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CN111471681A - 一种抑制猪繁殖与呼吸综合征病毒的干扰片段 - Google Patents

一种抑制猪繁殖与呼吸综合征病毒的干扰片段 Download PDF

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CN111471681A
CN111471681A CN201911225994.6A CN201911225994A CN111471681A CN 111471681 A CN111471681 A CN 111471681A CN 201911225994 A CN201911225994 A CN 201911225994A CN 111471681 A CN111471681 A CN 111471681A
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mfn2
respiratory syndrome
porcine reproductive
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张淑君
王凯
蒋金河
孙玉梅
许苏童
张敏
上官爱哨
阮健
肖世泽
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Huazhong Agricultural University
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Abstract

本发明属于动物基因工程技术领域，具体涉及一种抑制猪繁殖与呼吸综合征病毒的干扰片段。所述的片段具有如序列表SEQ ID NO:1所示的核苷酸序列。本发明发现MFN2基因在猪繁殖与呼吸综合征病毒增殖中发挥重要作用，通过本发明所示的干扰片段可以抑制其在细胞中的表达。进一步研究其功能发现，抑制MFN2的表达可以有效抑制猪繁殖与呼吸综合征病毒(PRRSV)的复制。本发明基因MFN2及其特异siRNA干扰片段，为培育抗PRRSV猪及制备防治PRRS病毒抑制剂提供新的分子靶标。

Description

一种抑制猪繁殖与呼吸综合征病毒的干扰片段
技术领域
本发明属于动物基因工程技术领域，具体涉及一种抑制猪繁殖与呼吸综合征病毒的干扰片段。所述的干扰片段是MFN2基因siRNA，其序列如序列表SEQ ID NO:1所示。该片段能够有效降低目的细胞中MFN2蛋白的表达，从而抑制猪繁殖与呼吸综合征病毒(PRRSV)的复制。
背景技术
猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome，简称PRRS)是由猪繁殖与呼吸综合征病毒(porcine reproductive and respiratory syndromevirus，简称PRRSV)引起的以母猪繁殖障碍、仔猪呼吸系统疾病和高死亡率为主要临床特征的重大病毒性传染病(Chand et al 2012；Van 1997)。自1987年首次在美国爆发以来，已成为影响全球养猪业发展的重要疾病(keffaber et al 1989)。据报道，美国每年因为该疾病造成的经济损失约为6亿美元(Du et al 2017)。由于PRRSV与宿主免疫系统之间复杂的互作关系以及病毒的高突变率，目前生产上仍然缺乏有效保护猪免受PRRSV侵害的方法。内质网与线粒体物理上紧密连接形成的连接区域叫作线粒体相关膜(mitochondria-associated membranes，MAM)。申请人前期研究发现PRRSV感染可以引起MAM区域结构、功能改变。MAM的正常结构是通过内质网和线粒体之间的连接蛋白来维持的。目前，关于内质网和线粒体连接蛋白的研究已经取得了一些进展。最新PNAS报道，利用透射电镜和检测细胞器偶联的荧光探针试验发现缺失MFN2会导致内质网和线粒体之间偶联减少，证实了MFN2作为内质网和线粒体之间的连接蛋白(Naon et al 2016)。抑制MFN2增加内质网和线粒体之间的距离，减少激动剂诱发的Ca2+从内质网到线粒体之间的转移(de Brito et al 2008)。登革热病毒(DENV)感染促进线粒体融合，融合的线粒体参与形成病毒复制区域CMs，造成线粒体与内质网之间的耦连减少)，抑制抗病毒天然免疫应答从而有利于病毒自身复制(Chatel-Chaix L et al，2016)。因此，本发明进一步探讨MAM区域连接蛋白MFN2在PRRSV增殖中的作用和效果具有重要意义。
发明内容
本发现目的是提供一种MFN2基因的siRNA干扰片段；本发明的另一个目的是提供MFN2基因的siRNA干扰片段在抑制猪繁殖与呼吸综合征病毒复制中的应用。
本发明通过深入研究PRRSV与宿主的互作机制，筛选出了影响PRRSV复制的宿主MFN2基因；然后通过合成靶向MFN2的siRNA干扰片段，能够有效降低细胞内MFN2蛋白的表达。利用Western Blot和qPCR检测，结果表明，干扰MFN2片段，可抑制PRRSV的复制。本发明为研制有效安全防治PRRS的新型疫苗药物提供了新的靶标，也为揭示PRRSV与宿主的互作机理以及抗PRRSV感染的机理等研究提供了候选基因(片段)。
本发明通过以下技术方案实现：
本发明首先参照文献选择针对MFN2基因序列特异性的siRNA干扰片段(Sugioka Ret al 2004)，该siMFN2干扰片段的核苷酸序列如下：5'-AAGAGACACAUGGCUGAGGUG-3'(如SEQ ID NO:1所示)。通过人工合成该序列并转染细胞，利用Western Blot方法验证该段能够有效降低细胞中MFN2蛋白的表达水平。
进一步，本发明探讨了干扰MFN2对PRRSV复制增殖的影响，结果发现细胞转染上述干扰片段能够抑制PRRSV核衣壳蛋白(简称N蛋白)和ORF7的表达水平。表明siRNA干扰片段在制备PRRSV病毒抑制剂中具有广泛的应用前景。
本发明的有益效果在于：
本发明首先发现了MFN2基因在PRRSV病毒的复制中发挥非常重要的作用，通过siMFN2干扰片段靶向抑制MFN2蛋白的表达会造成PRRSV病毒的复制被抑制，从而验证了所述siRNA干扰片段的功能。
附图说明
图1：利用Western Blot方法检测MFN2的干扰效率。附图标记说明：图1中的A图是用siMFN2和NC分别转染Marc145细胞24h后，检测MFN2蛋白的表达水平；图1中的B图是A图中MFN2蛋白的灰度值。
图2：利用qPCR方法检测感染12h、24h、36h和48h后PRRSV ORF7 mRNA的表达水平。
图3：Western Blot检测干扰MFN2后PRRSV N蛋白表达水平。附图标记说明：图3中的A图是Western Blot检测感染36h PRRSV N蛋白的表达水平；图3中的B图是统计图3中的A图中PRRSV N蛋白的灰度值。
具体实施方式
对序列表的说明
SEQ ID NO:1是本发明人工合成的抑制猪繁殖与呼吸综合征病毒的干扰片段(序列长度为21bp)。
下面实施例中所使用的技术，包括细胞转染、RNA提取、cDNA合成、qPCR和WesternBlot检测等生物学技术，除特殊说明，均为本领域已知的常规技术。所使用的仪器设备、试剂等，除非本说明书特别注明，均为本领域技术人员可通过公共途径或商业途径获得的材料或得到的制备方法。
实施例1：干扰片段的转染
(1)MFN2蛋白由MFN2基因(该基因的序列见说明书附录部分，序列长度为30756bp)编码，根据其mRNA序列(登陆号XM_007980521.1)选择特异干扰片段，该干扰片段序列由苏州吉玛公司合成。
(2)转染前一天将生长良好的Marc145细胞以1×105个/孔的量加入6孔细胞培养板，在37℃5％CO2培养箱中培养，待细胞汇合度达到50％-60％时准备转染；具体细胞转染步骤参考见Lipofectamine)3000(ThermoFisher scientific,USA)转染试剂盒上说明书。简单叙述为，配制细胞转染试剂，取2个1.5mL无菌离心管，分别编号为A管，B管。其中在编号为A管中加入125uL无血清培养基OPTI-MEM和4uL Lipofectamine 3000；在编号为B管中加入125uL无血清培养基OPTI-MEM和5uLsiMFN2干扰片段(20uM)或者5ul的NC对照片段(此片段没有干扰作用为实验对照)。A管和B管分别混匀室温放置5min；
(3)将编号为A管中的混合液与编号为B管中的混合液混合均匀，室温放置20min。期间，吸弃待转染细胞孔中的培养基，用PBS(SH30256,Hyclone)清洗2遍，加入800uL无血清培养基OPTI-MEM；
(4)将步骤(3)得到的混合液轻轻加入细胞培养板中，并轻轻摇匀，然后将细胞置于37℃，5％CO2培养箱中培养，4-6h换一次新鲜的步骤(3)的混合液。
(5)利用Western Blot方法检测siMFN2的干扰效率，具体操作步骤与实施例3中操作步骤基本一致，所用的一抗分别为MFN2鼠单克隆抗体(Santa Cruz Biotechonlogy,sc-100560)用一抗稀释液体积比为1：400稀释和GAPDH鼠单克隆抗体(Proteintech，60004-1-lg)用一抗稀释液体积比为1：5000稀释，二抗为HRP偶联抗鼠IgG(Proteintech，SA00001-1)用TBST体积比为1:5000稀释。结果如图1所示，表明siMFN2的干扰效率为超过70％。
实施例2利用qPCR检测干扰MFN2对PRRSV ORF7 mRNA的抑制效果
(1)转染特异siRNA干扰片段24h后，弃培养基并用DMEM洗2遍，加入PRRSV(MOI＝0.5)混匀，37℃孵育1h后，再加入1ml DMEM+10％FBS完全培养基，继续培养。
(2)分别于感染后不同时间点收集细胞并提取细胞总RNA。提取RNA方法按Omega公司总RNA提取试剂盒(R6834-01)的操作说明书操作。具体步骤如下：
a)提取步骤之前在每1mLTRK裂解液中加入20μLβ-巯基乙醇。弃去培养液，用磷酸盐缓冲液(PBS)清洗2遍，加入0.35mL TRK裂解液使细胞完全裂解，用细胞刮板刮取培养皿表面的细胞产物；
b)将裂解产物转移至Homogenization spin column，于10000g下离心2min，收集滤液；
c)向滤液中加入等体积(0.35mL)70％乙醇，混匀后转移至HiBind mini column，在10000g下离心2min，弃去滤液；
d)将column放在一个新的2mL收集管中，加入0.3mL RNA wash buffer I，于10000g下离心1min，弃去滤液；
e)向column加入0.5mL RNA wash buffer I，于10000g下离心1min，弃去滤液；
f)向column加入0.5mL RNA wash buffer II，于10000g下离心1min，弃滤液，重复此步一遍；
g)将column放在新的2mL收集管中，与10000g下离心2min，将column的盖子打开，晾干5min，减少吸附膜上的乙醇残留；
h)向吸附膜中央滴加40μL预热的DEPC水，室温下静置3-5min，于10000g下离心2min，获得RNA(RNA浓度低时可选择性进行第二次洗脱)。
(3)测得RNA浓度后，利用反转录试剂盒(Thermo Scientific，00238582)将RNA反转录成第一链cDNA。反转录反应体系见表1。
表1 反转录反应体系
Figure BDA0002302217430000031
(4)qRT-PCR检测PRRSV保守基因ORF7 mRNA表达水平。具体步骤如下：
cDNA经RNase Free H2O 5倍稀释，建立SYBR扩增体系如表2。
表2 SYBR扩增体系
Figure BDA0002302217430000032
Figure BDA0002302217430000041
cDNA样本通过Bio-Rad CFX96荧光定量PCR仪进行扩增，检测不同样本中特定基因表达的差异，同时设置内参基因，每个样本设置3个复孔。三步法的扩增反应条件为：预变性95℃4min，循环反应为变性95℃5sec，退火60℃20sec，延伸72℃20sec，并收集荧光信号，共进行40个循环，最后延伸72℃2min。65℃-95℃升温，升温速度0.5℃/5sec以检测融解曲线。目的基因的相对表达量按照2-△△Ct进行计算。所述的qPCR引物信息见表3。
表3 qPCR引物序列
Figure BDA0002302217430000042
结果如图2所示，经qPCR检测证实PRRSV感染12h、24h、36h和48h后siMFN2处理组胞内ORF7的mRNA表达水平低于NC组。
实施例3利用Western Blot方法检测干扰MFN2对PRRSV病毒蛋白的抑制效果
具体步骤为：
(1)蛋白样品的制备
a)Marc145细胞细胞经相应处理后，吸出培养液，用预冷的PBS洗1-2次；
b)每个30mm平皿中加入200μL蛋白裂解液(每100μL RIPA裂解液中，加入1μL蛋白酶抑制剂蛋白酶抑制剂Cocktail(P8340，Sigma)、磷酸化蛋白酶抑制剂PPEi(G2007，Sigma)，在冰上裂解，并用细胞刮板刮取细胞；
c)吸取裂解产物至1.5mL离心管中，反复吹打至清亮；
d)加入相应体积的5×SDS-PAGE上样缓冲液，沸水煮10min，-80℃储存。
(2)SDS-PAGE电泳
a)配胶：按照SDS-PAGE凝胶配制试剂盒(P0012A，碧云天生物科技有限公司)说明书制备12％分离胶和5％浓缩胶；
b)倒胶：夹好灌制SDS-PAGE的玻璃板，将12％分离胶约4-5mL灌入两玻璃板的间隙中，留出灌注浓缩胶所需的空间，在分离胶上缓慢灌入无水乙醇，室温下聚合45-60min，分离胶聚合后，倾出无水乙醇，用滤纸吸净残留液体。再将制好的5％浓缩胶灌入分离胶上，立即插入梳子，在室温下聚合45-60min，待胶聚合后，将SDS-PAGE玻璃板放入1×Tris-甘氨酸电泳缓冲液中，移出梳子，并用针头调整胶齿的曲直，固定电泳装置；
c)电泳：向上样孔内加入相应体积的蛋白样本及蛋白Marker，接通电源，浓缩胶用80V恒压电泳，待溴酚蓝指示剂平直跑到分离胶时，将电压调至120V；待溴酚蓝指示剂跑到分离胶底部(约2h)时，结束电泳。
(3)利用Western Blot方法检测分析
a)电转：电泳快结束时开始准备与转膜夹板尺寸相当的滤纸，当SDS-PAGE电泳结束后，量取目的蛋白所在SDS-PAGE的条带尺寸，同时裁下相应尺寸的PVDF膜，并作好标记以区别条带，甲醇激活PVDF膜15-20sec，遂将PVDF转移至1×转膜缓冲液中平衡10min；
b)湿转三明治的制备：负极/海绵/3层滤纸/SDS-PAGE/PVDF膜/3层滤纸/海绵/正极，每层放好后，用玻璃板赶去气泡。将转膜仪放入冰浴中，加入1×转膜缓冲液，100V恒压转印1h；
c)封闭：将刚从转膜仪中取出的PVDF膜放入1×TBST中漂洗，以洗去膜上的转膜液，然后加入封闭液，于摇床上缓慢摇动，室温封闭2h；
d)一抗孵育：一抗分别为PRRSV N蛋白抗体，为鼠单克隆抗体(商购)用一抗稀释液以体积比为1：500稀释和GAPDH鼠单克隆抗体(Proteintech，60004-1-lg)用一抗稀释液以体积比为1：5000稀释，4℃孵育过夜，第二天于摇床上室温放置1h。回收一抗，摇床上洗膜：1×TBST洗2次，1×TBS洗1次，每次10min；
e)二抗孵育：按照HRP偶联抗鼠IgG(Proteintech，SA00001-1)使用说明书，用1×TBST稀释HPR标记的二抗。于摇床上缓慢摇动，室温孵育2h。摇床上洗膜：1×TBST洗3次，1×TBS洗2次，每次10min；
f)蛋白检测：采用ECL发光法，将A、B显色液按照体积比为1：1混合配制成显影液，用滤纸吸取膜上的液体，滴加显影液，最后在化学发光成像系统中进行显影分析。得到如图3中的A图和图中的B图所示的效果，表明抑制MFN2的表达导致胞内病毒N蛋白表达量减少。
附录：
本发明涉及MFN2基因编码的蛋白基因序列：
绿猴Chlorocebus sabaeus(green monkey)
2MFN2基因序列(30756bp，来源见实施例1的具体描述)：
TACAGGTGTGAGCCACCCCACCCGACCTGAAAGTCTTTTAAAAGCCAGGCACAGTGGCTCATGCCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGCGGATTGCCTAAGGTCAGGAGTTTGAGACCAGGCTGGCCAACGTGGTGAAATTCTGTCTCTACTAATAATACAAAAAAATTAGCTGGGTGTGGTGGCATGCACCTGTAATCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATCGCTTGAACTTAGGTGGCAGAGGTTGCAATGAACCGAGATCGGGCCACTGCACTCCAGTCTGGGCGACAGAGCAAGACTCCGTCTCAATTAAAAAAAAAAAAAATTTAGTCTTTTCATTTTTAAATTTGACTCATCATAAATTCAAATAGCACAAATCTTAATCTTTCTCTCATCCTTTCCCCGCAGCTGCTCAGTTCCTCTCTCTGAAAGCCACCATGTCTAATCTGTTTTTTGAATGTTCTTGAGCTGGTTCGTGCCGACACAAGCCTGTGTATTTGTGTATATTAGGGTTGTTTTTGTTTTTGTTTTTGAGATGGAGTCTTGCTCTGTCGCCTAGGCTGGAGTGCAATGTCGCGATCTTGGCTCACTGCAACCTCTGCTGCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCATGACTAGCTGGGATTACAGGCACCCGCCACCACACCCAGCTAGTTTTTGTCCTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGGTGGTCTCGGACTCCTGACCGCAGATGATCCACCTGCCTCAGCCTTCCAAAGTGCTAGGATTACAGGCGTGAGCCACTGTGCCCGGCCTAGGGTTGTTTTTAACTGTTAACTTTTTTCTGATTACAAATGCATCTTCATCATGAAAAAGTCAAACAAAATAGGTAACATGGGGGGCCGGATGCTGTGGCTTACGCCTGTAATCCCAGTACTTTGGGAGGCAGAGGTGGGTGGCTCACTTGAGGTCAAGAGTTCGAGAGCAGCCTGGCCAACATGGCAAAACCCCCATCTCTACAAAAAATACAAAAATTAGCCAGGTGTGGTGGCAGGTGCCTGTAATCCCAGCTACTTGGGAGGCTGAGCTGGGATAATGACTTGAACCTGGGAGGTGGAGGTTGCAGTGAGCCAAGATTGCACCACAACACTCCAACCTGGGGGACAGAACAAGAACCTGTCACCAAAAAAAAAAAAGTTATATGGACCAGGTGTGGTGGGAGGCCGAGGTGGGAGGATCACTTGAGGCCAGGAATTCTCAACCAGCATGGCCAACATGGCAAAACCCCATCTCTACTAAAAATACAAAAATAAGGTGGGCTTTGTGGCACATGCCTGTAATCCCAACTACTCAGGAGGCTGCAGCAGGAGAATCGCTTGAACCTGGGAGGCAGAGGTTGTAGTGTAGTGAGCTGAGATCACGCCACTGCACTCCAGCCTGGGTGACAGAGCAAGACTCTTGTCTAAAAAAAAAAAACAAGGTAAGGTAAGAAAGAAAGGTCTCCTGGAGTTGTACATGAATACATCAATGTGCTCTGCACCCATTCCCGATAGCACTTAGCACTGCTAGGTAAGTTTTGTGATCCAGAGGGTTGAAACGAGAATTCAGCCATCTCAATGAGAAACACATGTTGATGGGTTGAAACATATCTACAAACTGCGAGATTTCCTCTACTTGCCGGGCAAGTTTGCAGATGTGTTTCAGAGAGAGCCTAGCTGCTGGGCAGAGTACAAGACTAAATGCCTGGGAATGGATTCAGAGGCTCCTACAGCAGTCCAGGCAAGTGTCCAGAGCCTAGACCAATGCTTGCAGGGAAGTTAAGGGAAGAGGAGGGAGTGGGCAGGAGAGATGTTTAGGAGAGAGAATACCCAGGCTTCCACCTGACTTGGGCAAGATTATGTTAAAGAAGGAAAAGGAAGGCTGGGCATGGTGGCTCACGCCTGTAGTCCCATTACTTTGGGAGGCCGAGGCGGGTGGATCACCTGAGGTCAGGAGTGAGACCAGCCTGGCCAACATGGTGAAACCCCATCTCTACTAAAAATACAAAAATTAGCCAGGCATGGTGGTGCACACCTGTAATCCCAGCTAGTGGGGAGACTGAGGCGGGAGAATTGCCTGAGGTGGAGGTTGCGGTGAGCCAGGATGGTGCCACTGCGCTCCAGTCTGGGCAACAGAGCGAGAGCGTCTGGAAAAAAAAAAAAAAAAGGGAAATGGAATTTATAGTTCCTGCCCTCAGAATGGAAGTGGAATTTAGAGTTCCTCCCTTAGGGAGTCCAGAGATTGTGGAGGAGGAAACAATCCGACCCAGTACAAGGCGCTAGCAACTGAGGCCCCAAGACGTGCCACATCCCAGACAGGGCCCTGTCTGCATCCTGAGGCCAAGTCTGCGTGTGGCCCTACACACAGACCCGCGAGGCACTAGGCTACTCAGAGGGCAGTGTGGGCAGGACAGCTCCCTTTGGCCATCCTCACAGGAGACTGATAAACCATTCAGTTTGATATCAGAGTACTGATTCGCCTCTTGAGGTTATTTGTGGCAATAAATTGAAGTTGTTCTACCGTGGTGATACAGCAGCACCATCCCATAGCATTACAATTTGAGCCACAAACGCAAGCTACTTATGTAATTTTTGAAGGGATGGCCTACCTCGAAAGGCGGTTTTCTCCTTTCTCAGTAAACAGAACATACATTGGGTTTTATACCGAGATGTTCCATTGCCCAGGGACGGGCAGGAGACAGATGCTTTTCTCTATCTCAACTGCGAACAACTGCCAAGAGGCCTTCTTTCCTCTTACACTAGTCCTCCTCAGCACAGACCCTTCACGGGTGTCAGGCTGGGGGATGATCAGGTCTTTCCCATCCAAAGAGGCCATATTTCAGACTATCACATGGGGAGAAACCTTGGACAATACCTAGCTTTCCTAGGCAGAGGTCCCTGCGACCTTTGGCAGTGTGCGTATCCCTGGGTACTTGAGATTAAGAGAATCGTGATGACTTTTAACCAGCAAGCTGCCTTCAGGCACTTGTTTAACAAAGCACACCCTGCATAGCCCAAAATTCTTTAAACCTTGAGTCACCACAGCACATGTCTCTTGCAAGGACAAGGTTGGGGGTAGGGTCACAGATTAACAGCATCTCAAATACAGAACAAAATGGAGTCTCTTATGTCTACTTCTTTCTATATAGACACAGTAACAGGCTGATCTCTCTTTTCCCCACAATTTTACATTTTCTAGTAGCCACATTTTTAAAAGTAAAAAGAAACAGGTGCAATTAATCTTAGTAATGTATTTAACCCAAATGATCCAAAACATTATCACTTCAACATGTAATCAGCATATAAATATTAATGAGCTAATAGCCCTGCACTGTGGCAGGTACCTGTAATTCAGCATTTTGGGAAGCTGAGGCAGGAGATTGCTTGACTCCAGGAGTCCAGGAGTTCAGGAGACCAGCCTGGGTAACAAAGTGAAATGCTTACCCATGTCTACAAAAAATAAATATTAGGCATGGCTGCAGCGTCTGTACTCCCAGCTACTCAGGAGGCTGAGTGAGGTGGGAGGATTACTTGAGCCCAGGAGTTCGAGGCTGCAGTGAGCGGTGATTGCATCACTGCACTACAGCGTGAGTGACAGAGTGAGACTCTGGCTCAAAAATATACATTTTTTTCTTTTTTTGAGACGTAGTCTCTGTTGCCCAGGCTGGAGGGCAGTGGCACGATCTCGGCTCACTGCAACCTCTGCCCGCCGTGTTCAAGCCGTTCTCCTGCCTCTTCCTCCTGAGTGCACCATCACACTTGGCTAATTTTTGTATTTTTAGTACAGACGGGGTTTCACCGTGTTGGCCAGGCTGGTCTTGAACTCCCGACCTCAAGCGATCCACCCGCCTCGCCCTCCCAAAATTCTGGAATTACAGGCATGAGCCACCGCGCGCGGTCGATATTTTTTTTTCCTAAAAATTGAGATAGTTTAACAGTCTTTTTTGTCCTAAGTCTTATAGCGCATCTTCGTTTGCACTGGCTCAGGACTCGAGAGCCACATGTGGCTAGTGGCTACTGTTCTGGCGCCCTCAGGCTTGGAGCCTTCCCTGCGGAGGCCAAGTCCTGCAGGGGACCTGTTTGCTGCGGTTTCCTAGTGTCTGGCGCATCACTGGGGCCGAGTGATCTGGGTAACCATGTGAGCTTGGGTGCCCTCCCCTCTCCGTCTCCTGGGACTGCCGTGGAGATTAAACACAGCGGTGGATGTTAGAGACGCAGTTCAGTCACTAGCATTGATTCGCCGTTGTTGCTGCTGTTCTTTGCTCAAAGGCGACTGAAGGGCAGCAGGTCCAAGCTCGCTTCTGTCTGCCCGATGAGTCACTTCACCCTAGGCCTGGCCCTCCAGAGAGCACTCCTCTAAGATTACAGAATGCAGATCTGGATTCCAGAGCTGGCAGAAGTCCCAGGGTCAGACTCTAGGACAACCGGCGAAAGAGCGGGAACCTCTACTCCACGCCCTTGGTTTTTCAAAGCCGCTTGACAGCTTGAGGCTCCGCCCCGCTCTGCCCCGCCCCCCAGCTCCGGCCGACGCCCACCGGAACTACAGCCCCCATGATGCAGCGGGAGTCCGAGCCGCTGCGTCATCGGCTTAGGACGCGCCGGCGGAGGCGTCGCGCGCTGAGGCGCCGCTCGAAGTGTCCAGTCGCGGGGCCGCAGAGGCGTAAGGAGTAGGCGGGGCGAGCCAGCTGGGCTCAGGGTCCACCAGCTCACCCGGGTCGAGGGGCAATCCTGAGGCGACTGGTGACGCGCGCGTCCCTTTCCCTCCTCCCGGCTCCCCCCGCGGTGGCGCTTGCTGGTGACGTAGTGAGTGTGATGGCCACCGCGAGGCCGGGAAGGTGAAGGTGAGAGGGCGAGCAAGCCGGGGTGGCGGGGACTGGCCCGGCCCGGCCGGGCAAGTCCTGAGGAGCTCGGTGTAGGCCGACGGGATTGTGGGGCCACGGAGGTCTGAGATGCAGTCAAGTGGGGGTCCCCACTCTCCGGCCTCGCCGCAGGCCTGGAGAGGAGGGGGCGGGCCACGGGACCTGGGACTGGGAGGTGGGCTCCCCTGTAGCTTCTCACTGTCCCCGCCTTTCCACCCTTCACGGCAACGTTCTACCCCTGCCCCTTTTCTGTTTTTCATCGAGTCTTCGTTTTGCCAGCATTTACTCAGCACCTGATAGGTGTACACGTTCACCGCGGGAGCGCCCTGCCCTCCCCACTTTGGACACTTACTACCCTCTCGTTCAACCCTGTTTACTTGTATGACTTCTCACTGGACTCTAAGGCCCTGGAGGGCAGGACTATTTCTTATCCATTCCTAACGTGGCGCCTGACACATATCCAGAAGGCACACAGAAAATGTTGAATAAATAGCTGTTTAGACGCACGAAGGCAGAACTGCCCGTTACCCCCTCTCGTACTCCTTCATTGCCCTCTTGCTTTAAATTCTCAATCATCTCTCTCTCTTCTACCCAGAAAAAGGCTCTGGTTATCCGCCTCTGGTGTCCCAAGGTTTAGGGCTTGGGGGAGGAAGGCAGCTTAATGTAATTATAAAAGGAAGGAGGCCGGTCGCAGTGGATCACACCTGTAATCCCAGCACTTTGGAAGGCGGAGGCGGGTGGATCACCTGAGGTCAGGAGTTCGAGACCAGCCTGGTCAACATGGTGAAACCCGGTCTGTACTGAAAACAAAAATTAGCCAGGCTTGCTGGCACACGCCTGTAATCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAAGTGGAGGTTGAGCTGAGATCGCACCACTGCACTCCAGGAGCTTTGGAGTCAAAGGGAGCTAATATTTCAAATCCCAGCTTAGACAATCGACTAACAGTCTATAAATCTTCAAGTATTCTAGCCTTGTGTTTGCCTCATAGAATTGAGAATTAAGTGAGATACTTTACAGTAGCTGAACACATTGGCTTTCAGTCAGTTCTTCTGTTGCTCAGTAGTCAGCTGTTCCACAATCCTCAAGCCCAGTTTTTTTCCTGGTTTTGCTCTGTCCTCCTCTCCCCTCTCTAGATGTACCTTTCCCAGGGCTCCTGCAGGGACCTCCTTGGTGCCTCAAACCCGCACATGGCAGAACACATGTTTGTGCATAGCCAGGATTGGTCCCACACCTCTGTGTTTTTTTCCCCAGGGCACTTCCTCACATGCCAGTGCAACTCCACAATACCTCAATGAGTAAGCTTCCCTCAGGGATGAGTTTGTTTGGCTTGAAACCTGATTAGACATGTTTTGTACACCAGTGTTTTTTTTGCGGGGGAGGACACCTGATCTATCTCCCTTTTCTTTTCTAATAAGTCAGGACTGGTGGAGTCAACACAGTCAATCAATAACCAACCTCAACCTGAGACAAGACAGAAGAGAACTCAGAATCTTTTTGTCTTTTGGACTCCAGCCATGTCCATGATGCCTACCTTGTGAAGATCTCTCACCATCCAAAAAAGTGAGTAGTAGTTGTGACTTCTGGCACCCTGGATTTGAGTTGACTGGACAGGGGCATGTTCTCCTGGACCTCTGGTGGCAAGTCTCTTGGATGGCATCCTCTTTATATAGAGCCACAGCTTTCTAACTTCTGTTGAAAAACTGCTGTATCTCTTTTCTGCTTCCGTGAAGGATTTCGCTGGTTGCTTAGAGAGGCTGTTTAAAAGTTGACAGTTCAGGAAGTGGCTGGGATCTCTTGGGCTCCCCGAAGAATTAGTCTGTGTCTCCCTCATTTGGTCTTCTCTGCAGTGCTGGCTCTTCCCCGTCTCTGCCCAGCACAGATTGGCTCCTTGTCACTGCTCTTCTGAGATTCTGCTCTGTCCCCTTTACCCTGTATCCTCTTTTGTTTCGATTTCTCAGCTTCTCCTCTTTTCCAGTTGTGCCAGCAGACGATCCGTGGATCCATTTTATTTTCCCTGCTAGGAGTTGCTGTACATAACACTGGGTAAGGAGGTGGTGTGGATTCAGGGACTGTGCTTGGGATCCAAACACACCTTGCAGTGAATTTTGCTACTGTTGTCTCTACTCCCAAGATACCTAATACTCCGTGACTGATAGCCTAGGTTGAGGCAAAACTTTGGACCACCTTGGAGGGCTAATAGCAGTTTCCATTTAGAACTAGGTTCATTTCTGGTCTTAGGTTAATATTTGTCAATACCGTGCCCCTGGCGCACTTTTTGGGGGTAGGGGTGAGAGAGTACCAGAAAGAATGGGCCTTTCCCTAAAGCGTTATCCTCGGGATTTTTATTTTATTTTATTTTTTATTTTTATTTTTTATTTATTTTATTTTATTTTATTTTATTTATTTTATTTTATTTTATATTTTATTTTATTTTATATTTTATTTTATTTTATTTTATTTTATTTTATTTTATAATTTTATTTTATTTTATTTTATTTTATTTTATATTTTATTTTTTTTGAGACGGAGTCTCGCTCTGCCGCCCAGGCTGGAGTGCAGTGGCCGGATCTCAGCTCACTGCAAGCTCCGCCTCCCGGGTTTACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGCGCCCGCCACCTCGCCCGGCTAGTTTTTTGTATTTTTTAGTAGAGACGGGGTTTCACCATGTTAACCAGGATGGTCTCGATCTCCTGACTCGTGACCCGCCCGTCTCGGCCTCCCAAAGTGCTGGGATTACAGGCTTGAGCCACCGCGCCCGGCCTATTTTATTTTATTTTTGAGATGAAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCCGTGGCACAATCTCGGCTCACTGCAAGCTCCACCTCCCGGGTTCACGCCATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTATAGGCGCCCACCACCACGCCCGGCTAATTTTTTGTGTTTTTAGTGGAAACAGGGTTTCACTGTGTTAGCCAGGATGGTCTCGATCTCCTGACCTCGTGATCCGCCCACCTCGGCCTCCCAAAGTTCTGGGATTATAGGCGTGAGCTACTGCACCCGGCCCGGGATTTTAAAAACTATTACACAGAAGGGTTTTTTAATTGAATATATTTGTGAAATGCTGAATTCAATACAGTTAAACTAGTGTCTTTACAACAAGCCTTCCTTCACAAACTCTTTGCTGTGCTGGTGTGCTCCACTGAGGGGAATAGTAGCGTATGGTGTTTTACAAACTGATTCTACTATTGAACCTTCTTTAGGTGGAATATTGGGTGCAAGTAGAGTTCTGTGGACCACACTTTGGGAAGTGCTGCCAGAGACTATCCAAAACTGTAATCTCCAGAGCACTTATGAGGCAGTCACAGCTTGAGGTGTGACGCTTAGTGTGTAACTGATACTGGAGCCAGGCTCTCAGGCTATGCTTGTGGGATGGTCCCCGCGTGCCTGTGGCGAGCTCTGTGGTGAAGCAGAGATTATCAAGGCTTTCCAGAGGAGGAGCAGGAACCCAGCTGCCCCGCATCACTCCGGAGGAGTAGGGAGTCAGCAGTCAAAGCCTTGAGATAATTGGGGGACACAGGATGTGATAAAAACCGGGTCTGTGATGTTGTTTGTTCTCTATGAGGCATCAGAGGAGGCTTCATGGAGGGAGTGACACTTGAGTTGGGTGTAGAAGGACAGTAGGAGTATGCCAAATGGGTGGAAAAGGAAGGAGGACTCCAGGCAGAAAGCAGCTCATGCAACACAAATGTGGAATCTTAGCTTTGCAAGTTAGCAGAGCCCGATAAATCAGGCAGAGTTTGGAGGGGTTGACGGTAGGTTTTACGCCAGGGGTTTGGAGGCTGCTGCAGGCTGGTTATTGGTGAGAGCAGGGAGGCTCCTGGATCGGCTGTGCTCCTAGCAGACCTTGTAACCTGGAGCTCCCAAGCTTCTACTGGTGTACACGCTGTTAAGTCTTGCAGTTAGTGAGCACTAATGCTTTTGGTTTTATATCCCCTGGGCTTGCAAACATTGACCAGAACTTAAAGGCATCTGCTTCTGATAGTGGGGAACTTGCTTCCTGCAGCAAGTCACTTCTGGGGTGGGGTATCGCTGATCCTTCCCCAGCTGACCTGTGTATTTGATCTCCGTGGTAGCGGATAAGGGAGTGGTGTGTGTGGGGTGAGTTGGCAGACTTGCCTCTGCCCTCCCTGAAGGCTGGATTTTCATGGAAAGAGTCTTGTGATCTTGCTGGTAGAGTTTGTTGTTATTGCTTTATAACATGTAAAGCTGCTTTTAGCTCTTACTGTATATGAAGCACGTTGCTTGCATCATCAGCTTTAGTCTTCTCACAGTTGAGATGGTATTATGGTATGAGGATATGGGTTTCATCCACGGACCCAGGCCTGTAACTCCTATCTATAGCCCTTGTTCTGGTTTCATGTTATAATGTTAGATGTGGTAGGCCTCAGGGGCAGGCCTCTGACCTTCTCCTGCTCTCCTTCCACCCTAATTTTCCCCACCTTTCTGTGGGGCCTTAAAACCCTTCCCTGAGACTGTGGGCCTTAAAACCCTCCCCTGAGAGGGTTGGATCCTGTATCCTGGGGGAAGGAATGCTGATGTCATGAAGCTTCCATAAAAATCCAAGAGGACAGAGTTCAGGGAGCTTGCAAATACCTGAACACGCGGAGGTTCCTGGAGAGAGGCCTGCTTAGGGAGGGCACAGAAGCTACACGCCTCTTCCCCCGTACCTCGCCCTAAGTATTTCTTTATCTGTATCCTTTGCGATAGCCCTCATAACCCAGTAAACATAAGTAAGTGTTTCCCTGGGTTAGGTAAGCTGCTCCATCAAATTAATCGAACTCAAAGAGGGGGTCATGGGAACCCCAGCCTAAAGCCAGTCGGTCAGAAGTTCTGGAGGCCCAGACTTGTGACTGGTATGTTTGAGGCGTGGTGGTGGAGAGGTGGGGCAGTCCTGGGGACTGAGCCTTCACCCCGTGGGATCTGACACTCTCTCCGGGTAGACAGTGTGAGAACTGAGTGAGAGGACACCCAGCTGGTGTCCGGTGCTTGGGGTGGGGAAGAAACCCCCTCACATTTGGTCACAGAAGTCTCTTGTGTTGATGATTATGGTGTGAGAGTAGAGAAAAAAGACAGTTTGAGACAGTTTTTCTCTCCAATAGGTGTCTTGTTTGTTGTACACATGAAGAAACCAAGGTTCTGTGATGAAATAACTTGCTTAAGTTTCAGCAGCCCAGAAATAATATAGTCACAATTTGAACCCAGCTCTGCCTGATTACAAAACCTGTCATAGGTAGTACTATACCATGCCCCATCCCTGATCCCTTTTTTTTTTCTTTTTTTTTCTTTTTCTTTTTTTTTTTTAAGAGAGAGACTCTCGCTCTGTCGCCCAGGCTGGAGCACAGTGGTGAGGTCTTAGCTCACTGCAACTTCCACCTCCCAGGTTCAAGTGGTTCTCCTGCCTCAGCCTCCCGAGTGGCTGGGACTACAGGCATGCACCACCACACCCAGCTAATTTTTGTATTTTTAGTAGAGATGGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTAGGTGAGCTGCCTGCCTCGGCTTCCCAAAGTGCTGGAATTACAGACATGAGCCACCGTGCCTGGCCCCTGATCTCTTTTCTTTTCCTTTTGGGTGGCTTTCCTCTTGTGCCTGCCACATCCCCTCTTTCTCGAACAGTCTGATCTGCCAGGCCATGGCAAGCCTGATTTCAGTACACTTTTCCTTTGCTGCTTTGGAAGAGTGAGAGTAGCATTTTCTTTGCAGTTTCCTTCCTCAGAGCTGTACTGTCTTTAAGCCCTGCACATATGGCTTCCATGCTGAGAAAACATCTCTCATAGCTGTGGCGTCATGCTGAGGAGGAATGTAGAAATGGAAGTGGGGTCACTTCCTTCCCTCGTCTCCAGAGGAATCATTAACATTGCACAAGAACTTGTTGGTTTAGGCACACATGTGGGTCCCTATCCCACTTTGGGTCGGTCTGTCTTCTGCCCACTCTTTGGGATGGTTAGTTCCTAAACGTCCGTTTTGGCATTTGGTCTTCATGTACCTTTTCTGGGTGTTCTTTCTGCTTTTCAGGACCTTCTGCCGCATACTTTGGTCCCATTTGGAAGTTTCTCGTGTGCTTGCTCGGGAGCAACACCTCATTTCTCGTCTAGAGAGAGTGAGCTTGCTGCTGGGTGGAGAACTGAGGGCCCTAAGATTAGGGTGACTGGGGACGCAGTGACAGACCTAGGCACGGTCAGCTGTGCACACGCCCCCTCATCCAGGAGGGCCTTTGCCCTTTGACTTCCTAGGATCGGTGGGCTCTGCTCAAGAAGTACCTTTCGCCATGGTGGGAACTCTGAGGGACCTGGCAGGGATCCACTTTTGGGGTCAGAATTCCCAGTCTTACCCTGGACACTACCTTCTGGTGTGTCTGCCTCTTTTTGAGCTTTTTTTTTTTTTTTGAGATAGAGTCTTGCTCTATCACTCAGGCTGGAGTGCAATGGCGCAATCTTAGCTCACTGCAACTTCCACCTCCCTGGTTCAAGCAATTCTCATGCCTCGACCTCCTGAGTAGCTGGTATTACAGGTGCCTGCCACCATGCCTGGCTAATTTTTGTATTTTTAGTCGAGGTGGGGTTTCACCATGTTGGTCAGGCTGGTCTTGAACTCCTGACCTCAAATGATTCACCCACCTCATCCTCCCAGAGTGCTGGGATTACAGGCATGAGCCACCGCGCCCGGCCCAACACTTTGTTTATTGTAAGTCTTTTTTTTTTTTTTTTTGAGACAGAGTCTCACTCTGTCACCCAGGCTGGAGTGCAGTGGTGCAGTCTCAGCTCACTACAACCTCCACCTCCCAGGTTCAAGCGATTCTTCTGTCTCACTCAGCCTCCTGAGTAGCCAAGATTACGGGCATGCACCACCACACCCGACTAATTTTTGTATTTTTTGTAGAGACAGGGCTTCACCATGTTGGCCAGGCTGGGCTTAAACTCCTGACCTCAAGTGATCCACCCACTTCAGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACTACACCCGGCCATATTATCACAGTCTATTTTTATGTGACTATCTCTTGCTCTCACTCAATCCCACACATCTTTGTAGCTTATCTTTCCATATAAGAATAACATTTTAGAGGTCATGAGACTTGACTCCAAAGTCCTAGAGGTCTTTGGTCAAAGGTCAAAAGAGGTGGGGCATAGTGGGAGGGCTGGGCTGGGAGGGCTCCGCCTCTCAGGTTCAAGCAATGCTCTACTTCAGCTTCCCAAGTAACTGGGATTATACATGGCCGCCACCACAACCGGCTAAATTTTTTTGTATTTTTAGTAGAGACGGGGTTTCATCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTTGTGATCCACCTGTCTTAGCCTCCCAAATTGCTGGGATTACAGGCATGATCCACCATGCCCGGCTGGGTTTTTTTTTTTTTTTTTTTTTTTAAATAGAGACGGGATCCTGTGTTGCCTAGGCTGGTCTCAAACTCCTAAGGTCAAGCGATCTGCCTGCCTTGGCCTCTCAAAGTGCTGGGATTACAGGTATAAGCCACTGAACCCGCCCCCTAACCAGTAGTGTTTTTATGTGGTCTTGCTCTGTCTCCCAGGCTAGAGTGCAGTGGACCAATCATAGGTCACTACAGCCCAAACTCCTGGGCTCAAGTGACCCTCCCGCCTCAGACTGTCAAGTAGCTAGGACTGCAGATGCATGCCACTGTGTCAGGCTAATTTTATTTTATTTTATTTTATTTTATTTTACTTCATGTTTTGAGACAAAGTTTCACCCTGTCACCCAGGCTGGAGTACAATGACATGATCTCAGTTCATTGCAGCTTCCATCTCCCAGGTTCAAGCGATTCTCCTGCCTCAGTCTCCCGAGTAGCTGGGATTACAGGTGTGTGCCACCAGGCCTGGCTAATTATTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAGGTGATCCACATGCCTCGGCCTCCCAAAGTTCTGAGAAACAGGGGTGAGCCACCGTGCCCAGCCTGAGACACTTCTTATCTCATCATCCTTTGTTCTAGTCCTGGAGATTGCAGTGACCTGAGATCCCCATATTCTTGATTCTCCCCCAAGCATTCCACCATCCTTCCAGCAGGATGTCCCGAGGTAGGTATCACCGTGTTCGCTCACATTGGCTTCTCTTGCAGCGCAATGTCCCTGCTCTTCTCTCGATGCAACACTATCGTCACAGTCAAGAAAGATAAGAGACACATGGCTGAAGTGAATGCTTCCCCACTCAAGCACTTTGTCACTGCCAAGAAGAAGATCAATGGCATTTTTGAGCAGCTGGGGGCCTACATCCAGGAGAGCGCCACCTTCCTTGAAGGTAAGGGGGCACCTGCTCAGCCAGGCCCACTCTTACCTGTTCAGATGTTTAGCTAGTGGGATTTATGGGTGGGGAAGTATAGCTCTGCTTTTAGGGAACTCATGCCCAGATAGCCTCAGAAATGCTCTGCTCTTGAAATCATGTGGAATTGGAGTCTGGGGAGATTAGGCCAGGAAGACAAGGGAACTGCCATTTAGTAATTACTTTAGAAGTCCACAGGTTTCGTTTCATTTAATCATGAAAGATAGGTGAATCTTATCCCCATCTACAAGGCAAGCAAATTGAGGTTTAAGAGTTGTCGGATACCTTGTAAGTAATAGATCAGGGCTTTGCTCAGGTGTCTCCAACTCCAGAGCCCACGTGTTTTCCTTTGCCTAGTTCTGGCTGCCATTAATACCAGGCACTGTTTGGGTGTCAGTGATCCCTGACCGGGGCGCCCTGTCTTTTATATTTTTAGATTGGTGACAGAACTATAGGAAACTCGTGTCACTTGTGCCTTTTCTACTAGTTACTAATTTGAGCCTAACCTCTAGTTCCTTGTTTATCAGCAGAGAAGGACTGGTTCCCGGGGCCCCGCTCGTCTGAGTGACAACTCCAGCGCCTGCTTCCTCAGGCTCCCTCTTTAGAAGGGCACTTTGGCGCCAGGTCCTGAAATTGTCTGTGGAGACCCAGTAGCCGAGTCTTATCTGCCCTTGGTTTCCTCGCTTGTGAAAGGGGGCTCAGGTGTGAACAGTGGTTTCGGATTCTAGTCTGCAGAACCCTAGAAGTTCAGTGGTGGGGCTTCCGGGGTTGGCCCCTGGCTCAGGGGGGCCACCTTCTTTCCATTCTGCTTTCAACGAGAGCAGCTTGGCTTCTCGTTGTTTTACATGCCAGGCTGCTGCACAAGGCTCCCTTTGAAGAAAGAGACTGTCCAATTTCATGCCCTCATCTGTGCCCTGCCCTTTGCATGGCACTTGATGTAACAGAAGGTATTTCCTAAATACTTGTTGAACTAAAATCTGAATTTTAAAGTAGTATGATTGGTAGTTGAAATAATCCCAACTCATTGCCTGTTTATCAGTGGCTGTGGTGTGCCAGCCCTGTGGCAGGTGCTGGGCATGCAGAGATGGGGTAGATCAGGCCACTGCCCTCTGGGAGCCCAGAGTAACCCTCTTGGAAGCTCTGAGTCACCTACTCAGATGACGTTGTGGACAGACACATGCTTAATGCAGATGTTTATGTGATGCATGCTTTGATTTCTCTCTGTGTGAAAACATGAGGCCTTGCCGGGCGCGGTGGCTCAAGCCTGTAATCCCAGCACTTTGGGAGGCCGAGACGGGCGGATCACGAGGTCAGGAGATCAAGACCATCCTGGCGAACACAGTGAAACCCCGTCTCTACTAAAAATACAAAAAATTAGCCGGGCGAGGTGGCGGGCGCCTGTAGTCCCAGCTACTCGGGAGGCTGAGGCAGGAGAATGGCGTAAACCCGGGAGGCGGAGCTTGCAGTGAGCTGAGATCCGGCCATTGCACTCCAGCCTGGGCGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGGGGGGGGGGGNNNNNNNNNNAAAAAGAAGGGGTCCAGGGTCCTGACTCAGGTTGCAGGGGTGGTCAGAGAAGGCTTCCTGGGGGCAGTACTGTCTAAGCTAGACTTGAGGTCAAGTCAGAATGAGTCAGGCTGCCCCAAGGCAGTGTAGTGTGTCAGTTCTTCAGAGAGTGGGAAGGGGTTTGGGCAGCGGGAGCAGAGCCAGAGTGCCGAGGAAGCTGGTGAGTTTGGGGATATGGTGAGGGAGGACTGTGAGGCAGCGGGGGGCAAGAGGCCAGCAGGGCCAGGGCAGGGAGGGCCTCGAGAAGTGACACAGAGTGGTTAAGAGCCTGGACTCCAGAGCCAGGCTGCCTAATTCAAATCCTAGCTCTGCCTTTTACTACCTCTGGAGCCACGTTAATAAAGAGGACCAAAGGAGAGAATGAATTTGAAAAGTGAAAGGAACAGTGCCTGGCATGGGGTAAGAACTTCGTAAATATTTGTTAAGAAAAAATAAACAAGGATGGCAGTTAACATTCAGGTATTCATCTTACACCAACCGCCATTGACAGCATTTGTATTTTAATTCATTTGATTCTCACAGTGGTCTCATGAAGTAGATATTGTCATTATCTCCATTTTGTGCCTAAGGAAACTAAGACCCAGAAAGGTTCTTTTGCTTGAGGCCACAGAGCTAGAAAGTGGTAAGAACCCAGCTTCAAAACCAGGCAGTGTTGCTTCTTAGCCATGTTCTTAACCACCACACCATGGGGCCACCATAGAGGATCTGGAACTCAGCCTGTCGGCTGCAGGGAGCCAGAGGTAGACTCATTTAGGGTAAGCAGGGCCGTTGCTCTGGCCCTTCCAGACTTATGACTGTGAAACTCCTCTGACCACGTGGTGACCCATGTTCCATCCCCACCTCCAGACACCTACAGGAATGCAGAACTGGACCCCGTTACCACAGAAGAACAGGTTCTGGACGTCAAAGGTTACCTATCCAAAGTGAGAGGCATCAGCGAGGTGCTGGCTCGGCGGCACATGAAAGTGGCTTTTTTTGGCCGGTATGTCCTTGTGGCACCCACCCTTTCTTTCTTCCTGGCTAGGAGGGAGGGAGGCACTTTGTGCAAGGCCACAGAACGTTCCAGGGCAGGGACACGCTTCAGAATTCATTGCTTTCCTTTTCATTTATATTTTATTTATTTGATTTTTGAGACAGCATTCTCTGTCACCCAGGCTGGAAGCTCGTTACAGCCTCGACCTCCCTGGGCTCAGGTGATCCTCCCACCTGAACCTCCTGAGTAGCTGGGACTGCAGGTGCATGCCATCAAGCCTGACTACTTTTGATTTTCTGATTGTGGTTAAAAGGATATAAAATAAAATTCACCCCCTTAACCGTTTTAAGCATATAGTTCACTAGTAGGTATATTTGCAACCAGTCTCCAGAGCTTTTTCATCTTGCAAAATTGAAGCTTGTATTATACTCATTAAACAATAACTCCCTGTCCTCACATGTCCCTCACCCTGGCCACCACCATTCTACTTTCTGTCCCAATGAATTTGACTATGCTAGGTATCTCACGTAAGTGGAATCATACAATACTTGGTTTTCTTTTTTCTCCATTCATAAAACTTTTATTCCACTTACATGAATTTAATACACATGTTCTAAACAATTATGCTTGTATTGTTCATGAAAATTTCGTAAGACATTAAACAAAGCTAGCCATCATCTCAAGTTATTTCCCTGTTAACTATTTTTACAGCACATGCATGTTAGGCAAGCATCAAAAAAAAAAAAATTCACAAAAGCATAAAACCTAAAAATAAGTTAAATACATGTTTTTTTGTTTTGTTTTAGTGCTATGCTTGTTGATACACATGAAGTAATGAATATCAAGCAATTCATTTTTATTACATCTTTACTTTTACATTTGTTCTTAGGTTGCCTAAAGCATTTTAAATACAAATAAAATGAGTGTAGCAAAAATAATGAAAGCTAACAGAAGATAACTTTACAAATAATGGAATGTGAACCGTTTCTGTCCTTATCCAGAGTAAAATGGGCCACAGCTTTGTCTAAAGGAACGCTTCCACAGCGGTAGTCAAAGGTGTTCACATTGAGATTGAGTATTCCACAGATACACATGGTTTAACATGTGATATCCATGGGGTATCTGTTTCTACCACAGCCTTGTAAGTGCCCCAAACCTTAAGGTACCCACAGTTACTACTCCTGTGACTGGAACCAGTGATCCTTTTTATTCCCCACCAGGACAAACCAATATATAGGCAGTTTTCTTTGCTTTGACATGGAAGCAGTTTTAACACTGGCCCTTATGAAGCCACAATGTACCCAAAGTACTATGCCAAACATTTATAACTGGTATAATGGCCAAGTGCGGCGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGTGGATCACAAGGTCAGGAGGTCAAGACCATCCTGGCTAACGGTGAAACCCCGTCTCTACTAAAAATCCAAAAAAATTACCCAGGCATGGTGGCAGGTGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAATGGCATGAACCCAGGAGGCAGAGGTTGCAGTGAGCTGAGATCATACCTCACTGTACTCCAGCCTGGACAACAGAGCGAGACTCTGTCTCCAAAAAAAAAAAAGTGTACACACACACACACACACACACACACATACATCTAATACAGGAAAAAAGACATCTAGACAGATTGTATGCTAGGCACCTAGTGGTAGCCTGGGGAATAGGCACTGTGCCTAGAGTCTTGTTCCTGATGACCTGTAACCTCAGGTGTGGTCCTCAACTCTGAACTATCATTTCTTTATCCTCACCTGGGAGGTATGACCCCTACTTAGGTGAGCTAGTGGGCTGCCTGTCTCAGTGGGTGCAGGTAAGTTCATTGTAGTTCTGCTCTAGTGATGTGGCTGGGAAACTTTAACTCAGAAACACTGTATTTAGCACAGGTTGAAAGCTCTGAGACTCAGAGCAGATGAGTCTGCTGAGTGGAGCTATTTTCAGGAAGCAGAAATGGCCGGGTGCAGTGGTTCACACCTGTAATCCCAGCACTGGGAGGCTGAGGCAGGAGAATTGCTTGAACCCAGAAGTTTGAGACGAGCCCAAGCAACATGGCAAGACCCCATCTCTACAAAAAATTTAAAAATTAGCTGGGCGTGGTTGCACGTACTTACAGTGTAAGCTACTTGGGAGGCTGAGGTGGGAGGATCATTGAACCCTGATGGTCGAGGCTTCAGTGAGCTCTATTCATGCCACACGCCACTGCACTCCAGCTTGGGCGACAAAGCAAGACCCTGTATCAAAAAAACCCAAAAAAACCCCACCAGATTACATGAAAGGAGTGTGATAGGTAAGAATGAGAAAGTAGGCTGGGCATGGTGGCTCACATCTGCAATCCCAGCACTTTAGGAGGTTGAAGCAGGAGGATCACCTGAAGTCAGGAGTTCGAGACCAGCCTGGCCAACATGGTGAAACCCTGTCTCTACTAAAAATACAAAAGTTAGTTGGGCATGGTGGTGCACGCCTGTAGTCCCAGCTACTCAGGAGGCTGAGGCAGGAGAATCGCTTAAACTCAGGAGGCAGAGGTTGCAATGAGCTGAGATCATGCCACTGCACTCCAGCCTGGGCGACAGAGCAAGACTCCATGTCAAATAATAATAAAAACAAAGAATGAGAAAGTAATCTTCCCTTTAGCCTAGCAGTTGTCAGCTGGGACTGACAAGGTAAAGCTTCATGTGTAAGGAGGAGCGTGATGGGAGAAAAGCAATGAAACATGCCCTGTGTGGGAAACACTGGTGTCATTTAGACTGAGAAAGACGCTTTGGGGGTGGTGAGTCGGTGTTTCAGGTAGGCCTGAGTTAAACTTTGGAGACTGTCTGACTGTCTCAGACAGTCTCTATAAGTGATGCTGAGGGTGGCTGTATGATTCAAGGGGCAGACAGTGCGCAGTGAGCCTCTTGCAGATTAATCTGAAATGATGAACCAGGGGTATGTCACAGGTTTGGGCCTGTGGGTGTTTATTCATTTTAATAAACAGTGCCTACTCTGTGTCTCAGCACTGTCTGGGCACTGGCAACGTTGCAATGAATAGGGCTTTGGCCTTCCAGTCTGGTGTCTGCATTGTGAAAGTAATTCAGGTCAGATATTGGTGGCTTTGCTGACAGCTGTTACTTCCTTCTAGGACGAGCAATGGGAAGAGCACCGTGATCAATGCCATGCTCTGGGACAAAGTTCTGCCCTCTGGGATTGGCCACACCACCAATTGCTTCCTGCGGGTAGAGGGCACAGATGGCCATGAGGCCTTTCTCCTTACCGAGGGCTCAGAGGAGAAGAGGAGCGCCAAGGTGAGGGTGCCAGGCTGGCTGTCAGCCCTGTGCCTGCTGGCAGAGTAAGTCCTCCATTGTGACATGGCTGACCTCACCTCTAGGGAGAGGGCAGCACTGCCCTGTGGAGGTGAATGGGAGACCCTATTTTAGACAAGCTTTTTGAAGGATGTGAGAGCTTCTGGGGGCTGGCGGGGGCGGTTTTCAGAAATGTGGATCACCAAGAATGAGGACCCTGCTGTTCCTCATACAGCTCTGAGCGGCCACTGGCCTCTCTTCACTAGTTAAGGAAGACAGTATGTGCTGTGTTCTTCCTTGGGGTATTGCCAGAGAGGGATTCCTGCATTTGCTCTGTGTCTCTGACCTATCATTCAGGGGCCGACACTCTTGAGTTTTCTGAGAGGCAGCTGTATTCTTGTTTGGGGCCAGATGCTCAATTTGCTTCATAAAGGATCAATCGGCCAGGCACGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGACCGAGGCAGGTAGATCATTTGAGGTCAGGAGATCAAACCAGCCTGGTCAACATGGTGAAACGCTGTCTCTACTAAAAATACAAAAATTAGCTGGGCATGGTGGCAGGTGCCTGTAATCCCAGCTAGTCGGGAGGCTGAGGCAGGAGGACTGCTTGAACCCGGAAGGTGGAGGTTGCGGTGAGCCAAGATCACGCCACTACTCCAGCCTGGGCGACAGAGCGAGACTCTGTCTCAAAAAAAAAAAAAGAATCAATCAGTGCAGAACAGACGACTTTAGCGTGATATCCAGGAAAGAAGCTCAAAAGAGCAGTGAAGAAAGTAGGTTCTTAACTGACAACTCCCAGCTGTTAACTTTTTCTATGTGGAAGAAGAAAGGGTAGCAGATGGGCAGCAGTCCCAGCCACTGTGCTGTGATGCAGCGGCACAGGAAATCTGGCTTGGTGTTTCCTCCTCAGCCTCTTTCTTTCCTTCCTCTGCCATCAGACTGTGAACCAGCTGGCTCATGCCCTCCATCAGGACAAGCAGCTCCATGCCGGCAGCCTAGTGAGTGTGATGTGGCCCAACTCTAAGTGCCCACTTCTGAAAGATGACCTCGTTTTGATGGACAGGTAAGAGGGAGGTACCCTCCTAGGAGGTCCAAACCGGGAGGCGCCAGGTTTCCATTTCTGGGTAATCCGGGCCAGAGTCCCTGGGCCCCATCCATGCTCTACTTAAGTACTACTGCCTTTCATATGGGCTCAACCAAATCCTCTGGCCTCTTGGCTTTCCTCTAGAGTCTGGTACCTATACCTGTTTACCAGTACCCTGCAGATGTCCCAGGGATGGTCTGTAAGAGATAGGAGGGATGCTTAAAAGTTGGGGAGGTTCTCAACCTCAGCCCAGTGCCAGCACGTGTACTGGGGGTGGGGAGTTGTGGCGGAGTCAGGGGTGTATTTTATCTCCCCTTGAAGAAACACTTTGCCCTGGCTTGGTGGACAGGTTATAACTATACCAAGAAGTGCAGTATTGTAGCATTTACCTGGACTCACATCAAGTCACGCCTTCACCTTAATTATATATTGTATATTGTATGTCATCTTTTTTTTTTTTTTTTTTTTTTTTAGATGGAGTCTCACTCTGTCGCCCAGGCTGAAGTACAGTCTCGACTCGCTGCAACCTCCGCCTCCCGGGTTCAAGCAATTCTCCTGCCTCAGCCTCTGGAGTAGCTGGGATTACAGGCACCCTCCACCATGCCTGGCTAATTTTTGTTTTTTTAATAGAGACCGGGTTTCACCGTATTGGCCAGGCTGGTCTCGAACTGCTGACTTCTTGATCCACCTGCCTCGGCCTCCCAAAGTGTTGGGATTACAGGCGTGAGCCATTGCGCCCGTCCTGGTATATCATCTTTAAAGTAGAATTACAAGTAATTCTGTTTTTAAAAAAGCAATAGATGTATGTGTTTTTTAAAAACACTCATTTCCTCCACGCGTGGTGGTTCATACCTGTAATCCTAGTGCTTTGAGAGGCTGAGGTGGACAGATCAGGAGGTCAAGTGATTGAGACCAGCCTGATCAACGTGGTGGAACCCCGTCTCTATAAAAATACAAAAAAATTAGCTGGGCATGGTGGTGCATGCCTGTAGTACTAGCTACTTGGGAGGCTGAGGCAGGAGGATCACTTGAACCCGGGAGGTGGGGGTTGCAGTGAGCCGAGATCTTGCCATTGCACTGCAGCCTGGGCGACAGAGCAAGACTCCATCTCAAAAAACAAAACAAGGCCGGGCGCAGTGGCTCACGCCTGTAATCCCAGCACTTTAGAAGACCGAGGCGGGCGGATCACGAGATCAGGAGATCGAGACCATCCTGGCTAACACGGTGAAACCCCGTCTCTACTAAAAATGCAAAAAATTAGCCTGGCGTGGTGGTGGGCGCCTGTAGTCCCAGCTACTTGGTAGGCTGAGGCAGGAGAATGGTATGAACCCGGGAGGCGGAGCTTGCAGTCAGCCAAGATTGCGCTACTGCACTCCAGCCTGGGCGACTGAGCGAGACTCCATCTCAAAAAACAAACAAACAAACAAACCAAAACTCTAATTTCCAGGCAATCTGGAAGGATAGGAAATGGAAAGTAAAAATCGGTTAATGAGGGCCGGGCCTGATTTCTCTCCCCTCACTGACTTTCTCCTCCATGACCTGCCTGCCTCACAAGTCTCAGGTCTGTTCTCAGCAAGAAGAATAGGGCTCCTGCTCTGCCTGATGATTTGGTTTACTCCTGTCACCTTTAGCCCTGGTATTGATGTCACCACAGAGCTGGACAGCTGGATTGACAAGTTTTGTCTGGATGCTGATGTGTTTGTGCTGGTGGCCAACTCAGAGTCCACCCTGATGCAGACGGTAACTCCTCCTCTGCCTTCTCCCAAGCTCCCACACCCCCTGGGCAGGCAGCTGATGGGGCCTTGGCTGTCAGGCTCCCGCTCCACCGAGGTCTTACCCTTTGTCTAGGAAAAGCACTTCTTCCACAAGGTGAGTGAGCGTCTCTCCCGGCCGAACATCTTCATCCTGAACAACCGCTGGGATGCATCCGCCTCAGAGCCTGAGTACATGGAGGAGGTTAGTGCCTCTGTTTGGCAGTTGGGGAATGCCACCCCGAGGGAGCACTGCCTGCCACTAGGGCCACTTCATGCCCTCCTGGATCTAGTGACTTCCCTGAATTAACTTTATTGCCAAATAATTGTCCAAATACTCTTCTATGGTATCTTCTGGTGGCTTTTCCCCTCTGCCATCCACTTTGCTGGACGCACCCCCTCTTCAAGCCACTCTCCCCTAGGCTTAAAAAGGATCCCTGTAGCATTAGTGCATCTTCGGGACCAGTGCTCAGTTTGGTTTCTGAGGAATTAGTATGGAACAGGCTCAGCTCTAGGGAGGCCTCTGTGAAAGAGGCTGGAAAGAGGAATGGAGAATACTGATTCTAACCGGTGACTCACAACTCTTAGCTGTTTACTTATTTATTTAGAGACTGGGTTATGAGGCTGCCTAATTTTTGTATTTTTGGTAGAGACGGGGTTTTGCCATGTTGCCCACTCTGGTTTTGAACTCCACGGCTCAAGTGATCCACCCGCCTCAGTCCCCCAAAGTTCTGGGATTACAGGCATGAGCCACTGCACCTGGCCTCACAACTCTAAATATGTGAAATGAAAGACAAACTGGGCATGGTGGTGCACACCTATAGGCTGAGAGAGGATCCCTTGAGACCGGGAATTTGAAACCACCCTGGGCGACATAGTACAACGCTGTCTCCAAAAAAAATAAAGGGTAATAAGATTGATAATATAGGATGATAAAACATGCAGTTGCAGTCAATAGGCCGGAGTCCAAAACCTTTTTTTTTTTTGAGATGGAGTTTTGCTTGTCACCCAGGCTGGAGTGCAGTGGCGCAATCTCGGCTCACTGCAACCTCCGCCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCAAGTAGCTGGGATTACAGGCACCTGCCATCACGCCTGGCTAATGTTTGTATTTTTAATAGAGATGGGATTTCACCATGTTGACCAGGCTGGTCTCAAACTCCTGACGTTAGATAATTCGCCCGCCTTGGCCTCCCAAAGTGCCAGGATTACAGGCATGAATGAGCCACTGTGCCTGGCCCAGAGTCCAAATTCTGACTGACACTAGGCAGGCAGTGTGACTTTGGCAGATCCCTTGACCCTTCTGGTAAATCCAATTTCCTCAGCAGTGTGATAAATAGACTTGTTGAGAGGATGAAATCTGTCAAAAACACCTAACACATGGTAGGTGTCTACAAGAAGCTGTTGTCACATTTTATCGGTAGAACCCCAGGTGTCAGAGCCAGGAGGGATTTTCTAGTGATGCCCACTCTTTTTGTAAACAATGCCCCAGGAGGACACGGCTTGTCAGAGTACAGCTCCCAGATTTAGGTTACTTGACCCCAGGGTTCCTCTCCTTCCAGGGCACAAAAGTGAGGCACTATATAAGCTTAACAAGAGGCAGTCTAGGGCATGTTTAAGCATTTCTGCTTGTATATGCATAAAATACCTTTGGCAGGCTCTACGCCAAGTCACCCCCACACATTGGTCTGGGAAACTTGGTGGTTGGAGAGCAGAATAGTGGGAGACATTGCCCTGTGTATGTGTCTGTGCCTTTGGCTGTTGAGCTATGTGAATATATTGCCTATTCTTTTTTTTCTCACTCTGTTGCCCAGGCTGGAGTGCAGTGGTGCTATCTTTGGCTCACTGCAACCTCCGTCTCCAAGTTCAAGCGATTCTCCTGCCTCAGCCTCCTGAGTAGCTGGGGTTACAGGCGCCCGCCACCATGTGTGGCTGATTTTTTGTGTTTTTAGTAGAGACGGGGTTTCACCGTGTTGGCCAGGCTGGTCTTGAACTCCTGACCTCAATGATCCACCAGCTGTGGCCTCCCGAAGTGCTGGGATTACAGGCGTGAGCCACCGTGCCCAGCCTCTTATGACCTATTCTTTTAATGAAAGAGGCAGGTGGGGGCTGTGGGGCCACCTGCACCCACTCTGGACACATTTGTTTGGGCTCCAGGTGCGGCGGCAGCACATGGAGCGTTGTACCAGCTTCCTAGTGGATGAGCTGGGTGTGGTGGATCGATCCCAGGCTGGGGACCGCATCTTCTTTGTGTCTGCTAAGGAGGTGCTCAACGCCAGGATTCAGAAAGCCCAGGGCATGCCCGAAGGAGGTAATGATGAGAACAGATGTCCTTCTTTTCCCTGATGTTTGAGACATTTTGTCTCGTGCTGAGTCTGTCAGTAGAAAATCCTTCTGAGAAGAGGCTGCTGAGAAGAGCAGAGTGAGAGGCTCTTGCTCTTCAGCCAGTGGCTTGGTTTCTGGGGATTTCAGCCTTTTCCTCTGCCGCCAAGTTGTTTCTGGGCTAATGCAGTACAATTCTCCTAGGGGGCGCTCTCGCAGAAGGCTTTCAAGTGAGGATGTTTGAGTTTCAGAATTTTGAGAGGAGGTTTGAGGTGAGTCCTTTGATTCTGGTACATGGTGATTCTGTGCCTTCCCAGCTCCCACTGGCTGTATCCCTGGCAGTGAAAGCCAGAGTCTGGCCCTTGGCCATAGGCCCCTGGTGGCCCCCACCTCCCTCTGTGCCTTTGTGTGTTCCAGGAGTGCATCTCCCAGTCTGCAGTGAAGACCAAGTTTGAGCAGCACACGGTCCGGGCCAAGCAGATTGCAGAGGCGGTTCGACTCATCATGGACTCATTGCACGTGGCAGCTCGGGAGCAGCAGTAAGAGTCCAAGACTGCAGATAGGTGGAGAGGGCTGCCGAGATGAGGTTGTTCCTCCCCTGCCTTGGGCAGTTAGGACATGCCTTGATGGCAGGGCTGAGTCTCTGGGCTCCCATCCAGGGCCCTTTCCCTGGCCACCAGACCATGTTAGAGCCACATAGACAGCCTGGTAAGGAGGGCGGCTGGGTGCTTCATCACCCCACCTGGTCTGTGCAGCACGATTCCCTGTGTTGGAGGCCTTGGCCCGTACCCTGCTGAGCTCTGCAGCCATTCTCAGAGCCTGAACACATTTTTAAAAACAACATTCCAGCCTGGAGCAGTGGCTCACACCTGTGATCTCAACACTTTGAGAGGCCAAGGTGGGAGGACTGCTTGAGGCCAGGAGTTTTGAGTCCAGCCCAGATAACATAGTAAGACCCCATCTCTACAAAAATTTTAAAAACCAGCTGGGCGTGGTGGCCGCGCACCTATAGTTCCAGCTACTTTGGGAGGCTGAGGCAGGAGGATCACTTGAGCCCAGGAGTTCAAGGTTCCAGTGAGCTGTGATTACACCATTGCACTCCAGCCTGGGCAACAGAGTGAGACCCTGTCACTAAAGAATGATATGAAAACACCTTGCTTCTGTCACACAGAATTGACTTTCTTGATTGTTTATTTTATTTATTTATTTATTTTTGAGACGGAGTCTCGCTCTGTCGCCCAGGCTGGAGTGCAGGGGCGCGATCTCGGCTCACTGCAAGCTCTGCCTCCCGGATGCACACCATTCTCCTGCCTCAACCTCCCGAGTAGCTGGGACTACAGGTGTCCACCACCACGCTCAGCTAATTTTTTGTATTTTTTTAGTAGAGACGGGGTTTCACCGTGTTAGCCAGGATGGTCTGTTGACCTTGTGATCCACCCACCTCGGCCTCCCAAAGTGCTGGGATTATAGGTGTGAGCCACTGCACTGGGCCTCAATTGTTTATATTAAGTTAAAATTTTAAAAAGTTAAGTTCCCTTTTGATTCTTTTGCACTTGTCCCAGTGTCACTCTGCCCAGAGGCACCATAATCATGAATTTACATTGTTCCATCACCCATTCAAACAGTACTTTCACATAAATGCTGAAGAATATGTCAGGTTTTTACATGCTTCAGACGTACAGATGTGGCATCACATTGTCACTGTACCTGGCTTTACTTTCTACTCAACATCGTTTTTCCCTTTATCCTTGTTAGTATGTGTAGTTTGAGTTTATGAATAGGAACTGCTGTAGTATATTCCATTGTGAATATGCCTCATTTTCCTATGGAGGGAATTTTAGATTTCTTTTTTGCTATTGGAAACCTTACTGCAGTGGACATGTGTTTATGTGTCCCCTTGTGCACCTTTGTAAGAGTCTTCCTGGGGTGAATTCCTAGGGGTGTGGCGATTCTGTTTTAGGGTATACGCGTTTCCAGTTTTAGTCAGTTTTACTTGATTGCTCTCCAGAACAGCCGTACCCATTCGTTGCATGACCAGTACTGTCCATAGGCCTTCCATTTCCTCAGCTCTCACCAGAACTTCAGGCCTGGTCATTTTTGCCAGGCTTTGGTAGAAAATTGTGCCTTTAAAAATTTCATTTCCGACTGGGCATGGTGGCTCACGCCTGTAATCCCAACACTGTGGGAGGCCAAGGCGGATGGATCACCTGAGGTCAGGAGTTCAAGACCAGCCTGACCAACATGGAGAAACCCTGTCTCTGCTAAAATTACAGAATTAGCTGGGTGTGGTGGCACATGCCTGTAATCCCAGCCGCTCGGGAGGCTGAAGCAGGAGAATTGCTTGAACCTGGGAGGCGGAGGTTGCAGTGAGCGGAGATCGCGCCGTTGCACTCCAGCCTGGGCAACAAGAGTGAAACTCCCATCTCAAAAAAAATAATAAATTCATTTCCGTGATTACTAGTGAGGTTGAGCTTTTTTCCCTATATTAATTGGCAGAATTTGTTTAAACCCCCTTACAACACCCTCCCTGCTCTGTCCCCACCGCCTGTCCCACATGGAAGACTGAGGAGTGCATGGTTGGCTGCGGGGCTCCCTTCTTACCTGGGAAGGGGAAAGCTCATGCCCCTGGGTACGTGTGTGCAGCCCTGCCAGGCAAGAGAGTGGGCGGGGCAGCCTGGGACTTCTGGTATCCCCTCTTGCTCCTCTGCTTAGTCAGACAGGAACATGGATTTCTCACCAGTACTCTGCTTTCAGGGTTTACTGCGAGGAAATGCGTGAAGAGCGGCAAGACCGACTGAAATTTATTGACAAACAGCTGGAGCTCTTGGCTCAAGACTATAAGCTGCGAATTAAGCAGATTACGGAGGAAGTGGAGAGGCAGGTGAGAAATGAGGAGGACGCATTCTGGGGAGATTTGGACTCCCGAGTAGAGTCCAGAAGAAAGCAGACCTCCTCTTAGGGACTTCCCAGCCTTCCAGAAGGAAGTTGTGGCCCTGCTTCAAGAATACAGAGCTGCCCTTTGGGTTCTGTTATTGGGTTGTGTGATGCTGGGCATGTTCCCCTTCCTCTGGACCTCCACAGATCACGTGAGCGTTTGATCAGCCAGTTTCCCAGACCCTCTCACCTCAGAGGCTTTAACTCCATAGAGGAGCTGAGGAGGCTGCTGGTTTGAGAGGAAAGATATGCCATCTGCTGGACTGAACTTTGGTCTTCCTTGATACTTAACAGTGTGCTTCCTTTTGCCGTAGGTGTCAACTGCGATGGCCGAGGAGATCAGGCGCCTCTCTGTGCTGGTGGATGATTACCAGATGGACTTCCACCCTTCTCCAGTAGTCCTCAAGGTTTATAAGAATGTGAGTCATGGAGCAACAGGTCCTCTTGGCAGGAGGCCCCCAAAAGTGATTCAGTCCCTGCTGGTCTGGGTCAGGGCCCCCCAGCAGTGAAAGTGGAAGCCACAGAGACGAGGCCCAGGCTTCCATCAGCCTTCTGGGGTCACCTGGTGGATGTGGCCTGAAGGGAATTTTGATGGACGTGCTGCTTCTTTTAACCTCCCTCTTCTGGTGGCAGGAACTGCACCGCCACATAGAGGAAGGACTGGGCCGAAACATGTCAGACCGCTGCTCCACGGCCATCACCAACTCCCTGCAGACCATGCAGCAGGACATGATAGGTCAGTGCCCATAGGGAACTGGGGAGCTGTGGCCTGGGCTGCCCGAAGATCAGATACAGAGGCCAAGCTAAAGAAATCAGGACTTTCCTTATCTGTCACTTTTCATGATGATTCAACTCAATACCTTCAGGTTCTGGGTACGTGTTTGGTTTTTGAGACAGAGTCTCATTCTGTAACCCAGGCTGGAGTGCAGTGGCATGATCTTGACTCACTGCAGCCTCCACCACCTAGGTTCAAGCGATTCTTCTGCTTCAGCCTCCTGAGTAGCTGGGACTATAGGCATGACCATCACACCTGGCTAAATTTTTTGTATTTTTAGTAGAGATGGGGTTTTGCCATGTTGGCTGGGCTGGTCTCAAATTCCTGACCTGAAGTGATCTATCTACCTTGGCCTCCCAAAGTGCTGGGATTACAGGTGTGAGCCACCAGGCCTGGCCTTGTGAACTCATTCTGTGCGCAGGGTTGGGGAGCCCATCTGGTGCTACTACATGGGTCTATCCTAGGCTGAGAATTGGGCCTGCTCCTATAAGTCCATGTGAGGCAGAAGTGTTATCAGTATGGCATCTGGGTCCACACTTGCCTGGGGGAGCTGGCCTCAAGTGAGGCATGCTCAGTCTCATGGGCCAACTTGACCGGGGTGTGGTTCCCAGGCAGAGCTATTCAGCATGCCTGGCAGTAGCTGGTGGAGCCGTCTCCAAAGCTTGGTGCTGCTGTGGTGCAGGGCTGAGCTGATAAGCTTTTCCTCCATTTCTCCCCCGATAGATGGCTTGAAACCCCTCCTTCCCGTGTCTGTGCGGAGTCAGATAGACATGCTGGTCCCGCGCCAGTGCTTCTCCCTCAACTACGACCTGAACTGTGACAAGCTGTGTGCTGACTTTCAGGAAGACATTGAGTTCCATTTCTCTCTCGGATGGACCATGCTGGTGAACAGGTTCCTGGGCCCCAAGAACAGCCGTCGGGCCTTGATGGGCTACAATGACCAGGCAAGCAAAGCTCCTCACCTCAGGGGCATCGTGGGGGATCAGTCTGTACCCCGCCTCCAGACACAGGAACCATTCTGAAACAAGGTAAAGGTCGGCAGCTGTGGTGGTGTGCCCCACCACACACTACACCACAGCCAGAATTTCTGCTGCTGATTGAGCTGATCTGTTCTAAGCAGTGATATTTGTGAAATGACAGATGCTAGTATTTTTCTCTAATACATATTAAAGTAAGTATGTAACTATTAAAAATAGATGACTTTAAAAATAACCACCTACATACAACTATTTTAAAGTTCAGATTTTAAAAACATTCATCTCTAAACCACTTGAGTTGTCACACGTACTGCCCTCTGTACACATCCCACGCTCGGGGAAGCTGCCAGGCGGGCCAGGATGACTCCTGCTCACATTTTGCACATCCTGTGGTGTTCACCTGAGGGTCTCCTCTGCTACATCCGAAGCTAGTTGTCCTCTTTTTGCCTTTCGGAAATTGAAAAGCCACTCTACGTCCCTGTTCCCCAGACTAGGGCGACACTGAGGGTTCACGTGAATGAAAGACTCACCACGTCCCTGTCACCCCTCTCATCTTTCTCTCCTCAGGTTCAGCGTCCTATCCCTCTGACGCCAGCCAACCCCAGCATGCCCCCGCTGCCACAGGGCTCGCTCACCCAGGAGGAGTTCATGGTTTCCATGGTTACCGGCCTGGCCTCCCTGACATCCAGGACCTCCATGGGCATTCTTGTTGTTGGAGGAGTGGTCAGTGACCAGTTCAGCTTGGGAAGGTGGGGGTGGAGGGCAGGTGGGTGGGGCCTGAGGGCTAGGTTCCTGTGGTGGCCCCTGCGTTGGGCCACACTCTACAGACCACTGTATACTTTCTCCTTTCAGTGACTCTTCACTGTGATGGCATGGAGAGTGCTTACTCCTTCACCAAGTTTCCCTCACTTCCCGCTGGAGATGGTCCCAGCCTTTCTGGCCTCAGTGGGATCAAAGGAGACATAGAAAGAGGAACTTAAGAGTAGAAACATGAGGGCTCGTTGGCTGGGGCCCTTCGGACGGCCCTGGTAGTGACGGACCCTAAAGGAGGGTGGGCAACAGAGAGGCTGTCCTCCAGGCTGACTATGTGCTTTGCAGGTGTGGAAGGCAGTGGGCTGGCGGCTCATTGCCCTCTCCTTTGGGCTCTATGGCCTCCTCTACGTCTATGAGCGTCTGACCTGGACCACCAAGGCCAAGGAGAGGGCCTTCAAGCGCCAGTTTGTGGAGCATGCCAGTGAGAAGCTGCAGCTCGTCATCAGCTATACTGGCTCCAACTGCAGCCACCAAGTCCAGCAGTGAGTGGCCCCATCGGACCCCAGCAGGAGACTTTCTTTAGGCAGCGTCAGCCCCATCTCCCTTCCCCATCTCCCTTCCAACCTGGCCTGGAAGCCACTGGGTCCTGAGCATCATAGACCCTGGGCCTTCAGGTGTGCAGGGCCAGCTTTGAGGCCAGTGATGGGAAGGGACACTTTGTGTCTTGGGCTGGGACTGGGTAGGCCTCTGGGCCCCTTGGTGAGGAAGAAATGGGAAGGCAAGAGATATTCCTAGTAGGAGTTTTAGAGGTTGCCTTGAAGAGCTTGGGCTGTGGATGTAGCTTTGAAGTTTTGAACCCAGCCTGATCTTCATGCACATCTTAGGTCAGGTGGGAATCGATTGCTTAATCTGTGGTGGTCTGGACGAATAGGCTGGCTTCCTTTTTTTGGTTGTGTGTGGCAGACCCTTTGCTCTAGACATTTGGCATCGAATTTATGTATTTCTCTGATTCTTTGAGGTAGATGTTGTCTTATAGGCATCTCCTTTTTCAAACTGAAGAAACTGAGATTTGAAAAACAGTCACGTGTTCCCTGCTACTCGGCTGATGAGCAGCAGATCAGGATCTCTGCGTGCTGTTGCTTTTCAGGCCGTGGTGATGCCAAGTGTGTTTGGTGGTTATGATTATTCACAGTCTCAGCAGGATGTAGGAGATTCTGCCAAACCAGGGCTGGCTCAGGGCAGAGCTGCTGGCAGGGATATCGACGGGCCCAGCTGCTTCCCACTCTGGGCGGGTTAAGCCACCAGTGGCATCTTGCTCCACACACCCCAACTGATCCCTTCTCTGTCCTCCCCAGGGAACTGTCTGGGACCTTTGCTCACCTGTGCCAGCAAGTTGACGTCACCCGAGAGAACCTGGAGCAGGAAATTGCTGCCATGAACAAGAAAATTGAGGTTCTTGACTCACTTCAGAGCAAAGCAAAGCTGCTCAGGTGAGGCTGGCCTGGGTGGCCAAAGGAGGGTTCCAGGGTGCAGCACAGGCGCACTGGGGCTCAGCTGCTGGGCTTGTGTCTTGGGTGTGGACACAAACTTCTCTGGTGGGGATTTAATTTATTCGTGTTAGATGTGTACCACAGGCCAGGCACAGTGGCCAACACCTGTAATCCCAGCACTTTGGGAGGCCAAGGCAGGTAGATCACCTAAGGTCAGGAGTTTGAGACCAGCCTGGCCAACATGGTGAAACCCTGTGTGTACTAAAAATGCAGAAATTAGCTGGGTGTGGTGGCGTGCACCTGTAATCCCAGCCACTTGGGAGGCTGAGGCTGGAAAATGGCTTGAACCTGGGAGGCGGAGGTTGCAGTGATCAGAGATCGCGCCATTGCACTCTAGCCTGGGCGACAAGAGCAAAACTCTGTCTCAAAAAAAAAAAAAAAAAAAAATATGATGTGTACCACGTACCTCATGCGTGCCAGATGGTGCTGGTGCTGGAGGCTCAACAAGACAGCATCACTGCCTTCAAGGCACTTGCAGCTGAAGGCATGATGGCATCACAGTACTGCGAGACTGCAGTTCACAGCGCCCTGTGCCGAACCGCAGGGCCTACAGTGCTTTACATGTGCTGTTTCACTCAGTCTTCCCTATCCCTCCATTTTCTACGTACAAAAATTGGGGAACTTGTCTGGCATCTCAGAAAATGGCCAGGCTAGGAGGGGAGCCCAGGTGTACTTGGCTCCAGCAGCCCTTGCACTCTGATTCTCCTTACACCTGCCGCTCTTTGGACCCATGAAGAGGGCATAGCCCCCCGCTTGTCCACAGAGAAGCTGGTTACTTGGCTGGGGACTCGCAGAACTGGGATTGGAACTCAGATCTCCAGGGAGTCTCTGTGGAGCTTTCATTAACAGTTTGCTGGAAAGAGCATCAGTCTGATGAATCTGGAGTCCTGGGTTCTCTCCAGCTACTCATGTTTGGCTCAAGCTCTTAACTCTGCTATGTCTCATTATTTTCATCTGTAAAATGGGAGTAAGATTTCATGACACAGCTGCACGACTGAATGTAAAAGTGCCCTGCAAAAATCCTTGGCCTGCCCGGCTCATGATAGCATCAGCACACGGGGACCCTGCAGAAGTCCTCGGCCTGCCCAGCCCGTGATAACATCAGCACGTGGGGACCCTCCACTTCCTGCCCTCACCCCCTGACGTCAGAGCCTGCAGGGTGGCAGAGCCAGAGGGGAGATGCATCAGAGGGTGTCAGCTCCAACTGTCACCAGATCAGTGGGCTTTCCCTGCCCTCGACTGTGCGGATGTGCCCCCCCCGCCCCGCACCCACCTGGGGACAGCTGCTTCCTAGGGACACACTGCTTGCCCTCTGGGGACTCTCAAAAAGGGGGAATGTGGTTGGGGGGGGAACTGCTGCTTGTCAGCCACAGTGACATGGTTGAACCTGGAAAGGGTCTCCTGCCAAGGAAGCACCTCAACCCCTTCCTTGACAGGCAGGGAAACTGAGTTCCTGACAGCTCAGGGACTTGCACATAGTCCTGGTGAAGCAAACACAGGTCTTGAGAATCAGAAAAGCCTGACTTGCAGCTCCCTGGCTTATTAACCGTGTGACCTTGGGAGGTTACTTAACCCCTCTGGGCCTCCTTGTCCCCATCTGTGAAACAGGAATAAATATGATTGTTGGAGGATTACGTAAGGGTGTGTATCAAGTGTCCTTAGGATGGTGCCTGGTGGGTAGTCCTAATACTGCCTGTCATCAGCTGTCATGGTTACTAAAGAAACATTTCTTTGCAGGAATAAAGCCGGTTGGTTGGACAGTGAGCTCAACATGTTCACACACCAGTACCTGCAGCCCAGCAGATAGTGGGCACCTGAGGCGGAGCCTGCGTGGAGAGGGGTGGTGCTGCCAGCCCTGAGTGCCGTGTGGGCTCCCCCAGGGGCACGTGTGGCTCCTGCCCCTGGCCACCGCCAAGAGAATGAAGCACCCAGTCTCGTACCATTTTGAGCCCTCCAGCACTAGTTATTTTCCCCCTCCTTTGCCTGCTGTTGCAGGAAGATCTGGCTCATACCCCTAAAGGACACTTTCAATGACAGTTATGGACAGCATGGTACCAAGGAGTTGAGGCTTTTTCCAGCTTTCTCGGGTTCATCTGAGTGCTTGATAAGACCTCGGGATCTCAGCATTGCACAATGCCTCATGGAAGCCTTTGAGGGTATCACGCAGACACCCCCACCTTTCTCCAGCCTGTGCGCACCTGCCCTCCTTGCAGCCCAGCACACCTACAGGTGTAAGGGATGATTGGAGTTTCTTCCCAGAGAGTCTGTCCCAGAAGGACTGTGGCTTGTGTGTGTTCGTCTCGCCTGTTGGCTCAGTGCTTTATCCCATTTGCAGAGCCTCTCAGACACGTCTTGGTGTTGAGGCTCAGTTACCCCTGGGCTTAGGCTGAGGTGGGCCCTGTGCTGTGGCCGAGGCAGCTGGAGGAGTGGGAGTAGCTCAGAGGAGAGGGCTGTTGGACGTATGGGGAGCTGGCAGAGCAGGTGGTGGTCACTGGGACAAGGAGGGACTTGCCTCTCTTCTCATTATTGTGCCCCCTGCTTTAGTGTCAGTCCTGGACTTGTGCAGGCCTGTTTTGTGTAGATGTGTTTTGGAAGATGGCTTGGTCTAGGTGGTTGAAGGATGTAGTAGAAGGAAGGATGGTGGAAGGTGGGGACCTTGGTAGCTGAGGAGCGTGGGCCCCACACAAGACAGCTGGAGAATGGGCCGTCCATTTGACCTCGTTCTCAAGGGGCTCATGGGTCTGAGAGACCCCACCCACTAGGCTTGATTGCATCCCTGTTGTGCCCTTTAAGACACATGTCTCCACCCCACCCCCCAACCTTGCCCCAAGTGCCCTGGACTAAATTTCCTGCGCCAGTGACTGCAGTTGGCCAAGGGACAATGTGGAAAATCCAGTGCCCATCCTTCCACCCTCCCTGATCCCCAGAAACTTCGACTGACCCCTTTGTCCTTATGCTGATGTTGAGTTTTGGGATTGTTGCTGGTTGAAGTGGGGGCAGATACCTGTCACCAAGGTGTTGACTGTGTGAGAAAAGCAGTTTGGGTGACAAATCCTGTGTGGCACAAGTTGGATCGCTTCCTAGAAATAAGCAACACCTCTCCCAAAAAGCAGCCCAGAGGGCAGGGGCCCAGCAGCCCAGCCATCACTCATCTTTGAGGAAATTAGTTGGTGGCCTCTGTGCACTGGTTGGTGGCCACATCACAGGTGATGTCCTGTTCACATACCTGCTTGTACTTAAAACCCTCAGTCCTGTTGTGTGGGGCAAAGTGATGGACTCTGCCAGCTGGACGTGCTGTGGGTGGGTGTTCCCGGTGTGTGCTGGGCCTGAATGGATAGGCGCCACTTCACAGCATGTCAGGGAAAATCAGTGTCACACAATTCCAATGGATTTTATGCTCTTTTTGGGGAAAAACAAATTCTTTAGCATAAACATGAATTTTTTTTCAATGTAGCCCCTGGGGAATGAATGAAATTTTGAGCTTCTTCAATAAGTAAAATTAAATTAAGACCACTGAGGGAGAGACCCTTTCTGAAAGAAGTCTGGCCAAAAGCACTTGGAATGCTGCTGACATTGTTCTGTTTTTACGTTCATTTGCTGGAGCGCAAGACGTGCTGACACAGTGAGTTTTCTCTGATGTATTTAAGGTGATGTATTTGCCTGAGTTACTCCTGTATCATTCTCATAATATTGGAAACTAAAATAAAACCTAGTTGGAAATCCTCTGTGAGATCTCCACCATGTTTTCGGGTTCTTGTACAAACTCCTCTTTGACATGGCCAGCTGTGTTCTGAAGTGACCACAACTATAGTTCCCACTATAATGTGGCTCCTTATTCTTGGAAGGAATTTGATACTCTCAAAAGAACAAATGGTGTAACTGATCTTTAAGGATCAGGAAATCGAGACTGATTTCTTTATAGATTTCTCTATAACTCCAGACCATTTTTTCACTTAAAAAGTAAATGACTCACTGTTCCATCCTGCTTTGCCTTCTGAGAATTTGGTTGTAACCATTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTGAGATGGAGTCACGCTCTACCGCCCAGGCTGGAGTACTGGAGTGCGGTGGCATGATCTCAGCTCACTGCAATCTCCACCTCCTGGGTTCAGGCAGTTCTCTGCCTCAGCCTCCCAAGTGGCTGGGATGACAGGCGCCCGCCACCACACCCGGCTAATTTTGGTATTTTAGTAGACGGGGTTTCACCATCTTGGCCGTGCTGGTCTGGAACTCCTGACCTTGTGATCTACCTGCCTCGGCCTCCCAAAGTGCTGGGATTACAGGCATGAGCCGTCGCGCCCAGCCCTTTTCTTTTGTTTAAGGGTCCTCAGGAGTAGATTCGGGGGTGGCTAGTCAGTTTCCCCACCCCTACAGCTGTCCTCTCGGCTCTGGTGCCGCCCAGTGGCCGCCCTGCTCTCCCACAGAGGCCACAGCTCAGCTGATAGCAACCTCATCCTTCCAGATTCCCAGGTCACACACTTGGGGCATCACCCATGAGTCCGCGGTCACACGTGCTGCACCTAATCTGTCACCTGCTGTCTTCCAAGTCTATCAACCCTGCCCAGTTCCTCACCTCCATCACAACCATCAGCCCGGATCCGGGTTCCTGCTTCTGCCTTGTCCCTGCCTATCCTTGCCATCCTGTGGCTGTTTCAAGGCAGCAGCCAATGACTTTCTTAGGTAACAAAAAAAATGAATCATTATCCTTTTTTTTTTTTGCAATCCACAAAACAAGTAGTTTCTGTCATCAACAAGATGTTATGTACAACCCACTAAATTATTCACACCACCATCATCCTGGGTGATGAGCCATAGTTTGAAAAAGGGTCTTTTTGCCTTATGCAAATGCTGCTTAGTTATTCCCAGTCAAGGCTAGCCTCATTCTTGTGCTTTCTCTGACTACCTCATGTCCACTGGGGAATCTGAGAGGCTGGTAGTTTTAACCCTCTAGTAACAATGTCCCCATTTAATCACAAAAAGTAGGTAGTCAGGAATGTATCACTGTGTCCCAAGCACTGGGGTGACAGCCGAGGCAGCTCTCTCAGAGAGCATCTGGCTCATACATGGCTTCAGAAGCAGGGCCCTCCTCGCAGATGACAGCTAAGTTGTTCACTGGGTCCTCAAGTGGATGGATGGTTTCCTGTCTCTAAGCGGGACTCAGGCTTGAGATAAGAACAGCCAACTTTTTTGAGTTTAGCATGTGTCAGACATTCATATGCATGAGTCCTAATCTCAAAATCCAATGGTCTATTTTCCCTAATTTCCTCAAATGAGGAAGTTCATGATTTCAGCAAAGTATTTGTGCACCTACTCTGGGCTAAGCATGGTTCTAGGCATTGGGAATATAGCAGTAACTAAGGTAGATAAAAATCTGCCTCTGGGGCTGGGCGTGGTGGCTGATGCCTGTAATCCCAGCACTTTTGGGAGGCCAAGGTGGGCGGATCACAAGGTCAGGAATTAAAGACTAGCCTGGCCAACATGGTGGAACCCCATCTCTACTAAAAATACAAAAATTAGCCAGGCTTGGTGGCGTGCGCCTGTAATCCCAGCTGCTAGGGAGGCTGAGGCAGGAGAATTGCTTAAACCTGGGAGGAGGAGGTTGCAGTGAGCCGAGGTCGCGCCATTGCCCTCTAGCCTGGGCAACAGAGGGAGGCAGCCTCTCAAAACAACAACAACAACAGAAATCTCACTCACTGATTAATAACCTTTTTCAGTTACAAAGAAAACAACATATTCGCAAAACTCCCTAACATCAGGAGCCATCAGGCAAATGATCCCAAGTCTTGATTTACAAACCAAACCCTTATGCCTCTGATCACATAGAGGACTGGTTCTTACAAACATTTTTTTTTTTTTTTGAGATAGATTTTTGTTGGCTGGAGTGCAGTGGCTCTATCATGGCTCACTGCAGCCTTGACCTCCCAGCCTCAAGTGATCCTCTCACCTTAGCCTCCCGAATAGCTGGGATTAAAAGTGTGCGCCGCCCATGCCCGGATAAGTTTTTTGTTTTTTGTAGAGACGAGGGGTCTCACTATGTTACCCAGGCTGGTTTCGAACTCCTGATCTCAAGTGATCCTCACATCTAGGCTCCCCAAAGTGCTGTGATTATAGGCGTGAGCCACCATGCCTGGACACAAACATTCTTTTTCTTTTTAAAATTTAATTTAATTTTTAAAATAGAGAATGTATGTTGGCTGGGCATGGTGCCTCATGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCGAGACGATCGCTTGAGTTCATGAGTTTGAGACCAGCCTGGGCAATCTGGCAAAACCCTGTCTCTGCAAAAAATACAAAAATGAGCCCAGCATGATGGTGTGCACTGGTAGTCCTAGCTACTCCAGAGGCTGAGGTAGGAGGATCACTTGAACCTAGGAGGTGGAGGCTGCAGTGAGCTGAGATTGCATCACTGCACTCTAACCTGGGCAACAGGGAGATGCTGTCTCAAAAAAAAAAAAAAGAGAGATGGGATCTCACTGTGTTGCCCAGGCTGGCCTCGAATTCCTAAGCGCAAGTGATCCTCCCGCCTCAGCCTCCCAGAGTGCTGGAGCTACAGGCATGAGCCACCACACCCAGCCAACAAACATTCTTTTCTGATAAGCAACTGCAGAACTTAAGCTAGTTTCAGCCAGCTTACGGAAGCTGTGAACAAACTATTTCGCCATTGCACATGAGCTCACCTCTTCTCATAAATATGTATACAGCTTTTCCCTCAAACCTGCTAAATATGTATGAATACGGACCCTGTGAGGCATAAAACCCAACCTGTCTCTCCTCTCTTGGAAGAGAGCACCTTCCGTGCATGCTGCAGACTGTCTCTTCCTGGTTTGCAAACAAGTATCAACAAGAAAGTGCTCCTTTCTACTATTTAGCCATCTTGGTGATCTTTTAGACAACATTGTGAGTATTTCTGCTAGGAACATTAGTGTTCAAATCTCTGTGTAGGCAGATGTTTTCATTTTTTGCAGGTAGATATCTCAGGCTGGACTTGGGCCATATAGTAAATCTATGTTTAACTTTACAGATTTTGCGAAGTGGTTATACTGTTTTATATCCCCACCAGTAATGCACATGGGTTCATATATATATATATATATATATTTTTTTTTTTTTTTTTTTCTTCTTTCTTCTTTTTTTTTTTTTTTGAGACAGTCTTACTCTGTCATCCAGGCTGGAGTGCCGTGGCGCAATTGTGGATCACTGCAACCTCTGCCTCCCAGGTTCAAGCCATTCTCCTGCCTCAGCCTCTCGAGTAGCTGGGATTACAGGCGCCTGCCACCACGCCTGGCTAATTTTTTTGTATTTTTAATAGAGACAGGTTTCACCATGTTAGCCAGGCTGGTTTCGAATTTCTGACCTCAAATGATCCACCCGCCTCAGCCTCCCAAAGTGCTAGGATTACAGGTGTGAGACATTGTGCCCGGCCTACAGTGAATCTTTTAACTTTACACACCATCACTTTAGATATATAAATCGATATGTAGTGGTATCTCATTGTGGGTTTAATTTACACTTGCCTAGTGAATGGATCCCAGAGCAGGAGAAAACTTAGAAAGGCCTAGTGAATGGATCCCAGAGCAGGAGAAAACTTAGGCAGACTGGGATGGAAGCTGGAACTAGAGGCATTTGTGGTGGATAAGGATGCTGTATGGAGTCTCTGGCAAGCCTCAGTTTTTATGATGTTCAGCATCTTTTTATGTGCTTGTCATCTGTGCATTTCACCTTTGGTGAAATGTCTGTTCAATTATTTTGCCCATTTTAAAAACTGGGCTGTTTTATGAGTTTTGAGAGCTCTTTATGTAATCTGGATATAAATATTTTCTCCAATCTTGTGGTTTGACTTTTCACTTATTTTTTTCTAAAAATGGAACAAGTTCCAAATTTTTAATCTGGACAAAGAGAATTAAAACCAAGCATATCATACTTCCAAAACACATATCCATCGGCAAGTCAGCAAATCTGGGGAAAAGCAACTGGAATTGACTATGAGGCTGCTGTAATTAATCTTCATCGTCTCATTGCAAAGGTACTACCTGAATTCCTTTGGCTATCCTTTCTGTTAATATCATTTGCAGTAATTACTCTTAGTAACATCAAATCAAATGGTCCACCTGTAACATCCATTAGCATGCCACCAGCTTCAGTAACAATAATGCTAACTCCTGCAAGAGCCCAGCAGTGAATTCCCATTTCATAATATACATCTGCTCCTCCAGTTGCCACAAGGCAAACAGCTGCCGTTCCAATACTCCGGATCCCATGAACAGGAATGCAAAAAAACTTTTGCA。
主要参考文献
1.Chatel-Chaix L,Cortese M,Romero-Brey I,Bender S,Neufeldt CJ,FischlW,Scaturro P,Schieber N,Schwab Y,Fischer B,Ruggieri A,Bartenschlager R.DengueVirus Perturbs Mitochondrial Morphodynamics to Dampen Innate ImmuneResponses.Cell Host Microbe,2016,20:342-356.
2.Chand RJ,Trible BR,Rowland RR.Pathogenesis of porcine reproductiveand respiratory syndrome virus.Curr.Opin.Virol.,2012,2:256-263.
3.de Brito OM,Scorrano L.Mitofusin 2tethers endoplasmic reticulum tomitochondria.Nature,2008,456:605-610.
4.Du,T.,Nan,Y.,Xiao,S.,Zhao,Q.,and Zhou,E.M.Antiviral Strategiesagainst PRRSV Infection.Trends in microbiology,2017,25,968-979.
5.Keffaber KK.Reproduction failure of unkonwn etiology.Am Assoc swinepract Newsl,1989,1:1-19.
6.Munoz JP,Ivanova S,Sanchez-Wandelmer J,Martinez-Cristobal P,NogueraE,Sancho A,Diaz-Ramos A,Hernandez-Alvarez MI,Sebastian D,Mauvezin C,PalacinM,Zorzano A.Mfn2 modulates the UPR and mitochondrial function via repressionof PERK.EMBO J.,2013,32:2348-2361.
7.Naon D,Zaninello M,Giacomello M,Varanita T,Grespi F,Lakshminaranayan S,Serafini A,Semenzato M,Herkenne S,Hernández-AlvarezMI.Critical reappraisal confirms that Mitofusin 2is an endoplasmic reticulum-mitochondria tether.Proc.Natl.Acad.Sci.U.S.A.,2016,113:11249-11254.
8.Rizzuto R,Brini M,Murgia M,Pozzan T.Microdomains with High Ca2+Closeto IP3R-Sensitive Channels That Are Sensed by NeighboringMitochondria.Science,1993,262:744-747.
9.Rowland AA,Voeltz GK.Endoplasmic reticulum-mitochondria contacts:function of the junction.Nat.Rev.Mol.Cell Biol.,2012,13:607-625.
10.Sugioka R,Shimizu S,&Tsujimoto Y.Fzo1,a protein involved inmitochondrial fusion,inhibits apoptosis.Journal of Biological Chemistry,2004,279(50),52726-52734.
11.Van RK.Van Reeth,K.Pathogenesis and clinical aspects of arespiratory porcine reproductive and respiratory syndrome virusinfection.Vet.Microbiol.55,223-230.Vet.Microbiol.,1997,55:223-230。
序列表
<110> 华中农业大学
<120> 一种抑制猪繁殖与呼吸综合征病毒的干扰片段
<141> 2019-12-04
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> 绿猴(Chlorocebus sabaeus)
<220>
<221> gene
<222> (1)..(21)
<400> 1
aagagacaca uggcugaggu g 21

Claims (3)

1.一种MFN2基因siRNA干扰片段，其具有如序列表SEQ ID NO:1所示的核苷酸序列。
2.权利要求1所述的MFN2基因siRNA干扰片段在制备猪繁殖与呼吸综合症病毒抑制剂中的应用。
3.一种猪繁殖与呼吸综合症病毒抑制剂，其活性成分是权利要求1所述的MFN2基因siRNA干扰片段。
CN201911225994.6A 2019-12-04 2019-12-04 一种抑制猪繁殖与呼吸综合征病毒的干扰片段 Pending CN111471681A (zh)

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