttacctaat tccagtttta gagaactttt 19140
aataaaaggt cttagaatat attaaaaatt tttttgcatt tcttgaaatc atgctatttt 19200
attctttaag ttctttatgt gggaaaatgg actctgatgt tgaattattc ctgcatttgt 19260
gggataaagt ttatatgatc atgatgtgtt atgccttaaa tacacagcta gattctatgt 19320
tcctaatatt ttcttttaaa attttacctt tgttcacgag attggcttcg ttttcctaag 19380
gtcatttttc aatgctgtta tactagttaa tttaactttg taaaatgact agggtagatt 19440
acaatatttt tatatgttca acaacagtta tgtacgattg gaattatata tccttttttc 19500
ttttttttga gacagggtct ccctctgtca cttaggctgg agtgcagtgg cctgatcaca 19560
gctcactgca gcctccacct ccctgagccc aggtgatcct agttgagact acaggcatgt 19620
gccaccatgc ctggctaatt tttgtagttt ttgtagagat gaggttttgc cacattgccc 19680
agactagtct cgaactcctg ggctcaggca atctacctgc cttggcctct caaagtgctg 19740
ggattacagg tataagccac cacacccggc ctatatattc tttaatgttt ggataatatt 19800
gccagtaaac ctaccagtca ctggaatcta ttgccttgaa tattagatat aataggtact 19860
atgaccagca gatacaaatg atcatcagtc ataggtatct attgcctttt aggagcacag 19920
acactgagct caaatttaaa ttttttatgt ggttattgct gtatatcagt tattatatct 19980
tcatgggtta tttttcatgt ttttcatctg tttcatccag ttctaattta ctgttgcgtt 20040
aataggtttt ttcataaaca cttttaaaag ttttgtttta ttttagttga aacataatgc 20100
acatatttat gggctaccat gtgatgtttt atacatatgt gtattgtgta attatcaaat 20160
caggataatt agcatatctg tcacctcaaa cacttgtcat ttctttgtgg ttaggacatt 20220
caagatgctc tcttctagct atctgcaggc attcaggaca tttttgttga ctatatagtc 20280
accttattgt gcaatagtac atcagaactt atttctccta tctaactata actttgtacc 20340
catagaccaa tctcttccca gccaccccct cagcatctgg taaccactgt tctactctct 20400
acttatatga gatcaacttt tttaggttcc acatatgagt gagatcatgt gatatttgtc 20460
ctgttcttgg cgtatttcac ttaatataat gtcctctagc ttcatccaag ttgtcaaaaa 20520
taacaggatt ttattcattt ttataggtta atagtattct attctgcata tataccacat 20580
ttttgaaaat ccatttatct tttgatggac actgagttcg agtccatgtt attgctattg 20640
tggatagtgc tgcagtaaac atgggaatgc agatgcctct ttgacatact gatttccttt 20700
cctttggatg tatacccagt agttagattg ttggatcata tggtagttct atgtttactt 20760
ttttgagaaa cctccatact attttccata atggctgtgc taatttacat tcccaccaag 20820
agtgtgtaag agttccccct ttttccacat ttttaccagc atttgttatc ttttgtcttt 20880
ttaatagcca ttctaacagc agtgaggtga tatctgattg tagcttcgat ttgcatttcc 20940
ctataattag tgatattgag cattttttca tttacctgtt tgccatttgt atgtcttgag 21000
aaatgcctat tcaaatcttt tgactatttt ttaatcagat tatttgtttt tttgctgttg 21060
agttgacttt cttatatatt cttggtatta acaccttatt agatgcatag tttgtgaata 21120
ttttcttcca ttctgtaggt tgtctctttt ttgtttgctt tgagatggag tctcgctctg 21180
tcacccaggc tggagtgcag tggtgcaatc tcggctcact acaacctccg cctgccaggt 21240
tcaagtgatt ctcttgcctc agcctccaga gtagctggga ctacaggcac atgccaccac 21300
gcccggctaa ttttttgtat ttttagtaga gacggtgttt cactgtgtta gccaggatgg 21360
tctcagtctc ctgaccttgt gatctgcccg cctgggcccc ccaaagagct gggattatag 21420
atgtgagcca ccatgcccag gctgtaggtt gtctctttac tctcatttcc tttgctgtac 21480
agaagctttt tactttgatg tagtatgttt atctgttttt gcatttgtca gtgcttttga 21540
ggtattatta aaaaatcctt gcccagatca gtgtctgttt ccctgtgttt tcttctactg 21600
gtttcatagt ttccagtctt acatttaagt ctttaatcca ttttgagttg agtggtgaga 21660
caggggtcta gtttcattct tttgcaggtg gatattcagt tttcccagca acatttactg 21720
aagaaagtat ccttttccca atgtgtgttc ttggccttct tgttgaaaat cagttggctg 21780
caaatacttg gatttttttt ttcctgggtt ctctatcctg ttccatttgt ctatgtgtct 21840
gttttcatgc caataccata ctgtttggtt actattgttt tgtagtatat tttcaagtca 21900
tggagtttga tgcctccagc ttggttcttt ttgtacaaga ttgctttggc ggcctggcgc 21960
agtggctcac acctgtaatc ctaacacttt gggaggctga ggcaggtgga tcacctgagg 22020
ccaggagttt gagaccagcc tggccaacat ggcgaaaccc catctctact aaaaatacaa 22080
aaaattgctg ggtgtggtgg caggcgtctg tcatcccagc tactcaggag gctgaggcag 22140
gagaatcacg tgaacctggg aggtggaggt tgcagtgagc caagagtgtg ccattgcact 22200
ccagcctggg cgacaagagc aaaactccat ctcaaaaaaa aaaaattgct ttggctgttt 22260
ggggactttt gtggttccat atgaatttta ggattcttct ttgtatttct gtgcagtgtc 22320
attggtggta ttttgatagg gattgcatag catttgtaga ttgttttggg tagtatggac 22380
attttaacaa tgttaattct tctgatccat gaacatggaa aatatttcca cttattagtt 22440
tcctctttaa tttcttttgt caatgtttat agttctcatt gtaaaactct tgcctctttg 22500
gttaaattta ttcctttttt tttttttttt gctatctatt ataaatggga ttgatttcct 22560
tttcacagtt tcctgttggc ctatggaaat gctacttatt tttgtacatt gattttgtca 22620
gtcattagca ccttattttg ttcatttgtt gagctcatat tttcctgaat gttcttgatg 22680
tttatggaca cgtgatgtct gtgcactgag caatttattc aagagtttgc aggccggaac 22740
ctggtatctc acgcctgtaa tcccagcact ttgggaagct gaggcgggtg gatcacctga 22800
ggtcaggagt tcaagaccag cctgaccaat atggtgaaac cccatttcta ctaaaaatac 22860
aaaaattagc tggccatggt ggtgtgtgcc tgtagtccca gctactcagg aggctgagac 22920
aggagaattg cttgaaccct ggaggcagag gttgcagtga gctgagatca tgccactgca 22980
ctccagcctg ggcagcagag caagactcca tctcaatcaa tcaatcagtc aatcaatcaa 23040
gttcacagtc tagctttgtt tgtgcccatc cttcttccaa aggccttcca gacattctaa 23100
gcagactatc tatctgttgt attcccttga gcctgtaacc actgcatcca tctcagcatt 23160
aaagggcatt aagcccaggc ttcctgctag ccttgcaagg gctccaaggt tgttgtggct 23220
ctccacctgg gtggacctag agaaaaccca agaaggatac tggtgctgta tgggagtcca 23280
gacagggacc tgagcccaga agactgtcct ggtggcccag atgggtgtgc cttcagcagg 23340
tccttgcaca ggcaagatag gtctctaatt acagtgagag ggctggagct gagactgggc 23400
cccctcagga tttgctgtcc tacaggacag aggctggcaa gcctatcttg ttggcttaga 23460
caggtgtaca tctcccagca tgtctctgca cagatggggt agttctatga ctgcaacagg 23520
aggggctaga gcttagactg ggccctctcg ggatctgctg tgggacagag gcttccatgc 23580
acagatggaa tagttccctg actgcagtgg gaggtgctgg agctgagcct ggaccccctg 23640
aggatgtgct atgggatggc agctgatgag cccatctcag cccacatggg tgtgtctccc 23700
accgtgtcta cacggagggg atagttctcc agttattgca ggagggccca aggcaaagac 23760
tgggcttcct caaggtctgc tgtgggacag aggttggtga gcccatcgtg aagactccaa 23820
ctcccaaacc atgagatacg gatgaatctt actctggatc cttgtatgag cggttctgag 23880
ctggcacccc cagctgagga ggctggagct aagccacaga gcaactgtga ggttcactac 23940
caggactgtt gtcagcaggc ataagagact ctccacctag acactagtgt gcatgattcc 24000
tcctggacct ttaggcagat ggttttgatt gcacgcccaa ggccaaatgg ggctgtagcc 24060
aagccctctg gggaatgggg ccattttcaa gctttgaaca tgggatcagc atgggagatc 24120
agccacctgc gtgccagtct gcactctcaa aatgactttc ctaggtcttg ggctttactc 24180
aagttttaca acctcctacc tgaattccaa agcttctgca aagagacttt tatctgtgga 24240
tgggtgcaga attctcgtga ggggatatga gtgggttacc tcctatttct gccatcttgc 24300
tgacgtcact cccataatct ctcataaaca ctttttaact cctcaattta tggttacctt 24360
tttctctcca gtgctttgga tttgtgctaa atctaatttt tttctacacg tcactttttt 24420
tgtattattt atggatattt tcaaataatt ttggtatttt gattttgcta attatttctg 24480
ctgtttctac gtttctaatt tactcatttt ctgatttttt atctcactta tattttggtt 24540
tttagttatg ctttttgctc acttcttctt gaggtgaaat cttaattctc ttaactatag 24600
tatttcttgt tttttaataa atgcacttaa gactgtaaat ttccctgggt acaattttag 24660
ctgatttctt gggatttcac acaaagtttc ttttgctatt tggctctaaa ttgttttaaa 24720
attttatttt tatttccttc tataacaaac atcatataga attgtcttag taacttaatt 24780
ctaggattat atgttttttc tatgttttta taatttttag ttttatgcta atgtgataat 24840
taaaaacatt ttatgtcagt tttatctatc ttcctatatt actaattatg ttatagtctt 24900
ccttgttttt atttctggtc tgtttatatt ctattctcta ctatgacggt gattaatttc 24960
tctatatgtc tgctagtttt tgtttattta ggagctaagt tgttggttac ataatggtca 25020
tgactattca tcctcttgcc tggctcttag atgatgtccc tgagctgatc tcactgagaa 25080
aactgaagct agcagaagag aagagagatt cttgcttcct tgtctgttta cctaccttca 25140
tctatactaa ttcaacctct tttcctatta acatgtaacc tttttgtgcc tttacttaag 25200
cccagtaccc tcacttgtat actgaattcc attccttctc atatttttaa ggacactgtt 25260
gaagcaattg ccctttgtct cttctaacat gaacttgtcc atatctttta caataatttt 25320
catcagcata caaacatgcc ttaatttctt ctatcttaaa aggaaaaaaa acaaaaccaa 25380
atattttgac acacattttc ctcttgatat agctccattt ctttgctact ttttacagaa 25440
aaatacaaaa gaattttcca tattccctaa ctccagctct tctctcattt tttttttgtc 25500
tttttttttt tttattatac tttaagttct agggtacatg tgcacaacgt gcaggtttgt 25560
tacatatgta tacatgtgcc atgttggtgt actgcaccca ttaactcgtc atttacatta 25620
ggtatatctc ctaatgctat tcctcccccc ttccccctcc cccgacccca caacaggctc 25680
cattgtgtga tgttcccctt cctgtgtcca agtgttctca ttgttcaatt cccacctatg 25740
agtgagaaca tgtggtgttt gtttttttgt ccttgagata gtttgctaag aatgggtttc 25800
cggcgtcatc catgtcccta cgaaggacat gaactcatcc ttttttatgg ctgcatagta 25860
ttccatggtg tatatatgcc acattttctt aatccagtct atcattgatg gacatttggg 25920
ttggttccaa gtctttgcta ttgtgaatag tgctgcaata aacatacgtg tgcatgtgtc 25980
tttatagcag catgatttat aatcctttgg gtatatacca ggtaatggga tagctgggtc 26040
aaatggtatt tctagttctg gatccttgcg gaattgccac attgtcttcc acaatggttg 26100
aactagttta cagtcccacc aacagtgtaa aagtgttcct gtttctccac atcctctcca 26160
gcacctgttg tttcctgact ttttaatgat tgccattcta agtggtgtga gatggtatct 26220
cattgtggtt ttgatttgca tttctctgat ggccagttga tgatgagcat ttttccatgt 26280
gtctgttggc tgcatatgtg tctgttggct acataaatgt cttcttttga gaagtgtctg 26340
ttcatatcct ttgccgactt tttgatgggg ttgttttctt cttataaatt tgagttcttt 26400
gtagattctg gatattagcc ctttgtcaga tgggtagatt gcaaacattt tctcccattc 26460
tttaggttgc ctgttcactc tgttggtagt ttcttttgct gtgcagaagc tctttagttt 26520
aattagatcc catttgtcaa ttttggcttt tgttgccatt gcttttggtg ttttagacat 26580
gaagtccttg cccatgccta tgtcctgaat ggtattgcct aggttttctt ctagggtttt 26640
tatggtttta ggtctaacat ttaagtcttt aatccatctt gaattaattt ttatataagg 26700
tgtaagggag ggatccagtt tcagctttct acatatggct agccagtttt cccagcacca 26760
tttattaaat agggaatcct ttccccattt cttgtttttg tcaggtttgt caaagatcag 26820
atggttgtag atgggtggta ttatttctga gggctctgtt ctgttccatt gatctatatc 26880
tctgttttgg taccagtacc atgctgtttt ggttactgta gccttgtagt atagtttgag 26940
gtcaggtagc atgatgcctc cagctttgtt cttttggctt aggattggct tggcaatgcg 27000
ggttcttttt tggttccata ttgaacttta gtttttttcc aattctgtga agaaagtcat 27060
tggtagcttg atggggatgg cattgaatct ataaattacc ttgggcagta tggccatttt 27120
attgattctt gattcatcat tgattcagta tggccatttt acgatattga ttcttcctat 27180
ccatgagcat ggaatgttct tccatttgtt tgtgttctct tttatttcac tgagcagtgg 27240
tttgtagttc tccttgaaga ggtccttcac atcccttgta agttggattc ctaggtattt 27300
tattgtcttt gaagtaattg tgaatgggag ttcactcatg atttggctgt ttgtctgtta 27360
ttggtgtata agaatgcttg tgatttttgc acattgattt tgtatcctga gactttgctg 27420
aagttgctta tcagcttaag gagatttcgg gcagagacta tggggttttc taaatataca 27480
gtcatgtcat ctgcaaacag ggacaatttg acttcctctt ttcctaattg aatacccttt 27540
ctttctttct cctgtctgat tgccctggcc agaacttcca acactatgtt gaataggagt 27600
gatgagagag ggcatccctg tcttgtaatg cttccagttt ttgtccattc agtatgatat 27660
tggctgtggg tttgtcataa atagctctta ttattttgag atacatccca tcaataccta 27720
atttattggg agtttttagc atgaagggct gttgaatttt gtcaaaggcc ttttctgcat 27780
ctattgagat aatcatgtgg ttttgtcttt ggttctgttt atatgctgga ttacatttat 27840
tgatttgcat atgttgaacc agccttgcat cccagggatg aagcccactt gatcatgggt 27900
agataaactt tttgatgtgc tgctggattc ggtttgccag tattttatct tctctcattc 27960
ttttgaatct ggttcagtca gaacttgctc ccattattct gccaaaacta ttattgtgaa 28020
gattaccagt aatctccatt ttgctaataa tgccattttt aacttaggaa gcacgattta 28080
ggccgaacac ggtggctcac acctgtaatc ccaccacttt gggaggccaa ggcagggaga 28140
tcacctgagg gcagaagttc aagatctgaa ggggtggcct gcccctccat acctgtgggt 28200
gtttctcgtc aggtggaacg agagactgag aaaagaaaga gacagagaca aagtgtggag 28260
aaagaaaagt gggcccaggg gaccggtgct tactggtctc tgagttccct cagtatttat 28320
tgatcattat ctctaccatc tcggagaggg ggatgtggca ggacaatagg gtaatagtgg 28380
ggagagggtc agcgggaaaa catgtaaaca aaagtctctg tgtcgtaaac aaggttaaga 28440
aaaggtgctg tgccttgatg tgcacgtgta caaacatctc ggtgcattaa agagcagtat 28500
taccactagc atgtctctcc tccagcccta aggtggtttt ctcctatctc agtaaataga 28560
acatacaatt gggttttaca ctgagacatt ctattgccca gggacgagca ggagacagat 28620
gacttcctct tatctgaact gcaaagaggc cttcctcttt tactaatcct cctcagcaca 28680
gacccttttc gggtgtcggg ctgggggatg tcgggctggg ggacagtcag gtcttaccct 28740
tcccatgagg ccatatctca ggctatcaca tggggagaaa tcttgggcaa tacctggctt 28800
tcccaggcag aggtccctgt ggccttccac agtgtattgt gtcctgggta cttgagatta 28860
gagaatggtg atgactttta acaagcatac tgccttcaag cacttttttt taacaaagta 28920
catcctgcat agccctttgg taaatccatt aaaccttgag tcaacacagc acatgtctct 28980
gtgagcacag ggttggggct agggttacag attaacagca tctcaaggca gaagaatttt 29040
cttggtacag aacaaaacgg agtctcttat gtctacttat ttctgcatag acacggtaac 29100
agtctgatct ctcttttccc atttccccct tttcttttgg acaaaaccgc catcgtcatc 29160
atggcccgtt ctcgatggtc gctgtctctt tggagctgtt gggtacacct gcagactaac 29220
aacagacaga acaggcacac aaggattaat aggaaattta caatagtaga acttccgatg 29280
gtcttaaccc aagtgacagg gttaagattt gtgagaccat cagcaactcc atgattgcct 29340
cagttcctgg taccaaattt aatgggcttt gatccttcaa aaatttgttc ttttaatttg 29400
gaaatgtcta aagtgagatt atcttttctt ccctgtagac ggcatctaac catgtcccag 29460
tgatgctcag actcattata aactccaggt gtaatacaaa aatctgacgt attccagtca 29520
cactgtaact ggaaatgatg ttctaagctc atgagcctat ctcccatcca aatgacagtc 29580
taagatcatt aatttgattt gccaattttt gatcaatacc agattgtgaa ttccaccatc 29640
ttgtagaatt cttttgccaa tcattaacaa agtttactgt ccgaacagaa gagtgcaatg 29700
caactcctgc cacagcggct gtagttgtga ctgcgattaa tcccataatc actgcaatta 29760
aagtaaaaat gaatcttttg gatctattta aaatgacttt taatacttca gtcaaaatat 29820
ggatggatgg cgaggcctcc cactgtcggt ccatggacac agggatccac acgccttccc 29880
ttgctctcac tagcagaata cggtgctgcc aattgaaagt tgaatcaatg caagtaaaca 29940
atctgcaatt ttcacaggtt atatagtttg ggagtctggt ttaataacta tatttcctac 30000
aactagcata taagggcgct ttatacaact ttgtaaagaa accgttagac ttgaatttag 30060
gtcgatagta taaaatggct tatgatctcc tgtttctata gcttgatttc cagatcaaat 30120
tctaatgcgg tatgaggcca cagtaagcct ccataattct ggatgttcag gaccagaaac 30180
aggacttatt atttttggtc ttggagtaga gattcctttt tctccccatt cccaagggta 30240
gaaagactgt aattttttgt gcttatgttt gtctaaactt tctgttaagt cactatcaac 30300
agttggactc acttgtgcac tgggacatga ctgagtttgt cctgtgcaat tgtggtagaa 30360
ttgacctcga ggtgcccaat ctataatagt tccaaattca tcattttgta atatcaccac 30420
actattggcc acacattctt cctaaactaa aacttctgtg tcttttgata ccttgggaat 30480
ttccgtgggg caaggtttcc ctttaggcct acattttaat gatctttgat aagaaaagtc 30540
ctgtaaataa tttacctgtg gcctgagtga cattccgctt accatgtgat aagtgaatct 30600
actggtggta cagtaagtaa gtacttctac caaccaattt tggattgtag gcattaaaca 30660
tcctggtgtt ctccctagac aaataggagg ataacgatac ccagtagaaa tatttatcgt 30720
cattccttct tcctcaggtt tggcagggca gtgatcatct gtggggccag gtacccatac 30780
actgttatta acatatactt caataggatt atccatccat gcgactgccc gaattaaggg 30840
caggaaaggc acacaggccc agtaggtata attagctgca gctgctccta caggcatggg 30900
gagacttacc accattgata caatcatcaa agctgcaagc agcatactct ctggagtttg 30960
tgtcaccttt gtgttctcaa ggcctttttt agctaactgt gtcagcttct ttaattgtgc 31020
ccaagtcggc ggctctgcct tcttggtgaa tggcaacttc atctattctt ctgacatcac 31080
cattttatct tgtgagttga tggtgcttga ttgcagtgtt tctgtctccg tggaggtgct 31140
tttctttgca tctccgacag gttcattgta gaacttcaaa tgtctagttg gtatccaaac 31200
aggaagctga ttttctcctg gtgaaacaca agcaaaacct ctcctccacg ttaccacctt 31260
ccctatttcc catgtcttat tgttgttgtc tttccaccaa atcagttttc cttcatgtgg 31320
gctattcttt tttttaccag taagatgttg ttctgcagga gtagtagtct gatttctata 31380
aatgtttaaa aaatttaaag tatagagtgc tagattaagt tgcatctgag gagtggtaca 31440
ctccttactg tctccccctt ctttttgttt aactaattga gttttgtgtg ttctattagt 31500
tctttcaact atggcctgtc cttgggaatt atagggaatt cctgttgtat gtgtaatttt 31560
cttggaaagc tttactacag tatcctggtc cattgttagt tttaattttt tctggaactc 31620
ccattacagc aaaataagat aataaatgtt ttttaacatg ggaagtactt tctcctgtct 31680
ggcaggttgt ccatatgaaa tgtgagtaag tatcaactgt tacatgaaca catgataatc 31740
ttccaaatga aggtacatgc gtgacatcca tttgccataa cgcattagga cacagacctc 31800
tgggattaac ttctgcctct tgagtgggca ggtgtaggac ttgacactgg gtgcaatgtt 31860
gtacaatatt ttttgcctgt ttctatgtga catcaaattt gttttttaat cctgctgcat 31920
ttgcatgagc caaagcatga agttcttgtg cttttatgaa tgcagatgat accagtaagt 31980
cagcttgttc atttgcttta gtcaaaggcc ctggtaaatt agtgtgtgct cgaatatgag 32040
taatataaaa tgggaaattt ctttttctta cagtttgttg taataaattg aatagctggt 32100
ttaactgatc atccatgcta tatttgatta gagctgtctc aacatccctt gtagcctgta 32160
ctacatatgc agaatctgat acaatattga tagattgatc aaaatcttat aacactgtaa 32220
tgactgcaac caactctgct ctttgagcca attgatactg agttttgatt acttgctctt 32280
ttggctctgt gtaagctgct tttccattgt tggaaccatc agtatatact gtcagagcat 32340
tttctaaagg tccgtgactg gtaattttag gtaaaatcca agtagtcaat tttaaaaact 32400
ggaggatttt tgtttttggg taatgattat cagtaattcc cacaaaatca gcaagaccaa 32460
tctgccatgc accagaattg ataaaggctt gtctaacttg ttccttggtt aaagggacaa 32520
ctattttgtc tgggtcattt ccacacaatt ttattattcg taatcttgcc tgaccaatta 32580
atgtagctat ttgagccaag tataatgtaa aagtcttaat tgtactgtga ggaaggaatg 32640
accactccac aagatcagta ttttgaacaa tgatgcctgt tggaaaatgt gcagtagcaa 32700
aaatcaaaag ttggagtggg gctaagggat ctattctatt tattgcactg actgaatttt 32760
ttcttccact aatttaattt cttttgttgc ctctcgggtt aatattcttt tactatttaa 32820
gtctgggtct cctcttaaga tagagaacaa atttgacatg gcataagtag gaatacctag 32880
agttggccaa atccaattaa tgtctcctag caatttttga aaatcattta atgtttttaa 32940
tgtgcctttt cttatttcta ttttttgtgg cttaattttt ttattttcta tctgcatccc 33000
taaataatga aaaggagtag aggtttgaat cttaccagat gctattgcca gtcctgcgtt 33060
ggcaacctct gcttgcagaa atgtgtaaca gtcaattaat ttgtctctcg tttctgcagc 33120
acatcaaata tcaacgtaat gaataacagt ctgaaaactt atctctaact ggttgaagag 33180
cttgacctac gaaagtctga caaatagttg gactattaag cattccctga ggtaacactt 33240
tccactgaaa cctggtggct ggttctttat tatttatggc tggtatagta aaggcaaatt 33300
tttcacaatc ctgctccacc agaggaatgg taaaaaagca atccttcaga tcaattataa 33360
ttaaaggcca gtcttttggg atcatggccg gagagggcaa ccctggttgg agatgcccca 33420
tgggttgaat tatggcattt acggccctta agtcagttaa cattcgccat ctgctggatt 33480
ttttctgaat tacaaacaca ggagaattcc aaggcgagaa tgaaggctca gtatgtccgt 33540
tttctgattg ttactttgct aataaatgta aagcctccag ttttcgtttt ggtagcagcc 33600
actgatttac ccatacaggt ttttctgttt tccaagttaa tggagtgggt ttaggaggat 33660
ctacagtggc cgcccctaaa aaggataccc tattgctgct gtttcttgat ttccctcaat 33720
ctcaattggg actttaatgc cattttcatt ttttcctagt ccctttcctg gtatatatcc 33780
catcttagtc gtgatttttt gatttgtggg gctatataat ggggcaggca tagtgatttc 33840
ctcaccccat tgttgtaata aatctcgacc ccacagatta acgggaattg aagtaatcat 33900
tggctgaaca gtactttctt gattatctgg ccctaagcaa tgtaaaatct cagcactttg 33960
atacacttct gaggcagtgc ctacgccgac aagtcctgta acagcctttt gtttaggcca 34020
attttttggc caccgattga aagcaatgat agagacatct gctgcagtgt ctaccaaccc 34080
ctcaaactgt tttccttgaa taatggcctt acacacaggt ctgctctctg agacctgact 34140
tgcccaatat gcagcctagc tggatcagtg cttccaaacc ctcctgttct ttttatctca 34200
ctgttttcaa ccttaatata aggcaggaat aattaagcaa tcctgtctcc tggactggca 34260
ctccaaggaa ctgaggagct aataaccaat tgaatttcga ctttgtagtc tgaatcaacc 34320
acaccagtat gaatttgaac tccctttaga tttagactcg atcttcctga gattagtcct 34380
acagtcccct caggccgtgg gccatatatc cctgtgggga tttttttgtg ggggctccct 34440
ggaagcagag agactgcttg tatagtacac aaatctactg ctgcactgcc gcttgtggcc 34500
ggggacagtt gctgtattgt ggtaactggc ccattccctg aagcacttgt gacagtgggg 34560
gttgttgtcc ctgaaaaccc tgaggaacaa agggctaaat tgggaatgcc ccagtttgtt 34620
gcggggcctg aggctggccc ctcttctcgt ttcccgacaa tggttgccca tttttatcaa 34680
atttagaatg acattgacta gcccaatgtt ttcctttttt acctcttgga cataagtcag 34740
gtggctcttt atctgttctt gtagtagttt gaatagttat attttgttta tttgaggctg 34800
ggcaattctt tttttgatga ccaatttgtc cacaattata acattttccc ctaaatgttc 34860
taacttgtcc tcctaaagca agtcccgtta ttgcttgagc gataagcata gctttatgca 34920
tagctcctcc aattccatca caggctttta cgtactcttg agattacatc tgatcccact 34980
gggacctttc cttttaatgg tttaatggct gattgacact caggattggc attttcgtat 35040
gccatcaact ccactatgac cttacgggca ttctcattgg taattgactt ttgagcagca 35100
tcttggaggc ttgccgcaaa atcagggtag ggctccttta gcgccttgtc ttattgtatt 35160
gaatgagggg caggcggttc ctgggtcttg gcttttttcc caggctctaa ggcagatagg 35220
tctaacttgc tcagtggcct cattttacat tattgcttgt taattagtag tgctccaatt 35280
ttgacctgtt cctaatagtt gatctgcatc tatgttaact ggaggattgg cagccctatt 35340
tttttggacc tgttcttgtg ccccatcaat ccaccaagtc ttcaccaagt cttaaattgt 35400
aaacattgag agggtgagag cgatcatttg gccaaaatct cccaatcata aagaatgagt 35460
ctatgtccat gagcaatgga atctaataat gtcctcatat aaggggagtt gggtccatac 35520
tgttttactc cctctttcat atcttttagc atttttatgg aaaaggactc atatctggcc 35580
tcagctaggg gaggcgctcc ctcttgggcc tcttcttcag gtggtatcgg ttctaatatt 35640
actgggaatt gccatgcctc ggtatctcct tgttttcttg ccttatcaat aattttatgt 35700
aatgcactac cctgtccact aggtggtggt gtaggattaa atctcatagt gggctgctga 35760
ggatatagcg ccctgccctg tggtgctgga aacattcctg gctgtccata ctgattttct 35820
gggggcggct gatcctgaag ttcggctggc ggccagtatt gataggttac tggcggttgg 35880
gtcttatttt ccaccggctg atattgtgga tactgtattt ggattggcat tgccgtgaca 35940
gagactctat ctttccctat ttgatattct tttggggttt gtacttgtct aacctgcatt 36000
tgaggttgta atgttacagg catctgaact gctgggagag gagttggcca tcgtggttta 36060
gactctgatg gccccactaa ttctggacct ttttcttcta attttaacat ttcaggatat 36120
atcacttcct gtaattgatt atagtcaaca ttttgcattg accgaaccat taccggctct 36180
gctacatatt tacaatgtga actttccgtt cctttcccag attctatccc tgcctcttct 36240
tcacaatcta ttacacattt tccagggcat cagaaactga aatgctatct tctcctgttt 36300
gaaacagttc taaagctact ttaataatgg cccaatcatt ccatactgta agtgggatga 36360
ttttaccctc cctacttgct tgttttaatt ctttgccaat tttttcccaa tcttttagat 36420
ctaaagttcc ccgttctaga aaccatgggc agaattgttc tattgtttga aatagagtga 36480
ttagattttt tttgtagaga ctctgactcc ccctcttttt aaaagaattt taatggagct 36540
aagaggcata tttactttta gtttgtccca ttgttaccct ggcttcttct gagcgcacaa 36600
gtttaccgca aagggtgacc atagacgtac tcaggaatct ctcgttgact tgtcctcatt 36660
gaccacgctc gagcgtacct tcaccctaga gaaaagcacc tacgttggac acccgatgaa 36720
ggagtggcct gcccctccac acctgtgggt gtttctcgtc aggtggaatg agagacttga 36780
gaaaagaaag agacacagaa acaaagtata gagaaagaaa agtgggccca ggggaccggc 36840
gctcaccata cggaggaccc gcgccagcac tggtctctga gttctctcag tgtttatgga 36900
tcattatctc taccatcttg gagaggggga tgtggcagga ccgtagggta atagtgggga 36960
gagggtcagc aggaaaacat gtgaacagag gtctttgtgt cataaataag tttaaggaaa 37020
ggtgctgtgc cttgatgtac atgtatacaa acatctcggt gcattaaata gcagtattgc 37080
tgctagcacg tctcacctcc agccctaagg cagttttctt ctatctcagt aaatagaaca 37140
tacaattggg ttttacactg agacattcta ttgcccaggg acaagcagga gacaggtgcc 37200
ttcctcttat ctcaactgca gagaggcctt cctcttttac taatcctcct cagcacagac 37260
cctttacggg tgttgggctg gggggcggtc aggttttccc ttcccacgag gccatatctc 37320
aggctatcac atggagagaa accttggaca atacctggct ttcctaggca gaggttcctg 37380
tggccttcca cagtgtattg tgtccctggg tacttgagat tagagaatgg tgatgacttt 37440
taacaagcac gctgccttca agcacttttt taacaaagca catcctgcat agccctaaat 37500
ccattaaacc ttgagtcaac acagcacatg tttcagggag cacaaggttg ggggtagggt 37560
tacagattaa tagcatctca aggcagagga atttttctta gtacagaaca aaatggagtc 37620
tcttatgtct acttctttct acatagacac agtaagaggc tgatctctct ttcttttccc 37680
cacaaagacc agcttggcca acatggtgaa accctgtctc tactaaaaat acaaaaaaat 37740
ttagccaggc atgatggcac acgcagggat tcccagctac cttggagctg aggcaagaga 37800
attgcttgaa tccaggaggt ggaggttgca gtgaaccgag atcacaccac tgcacttcag 37860
cctgggcaac agagcaggac tccacctcaa aaaaaaaaaa aaaaaaaaaa gaatgcacca 37920
tttcgcctca tctctcagca tttgacataa ccgatcattc cttcttaaaa cacttttttt 37980
ggaaaatatg ggtggagcca agatggccaa ataggaacag ctctggtcta cagctcccag 38040
catgaacgac gcagaagacg ggtgatttct gcatttccat ctgaggtacc gggttcatct 38100
cactagggag tgccagacag tgggtgcagg acagtgggtg cagcacactg tgtgcgagcc 38160
gaagcagggc gaggcattac ctcactcagt aagtgcaagg ggtcagggag ttccctttcc 38220
tagtcaaaga aaggggtgac agacggcacc tggaaaattg ggtcactccc accctaatac 38280
tgcgcttttc caacgggctt aaaaaacagc acaccaggag attatatccc acacctagct 38340
cagagggtcc tatgcccatg gagtctcact gattgatctc agactgctgt gctagcaaac 38400
tgcaaggcag cagcgaggct gggggagggg tgcctgccat tgcccaggct tgcttaggta 38460
aacaaagcag cctggaagct tgaactgggt ggagcccacc acagctaaag gaggccttcc 38520
tgcctctata ggctccacct ctgggggcag ggcacagaca aacaaaaaga cagcagtaac 38580
ctgtgcagac ttaaacgtcc ctgtctgaca gctttgaaga gagtagtggt tctcccagca 38640
cgcagctgga gatctgagaa cggacagact gcctcctcaa gtgggtccct gacccccgag 38700
cagcctaact gggaggcacc ccccagtagg ggcagactga cacctcacat ggccgggtac 38760
tcctctgaga caaaacttcc agaggaacga tcaggcagca gcatttgcgg ttcacgaaaa 38820
tcggctgttc tacagccacc gctgttctgc agccactgct gctgataccc aggcaaacag 38880
ggtctggagt ggacctctag caaactccaa cagacctgca ggcgaggctc ctgtctgtta 38940
gaaggaaaac taacaaacag aaaggacatc cacaccaaaa acccatctgt acgtcaccat 39000
catcaaagac caaaagtaga taaaaccaca aagatgggga aaaaacagag cagaaaaact 39060
ggaaactcta aaaagcagag cacctctcct cctccaaagg aatgcagttc ctcaccagca 39120
acagaacaaa gctggatgga gaatgacttt gatgagttga gagaagaagg cttcagacga 39180
tcaaactact cccgagctac aggaggaaat tcaaaccaat ggcaaagaag ttaaaaactt 39240
tgaaataaaa attatacgta tgtataacta gaataaccaa tgcagagaag tacttaaagg 39300
agctgatgga gctgaaagcc aaggctcaag aactatgtga agaatgcaga agcctcagga 39360
gccgacacga tcaactggaa gaaagggtat cagtgatgga agatcaaatg aatgaaatga 39420
agcaagaaga gaagtttaga gaaaaaagaa taaaaagaaa caaacaaagt ctccaagaaa 39480
tatgggacta tgtgaaaaga ccaaatctat gtctgattgg tgtacctcaa agtgacaggg 39540
agaatggaac caagttggaa aacactctgc aggatattat ccaggagaac ttccccaatc 39600
tagcaaggca ggccaccatt cagattcagg aaatacagag aacgccacaa agatacttct 39660
cgagaagagc aactccaaga cacataattg tcagattcac caaagttgaa atgaaggaaa 39720
aaatgttaag ggcagccaga gagaaaggtc aggttaccca caaagggaag cccatcagac 39780
taacagctga tctctcagca gaaactctac aagccagaag agaagggggg ccaatattca 39840
acattcttaa aagaatttcc aacccagaat ttcatatcca gccaaactaa gcttcataag 39900
tgaaggagaa gtaaaatcct ttacagacaa gcaaatgctg agagattttg tcaccagcag 39960
gcctgcccta aaagagctcc tgaaggaaac actaaacata gaaaggaaca acctgtacca 40020
gccactgcaa aaacatgcca aattgtaaag accatcaagg ctgggaagaa actgcatcaa 40080
ctaatgagca aaataaccag ctaacatcat aatgacagga tcaaattcac acataacaat 40140
attaacttta aatgtaaatg ggctaaatgc tccaattaaa agacacagac tggcaaattg 40200
gataaagagt caagacccat cagtgtgctg tattcaggaa acccatctca cgtgcagaga 40260
cacacatagg ctcaaaataa agggatggag gaagatctac caagcaaatg gaaaacaaaa 40320
aaaggcaggg gttgcaatcc tagtctctga taaaacagac tttaaaccaa caaagatcaa 40380
aagagacaaa gaaggccatt acataatgtt aaagggatca attcaccaag aagagctaac 40440
tatcctaaat atatatgcat ccgatacagg agcacccaga ttcataaagc aagtcctgag 40500
tgacctacaa agagacttag actcccacac aataataatg ggagacttta acaccccact 40560
gtcaacatta cacagatcag caagacagaa agttaacaag gatacccagg aattgaactc 40620
agctctgcac caagcggacc taatagacat ctacagaact ctccacccca aatcaacaga 40680
atatacattt tttttcagca ccacaccaca cctattccaa aactgaccac atagttcgaa 40740
gtaaagctct cctcagcaaa tgtaaaagaa cagaaattat aacaaactgt ctctcagacc 40800
acagtgcaat caaactagaa ctcaggatta agaaactcac tcaaaaccac tccactacat 40860
ggaaactgaa caacctgctc ctgaatgact actgggtaca taatgaaatg aaggcagaaa 40920
tcaagatgtt ctttgaaacc aatgagaaca aagacacaac ataccagaat ctctgggaca 40980
cattcaaagc agtgtgtaga gggaaatata tagcactaag tgtccacaag agaaagcagg 41040
aaagattcaa aatggacacc ctaacaccac aattaaaaga actagaaaag caagagcaaa 41100
cacattcaaa agctagcaga aggcaagaaa taactaaaat cagagcagaa ctgaaggaaa 41160
tagagacaca aaaaaaaacc ttcaaaaaat taatgaatcc aggagctggt tttttgaaaa 41220
gatcaacaaa attgatagac tgctagcaag actaataaag aagaaaagag agaagaatca 41280
aatagatgca ataaaaaatg ataaagggga tatcaccacc aatcccacag aaatacaaac 41340
taccatcaga atactacaaa cacctctatg caaataaact agaaaatcta gaagaaatgg 41400
ataaattcct tgacacatac accctcccaa gactaaacca ggaagaagtt gaatctctga 41460
atagaccaat aacaggatct gaaattgtgg caataatcaa tagcttacca accaaaaaga 41520
gtccaggacc agatggattc acagccaaat tctaccagag gtacaaggag gagctggtac 41580
cattccttct gaaactattc caatcaatag aaaaagaggg aatcctgcct aactcatttt 41640
atgaggccag catcatcctg ataccaaagc ctggcagaga cacagccaaa aaagagaatt 41700
ttagaccaat attcctgatg aacattgatg caaaaatcct caataaaata ctggcaaacc 41760
gaatccagca gcacatcaaa aagcttatcc caccatgatc aagtgggctt catccctggg 41820
atgcaaggct ggttcaacat acacaaatca ataaatgtaa tccagcatat aaacagaacc 41880
aaagacaaaa accacatgat tatctcaata gatgcagaaa aggcctttga caaaattcaa 41940
caacccttca tgctaaaaac tctcaataaa ttaggtattg atgggatgta tctcaaaata 42000
ataagagcta tctatgacaa acccacagcc aatatcatac tgaatgggca aaaactggaa 42060
gcactcactt tgaaaactgg cacaagacag agatgccctc tctcaccact cctattcaac 42120
atagtgttgg aatttctggc cagggcaatt aggcaggaga aggaaataaa ggttattcaa 42180
ttaggaaaag aggaagtcaa attgtccctg tttgcagatg acatgattgt gtatctagaa 42240
aaccccattg tctcagccca aaatctcctt aagctgataa gcaactacag caaagtctca 42300
ggatacaaaa tcagtgtaca aaaatcagca gcattcttat acatgaataa cagacaaaca 42360
gagagccaaa tcatgagtga actcccattc acaattgctt caaagagaat aaaatatcta 42420
ggaatccaac ttacaaggga cgtgaaggac ctcttcaagg agaactacaa accactgctc 42480
aatgaaataa aagaggatac aaacaaatgg aagaacatta catgctcatg ggttggaaga 42540
atcaatatca tgaaaatggc catactgccc aaggtaattt atagattcaa tgccatcccc 42600
atcaagctac caatgacttt cttcacagaa ttggaaaaaa ctactttaaa gttcatatgg 42660
aaccaaaaaa gagcccgcat tgccaagtca atcctaagcc aaaagaacaa agctggaggc 42720
atcatgctac ctgacctcaa actatactac aaggctacag taaccaaaac agcatggtac 42780
tggtaccaaa acagagatat agatcaatgg aacagaacag agccctcaga aataatgccg 42840
catatctaca actatctgat ctttgacaaa cctgacaaaa acaagcaatg gggaaaggat 42900
tccctattta ataaatggtg ctgggaaaac tggctagcca tatgtagaaa gctgaaactg 42960
gatcccttcc ttacacctta tacaaaaatt aattcaagat ggattaaaga cttaaacgtt 43020
agacctaaaa ccataaaaac cctagaagaa aacctaggca ataccattca ggacataggc 43080
atgggcaagg acttcatgtc tcaaacacca aaagcaatgg caacaaaagc caaaactgac 43140
aaatgggatc taattaaact aaagagcttc tgcacagcaa aagaaactac catcagagtg 43200
aacaggcaac caacaaaatg gtagaaaatt ttcgcaacct actcctctga caaagggcta 43260
atatccagaa tctacaatga actcaaattt acaagaaaaa aacaacccca tcaaaaagtg 43320
ggtgaaggat atgaacagac acttctcaaa agaagacatt tatgcagcca aaaaacacat 43380
gaaaaaatgc tcatcatcac tggccatcag agaaatgcaa atcaaaacca caataccatc 43440
tcacaccatt tagaatggca atcattaaaa agtcaggaaa caacaggtgc tggagaggat 43500
gtggagaaat aggaacactt ctacactgtt ggtgggactg taaactagtt caaccattgt 43560
ggaagtcagt gtggtgcttc ctcagggatc tagaactaga aataccattt gacccagcca 43620
tcccattact gggtatatac ccaaaggact atatatcatg ctgctataaa gacacatgca 43680
cacgtatgtt tattgtggca ctattcacaa tagcaaagac ttggaaccaa cccaaatgtc 43740
caacaatgat agactggatt aagaaaatgt ggcacatata taccatggaa tagtatgcag 43800
ccataaaaaa tgatgagttc atgtccttta tagggacatg gatgaaactg gaaatcatca 43860
ttctcagcaa actatctcaa ggacagaaaa ccaaacaccg catgttctca ctcataggtg 43920
ggaattgaac agtgagaaca cagggacaca ggaaggggaa catcacactc cagggactgt 43980
tgtggggtgg ggggaggcgg gagggatagc attaggagat atacctaatg ctaaatgacg 44040
agttaatggg tgcagtacac caacatggca catgtataca tatgtaacaa acctgcacat 44100
tgtgcatatg taccctaaaa cttaaagtat aatttaaaaa aacaacaaca catttttcac 44160
atggcttctg gggttccgtg ctctcttaat ttttctcctt cctcaatggc tattccttct 44220
tagtctccat tgctgtttcc ttctcatcta cctgaagtga cccagggttc aatcctcaaa 44280
cttccctttt tcatccttcc ccttctgttt tgtatataaa ggtacgccct gggtgacctc 44340
atcaaatctt gtagctttaa atgttatttc gtgatgactt tcaaacttat atttgtaatc 44400
cagatacctc cccagagtca catacttaca catttgtgtc tattgaacat cttcatttga 44460
tatctaatag ttcaaaacca gacttttcac aatccctccc aggtctgccc catcacagtg 44520
aggagttctc agtaagtagc acttccattg ttccaacctt tcaggcaaaa cctgcatagt 44580
cttccttgtc ttctcttttc atgttacatg tccattccac caacaaatcc tctgtagtat 44640
accttcaaaa tatatccact atctaatgat acctcacttc ctccaccctt acctccacca 44700
tcatctggtc tgagtcaggt ggattattgt tgtagccgcc taatttctat ccatctagct 44760
tctgtacttt cccattaact atctttgtat ttactaattt tggctcccca actcagagta 44820
aaagtcgatt ttttaaagat ggctgagact gaggtcagat ggattttttc ctgtctctga 44880
actttaggcc ttgaataggt tccaattttg taaaatggtt tatttctaat ccctgccctt 44940
ttgtgggtga aggccttgtc tcttgaattt tttcagaaca ctaagataca accttaggct 45000
attgaagcca gcaaatgtcc caagggtagc tctaacttca gagcatgttc acctctccag 45060
tttctggctc tcagctctca cttcattgct tgacttcttt attttctttt actcttgtct 45120
tttttttttt tttttttttt ttgagcggag tctcgctctg tcacccaggc tggagtgcag 45180
tggtgcaatc ttggctcact gcaacctcca cctcctggtt caagtgattc tcctgcctca 45240
gcctcccgag tagctgggac tacaggcgtg tgccaccatg cccagctaat ttgtgtgtgt 45300
gtatttttag tagagagggg gtttcacgag tgttagccaa gatggtctca atctcctgac 45360
ctcgtaatcc gcccgcctca gcctcccaaa gtgctggtat tacaggcatg agccactgcg 45420
cccagcctta atcttgtcta tatttttaaa aggatgtttg tcatatttta cccaccattt 45480
attggtgttt gttaagaaat ctgatatacc attcagatac caactgtatc cacctactgt 45540
cacagtcata atatacaaca gttctattaa ccatccctaa attccctcat gctttcttct 45600
ttcattgtca acctctcccc tttaaccatt cccagcctat gaaaaactac tcaccaaatt 45660
ccatccatat agttttgctt tgtcaagaat gtcatataaa tagaatcaaa atatggcttt 45720
tctcacttag caaaatatat ttaagattta tccatattgt tgcatggatc aatttgtttc 45780
tgttattgtt gagtaatgtt gcatgaatca atactttgtt tctgttattg ctgagtaatg 45840
ttccagtacc tggaatggcc aaagtttgct gattcattcg ccttttgagg gacatctggt 45900
agttccagtt tggagcactt ctgaataaag ctgttataat ctttgacata tagatgtttg 45960
tgtgagcata agtgtaaata ctaatgtgac tgctgtatca tatgctaaat gtatgtttaa 46020
cattataaga aactaccaca ctctttttta ccatagttac agtgttttgc actctcagca 46080
acagtatttg agaatttttg tgttctgttt cttgatctgt atttctaact gaaatattct 46140
ttatcactat agactccttc ccttatattg gataattgtt ttttttttct ttttcttttt 46200
ttatgagcca gagtctcgct ctgttgccca ggctggcttg ctagtggtga gatcttggct 46260
cactgtaacc tctgcctccc tggttcaagt gattctcctg cctcagcctc ccaagtaatt 46320
gggattacag gcacctgcca ccacacccag ctgattttgt attttgtttt ttttgagatg 46380
aagtcttgca ctgtcaccca ggctggtgtg cagtggcatg atctcggctc actgcaacct 46440
ccgcctccca ggttcaagcg attctcctgc cttagcctcc cgagtagctg ggattacagg 46500
cacccgccac catgcctggc taattttttg tatttttagt agagacgggt tttcaccatg 46560
ttggccaggt tggtctcgaa ctcctgacct caaatgatcc acctgccttg gcctcccaaa 46620
gtgctgggat tacaggcatg agccactgcg cctggcatga ataattgcct tttttctctc 46680
caacattcca tgagattata gaaccctttc attacgttat agacctgtag tagtgcctgg 46740
gaactctagg atgtgttctt cccagaacac acagaattgt ccttgaatag acaagtaaat 46800
aagagaagac atgaggcaag gcatgaagaa acaaaggaaa ctgtgaagta aggataagta 46860
aaagcaactt agtatcctat ttgaaaggga tagagagagc taaagagcaa ttggagaggc 46920
atataggatc atagatttag tatggaggtg cttttgtttc tatgtgtaag gaagttatag 46980
ggaaaccaca gatcactggt ttctaggcat gtaccactag ggtggtatga agggttgatt 47040
acagagaaac aggacaaggg tatttaggag tgacagaagt atgacggtgg gtggatacat 47100
gatttgtgtc cttataagtc aaagtttctt ttgttttttc tcctgttttt cattcacctg 47160
gtttttattg agtgctttca ttacctagtt tttattgagt gctagacatc acaaagaaaa 47220
gttataaggt ttcagtcctt ggataacatt atacaaaatc caataaagat ttaagtttgc 47280
tatggcaagc agtcttgaat caccataatc caaatttttg aaatttgagc tgattcaaag 47340
atgattcaga cttcagactc tgtccagatt ggtctactgt ttctccttta ttcctcagta 47400
tagctctcag ggttcccaac ccaaagtata aagaattcat ggcaggccct atcctccagt 47460
tattgttgcc cagccctgtg acacagtcca aaacactgtt tagtttctcc ccctcttagc 47520
tttttctaga atcataagac ttctctaagt ggaaatgtgg acccagatgt tgggcttatc 47580
tctttgattt tcatattttt caatttttag ccacccactc tttattatgt aaacgctcca 47640
gtggcttcac tcaggtgcct tttataattt gcttaatttg tataattgct atcatctaga 47700
aggttggtct aaattaccta aattaccaga aattaatgtt taagatttag gggctgataa 47760
ttaaattaag acctgaagaa tgaggaagag tgtttgtata gtctcatcca gagcagtcca 47820
aagaaatgga ataggctctg gctagctttt aattgaaaaa ggtctgtgac tttttcagag 47880
ttgaaaggaa gctagttttt ctggcaaata aggagcgaag ctgagaaggc tcaacagata 47940
cataagcaag gagaagacca tggaagggga aataagtcaa agtcattgaa gagtttggat 48000
tttatattaa tatggaagct actaaaaagt attaagcaga taaagaaaat cattatctga 48060
atctgaatca gttgatagga aggattaaaa ttacagatct ccaggatctt tttctgattc 48120
actagcagtt agtctggtgt aatgttggac agctataatt ttaacatgct ccccaggtga 48180
ttttgatccc caccaaggct tgagaactac tcactttaaa attgttctta atctcttccc 48240
ttatcttctc aagtgagttt tcattattct ctaatctctc agatgcccct ttcatatatt 48300
aatatatgag atccttctta gctaattttt tttaaaattc tagataggaa cattactgta 48360
ccatgaaatc agtgtagtgg ggtttgtctg catgtgtatg agacagagaa attgaaaaaa 48420
gtagaaatat tgcagaatag aatagaaagc agggaagata ctgttttatg aaactgtata 48480
tatatagtag gttgtgatgt gaaaggtagg tattatatgt cataataatc aatatttgaa 48540
acaaactcat gtatgtctgt tatttcttat gggcattatt ttcctattat ttgtcttttc 48600
tatagaagtt atatttctca atttttttat tctctcttat ctttcagact tgccttcaag 48660
gtgtgcaagt aaggacttat ctccagaaaa gaacacttat gaaacagaat tatcccaatg 48720
ggaaatgagt gacagacttg aaaactgtga tcttgaagag tccaattcca gggattattt 48780
ggaagccaaa ggcaagatgg agaagcaaca agaaaatcag aaggaatatt tcaggcaagg 48840
gatgatcata tatgacaaaa tgtccatttt caaccagcat acttacttat ctcaacattc 48900
aagatgtcat tctactgaga aaccctataa atgtaaggaa tgtgggaaag ccttcagacg 48960
agcctcacac ctaacacaac atcaaagtat tcatactggt gaaaaaccct atgaatgtaa 49020
gcaatgcggg aaggccttta gtcgtgattc acaactcagt cttcatcaga gacttcatac 49080
tggtgagaaa ccctatgcat gtaaggaatg tgggaaggcc tttactcaaa gctcacaact 49140
tattttacat catagaattc atactggtga aaaaccatat aaatgtgaag aatgtgggaa 49200
agcctttatt cgtagctcac aacttacccg acatcaaaaa gttcatactg gtgagaaacc 49260
ttatgaatgt aaagaatgtg ggaaggcctt tactcagaat tcacaactta cactacacca 49320
gagacttcat actggtgaaa agctctatga atgtaaagaa tgtaggaagg tctttactca 49380
gctctcacaa cttattctgc ataagagaat tcataccggt gagaaaccct atgaatgtaa 49440
ggaatgtgga aaagctttta tttgtggctc acagctttct caacatcaga aaattcataa 49500
tggggaaaaa ccatatgaat gtaaggaatg tggaagggcc tttattcggg gctcactact 49560
gatgcaacat cagaggattc atactggtga aaaaccctat aaatgtgaag aatgtgggaa 49620
ggcctttatc cgtggctcac aacttactca acaccagaga attcacacca atgaaaagcc 49680
ctatgaatgt aaggaatgtg gaaagatgtt tagtcatggc tcacaactta ctcaacatca 49740
gagaatacac actggtgaga aaccctatca atgtaaggaa tgtggaaaag cgtttaatcg 49800
tggctcactc cttacacgac accagaggat tcatactggt gagaaaccct atgaatgtaa 49860
agaatgtgga aaaaccttta gtcgtggctc agaacttact caacatgagc gaattcacac 49920
aggtgagaaa ccctatgaat gtaaggaatg tgggaaatct tttattcgtg gttcccagct 49980
tactcaacat cagagaatcc atactggtga gaaaccttat gaatgtaaag aatgtagaat 50040
ggcctttact cagagttcac atctttccca acatcaaaga cttcacactg gtgagaaacc 50100
ctatgtgtgt aatgaatgtg gaaaggcctt tgcgcgtggc ttactactta tacaacatca 50160
gagaattcat actggtgaga aaccatatca atgtaaggaa tgtgggaaag cctttattcg 50220
tggttcacag ttgactcaac atcagcgaat tcacactgga gaaaaaccct atgaatgcaa 50280
ggagtgtggc aaggccttta gtcatggctc tcagcttact ctacatcaga gaatccatac 50340
tggtgagaag ccctatgaat gcagagaatg tagaaaggcc tttactcaga gttcacatct 50400
ttctcggcat cagagaattc atactggtga gaaaccatat caatgtaagg aatgtgggaa 50460
ggcctttact cgtggttcac agctaactca acatcagaga attcatatca gtgagaaatc 50520
ttttgaatat aaggaatgtg ggattgactt tagtcatggc tcacaagttt acatgtgaat 50580
tgtctgatta tttgagatca ctatgaagag gttctctggt tgttagcagc aaagaattct 50640
cacaaatgtg aatatgggcg cacatttgcc tcataaagca cagcatcaga taatttatgt 50700
gagagaaaat ggtagtgtca ttcatataga aaaacatcat tactggaaac ctattaaaca 50760
ttagcaaatt ggagaatagt tttaatatag taaatgtagg aagcccttta gccatattga 50820
aaacaaatat ctttttcaac gttatcttag ctctactagt tgatcttttt gttatatgta 50880
tcatgatact taacctctac cttggtttaa tcattttaag atagacctaa gtatattacc 50940
tttattgtaa gattcttgga agtattatgt aagttattac atgtaaaagc tcttagaatg 51000
gtgccttgaa cgtagcatac cacaaatatt agctaccatt ttcactagtg ttattttaga 51060
gaatttgcat gagaggagag tacttatgag tataatgaat attgagaaat cttttatcaa 51120
cacatccaag atgacttggt tggggggcac tgtatgccat aatgaatgtg agaaagctgt 51180
catttaaatc tcatccatta ttgctataag aggaaattca tactgttaaa aaaaaaaaaa 51240
cccagtggat ataatcagtg tattattaaa aaaaaaaaaa 51280
Claims (5)
1. KRAB型锌指蛋白KRANA及其活性片段在制备抑制p53转录功能的负调控分子中的应用;其中,所述KRAB型锌指蛋白KRANA的氨基酸残基序列如SEQ ID NO:1所示;
所述KRAB型锌指蛋白KRANA的活性片段选自下述氨基酸残基序列之一:
1)自SEQ ID NO:1氨基端第1位至70位所示的氨基酸残基序列;
2)自SEQ ID NO:1氨基端第1位至244位所示的氨基酸残基序列;
3)自SEQ ID NO:1氨基端第1位至514位所示的氨基酸残基序列;
4)自SEQ ID NO:1氨基端第240位至688位所示的氨基酸残基序列;
5)自SEQ ID NO:1氨基端第72位至688位所示的氨基酸残基序列。
2. 根据权利要求1所述的应用,其特征在于:所述KRAB型锌指蛋白KRANA的cDNA基因,是下述核苷酸序列之一:
1)碱基序列如SEQ ID NO:2所示的核苷酸序列;
2)编码序列表中SEQ ID NO:1所示的氨基酸残基序列的核苷酸序列。
3. 根据权利要求2所述的应用,其特征在于:所述KRAB型锌指蛋白KRANA的cDNA基因的开放阅读框架为SEQ ID NO:2所示的碱基序列的自5′端第225位-2291位。
4. 根据权利要求1所述的应用,其特征在于:所述KRAB型锌指蛋白KRANA的基因组基因,是下述核苷酸序列之一:
1)碱基序列如SEQ ID NO:3所示的核苷酸序列;
2)编码序列表中SEQ ID NO:1所示的氨基酸残基序列的核苷酸序列。
5. 根据权利要求4所述的应用,其特征在于:所述KRAB型锌指蛋白KRANA的基因组基因的开放阅读框架为SEQ ID NO:3所示的碱基序列的自5′端第10641位-50587位。
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