CN108893539B - 猪肉质性状相关的circRNA及其应用 - Google Patents

猪肉质性状相关的circRNA及其应用 Download PDF

Info

Publication number
CN108893539B
CN108893539B CN201810590056.5A CN201810590056A CN108893539B CN 108893539 B CN108893539 B CN 108893539B CN 201810590056 A CN201810590056 A CN 201810590056A CN 108893539 B CN108893539 B CN 108893539B
Authority
CN
China
Prior art keywords
circrna
mir
ssc
pig
pigs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201810590056.5A
Other languages
English (en)
Other versions
CN108893539A (zh
Inventor
苗向阳
李嫒
解领丽
黄万龙
张秀秀
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Institute of Animal Science of CAAS
Original Assignee
Institute of Animal Science of CAAS
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institute of Animal Science of CAAS filed Critical Institute of Animal Science of CAAS
Priority to CN201810590056.5A priority Critical patent/CN108893539B/zh
Publication of CN108893539A publication Critical patent/CN108893539A/zh
Application granted granted Critical
Publication of CN108893539B publication Critical patent/CN108893539B/zh
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/124—Animal traits, i.e. production traits, including athletic performance or the like
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

本发明涉及猪肉质性状相关的circRNA及其应用，具体的涉及circRNA_23437及其在选育具有不同肌肉品质猪中的应用。本发明采用circRNA‑seq技术分析大白猪和莱芜猪的皮下组织与肌内脂肪组织circRNA表达谱，预测差异表达的circRNA的miRNA靶标，并构建circRNA‑miRNA‑mRNA互作网络图，探究关键的circRNA_23437及其调控的基因ssc‑miR‑331‑3p、ssc‑miR‑769‑3p、ssc‑miR‑503或ssc‑miR‑370在脂肪沉积中的调控作用，为分子辅助育种奠定基础。

Description

猪肉质性状相关的circRNA及其应用
技术领域
本发明涉及动物基因工程技术领域，具体涉及一种与猪肉质性状相关的circRNA及其应用，更具体的涉及circRNA_23437及其应用。
背景技术
猪是全球肉类生产的重要来源。据美国农业部经济学家和经合组织——粮农组织预测，对比2014年，2023年全球猪肉产量将至少增长10.5％。随着人们需求量的增加，对猪肉肉质的要求也越来越高，主要包括肉色、嫩度、风味和多汁性等。了解脂肪沉积的分子机制有助于预防和治疗肥胖相关疾病和提高猪肉品质。大白猪，作为典型的瘦肉型猪，有较低的皮下脂肪。而莱芜猪具有肉色鲜红，持水性良好，脂肪含量高，肉质优良等特点。因此，大白和莱芜猪在脂肪沉积方面的显著差异为脂肪代谢的机制研究提供了良好模型。
环状RNA(circular RNA，circRNA)，亦称环形RNA，一些环状RNA具有miRNA应答元件，在基因的表达调控中发挥重要作用，这些环状RNA被称为miRNA海绵，具有此功能的环状RNA主要是由外显子组成的ecircRNA。再者，环状RNA既可通过碱基互补配对直接影响靶基因的表达，也可以通过与RNA结合蛋白形成RNA蛋白复合体(RPCs)，对RNA结合蛋白起调节作用。环状RNA还可作为翻译模板，在某些特定条件下其也可以被翻译为蛋白质。此外，circRNA可以通过顺式或反式作用影响亲本基因的表达。尽管目前关于调控动物经济性状相关的circRNA研究很少，但circRNA将成为动物遗传育种研究的新方向。
本研究采用circRNA-seq技术分析大白猪和莱芜猪的皮下组织与肌内脂肪组织circRNA表达谱，预测差异表达的circRNA的miRNA靶标；对circRNA的来源基因及靶基因进行注释和KEGG富集分析，并构建circRNA-miRNA-mRNA互作网络图，探究关键的circRNA在脂肪沉积中的调控作用。
发明内容
本发明的目的在于提供与猪脂肪相关的circRNA，命名为circRNA_23437，序列与SEQ ID NO.1具有90％以上序列同源性。
术语“同源”是主要是指序列上的同源，也就是用来说明两个或多个RNA或DNA序列具有相同的祖先。同源的序列一般有相似的功能。RNA和DNA的同源性常常通过它们序列的相似性来判定，相似性是指序列比对过程中用来描述检测序列和目标序列之间相同核苷酸顺序所占比例的高低。一般来说，当相似程度高于50％时，常推测检测序列和目标序列可能是同源序列；当相似性程度低于20％时，就难以确定其是否具有同源性。
优选的，circRNA_23437序列为SEQ ID NO.1。
进一步，脂肪为皮下脂肪或肌内脂肪。
环状RNA(circular RNA，circRNA)，亦称环形RNA，是最近几年研究确认的一种新型的非编码RNA(noncoding RNA，ncRNA)分子。根据RNA构成的不同，环状RNA可分为三类：外显子环状RNA(exon circular RNA，ecircRNA)、内含子环状RNA(circular intronic RNAs,ciRNAs)和外显子-内含子circRNA(exon-intron circRNA,EIciRNA)。
一种检测猪脂肪含量的方法，包括：(1)选取猪脂肪组织；(2)检测样品中circRNA_23437和/或其相关基因的表达量。
优选的，相关基因为ssc-miR-331-3p、ssc-miR-769-3p、ssc-miR-503或ssc-miR-370。
进一步，脂肪为皮下脂肪或肌内脂肪。
进一步，通过测序技术、核酸杂交技术或核酸扩增技术检测样品circRNA_23437和/或其相关基因的表达量。
本发明的目的在于提供一种检测脂肪含量的试剂，试剂通过测序技术、核酸杂交技术或核酸扩增技术检测样品circRNA_23437的表达水平。
优选的，通过高通量测序技术、探针杂交技术、基因芯片技术或荧光定量PCR技术检测样品circRNA_23437的表达水平。
优选的，试剂包含一对用于荧光定量PCR的引物，序列为SEQ ID NO.2和SEQ IDNO.3。
优选的，样品为组织，优选的，样品为皮下脂肪组织或肌内脂肪组织，更优选的，样品为猪皮下脂肪组织或猪肌内脂肪组织。
进一步，所述核酸扩增技术选自聚合酶链式反应(PCR)、逆转录聚合酶链式反应(RT-PCR)、转录介导的扩增(TMA)、连接酶链式反应(LCR)、链置换扩增(SDA)和基于核酸序列的扩增(NASBA)。其中，PCR需要在扩增前将RNA逆转录成DNA(RT-PCR)，TMA和NASBA直接扩增RNA。
本发明中的核酸杂交技术包括但不限于原位杂交(ISH)、微阵列和Southern或Northern印迹。
本发明中“探针”指能与另一分子的特定序列或亚序列或其它部分结合的分子。除非另有指出，术语“探针”通常指能通过互补碱基配对与另一多核苷酸结合的多核苷酸探针。根据杂交条件的严谨性，探针能和与该探针缺乏完全序列互补性的靶多核苷酸结合。
所述探针具有与靶点基因的特定的碱基序列互补的碱基序列。这里，所谓“互补”，只要是杂交即可，可以不是完全互补。这些多核苷酸通常相对于该特定的碱基序列具有80％以上、优选90％以上、更优选95％以上、特别优选100％的同源性。这些探针可以是DNA，也可以是RNA，另外，可以为在其一部分或全部中核苷酸通过PNA(Polyamide nucleicacid，肽核酸)、LNA(注册商标，locked nucleic acid，Bridged Nucleic Acid，交联化核酸)、ENA(注册商标，2′-O,4′-C-Ethylene-bridged nucleic acids)、GNA(Glycerolnucleic acid，甘油核酸)、TNA(Threose nucleic acid，苏糖核酸)等人工核酸置换得到的多核苷酸。
本发明中的术语“杂交”用于指代互补核酸的配对。杂交和杂交强度(即，核酸之间的缔合强度)受诸如以下的因素影响：核酸之间的互补程度、所涉及的条件的严格性、形成的杂交体的Tm和核酸内的G:C比率。
测序技术主要为高通量测序技术(High-throughput sequencing)，又称下一代测序技术(next generation sequencing)，是对传统测序一次革命性的改变，一次对几十万到几百万条DNA分子进行序列测定，极大提高了测序效率。高通量测序平台的代表是罗氏公司(Roche)的454测序仪(Roch GSFLX sequencer)，Illumina公司的Solexa基因组分析仪(Illumina Genome Analyzer)和ABI的SOLiD测序仪(ABI SOLiD sequencer)。
本发明的目的在于提供下述任意一项应用：
circRNA_23437在预测或辅助预测猪肉品质中的应用；
circRNA_23437在制备预测或辅助预测猪肉品质试剂中的应用；
circRNA_23437在选育具有不同肌肉品质猪中的应用。
上述试剂在预测或辅助预测猪肉品质中的应用；
上述试剂在制备预测或辅助预测猪肉品质试剂中的应用；
上述试剂在选育具有不同肌肉品质猪中的应用；
本发明的目的在于提供一种检测脂肪含量的试剂，检测circRNA_23437相关基因的表达水平，相关基因为ssc-miR-331-3p、ssc-miR-769-3p、ssc-miR-503或ssc-miR-370。
优选的，试剂通过测序技术、核酸杂交技术、核酸扩增技术或免疫测定的方法检测circRNA_23437相关基因的表达水平。
进一步，所述试剂包含特异性扩增ssc-miR-331-3p的引物；或特异性扩增ssc-miR-769-3p的引物；或特异性扩增ssc-miR-503的引物；或特异性扩增ssc-miR-370的引物。
本发明的目的在于提供下述任意一项应用：
上述试剂在预测或辅助预测猪肉品质中的应用；
上述试剂在制备预测或辅助预测猪肉品质试剂中的应用；
上述试剂在选育具有不同肌肉品质猪中的应用。
附图说明
图1脂肪组织总RNA提取图
图2是皮下脂肪circRNA-miRNA-mRNA互作网络图
图3是肌内脂肪circRNA-miRNA-mRNA互作网络图
图4是荧光定量验证候选基因皮下脂肪差异表达图
图5是荧光定量验证候选基因肌内脂肪差异表达图
具体实施方式
下面结合附图和实施例对本发明作进一步详细的说明。以下实施例仅用于说明本发明而不用于限制本发明的范围。实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。
实施例1circRNA测序及差异表达circRNA挑选
1.1实验动物：
该试验以脂肪沉积存在明显差异的大白猪和莱芜猪为材料，饲养于莱芜市大千农牧有限公司，在相同饲养条件和环境下生长育肥，参照营养需要标准(National ResearchCouncil,NRC,1998)饲喂日粮，选择180日龄、种内体重接近的大白猪(约100kg)和莱芜猪(约35kg)各3只，且健康、体质优良。
1.2样品采集：
屠宰实验猪后，迅速将皮下脂肪组织和肌内脂肪组织分离，每个样本设置3个生物学重复。为减少RNA的降解，所有过程在冰上进行。之后用消毒剪刀将组织剪成小块，分别装入5mL冻存管，放入液氮冷冻，后转移至-80℃冰箱保存，用于提取总RNA。实验设置分为2组，分别对大白猪皮下脂肪组织(D_PX)与莱芜猪皮下脂肪组织(L_PX)、大白猪肌内脂肪组织(D_JN)与莱芜猪肌内脂肪组织(L_JN)中的circRNA进行鉴定及分析。
1.3样品总RNA的提取及质控：
取出等量低温保存的脂肪组织，使用QIAGEN抽提试剂盒提取RNA，提取的总RNA保存于-80℃冰箱。对提取的总RNA进行质检，结果如1所示。用NanoDrop 2000紫外分光光度计测定RNA样品的浓度和OD260nm/OD280nm比值，比值在1.9～2.1间，Bioanalyzer 2100检测总RNA的质量，且RIN≥7、28S/18S≥0.7，用RNase-free DNaseⅠ除去基因组DNA污染。
1.4circRNA测序建库
1本试验共建立12个cDNA文库：大白猪和莱芜猪皮下脂肪组织cDNA文库，分别为D_PX_1、D_PX_2、D_PX_3、L_PX_1、L_PX_2、L_PX_3；大白猪和莱芜猪肌内脂肪组织cDNA文库，分别为D_JN_1、D_JN_2、D_JN_3、L_JN_1、L_JN_2、L_JN_3文库质检合格后，运用IlluminaHiSeqTM2500测序平台进行双端测序，对文库进行测序分析，下机得到的数据是原始测序数据。
1.5样品组间差异circRNA筛选：
经过一系列数据的加工和分析，在莱芜猪和大白猪皮下脂肪组织(L_PX vs D_PX)中共鉴定得到29763个circRNAs，对其进行差异表达分析(按照|FoldChange|≥2.0和P-value<0.05严格筛选)，在皮下脂肪组织中共鉴定得到275个差异表达circRNAs(70个上调，205个下调)。在70个上调的circRNA中，有17个intergenic circRNA，3个exonic circRNA，49个sense-overlapping circRNA和1个antisense circRNA；在205个下调的circRNA中，有7个exonic circRNA，21个intergenic circRNA，174个sense-overlapping circRNA和3个antisense circRNA。在肌内脂肪组织中共鉴定得到283个差异表达circRNAs(101个上调，182个下调)，其中具有2倍以上差异的circRNAs约占35.7％。在101个上调的circRNA中，有26个intergenic circRNA，1个intronic circRNA，73个sense-overlapping circRNA和1个antisense circRNA；在182个下调的circRNA中，有1个exonic circRNA，29个intergeniccircRNA，1个intronic circRNA，149个sense-overlapping circRNA和2个antisensecircRNA。其中，sense-overlapping circRNA——circRNA_23437在皮下脂肪组织和肌内脂肪组织均差异表达上调，进入我们的后续研究范围。
实施例2circRNA深度分析
2.1差异表达circRNA功能分析：
得到差异表达circRNA之后，利用circRNA来源基因的信息，我们对差异表达circRNA进行GO富集分析。统计每个GO条目中所包括的差异circRNA个数，并用超几何分布检验方法计算每个GO条目中差异circRNA富集的显著性。KEGG是与Pathway相关的重要公共数据库，利用KEGG数据库对差异circRNA来源基因进行Pathway分析，可以找到富集差异circRNA来源基因的Pathway条目，寻找潜在的不同样品的差异circRNA可能和哪些细胞通路的改变有关。
2.2circRNA-miRNA-mRNA互作网络图
皮下脂肪组织分析，结合生物学信息学分析和本实验室前期做过的大白猪和莱芜猪皮下脂肪组织miRNA和mRNA的测序结果进行整合分析，挑选出了与circRNA_23437结合的ssc-miR-331-3p，且这些miRNA在大白猪和莱芜猪皮下脂肪组织差异表达，进一步分析ssc-miR-331-3p调控的靶基因及其表达情况，将分析的数据进行整理，构建了circRNA-miRNA-mRNA互作网络图(见图2)。
肌内脂肪组织分析，结合生物学信息学分析和本实验室前期做过的大白猪和莱芜猪肌内脂肪组织miRNA和mRNA的测序结果进行整合分析，挑选出了与circRNA_23437结合的ssc-miR-769-3p、ssc-miR-503、ssc-miR-370，且这些miRNA在大白猪和莱芜猪肌内脂肪组织差异表达，其它miRNA也是circRNA_23437的潜在结合位点，进一步分析ssc-miR-769-3p、ssc-miR-503、ssc-miR-370调控的靶基因及其表达情况，将分析的数据进行整理，构建了circRNA-miRNA-mRNA互作网络图(见图3)。
2.3circRNA_23437靶基因GO和KEGG富集分析
circRNA_23437肌内脂肪靶基因富集于肌肉松弛、胆固醇生物合成过程和代谢过程调控、胰岛素分泌、平滑肌收缩和激素分泌等生物学过程。在KEGG通路分析中，靶基因富集于昼夜节律、胰岛素分泌、ECM受体相互作用、脂肪酸生物合成、蛋白质消化和吸收、肾素-血管紧张素系统和脂肪酸降解等通路。
实施例3实时荧光定量PCR验证
选择circRNA_23437，circRNA_23437皮下脂肪靶基因中的miRNA(ssc-miR-331-3p)和circRNA_23437肌内脂肪靶基因中的miRNA(ssc-miR-769-3p、ssc-miR-503、ssc-miR-370)，每个设置20个生物学重复(大白猪和莱芜猪各20例)，分别取皮下脂肪组织(皮下脂肪组)和背最长肌组织(肌内脂肪组)，以甘油醛-3-磷酸脱氢酶(glyceraldehyde-3-phosphate dehydrogenase,GAPDH)基因和5S rRNA为内参，qRT-PCR方法用于验证基因的表达水平。
3.1引物设计：
circRNA_23437扩增：
正向引物：5’-GGGCAGGACTATTGGGATCG-3’(SEQ ID NO.2)
反向引物：5’-CGTCTGGTCCCAAGTAGCAG-3’(SEQ ID NO.3)
ssc-miR-331-3p扩增：
正向引物：5’-GCCCCTGGGCCTATCCTAGAA-3’(SEQ ID NO.4)
ssc-miR-769-3p扩增：
正向引物：5’-CTGGGATCTCTGGGGTCTTGGTT-3’(SEQ ID NO.5)
ssc-miR-503扩增：
特异引物：5’-TAGCAGCGGGAACAGTACTGCAG-3’(SEQ ID NO.6)
ssc-miR-370扩增：
特异引物：5’-GCCTGCTGGGGTGGAACCTGGT-3’(SEQ ID NO.7)
3.2反转录体系：
将下列物质加入0.5ml的离心管中：RNA 5μl；Oligo(dT)2μl；dd H2O(DEPC处理)4.5μl；
以上首先70℃孵育5min，然后迅速放在冰上。
5Buffer 5μl；dNTP(10mM)2μl；Ribonuclease inhibitor 0.5μl；M-MLV RT 1μl；以上20μl体系42℃孵育60min，然后70℃10min。
3.3 Real-time RCR反应
(1)将所得的cDNA与下列物质配成20μl体系加入0.2ml的离心管中混匀。Real-Time PCR体系：cDNA 2μl；引物1(10pmol/μl)0.5μl；引物2(10pmol/μl)0.5μl；SYBR mix 10μl；dd H2O 7μl。
(2)Real-Time PCR程序设置：94℃预变性10min；94℃15s，60℃60s，45个循环；72℃10min。
3.4结果统计分析：
为了验证测序分析结果，选取目标分子circRNA_23437及其在皮下脂肪和肌内脂肪中的靶基因，进行验证。2-△△Ct法分析每组样本间基因的相对表达情况，用t-检验统计分析相对表达量，数据结果展示为平均数±标准差(Mean±SD)，P<0.05为差异显著。
结果显示，皮下脂肪组织中，circRNA_23437在瘦肉型猪大白猪组表达上调，ssc-miR-331-3p在瘦肉型猪大白猪组表达下调，具体结果见图4。circRNA_23437在瘦肉型猪大白猪肌内脂肪组织显著下调表达，ssc-miR-769-3p、ssc-miR-503、ssc-miR-370在瘦肉型猪大白猪肌内脂肪组织上调表达明显，具体结果见图5。荧光定量PCR结果与测序结果一致。显示circRNA_23437及其靶基因可以作为猪皮下脂肪含量和肌内脂肪含量检测的分子标志物，在预测或辅助预测猪肉品质、选育具有不同肌肉品质猪等领域具有很好的应用前景。
序列表
<110> 中国农业科学院北京畜牧兽医研究所
<120> 猪肉质性状相关的circRNA及其应用
<160> 7
<170> SIPOSequenceListing 1.0
<210> 1
<211> 57856
<212> DNA
<213> Sus scrofa
<400> 1
ggtctcacac cctccagagc atgtttggct gctacttggg accagacggg ctcctcctcc 60
acgggtacag acaggacgcc tacgacggcg ccgattacat cgccctgaac gaggacctgc 120
gctcctggac cgcggcggac atggcggctc agatctccaa gcgcaagtgg gaggcggccg 180
atgcggcgga gcactggagg agctacctgc agggactgtg tgtggagtcg ctccgcagat 240
acctggagat ggggaaggac acgctgcagc gcgcagagcc tccaaagaca catgtgaccc 300
gccaccccag ctctgacctc ggggtcacct tgaggtgctg ggccctgggc ttctacccta 360
aggagatctc cctgacctgg cagcgggagg gccaggacca gagccaggac atggagctgg 420
tggtgcctcc tggagaggag cagagctaca cctgccatgt gcagcacgag ggcctgcagg 480
agcccctcac cctgagatgg gaccctgctc agccccccgt ccccatcgtg ggcatcattg 540
ttggcctggt tctggtcctg gtcgctggag ctgtggtggc tggagttgtg atctggagga 600
agacgcgctc aggtgaaaaa ggagggagct acactcaggc tgcaggcagt gacagtgacc 660
agggctccga tgtgtccctt accaaggatc ctagagtgtg agacagctgc cttgtgggaa 720
ctgagggaca caagatttgt tcacgtccca ctttgtgaat ccagatcccc tgactcctgt 780
ttctgcagct gcctctgaaa gggtctgtgt tcctatgagc atcctgagag gaggtgggga 840
ccctggcccc cacgtcccct cctcaccctg acctgtgttc tcttccatga tcctctttcc 900
agttcttgca agtgggagcc gggcgggggg agggggggca ctggaccatc tccatcctta 960
cttaacttga ctgccctgag taatgacttc ctgttgaatt tgctttttct aattggtgcc 1020
atgaggagtt gaggggataa taaatgagag atttcctatg tttgaaagag gaaataaatg 1080
gaaggattga gaaccttcca gaatccacat gtgtgctgtg ctgtgtcagt tccggatggg 1140
atgtgaggag agaaacatgg acggggcctg tgcccagtga gagctcaggg catcatgggc 1200
ttcggtgtgg acactcccag gctgggtcac cttttcccct gtccctttgt ccttgtccct 1260
tcaggagaac tttcttccac caggccctgt gatctcaggg actgagaagt cccctgggcc 1320
ttgtcctgta ttcaggagct tggttagcaa gggcctcctg tcaccaggta gcctacgctc 1380
cagccttggt cccatcagta gtccctttat tctgtgtttg tatttggcct atttagattt 1440
ttttttaaaa tacggacttt tcatttacat gtgtgaaatt acagtctcat ttttctctgg 1500
gtgtccctcg tctgcaacag ccacaacagt catttgcctg tcattgttcc cacatgtcca 1560
gggggtccct gctcaaagat tctcagtggt atcaagagat ccattttcag cctcatccag 1620
ctcttgccct ccttccagag atgtttactg gatgcttctt tctttctttc tttttttttt 1680
tttttttttt tttttttttt tgacttttca gggccacact cagggcacat ggaggttccc 1740
aggctagggg tctaatcaga gctacagctg ctggcctata ccacagccat aggaacgtgg 1800
catctgagcc gcatctgtaa actacaccac aacacacagc aatgctggat ccttaaccca 1860
gtgagcatgg ccagggatca aacccaccac ctcatggttc ctagtggggt tcatttccac 1920
tgtgccacca cgggaacgcc tggatgcttc tttctaactt ttccccatcc ttttgtggga 1980
acgagattct ggaagtagca accaggaggg acccagtggt tttccatctt gacctcattc 2040
ttgctggatc tggatcttct ctgcagcccg ccccctcctc tgccctgaga tctcctcagc 2100
ccactgccgg ctccaatcca agcccatgca tttcgaaagc agagtctgat agactcacag 2160
cagtgggaat ctagggccag aagcgcagga gcatctttcc tggggagaga aagagcgctg 2220
tgaggggtga gcagaaggac tggtctggca gggaggggac atcagccctt gtgaggaaag 2280
gacaggcagc cctgatgtca ccgggaatgg acattgtgct ggagctgcca ccagacagca 2340
cgtggcccga ggtcacacga gtgaaggtac tgtccctaga agagggaggg gctgtgcact 2400
gatccgtcat tcaactctgt tgaacggata tttgttgtgg gccagacaca ggacacacgc 2460
tttcggcatc tgggagacct cagggacgtt gccagagacc aaacttgttg gccgtgtgga 2520
gtctctgtcc tgggggaggg gcagagtgga cactatatgc ctcgtcaggg agggacgtgt 2580
cacatcggcg gtgggagggg ccttggggaa ggtgaccccg agtagaagtg tgtggggaca 2640
gggtcggggg gtgggtgtgg gccttcctgg gagggtgacc tttgagtcca gatgggaagg 2700
acgtgaagga aatgcccgcg aggctgtgtg gggacgttct ctgcaggcag gggaccctca 2760
cagggcaaag gcacaaggac aggagggggt gtgtgttcag ggaggagcca agaggccggc 2820
gaggctgcag ggagggaggc gctgggatga gacccactgt ccggccagat cgtgtggcct 2880
ccggatggga ggagggtttt gattttgtcc cagatggtgg cgtttgtaag atggtttgac 2940
cgaagatcag gctggagtga gtgctctcta gaagcatggg tctggccgct gcacagactc 3000
aggaggggaa ggcgagcgac accgtcgagg aaggaaacag agcagcgtcc aggctggagg 3060
agatgctgct ggtcgcggtg agagcaggga acggacgagt cagtgactgg agtctcgaca 3120
cattctcagg atgaatttag agcatttcct cctggatcaa atctggactg tgagaaagca 3180
gaaagaagga tggcacccca aacttgagac tgtgcaagtt ggcctgtgga aatggggaga 3240
ttctgggggt tcccgtcgta gggcagtggt taacaaatcc aactaggaac gatgagattt 3300
tgggtttgat ccctggcctt gctcagtggt ttaaagatcg ggcattgctg tgagctgtgg 3360
tgtaggttgc agacgtggct cagatcccgt gttgctgtgg ctctggcata ggccagtggc 3420
aacagctctg attagacccc tagcctggga acctctatct gccacgggag tggccttaga 3480
aaagataaaa acaacaaaaa caaaaaagaa aagaaagaaa tgaggagact ctggaaggag 3540
cacatttgag gaggtggcag cacctgcact cggattagga acgtggccac tgagtggcat 3600
gtacccatct gggctttagg ggaggtgcct tggctggaga aatagatgga ggaggtgaca 3660
ccccagtgaa tgatattgaa agctctaaga ggagttcccg tcgtggcgca gtggttaacg 3720
aatccgacta ggaaccatga ggtcgcgggt tccgtccctg cgcttgctca gtgggttaac 3780
gatccggcgt tgccgtgagc tgtggtgtag gttgcagacg cggctcggat cccgcgttgc 3840
tgtggctctg gcgtaggccg gttgctatgg ctccgattcg acccctagca tgggaacctc 3900
catatgccgc aggggcggcc caagaaatag caaaaaaaaa aaaaaaaaaa aaggaagctc 3960
aagaacagac gaggtcacca ggggagaact gactacagaa gaaggacaag gattaaaccc 4020
tgaaccccca gcgctgggcg atctgatcag agcacacacc tgagcagact gtgcggatct 4080
gaccccgggt ccagacacca caggcccagg gagtccccat ccccacgtgc acaggactca 4140
gctggacgcc tcgttagata cagtcggtct tgataagagg gtgagcttct gcctttacag 4200
caaggttggt gctgatgccc tgctggtctc cgggtcacac tttgagcggc aagacggtcc 4260
ccaggattcc cacgtgcctg tgcctcagtc tcccctgtag agcttgttaa tcagggttcc 4320
tggatccatg cccagggtgc tgagatgggg ggtctgggaa gaggcctgag gatgtgtaat 4380
aatcccgacc agcaggcctg aggctctgtc agagtgagat ccacggaggt cagtggtcag 4440
agagggatca gcctggagtg ggctcttcaa tacacatttc caggtgtttt cctggaagca 4500
aatgacaaga cgtgtcctct ctaccctggg gtataaccag gtggctgact taggacgttc 4560
tcagctcagc ataaagaaag cctcagagtt gctaagagaa tcgatttaaa ttcacacaca 4620
cacacacaca aatggtaaag atgtgacatc acacagttgt cggctgaggc gatgacgata 4680
accactttgc agtacgtaag tgtttcctat cgacaatttg tacactataa acttaatatt 4740
tatatcaatc atatctcaat aaactagggg gggaaatctc tgaatcttgc tctctgagag 4800
tttccttgtg tgttattctt gcaaagttct ttgtgggcaa aaggatttcg aatcgttcta 4860
aattcgagtt cccatcatgg ctcagtggtt aatgaaccca gctagtatcc atgaggatgc 4920
gggctcgatc cctggcctcg ctcagtgagt taaggatccg gtattgccgt gagctgtggt 4980
gtaggtggaa gacaagtctc atgtggctgt gacataggcc ggcagctaga gctgcaattc 5040
aacccctacc ctgggaacct ccttgtcctg cgggtagagc cctaagaaga tataagacca 5100
aaaaaaaaaa aaaaaaaggt tctaaacaaa taaaggcaga gagacagatt cgtagttttg 5160
ttttatttca ggaacagccc tgaggccaca cagccaggaa cagaatccac agccctaccc 5220
agttcgggtc ccgcctggga accactgcct tgcaggtggc acagatgcca ccgttcacaa 5280
caccgtcagt ctgatggggg acagtggact cggggctaga cctgctgtcc ctgctgctcc 5340
tggaaactgg gtgacactac tgccacctct gccacatgtg ctaaaatcat tcagcacagg 5400
gccagcttcc ctgtgtgccc aggttctgag tcagagtctg gcccgtggtc ccctgcctgg 5460
tggcgcctgg ggctggctga acctgcggca atgtttagga agcaagcttt cctcttacgg 5520
ggggaggggt ctcagcctcc caccaacatc ctcaagtttg aacttgtcct cctctgggaa 5580
gaggatacag attcttgggt ggagggcaag ggaaggaaga gaaaggaagg acacatggcg 5640
atgtttggag cagtgcccca gcacagaccc tgacctggca catcacaggc actggggttt 5700
ggagggatga aggaaggact cagaaatgac accgtccgct tctgcgatgt tctgccacgg 5760
ggtccagctt ccgtctcctc cagtttacac tccggacaag cagagcgcat tttttgaaag 5820
atgacacaat tcctcaggat gttttctccc tgcctttgtc tctgtgtaaa tcttcatgcg 5880
atacctctat acatgaatat atacagacac gtgaatactt agatacacac accgacattt 5940
tacgttaagt tttatatttc tttggacact cccgtccttc ttggggaagt tcccatattt 6000
accagtttcc ctgggacccc cacaaaagcc cttaaaatcc cccgtttaga caccaaagtg 6060
ctcaatctca ggtccaggag aagctaatca tccaactggt ttagaagact caaggacgtg 6120
attttttggt ttactcattt atttattttg ctttttaggg ctgaactcga ggcatatgga 6180
gattcccagg ctagggatca aatcagagct acagctgcct gcctactcca cagccacagc 6240
cacacaggat ctgagatgca tcgctgacct acaccttgca gtgccacgaa gcgaggccag 6300
ggatcaaact cgcaacctca tggttcatag tcggatttgt ttctgctgcg ccaaaatggg 6360
aacgctgtca atgacatgct gtcctctctt tcttggcaat gacccctctg ggtcaagaga 6420
aagtagaggt tgacttgctc actctctctt acaatgtctg tctgtttctc atttcacaaa 6480
cattccacaa accagcagag agatcaagta ccattggttt aggtggactt ggattctagc 6540
acaaaccctg gcgccagata aacagcagga gtttaaaaaa tgagccctgg ggattctatt 6600
tccagtagca tggtaggcct tgctttaaag ctgcctcttc aagtgaaaac aaataaaaag 6660
aatagagaaa aggggtgccc ttgtggccca gtggcttcaa gatccagcac tgtcactgct 6720
ctgtctgtgg ttacttctgt ggcgcgggtt ccatccctgg cctgggtact ccagcatgcc 6780
tctggggttg ggcaaaaaaa atgatcaaat acggaaagct tcaggagttc ccatcatggc 6840
tcagcagtta acgaatccga ttagcatcca tgaggacatg gtttcaatcc ctggcctcgc 6900
tcagtgggtt aaggatccgg ggttgtcgtg agctgtggtg taggttgcag acatggctca 6960
gatcggctgt ggtgtaggct ggtggctata gctccaattc agcccctatc ttgggaacct 7020
ccatatgcag ggagtgtggc cctaaaaagc caaaaagaga aaaacaataa taaagaaagc 7080
ttgcttccat aagtacagta gcaaggaagc aattcttcag gtcaaaatat caatgtgggc 7140
agtaatccag aggaaaagtt ctttttattc tcaggctatt ggtcaaatct ggaaaaacct 7200
tccctttggg tttctcaagc ttatgaattg tactagaaaa tgtaacccag actgacatgc 7260
ggaggataag agacatcggc tccctgataa tctggggaac caaggttcac gtcctcagag 7320
aggatgtgga gatcctggaa ccatgtcatc cgcggggggg cgtatagaac ggtgccgcca 7380
ctttgggaca gtctggaagt tgctcagagt tgaacacagt gctcccggat gactgactcg 7440
acagtgacat tcctcgttgt ttacacagga gacatgacaa catcggtcca cacaaaaccc 7500
tgtgcatgat actcataaca ccgttgtcca tagtagccaa acgataagaa caactccact 7560
gtccatcact ggatgaatga ataaataaag gtgtacacgt gagcccgaga accttacttg 7620
gaactaaaac aagaatggag accagaggcc taacacagca tggatgaacc ttgaagacac 7680
catgttccac caaagaacag acaccaccgc tcatgttggt gatttaattg tgtttacagg 7740
attcataagc aaatcccttt aggcagaaag cagattactg gttgtctaga gatgagggga 7800
gcgggggagg gtaatgaggg ctgaccgcag aggaataacg gggtacaggg gatgacaatg 7860
gtctcaaagg ctttggggtg atgagcttct tcccagatga ctgtggttct ggtcacacct 7920
ctcaatgaat gtgcgaaaac caatcctctc caacgggtga attacatttg tgaattgtat 7980
ctgaatcggt ggttctcaca aaagtgatga cccaccttaa atgaagcaca gatgccagga 8040
ccaccacggc cattgcacag aaagacaagg aaaggcgcag agaagtgggc gagcagccac 8100
aaaagaaaac cggaccaaac cccttccttc gactatttcc cgaaggaagc catcggccca 8160
gaaggatgag cacaagccag agcagtgccc tcactgcgcc tgtcccttgg cggcagggct 8220
gactctagga aagagtatga ccgcagcaat ggtcagaaga gggtcccaga aggccagaga 8280
aactgaggcc atcctcccac catcctccag caaaagacgg gcgcccagct ctccgaccac 8340
aaggacccgc tcagtcggac tccctaagag cccttttctt tttgaggaca gacatgctgc 8400
tctctcttta ctggcataaa tgtcacggtc cctttctctc ctagttgttt tcctctcact 8460
ctcgccatga agccccagaa gggagcagca ggcagcaggg aggaagtgct gctgagcagg 8520
gctatggcca gacccctcct ctggtgtcac tttggagagc agacgttccc aggacgtcag 8580
ctggagcaga gccgtcagac cacagggatg caaaggacac gagtgatccc cccgtgactc 8640
tcaggtccac ggggggctgt gtttcgggct gtgaagtgac ccaggggtga gtaaacgctg 8700
ctcggcccct gctgctcttc acccacagca tctggaaagc ccagctcact ccccagatgg 8760
aaagaccttt gatgccctgc agccagctgt gacccaccct gcctgccacc cccagcaaac 8820
gcaatccatc agccagtgtg aaccaactct cctgtcactt ccgaccctgg ggactcgcct 8880
gtagggactg agcaatgcag tgggttcccg gccactccca gagctgggaa gtgctcagaa 8940
cagtgcctgg ctctgtgacc tcaggtgagt tactttccct ctgggcacct gtttcctcat 9000
ctactaaatg ggtgacgcat gagggctgcg gtgaggactg gctgccctga tacaagcaaa 9060
gggctcggaa caattcctgg catgtacctg aggctcaacc catctttgct ctggtggctg 9120
gaggctgatg acggagcccc gccccaagag ccctccttgc ctgctgtcgt aaccagaagc 9180
gaaggtgaac ttccattcgc agaaaggccc ctctgcccgc atgtggagaa gggactggta 9240
aggataagaa tgcacgtgag gggaccagtt aggaggccca gcttgaggcc tagattactc 9300
ctctccaccc caacaactcc tcctcccact gcaattcttg ttcacgttcc taactgcatc 9360
ctgctcctgt cccagagtca atattaagaa ggaattcttg gagatcccgt tgtggctcag 9420
tggttaagga aaccgactag tatccatggg gacacgggta caatccctgt cctccatcag 9480
tgggttaagg atgcggagtt gccctgagct gtgctgtaga ttgcagacac agcttgaatc 9540
ccgcattgct gtgcctgtgg tgtaggccgg cagctgtagc tccgattcaa cacctagtct 9600
gggaacctct acatggggca gttgagaccc taaaatttta aaaaagacaa aagacaaaaa 9660
aaaaaaaaaa aaaaaaaaaa aaaaaggaat tgcttggctg gagagaagga gagaacctgc 9720
gttttgtgaa ggaggaaact tggtgaaact acagtgaaat ctagagtaaa aagcaagttg 9780
gtcccagatg tcctgtggcc tgtaaaatca gggtgtcatt tgttttggtt catgttcaat 9840
gattttaatc actggaatgc acacattttt aatatgcaaa tcaaaggttt acaacttgag 9900
gaaaagaaaa gaagggattg ctgagcaagc ctgggatggc agtcagggta tcaggcccac 9960
tgctgggaac ctcctggaac acgtgacctc agggtctgtt gggagctcct gaaggaccca 10020
gtgagcatct cctgtggtcc cagctcttgc caggcctgca tccccaaggg ctgtgtcact 10080
ttcaggccca gggaggaagc cctgtgctgt ccaatctgca agaggacatt tcaaggggga 10140
ccctcgggcc caggtggttt ctggccagtt tggtccccag ctgcctagca ctggagccca 10200
ggggtgggaa tccaacacag aaagtttggt tctgtgagct gcacaaggaa agggcctttc 10260
ttctgtcacc ggaaggacca gcaattcccg tgtatgcttt ccaaagacct cctagagcac 10320
aactgccact cggcactaca gagggaggaa gatgctgggt cttgggaggt aggtagaccc 10380
ttttcctgtc ttgtctaaat gaggtacaag ccttggggtg actggcccta tccctggctt 10440
ttgctcaagg gcagaaaaat atacggccct cttctaaata ggattctaag tgcaccaata 10500
attgcttttt caatcctagc ttctcttggt aaaactctct gccagtgcct accatttcct 10560
tcgaagtaac ccagcaacag aaggcttcgc agcttctcta cattcggcat acacaaacct 10620
gggtgttgct ttgaaatgtc tcacaccctc cagaatcccc ttcagttttc tgggggtgaa 10680
gcaaacatga ttgaccatga agctgtgcaa gcaaaaaggc tctagataaa cagagctttg 10740
ccttcaggtt cgctctacta tttgggcaaa tcaaaacatg ttattgctgg aaaagtgaaa 10800
ccaggtggca ttgatccgtt ccattttggt tggaagtggc agaaattcaa ctcaggcttc 10860
tcaaaggagg agtccttgtc ttattgaacc aactaaggtg aagctggggt gagcttcagg 10920
tgtaactgga accaggggct tgagtacagc tgagactcct cacctctttc cccctcttgt 10980
ctccgtattg gcaccatttt caggccagtt acctggacaa gctgcagcct caaccatgtc 11040
tgggttcaaa cctcttatct tgccaacata gaaagactca gaattaccct ccctggtcct 11100
gacccgtcac aatttaggaa agatttttct ttttcttctc cttctccttc ttctgttctc 11160
tcttttttgc ttcttaggga tgcacccaca gatgacacat ggaagcttcc agactagggg 11220
ttaaattgga gctacagctc ctggcctaca ccacagccac agcaacgcgg gatctgtgcc 11280
atgtcttcga cctacaccac aactcagggc aatgcctgat ccccaaccca ctgatcgagg 11340
ccagggattg aaactgcatc ctcatggatc ctagttgggt ctgtccacaa ctgagccatg 11400
aagggaactc ctggaaagga ttctgaatca gtggcctgaa tcctggctta tctgtactgg 11460
gcagagctga gagactggct gaagagtgag ctcgcagtga gctttagggc cagaagccag 11520
tgtaggatca agtcctagtg catccgtgta acgcgacaga ccaggttatg aggagtaacc 11580
gagacctccc acacgccagt ggcctaaaga aaaagatttc tgtcctgtac agagctctaa 11640
aatacacgga gtactacgca atagaacaaa tgaggaaata aaattatttc ctttatattt 11700
atatgtcagt tacctttacg gaccaattgt tgtatagatc aataatagaa atggtaaaca 11760
aaaagcaagt aagggtagag agggcttaaa cccaagctaa cccgactaac cggcatgtat 11820
tgaccatcac atccaccaag aacagaagag acggtctttc tccagtgcac ctggaacagt 11880
ggccaaaaga gacaatattc tgaccctaaa caagtctcag gaagtttaaa gtgactggaa 11940
gtgtagatgg agttcccgct gtggtgcagt gggttaagca tctaactgga gcagcttggg 12000
tctctgtgga ggtgcaggtt cgatccccaa cctggcacag tgggttaaag gatccagagg 12060
cagagatcac agctgcagcc tggatttcat ccacggcagg ggacatccat gtgccatagg 12120
tacagccatt aaaaataaga aaaagaagga agggaagaaa gaaagaaaga aagaaaagag 12180
aaaaaaagaa agggagggag ggaagagtat agaccacaat tatattaact cagaaatggg 12240
gggatttcct gtcgtggctt agtggttaat gaatctgact acgaacgatg aggtttcggg 12300
ttggatccct ggccttgctc agtgggttca ggatctggca ttgctgtgag ctgtggtgta 12360
ggtggcagac acggctcata actggcgttg ctgtggctct ggtgtaggcc tgtggctaca 12420
gctctgatta gacccctagc ctgggaacct ccatgtgcca ccggagcggc gctagaaaag 12480
gcaaaaagac caaagaaaag aaaaaagaaa tgtgtaaatg aagacatctg gaaaatccac 12540
acatatttag aagttaacta acacacttca attatcacaa ctttgtaaat caactatact 12600
tcgataaaac ttaaaaaaaa agagtattcg aaagattaat ttttaaaaac tgtgggattc 12660
atcaaaagta gtttcaggag ttcccatcgt ggcacagtgg ttaacgaatc cgactaggaa 12720
ccatgaggtt gcgggttcgg tccctgccct tgctcagtgg gttaacgatc cggcgttgcc 12780
gtgagctgtg gtgtaggttg cagacgaggc tcggatcccg cgttgctgtg gctctggcgt 12840
aggccggtgg ctacagctcc gattcaaccc ctggcctggg aacctccata tgcctcggga 12900
gtggcccaag aaatagcaac aacaacaaca acaactaaaa aaaataaaaa taaaaataaa 12960
aaataaaaaa aagtagtttc agaaggaaag agactattac aaacaactta agcaaataca 13020
tttgataact agtatcaagt aaatcagttt ctttaaagac accacttacc aaactgaatt 13080
aagaagaaac aggaaagtta atgacttctt atgaattgaa gaaactgaat tcataattta 13140
aaaaagactc caggcagaaa attcctggct tggatggctt cattggtgaa ttctatcaaa 13200
cattcaacta ttaacggcag cacgtcacaa actcttggag aaaatacaag gggaagacaa 13260
tctacctccg tggccaccag ctgggcggct ctttttctga agctctgtgg ccagtctttg 13320
tgccacagta ctccatgctg tctgagctcc caatgagcca gggtcccagt agcccagggc 13380
tgtgtgctga ggacaggctg tcccttgact gtccctgtcc ccagcatcat gtgtgggggg 13440
gatgtggtgg ggagaggggt ggggtgtttc actggaggag ggggattcct tgaatcacct 13500
ccccactctc ctgtctgctc tgcccttcct tctgggtgag tctgagttat aacagtcctg 13560
taaggacaga aaaaggcctc acactgaagc agcatttacc agcccagccc agagcgaagg 13620
cctgtgtgat tatcatccag gggaaaagca gagcctcttc ccagcgccca ggccagaagc 13680
ccctgtgcct ctccccacac acaccccgcc ctccctgcac acgtgtcccc tgtgctgacc 13740
ctcccccggc cctgctctct ggtccttccc tcccctctcc tgtgcctcca tccccgactg 13800
tcaccgtttc ctccctctcc tcactttccc aggagactcc ccatcttctt aggcctttcc 13860
cctgggacct cctcctcatc ctgctccccc tacacccttc ctggaaggga gagtccaggg 13920
ttgactccct ggaggagact gtctctagaa gcagcattga aaacaagggt caggggattg 13980
ggcttcaagg gtcttcccag gagaccatga cacccactat ggttgtaggc tccaccctta 14040
ccctgggaac cagctgttag gcccttgagg gctggtcctc acttaaggcc aaatcccttg 14100
ctcatccagt gaggatacaa acttctgtgt cagcatgcat tcgggggaat caattcagat 14160
tttgaacctg acactggttc aaaccctgac acctggtgtg cacagcactg accccgctgc 14220
tgggagccca aggagaccct gggcaggtag gtgcctggta aggaggtcaa aagagagctc 14280
agctctcctg tcatcagccc tgtggccaca cgggggcacc actgctctgt tcactccttt 14340
cagcccagcc gggggagaag atgccctgtg gtctgaggca gagagatgct gccctggagg 14400
ctgggcaaca gtgggggatg aagatgcctt ctgggtagct ctggggtcag gctctgggat 14460
gggacagggt ttggaggaga ggagagccct ggagccctgc cctttgcctg aaggttccat 14520
cttttctccc tattactcct gtcttcatcc tttggtgaaa acaccctgga aaactcaatg 14580
tagaacattg atttttcagt cgcagacccg gctcccaacc tggcgttgct gtggctgtgc 14640
tgtaggccag cggctacagt tccgattcca cccctagcct gggaactccc acatgctgca 14700
ggtgctgccc taaaaagacg aaaaagaaaa aaaaaaaaaa agaaaatgga tgtttccact 14760
ccctgttcct ttctggggga tatgaatctt tttcaacgtc agtgtagttt gggattcacc 14820
aaaaagtagc ccagttagca gagagttacc gtctgtccca cacttggtat ctctgggccg 14880
acatctcatg tcgccatcgt gcatttgtca cagcccataa aacaatattg acacattctt 14940
accaaactcc aaaatttctt tgactgtcac tggtttttca tttttttcct gatccaggat 15000
cgtattgaag acacattata ttaagtccca atcgtgtctc cttccggccc ctctggctat 15060
gacagtctct cagcctttcc tcgactttga tgaccttggt gttttccagt cctgctcagg 15120
gaaatggcac aatgtctgtt tgagcttgga tgacggagcc aaagggtctg ggggaagagg 15180
acacggagat gaagggacca tctccccaca tcaagggtgt ttaccctcca cttgagaagg 15240
acccatacgc tcacctggca cctggctaag gtgtgttccc aggtctctcc gtgtgaactt 15300
tcattcttac ttcccccctt tccacacgcc agtccttaga aggaagtccc taagtgcagc 15360
ccacgcttaa gggccgctct gctctcaccg tttgtgcgca gggttctgtg caaatacgtc 15420
ttcagaggag ctggagaagg acccgtgtgc tcagcaggct gggtgctgag tccctgtgca 15480
gcttcccgag aacctgctga ccgtcaccca gtggctgtgc tgcttcgtat tcctgccccg 15540
gggatgaggg ttctgctgct ccacatcctg ctcagcacct ggtaccaccc atttgttttg 15600
gttttggttt tgggttgttt ttgttttgtt tttgtttttt tactcttttg atttaatttt 15660
ataaaaaatt ttgacaatat tcagtcaatt tctgctggaa agcaaagtga cccagtcata 15720
cacacacaca tacacacaca cacacacaca cacacacatt ctttttctca tattatgtgt 15780
tctatcacaa gtgactggat atggttccct gtgctacaga gcaggagctc atggcttagt 15840
cgctctaaat ggaatggttt gcatccactc agcccaacct ccccgtccat cccactccct 15900
ccccgtcggc aacctgaggt ctgttctctg caggtggata tcaccagttc ttggattcca 15960
gctcttctaa tgggtgtgtg atggttcatc agtgttttaa ttttcatttc ttaagaactt 16020
cagatgttga ttcgcttgcg tatgcatttc cattgataga tcttgttaaa gagatgtctg 16080
ttcagctctt cttcatttgt aaattatttg ttttcatttt gtggagttga agggtcctgt 16140
gtatattgac ttactagtcc tttattggat aggggaattg gcctagaatt tctgccagtc 16200
tgtggcctga gctcttttac tgacctaaca gtgtgtgtta atgaatagag agagttttct 16260
ggggagaaaa atgcagaatt actaattgcc atggtttctg agtagagcac ttgggcgaag 16320
ggggatgagg cagagaatat gaattcgtta tttgtgtatg ttccagagta tctttgcagt 16380
tctcttgcat gcaggtaaat ggcactttca gatttttttt tttttttttt gtctttttgt 16440
ctttttaggg ccgcatccac agcatgtgga gattcccagg ctaggggtct aatcggagct 16500
gtagctcctg gcctacacca cagctcacag caacgcctga tccttaaccc acaggtagag 16560
gccagggatc gaatccacca cctcatggtt cctagttgga ttcgtttccg ctgcgccaca 16620
acgggaagtc ccactttcag ataatgacat taaaatctct gacatcaaaa aagaaagaaa 16680
gctccctttg tggctcagca gtaccgagcc cgactagtat ccatgaggag gagggttcta 16740
tccctggcct cgctcagtgg gttaagggtc cagcattgcc atgagctgtg gtgtaggtca 16800
cagtcggctc agacgccatg ttgctctggc tgtggctccc attcaacccc tagcctggga 16860
acctccatat gtcacaggtg aggcccaaat aaagaactaa aatccccgac acatatgaac 16920
atgtgccaga ctgtcttcag atcacaagtg aggaagcaca tgctgatcct ttactccagg 16980
acccaccacg ttgggagtga gctactctgc cacagaaatt ccactactgg aaaagacggg 17040
ctggagtcta tggacgaggc tgcccacgga aaatagctaa aaatgcgatg taaacacaac 17100
aacagcctta agagcaacaa agaggtgacg aggtggtaaa aaatgagttg tggccaggag 17160
ttcccattgt ggctcagcag gttaagggct agacatctct gtgaggatac ggggacaatg 17220
cctgacctcg ctcaatgggt ggaggatcca gccttgccac agctgcccag cagagcacac 17280
gtgttgctgt gaccgcaggg taggcctcgg tgcagctcca gttcaacccc tagctctgga 17340
attcccagat gccagaggtg tggctgtaaa acaaatgaat ggaaagatcc tgcttggcct 17400
ccgcagaaaa tgtggtcccg agaagaggct gtgtgattcc ctgcccagag cccaggcatg 17460
gagcccagca cgtccagctc tgtgccccca gctcttggct tcttctaccc acctgccttc 17520
cttttaaaac atgcaaacaa agctttttat ttccaattat gaggtatatg cataatgtgt 17580
aatatacgga taatgtatac tcgctataaa acaagctgta aagtgttgaa tgtgataaat 17640
gacaacgtca aaagaatgtg gctaaaacta gcccgcctaa tcctcggggc agagcctgcc 17700
tgcatctctc acaagcatct tctcctaaga aatccattcc ttgggcgttc ccgttgtggc 17760
tcagtggtta acgaacccga ctagcatcca tgaggactca ggttccatcc ctggcctcgc 17820
tcagtgggtt gaggatccaa cgttgccgtg agctgtggtg taggtcacag acacggctca 17880
gatctggtgt tgctgtggct gtggcgcagg ccagcggcgg cagctccaat tagaccccta 17940
gcctgggacc ctccatgtgc cttggttgtg ggctgaaaag aaagaaaaag aaaagaaaga 18000
aaaagaaaag aaaagatatc tatttcctgc caatcagaaa aaaaaaaaaa aaagtcaaaa 18060
gaatagaaac ctcacactgg agatagttgc atgcgtttac tttcagttgc ttatacatcc 18120
tgctgcccac ttgctagaac tttctctaag gtgctgagca cagacctcag ggccccagcc 18180
tgcactctcc agaagccatt tcctgagtca gttcccctca cctgcccccc actctggggc 18240
aggtcctctg ccctcctccc tcctaatcct ctccactccc tccccacgcc ctgttcttga 18300
agggagcttc tttcttttgt cactcaggaa aaaagagact cagatgagaa ttacatcctc 18360
cacctccccc actctgccac ctgcacctgc accttcacct tcccaccagc aatgatgggc 18420
agccccctcc tgtctgaggc tgcccctcgg catgggcaca ggatccggcc cctctctcac 18480
tctctacttg ctcctgcctg cctccaaaca cacttaattc ctcccattaa aattaatcca 18540
acacacgaaa taaatcaccg agtttggggt tggtagatgc agactgctgc atttggagtg 18600
gataagaaat gagatcctac tctgtagcac ttgtgatgaa gcaggatgga ggatgaagcg 18660
agaaaaagaa tgtgcataaa tgcataactg ggccactttg ctgcacagta gatatggaca 18720
gaacactgta aatcgactgt aataaaaatg tgaaaactgg agttcacgtc atggctcagc 18780
agttaacgaa tccaactagg aaccatgagg ttgcgggttc gatccctggc ctcgctcagt 18840
gggttgatga tccggcatgg ctgtgagctg tggtgtaggt ggcagatgcg gctcggatct 18900
ggcattgctg tggctctggc tccgattaga cccctagcct aggaacctcc atatgccaag 18960
ggagtagccc tagaaaacgc aaaaggcaaa aaaagaaaaa aaaaagaaaa aactaaaaga 19020
aactaaataa atcaaaatac aaccgtcccg taaacatcac tactctatgt actttgacct 19080
tccaacttct gctctgtttc tatgactctt ggtccagaac aagccttggt ccatgctctt 19140
tcacctgcct attgtctttc atgtttcgcc aatggccaga ggcaggtagg ctgaaaaagt 19200
aaaatacttt acaagactcg ctgtcaggat ggctgcccca tcatcttcct gcccccgtct 19260
cttgaatctc cgagatgagc tgttttcaat ctttcagctg acgctttgtt ttgtctttct 19320
acgcctttaa aaccatgcct ataagaccct ttctctgtct ctcacagcac ttgactcagt 19380
tttgatttac tcctattcat ttttgttttc ttgttttggg ttttttgttt tcttgttttg 19440
tctttttagg gccacaccca tggcatatgg aagttcccag gttacgggtc caatcggagc 19500
catagctgct ggcctacaag tagcatcgat gaggacatgg ttccatccct ggcctcgatc 19560
agtgggttgg ggatccagca ttgccgtgag ctgtgatgta ggtctcagac ggggctcgga 19620
tcccgagttg ttgttttttt tggtttgcct ggggcatgtg gaaattccag ggccaaggac 19680
taacctgcgc catagcagtc cctgagcagc tgcaatgaaa ggccagatcc ttaaaaggct 19740
gggccaccag ggcactccga tttactctta tcatctttgt ctcaccacag ggcctgtcac 19800
atcaccaggg tcctgactgc tgttcttcat ttcatcacca gtgtctccag atctggagtc 19860
aaggggacct tgaaatagcc actccttaaa cctggagtct cccttccccc gtctgcacta 19920
gggatcacat gtcctgtgcc tcatgtgttg gctttgagga atacattcga ccactgtgtc 19980
aattacatgg catgatgcct ggaacacaaa gtgttcccct gttcatagtt actaacatta 20040
gagtgatgaa atcattttga ttatttccat tcttttattt atttttattt ttattttttt 20100
gtctttttgc catttcttgg gccactcccg aggcatatgg aggttcccgg gctaggggtg 20160
gaatcagagc tgtagccact ggcctacacc agagccgcca cagcaacgcg ggatctgagc 20220
cacgtctgca acctacatca cagctcacgg caacgccgga tctctaaccc tctaagcaag 20280
gccagggatc gaacctgcaa cctcatggtt cctcgttgga ttcgttaacc actgcgccat 20340
gatgggaact ccatttatta tttccattct ttgtaaaccg ccattaaaaa aaatccttga 20400
atatatattt ttgagaacac gaatgattat cataggatga ggttttagaa gacatttctg 20460
tgtcaaaatg taaacataat aaagttattg agaaatgttg caaaagtgac ctttaaattt 20520
tttgtaccag ttcttaatcc cacaacctta taacattatc atcttcctaa attcctgtca 20580
atactctcct catttacttc catctttggc aagatgacaa attttaattc ctttccattt 20640
cttatttcac attttcatgt ttttacatta attattacta taatatttgt ttaacacttg 20700
cacttcttct tttcaaatat gttttccagt attctctatt gggcggtgga atgtagttac 20760
ttcaaaatac tgaattcagg gggttcttat tgtagctcag caggtcacaa acatgactag 20820
tatccatgca gatgcaggtt tgattcctgg acttgttcag ttggttaaag atcctgcatt 20880
gccatgacct gtggtgcagg ccacagaccc catttgggtc tcctgttact gtggttgtgg 20940
cataggccag aggctgcagc tcagattcca cccctagcct gggaacttcc atataccgca 21000
tgtgaggccc taacagctaa agtacataaa caaaataaaa taaaataaaa tattgaattc 21060
aagagggaca aagcagccct ggacatcagg aactgctctg acacctccag gaggcctcag 21120
cagtgtcaga agcagggcag gtgctcacca cgcggtcggg ttgttctcct gttggacaga 21180
agtcacctca cagaacatca gcatcagacc aggacactga ccttgacaca gtgaaacaaa 21240
acaagaccac gacatcattc taaggccaga ccaagcaagg tggccctgtg acctcacagt 21300
gtgccaaact cctccctttg caatcacagc tcgaccctgg ctctagtctg ccctccctac 21360
aggtgagggt cactgagatg cccggtcaga gacctgcccc caccgcctga caccgacacc 21420
cgatctagac tgagcccctc ttccatccac cctcctcaga atcacccacc aaaagcccaa 21480
atcctatata tgttcttctt cttcttcctt tttttttttt tttttttttt gggctttttg 21540
tctttttagg gctgcaccca cggcatatgg aagttcccag gcgaggggtc taatcggagc 21600
tatagccact ggtctacacc agagccacag caacaccaga tccgagctgt gtctgccacc 21660
tataccagag ctcacggcaa caccagatcc ttgacccatt gagcaaggcc agggatcgaa 21720
cacacaacct catgattccc agtcatgctt gtttctgctg cgccatgacg ggaactccca 21780
aatcctgtat atgttctaac accttcacac taagacgccc tctgctccct gaggcagtgt 21840
gttctccctc gctgcatcca gacatacacc caacctcttt gcccgcatag ttgctcctgg 21900
tggctgcaga accttgagac atgttacttt tcatttgtca attttgttac atgtctttga 21960
gttgacatga gggcatgggt ttatattgct gtctgtgtct ggttgacaca gaaaagattt 22020
gtggcttttt atgggttttc atctttgggt gacccttaga gacgtcttca ttccatgtgt 22080
aaaacatttc atcaggagag aggggtccac tggtgaaggg aaacagcatt gactgaagtc 22140
gagggagggg gaccggagta gccgtcagta acattactgg gtattctaag gtggtggatt 22200
ctgaggtcac atcttctgtt ttataatttg acaaccatgt acaggtccta caagatattt 22260
gataaatgcc gtgttcaaca aacactttct gtggggctaa gtgaatgaag acttctgtgt 22320
ctatgcgatg tcttccgctt acatttatca ggttaaagaa aacgacacca agaacccaag 22380
gtaatataga caagcgttag ctccagtgat gggcagattt gagcagttat gaccttcaag 22440
ggataagtca tggtttggag cagaacctcc ttgcctgccc ccttgcatct ctcacaagtg 22500
tccaacttaa taaatgtttc ttgcctatca aaaaaaaaaa aatgtcatgg tttggttaat 22560
gtctgtacac cctaaataga tatgtaaata aatactataa atgctttcta aatactgtta 22620
tctttttttt ttcattcttg catttttttt ttttttttag agccacacct gtggcataag 22680
gaggttccca ggctaggggt caacttggag ctacagctgc tggcctgcac cacagccaca 22740
gcaacctggg atccaagctg tgtctgcgac ctacaccaca gctcatggta acgtcggatc 22800
cttgacccac tgagccaggc cagggatcaa acctgaaacc tcatggttcc taggtggatt 22860
tgttcctgct gtgcctgagt gggaactcct actctaaata ttctcaaaat aaatattaat 22920
taaactttct tggacatact gcattttaag gatatatcca agactcagac aaatccaaat 22980
gacttccata tgaacattac gtcaatatgc atcaatctat gaatcagacc ttatctttgg 23040
ggggaagagc ctgaaaatta agaacattct cgatatccac tgtcccatat gatagctaca 23100
ggtcacatat ggctacttta attaaattaa agaaacgtaa acatactcag cacaaaagcc 23160
atttctgagg tgctaaatag ccacatgtag ccagtgagca gctgatgaca cagacagata 23220
tgaacagttc cttcacacag aacgggctgt tggacaggac tgctcttcct gctttgtggg 23280
cctcacgtca gatacattag acccaagaga gaggattcct ctcagagact ctgtcacaga 23340
ttcctgtctg ggacgtgaat gcatatgaag ctgaggtgga acaaaaccaa ggcggtgatg 23400
cgtgggtctg gcaccttcag acataggctg ttcccagttc cagtggggaa acacctgtcc 23460
ccccacctcc cttcccagat ggaggactct ctctggcatc actttccagg ggctgagtcc 23520
tttcctggag gagcccaggg gcagggccct cagctgaagc tggggaggca gggagtccag 23580
ggcagggaga gggattctgg ggaatgaaga ggaggccaag gcaaaggtca gtttgaagac 23640
ttttccaggt gtattcagaa cagccgttct cagagtgtgg tctgtggaac ccttgaagtt 23700
atggagaccc tttcatgaga tccctgagtg aaaactacat tcatagcatt actaggatag 23760
catttgactt ttcactatgt tgacatttgc tttgaggtac aacttagcaa taatggggac 23820
tagggtagcc aactgtaata gtaattattt tcaccacaac ttacacgtag aaaaaaataa 23880
taataagcag gtttggtgtt tccgtcatgg cacagtggaa acgaacccga ctaggaacca 23940
tgaggatgca tgttcaatcc ccggccttgc ttgctcagtg ggttaaggat ctggcgttgt 24000
gacaagctat gtctgtgatg taggccggtg gctacagctc caatttcacc cctagcctgg 24060
gaacctccat atgccacagg tgaggcccta aaaacacaaa aggcaaatga caaaaaaaaa 24120
aaaagcaggc ttgttataaa acgtcctggt cgatgcaata aaaattccta tttacattta 24180
taaaccataa tggagaagaa tagacataga cacatatgtg tacaatcgaa ggacttcact 24240
gtccagcaga aatgaacaca tcgcaaatca gctatttgaa ataaaataaa atttttgaaa 24300
taaagaaatc aaaatgttta tcttccgtaa atcatctttt ttttttgtct ttgtatggcc 24360
gacccattgt atatgcagat ccccaggtta ggggttgaag aggttgcatc agagctgtag 24420
atgctgtcct ataccacagc cacaacacca ccagatccga acctcgtctg agacctacac 24480
cacagctcat gccatcatag catccttaac atactgatca aggccaggga tcgaacctgc 24540
ttcctcacag atgctactca gatttgtttc cactgaacca ggacaggaac tcctacatca 24600
tccttctaaa tccatgtttt aatagtatct tctctgatgt cgggggaagt acatatcaaa 24660
cactcttgca cattccaaag tagcatgtcc caaggaaaat cattctgtga gctgacctac 24720
cctctttttc ctggaataca acctttacta gaaagactga atgacaatgg aagatctatg 24780
taacttagtg aaacaatgta ttatttttaa aaaatcttac attagtacaa gaggcagtca 24840
atgtgcaaaa caggcttatg gagttaatgc aacagaatag aaaacattga atgatgttgt 24900
ttcagctcca cttggaaaat atttgagaaa ttaccataat gatagcatcc aaaattatct 24960
gaaaaggtta ttaaaaatac acatctacgt ttcatagatg tcagtcacaa aaaggactca 25020
gtgcagaagc agacataaac aaccagtgct cttctcctga gccagacagc agagagactt 25080
gccatagagt aaaatgcaaa aagctccact ttgggcacta cagcgtttct tatgggcaat 25140
aactgttttc atatgaaatg catggatggt tcatattcct ctaaaaaaat aaattcaaat 25200
gttaactatt atttctagta tggaaaatat cgattgaagt aacgacataa aatacatatc 25260
cctgaggacc taattcgtaa aatgtataaa tattcctact aaaatagttt gaaatcaccg 25320
agtacacatt ccttgggcca ggtgactcaa cggaaacaat gtgacaaaga gctttctgag 25380
cagaaggagc aggagcagga gcagggcctg gctctcgggg tcgcaggctc cagggcggta 25440
tgagggtggg caagctccat gttggagtcc ccgtgtcccc agtttcactt cgccatctcc 25500
caacctgtgt ggggccctcc tgtccggaca cttgtggcgc ggccccattt ctctctccta 25560
ttgagtgtcc ggtttctgga gacgccaatc ggcgccaccg cggttcccgg ttctaaactc 25620
tgcacgtacc ctcggctcga ctcagcttct ccccagactc cgcggctgag gatcatgggg 25680
ccccgagccc tcttcctgct gctgtcgggg gccctggccc tgaccgggac tcgggcaggt 25740
gagtgcgagg tcgggggaca cggccactgc ggggaggaga ggacaccgcc ccgtgggtcg 25800
gggtcggggt cggggtggcg gcaggaccca cggggaaggt gcgactcagc ggtccaggcc 25860
cagaccccac cccccgacct cgccccatcc ggtcccgtcc ctcccttgct tccagcgcct 25920
ctgctcgccc ccgactaaac ccggggactt tctccgacct ccagcccttt cgcgcctccc 25980
gagccccgag ctccctgccc ggtccctcgc acccggggcc ccgcgctgga agaagggtcg 26040
tgtctcaccc tccccgccgc caggtcccca ctccctgagc tatctcttca cctcggtgtc 26100
ccggcctggc cgcggggagc cccgcttcat cgccgtcggc tgcgtggacg acacgccgtt 26160
ggtgcggttc gacagcgacg ccccgaagct gaggaaggag ccgcgggcgt ggaagataaa 26220
gcaggagggg ccggagtatg ggacgaggag acgcggatca gcaaggacaa cgcacagact 26280
ctccgaggga aactgaacac cctgcgcggc tactacaacc agagcgaggc cagtgagcga 26340
cgcgggcccg gggccagctc acgaccccca tccccaggca ccggccgggg tcaccccgac 26400
ctccgggtcc gagggtcacg ccgaccttgc gggacccgcc aggccccccg acaaggctga 26460
gctggcgagg actttcgcct ggtttcagct tcagtttagg tttaacccct ggttggtcgc 26520
gacgggggcg tggctgactc ggggcggggt cagggtctca caccctccag agcttgtttg 26580
gctgcgacgt gggaccagac cggctcctcc tccgccggta ccgacgggac acctacgacg 26640
gcgccgatta cgtcgccctg aacgaggacc tgcgctcctg gaccgcggcg gacacggcgg 26700
ctcagatctc caaatgcaag tgggagacgg ccaatgtggc ggagcaggag aggagctacc 26760
tgcagggcac gtgtgtggag tggcttcaga aatacctgga gatgggggag gacacgctgc 26820
agcgcgcagg tatcaggggc cgcggggcct ccaccatctc ccctcgggag ggagctgcct 26880
tcccacaggg agaggaaaag ggggtccctg cgggaaagcc cccactgcct gtcctgagag 26940
ggaggagtcc acctaggttt ccagcttctc cacaagacag ggactccgca ggagcccctc 27000
ttccctaaag gacagttaag gaagcctttc tcttttgggg tgaaaggggg agaccatccc 27060
tgaaaggact gctcagcggt gccctttgac cctggccgcc atcgtgtgaa ccacgacttt 27120
ctcaagccta ttctgagcct gagaacagct ctgcatcctt gacaccacct tgcctgagtc 27180
attccgcctc caccaacgtc aggaacatga ctatattctc ccccttacag tcctggagct 27240
tcctccctgg actatcaccc tgagtctaga actttctaag gactaggagc tgttcccaga 27300
tacccaagtc caggctggcc aaatggtttt gtgcttcctt tcaaacccat tgacctggcc 27360
cttcccccac tggtcacatg aggctgcttc aggggcccaa gcaggggccc caaggggtga 27420
attttctgat tcttcttcct cagagcctcc aaagacacat gtgacccgcc accccagctc 27480
tgacctgagg gtcaccttga ggtgctgggc cctgggcttc taccctaagg agatctccct 27540
gacctggcag cgggaggggc aggaccagag ccaggacatg gagctggtgg agaccaggcc 27600
ctcagggatg ggaccttcca gaagtgggcg gccctggtgg tgcctcctgg agaggagcag 27660
agctacacct gccatgtgca gcacgagggc ctgcaggagc ccctcaccct gagatggggt 27720
aaggagggcc ctgggggcgg agcctcttct cagacacagc agaagccctt ctggagactt 27780
ccgcaaggtc aggactccag cctgaggaag gccctcacct tgccctttgt tcccagaccc 27840
tgctcagccc cctgtcccca ttgtggacat cattgttggc ctggtactgg tcctggtctc 27900
tggagccatg gtgactggag ttgtgatctg gaggaagaag cgctcaggta gggaagggag 27960
cggggatctg agtctccttg tgtcacgggg gtttcaagcc caggtggaaa tttgttctgc 28020
cttcttcctg ggacgtcccc tccacacaca tgtgctgagt ctggggctga gtgccaacac 28080
gtatccttct gtgaagcata tgtggaaatg aaagacaaag ttttccccag gatgattcca 28140
gttggcatct agtttctcag catttgacgg tcagagggga aggaccccac tgaggagagg 28200
cctccaggaa ggcagttgat ccaaactccc caacatttcc ttcctgctta ttcctggtcc 28260
ttttctggat ttttttctca aagttctgga accttctttt tgatccagaa ttagggtttc 28320
ctctggcatc tcatgactca cactactccc ccggcctctc acatgatttc tcttcccaca 28380
ggtgaaaaag gagggagctg cactcaagct gcaggtcagt gtggagggct gtgatccctg 28440
aggccctggt ggtacagaca ggaggccatg aggggagctc acccaccttc tctcgtcttc 28500
tctcttgggg gtcctgaccc cgtcctgctt ttgctctccc tcaggcagtg acagtgccca 28560
gggctctgat gtgtttcttc caatggatcc tagaggtgag accctggagg gcctagatgg 28620
gggaaggggt tggggcagag ggggtgccct gggtgatggg gatctttgag ggggacgttt 28680
ggagcatgtg gggctgttga gcatgtcagc ccttccttga ctgacctgcc tgtttcctgg 28740
tgattttctt tcccagtgtg agacagctgc cttgtgggga ctgagggaca caagatttgt 28800
tcacgtccca ctttgtgact tcagatcccc tgactcctgt ttctgcagct gcctctgaaa 28860
gtgtctgtgt ttctatgagc atcatgagag taggtgggaa cactggccta accctgtccc 28920
ccacatcccc tcctcaccct gacctgtgtt ctcttccctg atccactttt cagctccagc 28980
aggtgggggc tgggagatct actttgatcc ccactgtccc ctcctctggt ccccaagccc 29040
ctgtgtaatc ctgacttttg gggcatccgc tggccctagt gactcgctgg gaacaaacag 29100
aataggagat gtttggaaaa gtctattttg gtatccctgg ggtggggagg gtagagggag 29160
atacgaggga cctggggtca gtcgtctcag ggtggatggt ccaggccagg ctggggagca 29220
gagattcctg aagggtcccc gtgacaggtg acgctgcagc cctttcctac ccaaggggtt 29280
cctcctctag gtctcagcct ccacagcccc tgccatcgga caggaggagg aggagagacc 29340
agcagcagag actcccatcc tgcctggaat gtccggggaa ccgtgggtag aaaggacggg 29400
aagggaaggg gagagtcccg gtgaccccaa cactctgagg gaagccccag ggctggaaag 29460
aagaggaggg tgacaggttc caggctccct gtgccgcccc ctctggccca gctctctttc 29520
tcaactcaca ctaaggtccc ggagcccaca gtggtgaccc tgtaaatacc tctgggccca 29580
tctccagaac agcccaccct ggccgcacat cctgggaaat cctgtccgcc ctgcatgtgt 29640
gaactcagag gctgaagcag aaacccgcat accactggtc tctcaggacg gaagctggtg 29700
tgtgtcccat gtcagtgagg caggcagctc cagggctgct ggggtggctc tgcaccgcaa 29760
gatagtccag gcatccaggt ccctgtgacc gtccctccct gctgtctctg gggtggtgcc 29820
cttattagga tgacccaagg ttcccaaggc catgttcatg gaccatgagc aaccaggcag 29880
agcaggggag gaggcaaagt cacgtggcat gtgcttgggt ggccaaatgc atcatcaata 29940
agcaggagta ttttgtttgt ttgtctgttc gtttttaggg ccccacgcat ggcatatgga 30000
ggttcccagg ctatgggtcg aattagaggt gtagctgctg gccttctcca cagccacagc 30060
aacaccagat ccgagccaca tttgcaacct ataccacagc tcatggcaac accagatcct 30120
taacccactg agcaaggcca gggatggaac ctgcaacctc atggttccta gtcggattcg 30180
tttccactgt gccacgatgg gaactcctaa gcggtcatat cttgaaagga acttttcttc 30240
tgcgcagtag ttctcaacag tgggcatcgc atatacagtc cgccgtgttg taaacagatg 30300
tgctgtcatc taggctttgt tgttccattt ctagagcata ggcagggcgg ctataatatc 30360
catcttaacg gccctagggt tttctgaatg gcaaatgagc attggcttca atttcaggtg 30420
ctagttacct aaaaggagct tcccatagtt aaacttgcgg tttcaaaaca tgctgggaaa 30480
tgttcgctga tggttccgtt gtgaaagaac gttttgttgc aaacaatatg gccaagcact 30540
acaagattcc cgtatcctcc atctgtatct gtcaatacca gcagatacac gctgatttcg 30600
gacgttgtat gtttagctcc tcattgaggc agggacagag caattggcag ccagattttc 30660
cttctttctc ttttgtttct tttctgtggc cacacctgtg acatatggaa cttcccatgc 30720
caggagtgga attagagctg cagctgaggc ctatgccaca gccacagcaa ctctggatcc 30780
cagccgcatc tgccacctct gctccagctt gcagccatgt cggagcctta acccaatgag 30840
caaagccagg gatccaaccc gcatcctcac ggagacaacc ttggctcttt aacccactga 30900
tccacgacgg gaacgccgca gccagatctt ttctaggtgg tgagggccca aaggttttga 30960
ctaatatgtt ctattacagt tcaggccaca tttgacttat gggaccaaag gtaacgagtt 31020
ttgtgggtta aggatataga tagaggatgt tccaacaatt ttggttagaa atagacatga 31080
aatgaaatgc agattcttac taaagaacag ggtcagaatt taggaagata atggtgagac 31140
ccttcctctt tagaatattt tttcagtcaa cgattaatgt tagggaaata cattatcaag 31200
tgggtaggcg gatgactaga ataacaaatc agaaaaaaag aaattatgtt tggggtttcc 31260
gtcatggccc agtggttaac gaatccgact aggaaccatg aggttgcggg ttggatccct 31320
ggccttgtgc agtgggttaa ggatctggcg gtgccgtgag ctgtggtgta gctcccagat 31380
gtggcccgga tcctgagatg ccgtggctgt ggcataggct ggcagctaca gctccgatta 31440
ggcccctagc ctgggaacct ccgtatgccg agggaagcgg ccctagaaaa ggcaaaaaga 31500
caaaaaaggc agaaattatg ttcgatgctg caaccacagt caccagtaga tctgagtcaa 31560
ttgcctattt caaaagtgcc aacactccag tcttcaaaaa atatatcaaa aggccatgtg 31620
ctcctgttgc atgtatttgg tgaagaattt cggtcttata ttggcttgta cgggttccct 31680
gcagaaaaaa aataaatatt tttgctgtgt acatttctca ttgagcaaag acttgctcct 31740
ttgcaacaga agctggaacg ggtgtgccag tgaacctaac actgacgtgg tcattaccaa 31800
ggcaatgggt gcgtttccat tgtttgtgtg acagtaataa aagtttacat gaacctttca 31860
tcatgatgtt tacctgtcac acctgaaaat gttgagccat tttctgtcaa actcatagta 31920
gcaggtcaat ttctggcctt gtcgaaacca tttaaagctt aagaaatacc attctgtttt 31980
gttttctgat ttcaagtgtg cagggtgcgt tggagaatga agacacaaca cacccaaact 32040
tatggatgcc atgaaaccag cactaagagg gaaactgaga gctgtgaggg cttctgctaa 32100
aaagaagagg taggagttcc ctggtggcct accagttaag aacttggtgt tgtccctgct 32160
gtggcttggg ttctattcct ggcccaggaa cttccacagg ctaaaacttt ttgaaaagaa 32220
atatcttaaa ttaaccacct aattttcaat gacttaagca gttagaaaaa gatgagcaaa 32280
gtatacctca agctgggaaa acaaatgaaa taataaagat tagagcagag agaaatagag 32340
actagaaaag cactagagaa aacagtgaca ccaaaagagc tgctttgaaa tgacgactga 32400
tgaaaatgat gaacttatag ctacagtggc taagaacaaa tgagagcaga ctcacataac 32460
taaaatctga gaagagggga catcacgtgt gattcagaga aatgaaaagg attgtaagag 32520
aatatgtgaa cacttgtatg ccagcaacct agagaaaagt gataaacgcc tagaaacaca 32580
caacttgatg aagactgaat cacgaagaaa gagaaaatct gaaccaatcc ctaacaaaat 32640
tattaagaag ataaaataaa aaaaaaaaga agataaaatc agtatttaaa aacctcacaa 32700
aaagaaaaca ctagtagtag atgacttccc cagtgaattc tattcaatat tcaagaattc 32760
tattcaatat tcaagaatta gccccaatcc tcctcaaatt cttgcaagaa atctgagagg 32820
agagaattct tccccactga gtctacgagg ccagccttac cttgaaacca aagccagaca 32880
gtacaggaaa agaaaaccat agacaatact cttcatgaat attgatgcaa aaatcttcaa 32940
caagatacta gcaaacaatt caacatcacc ttcaaaagat tatgcaccac gatgaagttg 33000
gatttactcc tgcaacagaa gggaatttct ccaaggatgg ttcagcagac agaaaccaac 33060
cgcattaaca gaatagcaac aacaacaaaa cccccgtgtt catctccatt gatacaaaaa 33120
agcacttgag aaactgaaca ccctttcagg ataaaaacac ctaactcgca ggtcccgtgg 33180
tggctcagcc ataaggaacc cgactaggag ccatgaggat gcggttcagc tcttggcccc 33240
actcattgtg ttgaggatcc agctttgtca tgagctgcgg tgcaggtagc aggcggggct 33300
cggatctccc actgctgtgg ctgtggcgta ggctggaagc tgaggtttga tttcaccttc 33360
cctatgccac acatgcagcc ctaaaacaca aacaagcaaa cagacaaact aactaactag 33420
gaataaaggg aacgtcaaca taataaaagc catttatgaa gagcccgcag ctaactgaaa 33480
ggcatacttt gaagatactg caggtttggc tccagaccac agccatgaag caagtattgc 33540
aataaagaga gtcaaaccac atttttggtt tcccagggca tgcataagtc atgtttccat 33600
gataccgcag tctattgggt gtgcgatagc atcaggtcat gtacatacac taagacacaa 33660
tgggcaaaca ctaattagaa gaagacccaa tggataaaaa gtgctgacca tcaagtgagt 33720
cttcagcgag gtgtaatgtc tttgcaatag ttacatccaa gattgttgat cacagacaga 33780
cccccataac aggtctagtc atagtgacaa aagtgggaat cattatgaga agcaccaaca 33840
tgtgacacag agacatgcag ggaggagatg ctcttagaga aacagtgccc acagccttgc 33900
tgaatgtagg gttgccacaa accctcaatt cagaaaatac tccatatctg ccaagggcat 33960
taacgcgaag caaaatacaa cgaggcatca agtaagtctc ttgatggtga gagactgaaa 34020
gcttttcctc taagatcagg gacaagacaa agatgcccac tgtccctagc ttgttgaaca 34080
cagaactggg agtcttagta gtgagaccag caaaggaaat aaaaggcatc caaaatagaa 34140
aggaaaaagt aaaattattt ctctgcataa gcgacatgat cttatttgtc gaaaacccta 34200
aagagctccc atgcacaaaa aacgctctta caactgataa atgaattaag caaatggcag 34260
gacaccaaat caacacacaa aaatccatct gatttctata ccctcaacaa tgaacagtcg 34320
gaacatgaaa ttaaaaatat cagtgatgat atcatccaaa ataataaaat ccttagtaat 34380
acacttaccc atggagataa tgaaaactac aaaacgtggt tgaaagaaat agaggacacg 34440
cgtcgtctcc agccttgcca cctcgaacgc acccaatctc atctgaggtc ggaagctaag 34500
cagggtccgg cctggttagt ccgcagatgg gagaaattac aagtaaatgg aaaggtattc 34560
tgtgttcata gattcgaaga cttgatattg ttcagaaaac agcactaccc acggcagtcg 34620
acatatttaa tgcaatctct gtcgatactc catcagcatt tgtttgcaga aatcgcaaaa 34680
tccatcctca aattcatatg ggttattgac ggactcagaa tggctgaaaa cgattttgaa 34740
aaagaagatc aaaattgaaa tttgtgattt cagaacttaa tatgaagcta cagtcatcaa 34800
agtggtgtgg tactagcgga aaaacagaca taatatttac ataagttaat gggaacagca 34860
cggagagccc aaaagtaact cttgcacgtg tgggcaaatg attttggaca agggtgccaa 34920
gactattccg tgtggaaagg acagcctttc cacaaatggt atgaaaaact gggtatccac 34980
ataaacaaga aggaagttgg agtgctgcct tacaccatta catgcacatt aactccaaat 35040
gggccgaaga cctaaacgta agaacttcac ttgtaaagct cttaggagaa aacggaagag 35100
gaatattcat gatatggaac ctgacaataa tttcatggat atgacaccaa gcacacaggc 35160
aacaacaagc aaaggaggta agtcagacta cattaacgtt taaagcttca gtgcctcaaa 35220
ggacacagtg tggaaaggca gcctctggaa tgagagaaaa tatttacaaa tcgtctatct 35280
gatgatgctg aaaaaatgca caacttagga gttcctgctg tggcccagtg ggttttaagg 35340
atcctgagct gccacagcgg tggcacaggt tacagctgtg gttgggatgg atccctggcc 35400
ctggaacttc catgtgccgt gggtgcggtc aaaaaagaaa acacaaatgc acatcctaaa 35460
agttgaaaat tatgacttac tagattgtct atgttgcttt tataaactag aattcaatga 35520
acatattttg aagaacatac atgattaata gtcttaggat gaatttctac aagagagaca 35580
tctttgtgtc aaaatgtcaa cccatttaaa ctgctgagac atttgcaaaa ctgccctttc 35640
aaaagtttgt gctggttttc aatttcatca accctgtgat catatatttt ccctacccta 35700
tggccagtga tcaccccccc acacaccatg acagtgacca gagccacaga gtgacaaagg 35760
cagatcctta acccactgag ccaccaggga actcctacat atatattttt acttttgtat 35820
attatgactt ttgacagctt tgaaaacctt cctagctgag gagagactgc tttcccaggg 35880
ctagagccca gccaggagca tgcctttgat atgcaaacta accagtggag gccctacctc 35940
ctctgtgtgg tctgagtatc tgaggcaata ttctgcctta aggatcccag agctaggtac 36000
cagacacctt aagagcaccc ccatagcccc aaacctgctg aaactatcca caccagccag 36060
tcttcatctg tgtaccctgc cctgccttga ccttcccagg gagacctggt caaaatcgca 36120
gatttgtttg tggttgtttt ttttgttttt tgtttttttt tttttccttt ttttggccat 36180
gtggaagttt cctgggccag gggttgaatc ccagccagag ctgagaccta agccacagtt 36240
gcacctggct gggaatggaa cctacagtgc tgatcctgct gggccacagc gggaactcct 36300
aaagttgtag cctttatttt cctttcatct ttgccaagct gatagatttt aattttttgt 36360
tctatttctt cttccaacat tctcactttt gttcccagtc ttcattagtc atatatttga 36420
tatgtgcatt tcttcttttc caaaccattt tctaatagtc tttcattatt ttccaattga 36480
ggtgtaggaa ttttaatata ttgaatttaa gaaagaagaa aaatcagcct cccgcccccc 36540
agcgaacagg tgggaaactt gcagctggac ctcagtgttg tttcaggctg tccttttgct 36600
cacagctcgg ccatgttatt ctcccctttg aaataaccca tctcacaaaa caccaatatc 36660
agacagggac acgctgagac catgatgacg ggacacatag caagaccatg gcatcatttt 36720
gttgacgccc agagcaagac agggcactgt gctactcaca aggcagtgag catctccctg 36780
tcctgggtca catgctggct gcgactttac cgattgctgc tttatcctgg ctctagcctg 36840
tcctccttat aaataaggtt gataaggaca cccagtcatc tacttgactg tgcttcctgg 36900
cagtactctg ttttcttact catcccccaa attatccggc caaagcccaa ttcctataat 36960
agattctttc taactccctc attctaagat gccctgtggt tcctcgtggt gggcactctc 37020
cctgctgcat caaccagtca atacagtgtg ttcagctaag tgggttcctg ctggtctttg 37080
ggtgaagaat attgacacct gcccaccttt gcctgacacg tgtttttagg tgtccttgag 37140
tttgactatg gacatgggtg tgcatgcttg tgagtttgtg gttgccacag ataacttcta 37200
tttttttctt tagggttttt cgtctttggg tcatgtagaa aattctctat cctacaactg 37260
tgaaagtttc atgtatcttt tcctctagat acagatgatg aggggagtgg gtcaaagagt 37320
gaaagggagc agagaaacct gaattggggg gatggactgc ggaggatcgc aaggtaagga 37380
attctgtggt ggtggattac atctgctgct ttatcatttc ataattatgt agggcttaca 37440
aggtatttta tattcggggt attcaacaaa tgctttcttg tgggctaagc aaaggctcct 37500
gtgcttgctt ttaaaacgac ggccagtgag aggcgcagac agacatgtcg atcatgagcg 37560
cgcgtacggn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 37620
nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnng ccggcctaca 37680
ccagagccac agcaacacgg gatccgagcc gcgtctgcaa cctacaccac agctcacggc 37740
aacgccggat cgttaaccca ctgagcaagg gcaggaaccg aacccgcaac ctcatggttc 37800
ctagtcggat tcgttaacca ctgcgccacg acgggaactc cttttttttt ttctttttgc 37860
catttcttgg gccgctccgg agcagcatat ggaggttccc aggctagggg tggaatcgga 37920
gctatagccg ccagtctaca ccacagccac agcaacacag gatccgagcc acttctgcaa 37980
cctacacaac agctcatggc aacgccggat cgttaaccca ctgagcaagg gcagggaccg 38040
aacctgcaac ctcatggttc ctagttggat tcgttcacca ctgcgccacg acgggaattc 38100
ctaaatccat actttaatag tatcttctat gaggacgtag gaagtacata tgaaacactc 38160
ctgctacctt ccaaagtact gtgtcccaag gaaaatcttt ctgtgagctg cactagcccc 38220
tttttcatgg aatacaacct ttactggaaa gaatgaatga cactggaaga tccatataac 38280
ttagtgaaac aatgtattcg gtcttaaaaa tcttacatta gtataagcaa cagtcagtgt 38340
gcaaaacagg cttttaattt aacagaatag gaaatatgga gtgacactga tgcaggtgca 38400
cgttcaaaat aacctttgag aaattaccat aatgatagca tccaaaatta tctgaaaggg 38460
ttattaaaaa tacacgtcct acatgtgtgc ggggctttta catttcatag acgtcagcca 38520
ccaaaaggac tcagcgcaga agcagacaga aacctccagt ggttttccca tgagcccaca 38580
gcaaagagac ttgtgataga gtaaaacgta aaaagctcta ctcttcacac tacagtgttt 38640
cttatgcgaa ataattattt tcatagtaaa tgcatagatt atttatatct tctaaaaaat 38700
tgatgaaatt ttttttgntg tgtcttttnt gctatttctt gggccgctac cgcggcatat 38760
ggaggttccc aggctagggg tcgaatcgga gctgtagctg ccagcctaca ccagagccac 38820
agcaacgcgg gaccgagccg cgtctgcaac ctacaccaca gctcacggca acgccggatc 38880
cttaacccac tgagcaagcg cagggaccga accagcaacc tcgtggttcc tagtcggatt 38940
cgttaaccac tgcgccacga tgggaactcc tgatgaaatt tttaactatt atttctaata 39000
tggacaatat ccactgaagt atcaacacaa acatatcctt gcggtcttca ctaatttgta 39060
aaactgtagg aatattctga ctaaaaggtt tggaaatcgc tgggtacaca gccactgggc 39120
caggtgaggc acggagacac tgtgaccaag agctttctga gcagaaggag caggagcagg 39180
gcagagtccc agggccccag cagggcgtgg atctcggggt ctcaggctcc agggcggcgt 39240
ctgggcgggg aggcgccgtg gtggggagtc cccgtgtccc cagtttcact tctccgtctc 39300
gcaacctgtg tggggccgtc ctgcctggac actcgtgacg cggccccact tctctctcct 39360
attgtgtgtc gggtttctgg agaagccaat cagcgccacc gcggttcgcg gttctaaact 39420
ctccatccat tcgcctcgac tccgcttctc tccagactcc gaggctgagg atcatggggc 39480
cccgagccct cttcttgctg ctgtcgggga ccctggccct gactggtacc cgggagggtg 39540
agtgcgggat ccgggcacaa ggccgctgcg gggaggagcg agggcaccgc ctgggagtcg 39600
ggtgggggca ggacccacgg ggaaggtgcg actcagctgt tccggcccag acccgccact 39660
tcaccccgtc cggtcctgtc cctcccttgt ttcctgcccc tctgctcgcc gccccctaac 39720
ccggggccct tctccgacct ccaccccttt cccgcctccc cagccccgag ctccctgccc 39780
gggtcccggc cccaccacct tcccactcgg aggcccgcgc cgagaggagg gtcgtgtttc 39840
accctcccgc ccccagtccc cactccctga ggtatttcga caccgccgtg tcccggcccg 39900
accgcgggaa gccccgttca tctccgtcgg ctacgtggac gacacgcagt tcgtgcggtt 39960
cgacagcgac gccccgaatc cgcggatgga gccgcgggcg ccgtggatag agcaggaggg 40020
gcaggagtat tgggatgagg agacgcggaa cgccatgggc agcgcacaga ctttccgagt 40080
gaacctgaac aacctgcgcg gctactacaa ccagagcgag gccggtgagc gaggcgggcc 40140
cgggtccagc tcacgacccc catccccatc cccagggacg gaccggggtc accccgacct 40200
ccgggtccaa gggtcacccc ggcatttcgg acccgcccgg cccccgaccc ggaaggaccg 40260
gcggggactt tcccccggtt tcgttttcag tttaggttga accccgggtt ggtcgtggcg 40320
ggggcgtggc tgactcgggg cggggtcagg gtctcacacc ttccagagca tgtacggctg 40380
cgacgtgggg ccagacgggc tcctcctccg cgggtacagt cagtttggct acgacggcgc 40440
cgattacatc gccctgaacg aggacctgcg ctcctggacc gcggcggaca cggcggctca 40500
gatcaccaag cgcaagaggg aggcggccga tgcggcggag caaatgagaa gctacctgga 40560
gggcgcgtgt gtggtgtggc tccagaaata cctggagatg gggaataaca cgctgcagcg 40620
cgcaggtatc aggggccgcg gggcctccac catctcccct caggagggag ctgccttccc 40680
acaaggagag gaaaaggggg tccctgcggg aaagcccccc actgcctgtc ctgagaggga 40740
ggagtccacc taggttccca gcttctccac aagacaggga ctccccaggg ggtctgactt 40800
ctccacagga cagttaagga agcctgtctc ttttggggtg aaggggggag accatccgtg 40860
aaaggactgc tcagcggtgc cctttgaccc tggccgccat cttgggaacc atgactttct 40920
caggcctggt ctcagcctgc gaacagcttt gcatcctgga caccaccttg cctgagtcat 40980
tcagcctcca ccaacgtcag gaacaggact ctgtattctt ccccttaagt cctggagcct 41040
cctacctcaa tctcaccctg attctagact cttccaagga ctaggagcta ttcccagata 41100
cccaagtcca ggctggtgtc tgggttttgt gcttcctttc aaacccattg tcctggccct 41160
tcacccagtg gcccaggcag gggacccaca gggtgaattt tctgattctt cttcctcaga 41220
gcctccaaag acacatgtga cccgccaccc cagctctgac ctgggggtca ccctgaggtg 41280
ctgggccctg ggcttctacc ctaaggagat ctccctgacc tggcagcgcg agggccagga 41340
ccagagccaa gacatggagc tggtggagac caggccctca ggggatggga ccttccagaa 41400
gtgggcggcc ctggtggtgc ctcctggaga ggagcagagc tacacctgcc atgtgcagca 41460
cgagggcctg caggagcccc tcaccctgag atggggtaag gagggccctg gggggggagc 41520
ctcttctcag acacagcaga agcccttctg gagacttcca caaggtcagg gctcaagcct 41580
gaggagggca ctcacctcgc cctttgttcc cagaccctcc tcagcccccc gtccccatcg 41640
tgggcatcac tgttggcctg gttctcgtcc tggttgctgg agccgtggtg gctggagttg 41700
tgatctggag gaagaagcgc tcaggtaggg aagggagtgg ggatctgagt ctccttgtct 41760
cactggggtt tcaagcccag gtggaagttg acctgcctta ttcctgggac atcccctcca 41820
cacacatgtg ctgagtctgg ggctgagcgc tgccacgtac ccttctgtga agcacatgtg 41880
atagtcaaag actaattatt ccccaggata attccagttg gcgtctagtt tctcagcact 41940
tgaaggtcag aggtgaagga ccccactgag gagaggcgtc caggagggca gttggtccag 42000
tctcccccac atctccttgc tgtatactcc tgatcctgtc ctggattttt gttgaaagtt 42060
ctacactggt tgtgagatcc aggactaggt ttcttgtagg atcttatggc tcagttttct 42120
ttttggcctc tcacataaat gttatcttcc cccaggtgaa aaaggaggga gctacactca 42180
ggctgcaggt cagtgtggag ggttgtgatg cctgaggccc tggtggtgca gacaggaggc 42240
cgtgggggga gctcacccac cttctgtcat cttctttctt gggggtactg accccatcct 42300
gcttttgctg tcccccaggc agtgacagtg cccagggctc tgatgtgtcc ctcaccaagg 42360
atcctagagg tgagaccctg gagggcctag atgggagggg ggttggggca gagggggtgc 42420
cctgggtgac cgggatcttt gagggggacg tttggagcat gtggggatat tgagcatgtc 42480
agcccttcct tgactgacct gcctgtttcc tgatgatttt ctttcccagt gtgagacagc 42540
tgccttgtgg ggactaaggg acacaagatt tgttcacgtc ccactttgtg acttcagacc 42600
ccctgactcc tatttctgca gctgcctctg aaagggtctg tgttcctatg agcatcctga 42660
gaggaggtgg ggaccctggc ccagccctgc cccccatgtc ccctcttcac cctgacctgt 42720
gttctctttc ctgatccact ttccagctcc aacaggttgg ggggaggcag gcggcaatgg 42780
accatctcca tccttatgct aagttcattg ccctgactgg tgactgcggg gagccgagga 42840
aacacaggag gccaaggaag ggaactagcc tgacggcaca gttccaaagg cagggttcct 42900
aaccaaggga aaagagccta cccaccggcg cagttccaag ggtaggttta gaaataggga 42960
aaggggctta cccagtggca cagttccaag ggcaagttat aaaaataatg aggctcacct 43020
gctaacccga ctacaacggc aacaataaca aaacgaaggt tttgcacaat agtgcaagaa 43080
taaaaattgc ttcttggaaa attcagtctt tcctgttgtt tacttattac tcagcctttt 43140
ctgttatttg tttgttattc tttgttattt ttctatattg ttttgtgatt cagtaaaatt 43200
ggaacaacaa aaattggaat gagtttcacc ttggtgaaat tccccctatt cagatctaat 43260
acaagcaaaa acaaagtgtt cactggtttg ttacaagtaa aaataggttt tagagttagg 43320
tatttgtgga gtgtacaata ggttagaata ggttagacat acatcattct tgatggaaaa 43380
tataatcttg cttttaataa gttttgtagg gagcttttaa gttttatgaa attaagttgc 43440
tttgatatat aaaatatttt ctcatactgt cccctgactg taataccttg aagttagtac 43500
cttaagcttt aatctaaaaa atgagttttt gtaaatgtta aattcttgta cttgtgacct 43560
ttctagttgt taaagattag cttaaaacaa taacgggtgg tgcttgctaa aagtaaccac 43620
aagccgtttt atactgaatt gccttatttt tatataatga tttttcagca taatgttttg 43680
tacccattat aattttacat gatgcattgt atccattagt tttaactaaa attcttagag 43740
cacaatgtgt atctactgta ccacatagtc taaaatttaa cctttgaaag tatgatgtaa 43800
acattgtgat gtaagttaca gttaagctgt ctatataaac tgttacctat actaataaaa 43860
tttgagcagt ccagcgcctt gagagaaggg acaaagaggc tgtttccatg tacttcgccg 43920
acactgtcca tccctggggg gacaccctgg ctactggagc tggactccgg caggtgactt 43980
cctattttcc catagaaaat atagtatata ttaattttgc tttttctaat tggtgccatg 44040
gggaattcat gggataataa atgagagatt tcctaaagct tgaaagagaa aatgaatgga 44100
agcactgaga aattccagaa tccacctgta gcttagtttg gcccagaact tccatatgcc 44160
acgggcgcag ccataagaaa cctttttaaa aaggtgtcca gtcttgaaga gttctgcata 44220
ccctaagtcc tgaaatgaac agtgtgttcc cgtaacaatg gggtgaccgt caggagagtc 44280
caccccgaga gcaggagcac atgtgctgga gaaggccgcc caggcagggc ccgtgggcac 44340
aaagctaaga gccctaccga ggacccatgg tcagtgcaaa cagccctgtt tgcttcctgg 44400
tggcttctgt accagatcaa tgcatatttg taaataaaat cctgaaagac tctccagagt 44460
cagtgttggg aagcaacttt gcctaaagcc ggagacagag gaaagtctga aattcgcnac 44520
tggaggaaat aacaattcag acctatgact ttcgaaagtc ctgatgagtt ctatttcttg 44580
agggggcgtg gacatcaccc cagagctgga tcgaagcata aatagatctt tgtgaaccgc 44640
aggccaaggc atcctcgtca ctcagcaatg gcttctgcag cagccaccac tgaaagggga 44700
cacacaagac taaagcccaa agacatctag aaggtcccct ttcttcctgt ggagaagcag 44760
gaaacaggaa aagtaggtca acccccagtg gtcccttgga gctgctcttc tgtcaaggtg 44820
ccaagtgaca ggacacaggc accacctcac cttcctgaga ctcaaccttg tgcacttaaa 44880
ggagaagtaa catcttttct ttccttcttt ttcttttctt tctttctttc tttcattggc 44940
cacaccccca gtatgcagga gtttccaggc cagggattga acccacacca cagtggtgac 45000
aatgtcagct ccttggccca cttagctacc agagtactcc tgggtctcct atttttatct 45060
cgtggaaccc tgaccttcag tctctttctc tagcagcggg ggtggtcctg gaccaggacc 45120
ctgctcactc acagctagtg ggactgaggg tggggaacgg ctggctctga ggggctcaca 45180
tctggggccg cctgctcatc ctaagagacc aacccctatg tgctgggtct ggacggctgc 45240
tcccctggga cctgctgctg catggtggcc cctcctactg cagagcctgt gccctgggga 45300
tggttccaga gtcacctgga agagcgggaa ggatcttcca ctgccaggag caggggatct 45360
gggcggtagg gtcaccaggg gacgggactg agctctcaca cccccaaaac caaagggact 45420
cgggatgaaa attcccagat ggtagaggtg gccattaaat atcaggaagc ggtactgcag 45480
tttgttactg ggagacgtca tttttattat taatgatatt attgaaacag tatattgtct 45540
aacactgagc agaggtgccc agtctgccct gccctctgat gacttcctcc ctgcctgtgt 45600
gttcatatgg ttttcacacc cgaaccaata tctcaaggag atcatttgtt tcctatgctg 45660
cccttcatga gatatcttct gaagatcgac acgcagcaga agaggactcg gcattttcaa 45720
tactgggaaa tgagtcccag acctcagtag agatcccagc ctgggccagt tgtcctgggg 45780
ggcgggcagg aagtctaact gatgggaagc agctgtctgt cctccacgct cctcctggag 45840
caggtccttc tctccattag acccactggc cactggagtc atagtcattc atgaactttt 45900
ttttttttgt ctttttgtct ttttgtcttt tcaggaccgc attcatggca tatggaggtt 45960
cccaggctag gggtcaaatt ggagctatag cttctggcct acaccacagc cacagtaaca 46020
ccccatcctt aagccactga gcaaggccag agatcgaacc cacaacctcg tggttcctag 46080
tcggattcat ttccgctgca ccacaatggg aactcctcat tcatgaactc ttggtgaaaa 46140
tgattttttt tcttttttcc ttttcttttt cttttcggtt ttagggccac actctcggca 46200
tatggaaatt cccaggttag gggtcaaatc ggagctacag ctgccggcct gcaccacagc 46260
cacagcaacg ccagatccaa gtcacatctg tgatctacac cacagctcag agggatgcca 46320
gatccttaac ccactgaggg aggcctcgga tcgaacccgc attctcatgg atactagtca 46380
ggttccttac cactgcaccg caatgggaac tcccaaatga tatttcaaaa gaggcactgg 46440
gagttccctt tgtgtcccag gggaaacaaa tccgactagc atccatgatt atgcggatac 46500
cttcctgccc tggatcagtg ggtaagggat ccggtgatgc cgtgagctgc agggcagttc 46560
ggagaagggg ctcccatctg gcgttgctag tggctctggc tctgatttca acccctggcc 46620
tgggaacttc attatcccaa aggtgaggcc ctaaaagagg gggaaaaaaa aagaggcacg 46680
tgaccactct agagggggct gtccctcctc ccttgggtta cctgtcctgg cccaggaaga 46740
acaggtccat ggagagaggg gccaaggttt gtgccacaat cacacacaga tctgcccaca 46800
gaccacagcc ggggaagtac agcctcttgc ctgagtcacc actgcctctg tcatccagcc 46860
tgagtgtctt gtttggtcct aggttcctcc aaaagctata cgtaactagt tcccctgcac 46920
ggactttccc cagctgtacc ctgggtattg gcagggtagg ggggtgccta cctatagatt 46980
aactgaaatt caacctgaat cttaaagccc aatcaaaatc ctagaggttt tttttgtttt 47040
gttttttgtt ttaattgttg agcttattct acactccatg tgcaaaagca aaagatttga 47100
acaggcaaaa taattctgcg aaaagaggaa gcatcttggg ggaagcccga tacctgcttt 47160
caagcacaaa gtggtgtcag ataagagtgg tccagctcta tggaacagaa gaaagtgtgt 47220
gtgatcagaa cttcaaatat aggctcagaa aaaagaagtc aaggtcattc agtgggaaca 47280
aataatcttt tccatcaatg atgatgacag aaatagacac aaacaagaga atgaaaccac 47340
attcaaagac taatttgaag gaagcgtaga cctaaaccta agagctgcaa ccaaaccagt 47400
ctctcccgca caaggtcctg aaccataaac tgagccacat ttgtaatatt atattttcta 47460
gcagccatgt ttaaaaaatg ttttaattgg atgaaaataa ttctaacatt tactttaacc 47520
caaaacattc aataatttcc ttgcagaacc aaaacaagga taggtgatgg gctattttat 47580
accctttttc tcatttgtta cctttcttgc aattgttgct ttgagcctcc aggggtttag 47640
aaattctctc attttatacc ctgccacgtg gtgccttccc cccagatctt ggaattctgg 47700
aattattgaa gccaaaaccc acttccatct cacactggat gcaggagagg atgagctcag 47760
agataaagtg ggggcaggac acccaccccc agctccctcc tctccccaac ttcctttctt 47820
cttcttccag tctgaggaac tcaggaattc cacagcacaa atgggggagc cggttgtaaa 47880
cgctacaaac ccagatccat cctcatattc ctctgcagga agtccttcct gaagtctgcc 47940
cactcccctt cctgctgcag cctcagcggc tccccaggtc tgtgcagagg gcgataccag 48000
aactctcttc ctcccaaccc ctcagcgctc actccctggc ctccctcagg actcagctca 48060
aatggcatct ctcaggaaag ccgcccctga ggatactatt taaaatgacc accagctccc 48120
cgcgtccctc ccttgttctt tttctcgaaa tcagtctttg cttttttttt tttttttttt 48180
tttttcccca gtctttgctt tttgacaccc tgatctttga cttcaagagc cttgtgggag 48240
ttcccgtcgt ggcgcagtgg ttaacgaatc cgactaggaa ccatgaggtt gcgggttcgg 48300
tccctgccct tgctcagtgg gttaacgatc cggcgttgcc gtgagctgtg gtgtaggttg 48360
cagacgcggc tcggatcccg cgttgctgtg gctctggcgt aggccggtgg ctacagctcc 48420
gattcaaccc ctagcctgga aacctccata tgccgcggga gcggcccaag aaatagcaac 48480
aacaacaaca acaacaaaaa gacaaaagac aaaaaaaaaa aaaaaaaaaa gagccttgtg 48540
gtttatcggc agctcttcca tccgcgcgtg cgcacacagg gcggggactc gggcttagtt 48600
ggtctgaaac atccccaaag ggctagaatc ttgcctggcc tatagctggc gctcaaacat 48660
atctgtcgcc gaatgaataa gtctagggac cagaactcct taaaggtgag actggggcgg 48720
cacagggaga aggtccctgg taagagatgg ggagggggcg gggcctcgcc ccagtttgcc 48780
ccaggcccgc ggcccagcgc ccaggcagcc tcacgagacc gtccgcactt tgaatgtggc 48840
cacaagcaca ggagacagac tcaaccagac ccccagctgc ctccacagac tcttgccctc 48900
ctgcggggcg tccggctcag gtggtggggt tcctaggcgg ggacgtcctc caggtcccag 48960
tcttaggtcc agaatcctgg gtttctgcgc agctggtggc ggggctcagc ctcagcctgg 49020
gaggccctgg gactgggacc ccaaacctgc agggccagaa attggagacc aggattgtgg 49080
ggctttcagt tctttgtttg gaggtggagg gagcagacct gggggaggca gcctgcaggt 49140
ctgggtctct caggtgctca aaggggaaga agaggcggga tgctgggctc cttcctgtgg 49200
ttgtgaagtg gggccccccc cccacccccg gcctaggcac caaagcgtcg acagtctggc 49260
gcctgggcag aagcatccac ccttggcgac cttgggcgtc caggtcctga gccctgggtc 49320
ctgggaacct gagggggaac aagggctgca ttgggcttcc tgggaaaata tcaggccaag 49380
tccggggctg agactcacac agatctcctg gagtcactgt ggtcccaggc tgcagcctcc 49440
atcccactct ctgtgcccca ggaatgactc tgtggcatca gctgggcagc tctgtttctg 49500
cagccccagg gccactcttg ttgctgcggt ggttcccaca gtcctagaaa atagagacca 49560
tgtccaggca gcccagggct gtgagctgag gatggactca gatgaggccg tgacgtcctg 49620
gtgcacagag tgggacctgg agacctggga agcaggccca gggctgtgtc ctcaagggct 49680
gcacaggact ggctctccct tcctgattct ccctgcaccc cggcttttgt aggagggggt 49740
taaggtagga gggggagctc tcattgtaga aagaaggaat tcctttatcc ccctcctctc 49800
tctcctttat ggttttgccc tttcttctct gtgtgtgtgt gtgagttata ccagacctgt 49860
atatacagaa caatgctgcc aaactgaagc agagtttatc agccagtcat agcacgaaca 49920
cctgttatgc acccagcaga aaaacagaac ccccagacca ggagcccgag tgcccaccca 49980
gaccccatcc ctccctgcac aggtggcccc tacgtcgacc cccccccccg gttcctcctc 50040
ctcccctcct ctatgccttc gctaatacca ctgttccctc cttttcctca catctccaga 50100
gtctccccat ctccataagc tatcaccctg cggggctcct gcttcctctc ccccacttcc 50160
accccatcct aggatggaga ctccagagtg gattccctgg aggaagagcc tcccaccctg 50220
gaagcagcac caggaacaag tgaaccttgg ggtttaaggc tgcattccag gagactgcag 50280
cccacagtat gtctgtgggc tccaccctgg acccaggaac cagctgttaa ggctggagat 50340
ctgctcttcc ctgaggccaa agtcctgggt catttaacgg ggaggaaaac gtctgtgtca 50400
gtgtgtgttt ggggaaacag ttcaggctct gaacttgaca ctgcttgggg ctcccccttg 50460
ggggcacagc cctatccctc tgctgggagc ccaaggagac cctgggcagg tccctggtaa 50520
aaaggtcaaa ggggagctca ggtctcctgt catctgccct gtggccacac gggggcgcca 50580
gggctctgct ctctcctact ctgcgcgggg aagggagcgg atgggctgag gtctgagtcc 50640
cacggagatg gctgccctgg aggctgggca gcagtggggg agtgaaggtg cttgctgagc 50700
agctctaggg ggtggggcag agattggagg agaggagcgc tctggagacc gggcctgtct 50760
ccgaaggttc cctctttgct ccccaaacct ccccactcca gctcctttat gcaaacaccc 50820
tgggaaacta aaaccagaaa attgtttctt ccaaccccta ttccttctga agaacatctt 50880
ttaaataata ctttttaaaa ttagactagt gtgagattta caagacagct gcaaacgtgt 50940
aacagggctt cctcttactc tgtattcagt ttccccgtgg tgaacatctt atatgaacgg 51000
caggcatctt ttcacagcta atgcagcaat gttggtacat tattactgaa ctgcatactt 51060
tatttgcact tcataagttt ttctttctgt tctttttttg tttacatttt tatttttatt 51120
tatttattta tttttttttt tttttttgct ttttaggccc gcaccgcggc atatggagaa 51180
tccaaagcta ggggtcgaat tggagctgta gccactggcc tacaccacag ccacagcaac 51240
gccatatcca agctgagtct gtgatctaca ccacagctca cggcaatgac ggatccttaa 51300
cccactgagc aaagccaggg atcaaacctg caacttcaca gttcctagtc agatttgctt 51360
cttctgagcc acaacgggaa ctcttctaat tgtttttcta tgcctttaaa accatgccta 51420
tgatactctt cctctgtttc ttagtttatg tctcatcttt gcttaatttt tgtttgtcct 51480
cccaatataa ttataattta ctattttcaa tctctgaata gtataattct agttagctca 51540
atgttcaatg tttgaattat taggaggatg gaaatgcaag tctcaactga gtgtttagta 51600
gactatggtt tcttttttaa atggattttt caattttccc agaaatacac agcatcttag 51660
cattgttgta ctcaggtgaa accagtccca aactttcccc gagtctcgtc ttgctgagta 51720
tcttctagca cgtctgatct tccagcaacg tcatctccct gcacagccct cagtgctgcc 51780
acacgggacg ggtccccacg caactggggc agggctgaca gccccagtct ccttcgcctc 51840
gtcctgggca gccctctgcc tcccttgttc tggctccacc gcagcctgca tcccatgtct 51900
ccctattagt ggttttcccc atcatttccc cccactttgc taacagtctc aagaaatgga 51960
acagctagag ttcccatcgt ggctcagcgg ttaacgaaca tgactagcat ccttgaggat 52020
gcaggttcga acctggcctc actcagcggg ttaaggatcc agcgtggcac tgagctgtgg 52080
tataggtcaa taagctggct cccatcccct gttgctagtg gttatggtgt aggctggcag 52140
ctacagctcc tgtttaaccc ctagcctggg aacctccata tgccactggt gcagccctaa 52200
aaagacaaag aaaaaaaggg aacagcacaa gtaaaactct gtaactctga catgacttag 52260
gcaaccctag gactaacatg gtaactagat atagagctct gtgttggaaa cacttatttt 52320
tgccttggga ttcagaaacc atcacttcaa tgtcttctaa tctccaggac catggttttt 52380
gcaaggtttg cctcttgaag ttaacaaggt tgcttccact gtctttctga tatttcactg 52440
ccatgtgtct ggctgggggt ctgtcttcat ctcctgcggt aagaatttgg tcaggccatt 52500
caggatgtaa atttttgtct gtaaatctga caagttttcc tcaaattgaa attttcatca 52560
tttcctactc tccattagtt ttgaaattag tattattcac ttggaatctc ttgattgaca 52620
gatgaatatt tttatcagtc ttctctcctt cttgcgatga cttcgccttt cccatggctg 52680
agagtatgac cgtaaccttc caatccatct attggattat tcatttctat gacattattt 52740
ctaagttcca atagctcttt cttgttccat gtttgtatat gattgtatgg aattctatct 52800
ttgtttcata agctatatat ctcttacctc tttagatatt agtgataaca gtggtaggaa 52860
actgtcaggt ctccgggtta tctgctctca tgacatgaat gttttctcac agcacttgac 52920
tcagttttga tttaccctta atcatttttg ttttcttgtt ttgggttttt tgttttcttg 52980
ttttgtcttt ttagggccac acccatggca tatggaagtt cccaggttac gggtccaatc 53040
ggaatcatag ctgctggcct acaagtagca tcgatgagga cacggttcca tccctggcct 53100
cgatcaatgg gttggggatc cggcattgcc gtgagctgtg atgtaggttg cagatggggc 53160
ttggatcccg agttgctgtt ttctgggggt gttttttgcc tggcctgggg catgtggaaa 53220
ttctagggcc aaggactgaa actgcaccac agcagtgact tgagcagctg cagtgaaagg 53280
ccagatcctt aaaaggctgg gccaccaggg cactccgatt tacccttatc cttgtcatct 53340
ttgtctcacc acagagcctg tctcacatca ccagggtcct gactgctgtt cttcatttca 53400
ccaccagtgt ctccagatct ggagtcaagg ggaccttgaa atatccactc cttaaacctg 53460
gagtctccct tcccctgtct gcacgaggga tcacatgtcc tgtgcctcat gtgtttgctt 53520
tgaggaatat attcaaccac tgtgtcaatt acatggcatg attgcctgga acacaaagtg 53580
ttcccctgtt catagttact aacattagag tgatgaaatc attttgatta attccattct 53640
ttgtaaacca ccattaaaaa atccttgaat atatattttt gagaacacga atgattatct 53700
taggataaag ttttagatga aatttctgtg tcaaaatgta aacatagtga agtcattgag 53760
aaatgttgca aaagtgacct ttaacttttt tgtggcaaac ctctccctgt gctgatgtga 53820
gcaaccaatg cttctttgta atcacagctg gaccctggct ctagtctgcc ctccctacag 53880
gtgagggtca ctgagatgcc cggtcagaga cctttcccac cgcctgacag caacacccga 53940
tctagactga gcccctcttc catccaccct ccttgaaatc acccaccaaa gcctaaatcc 54000
tatatatgtt cttttttttt ttttgtcttt ttagagctgc actcacagca catggaggtt 54060
cccaggctag gggtccaatc agagctatag ccgctgacct actccacagc cacagcaaca 54120
ccagatccga gccacatctg ggacctacac cacagctcac ggcaatgcca gatccttgac 54180
ccactgagcc aggccaggga atcaaacctg aaacctcatg gtgcctagtt ggattcattt 54240
ctgctgtgcc ttaacgggaa ctcctactct aaatactctt aaaataaata ttaattaaac 54300
tttcttggac atgctgcatt ttaaggatat atccaagact cagacaaatc caaatgactt 54360
ccatatgaac attacgttaa tatgcatcaa tctatgaatc acactttctc tttggggaga 54420
agagcctgaa aattaagaac atgctcgata tccactgtcc catatgatag ctataggcca 54480
catgtggcta ctttaattaa attaaagaaa tttaagggtg ctcagcacaa tagtctcttt 54540
tgaggtgctg aatagccaca tgtagccagt gactagctga tgacacagac agatatgaat 54600
agttccttca aacagaacgg gctgttggac aggactgctc ttcccacttt gtgggcctca 54660
cgtcagatac attagaccca agagagagga ttcctctcag agactctgtc acagattcct 54720
gtctgggatg tgaatgcaca tgaagctgac tgggaacaaa accacggagg tgatgcgtgg 54780
gtctggcgcc ttcaggcaca ggctgttccc aggtccaatg gggagatgcc tgtcccccca 54840
cctcccttcc cagatggagg actctctctg gcatcacttt ccaggggctg agtcctttcc 54900
tggaggagcc caggggcagg gccctcagct gaggctgggg aggcagggag tccagggcag 54960
ggagagggat tctggggaat gaagaggagg ccaaggcaaa ggtcagttag gagacttttc 55020
caggtgtatt cagaacagcc gttctcagag tgtggtctgt ggaacccttg aaagtcaggg 55080
agaccctttc acatgagatg aacacaacat tcatagcatt actaagatag tatttgactt 55140
ttcactatgt tgacatttgc tttgaggtac aacttagcaa gaatggaaac tagggaagca 55200
aactgtaata gtaattatat cactatgact taacttgcag aaaaaaaaaa aggtttgctg 55260
taaaacatcc tggttgaatt ttaaaattct tatccttccc ttaaaaaatc ataaagggag 55320
aaaaatatga aagaaaagta taatatgtat gtgtgtaaac tgaaagcatt ttgtttgtac 55380
ccccaattta acccaacatt gtaaatcaac cgtattaaat aaatgttttg aaattaataa 55440
ataggagttc ccgtcgtggc tcagtggtta acgatccgac taggaaccat gaggtgcggg 55500
ttcggtccct gcccttgctc agtgggttaa ggatctggcg ttgccgtgag ctgtgctgta 55560
ggtcacagac acggctcgga tcccgcgttg ctgtggctct ggcgtaggcg ggtggctaca 55620
gctctgattc gacccctagc ctgggaacct ccatatgcca cgggagtggc cctagaaaag 55680
gcaaaaggac aagggaaaaa aaagaaatta ataaataaaa attcttatct ttcgtaaatc 55740
atcatccttt tttttggggg ggggcgcttt ttgcctgttc taggatcctt aacccactga 55800
gcaaggtcag ggatcaaacc tgcaacctca caattcctag tccgattcat taaccactga 55860
gccacaacag gaactcctaa atcatcatcc ttaaactcac atttttttct cctttttttt 55920
tttttcttaa actcacattt taatagaatc ttctatgatg acctgggaag tacataccaa 55980
acactcctgc tatattccaa agtactatgt cacaaggaaa atcatctgtg tgagcagaac 56040
taactgcttt tttacagaat acaatcttac ttcaaagaat aaaggacact gaaagatctg 56100
tataacagtg aaacaatgta ttactttttt taataatctt acattagtac aagggatagt 56160
caatgtgcaa aataggctta tggattttaa tgtaataaaa tagaaaacat tgaatgataa 56220
tgactcaggt ccactttgaa aataactttt tttttttttt tttttttttt ttgtcttttt 56280
gctatttctt tgggccgctc ccgcagcata tggaggttcc caggctaggg gtcgaatcag 56340
agctgtcgcc acccgcctac gccagagaca cagcaacgcg ggatccgagc cgtgtctgca 56400
gcctacacca cagctcacgg caacgccgga tcgttaaccc actgagcaag ggcagggatt 56460
gaacccgcaa cctcatggtt cctagttgga ttcgttcacc actgcgcctc gacgggaact 56520
ccctgaagat aacttttaac aaactaccat ttgtgtagtt ttagctttgt atcaaagatg 56580
agcatccata attatctgaa aaggttatta aaaatactct tgctacatat gtgtggggtt 56640
tttatcttca tagacatcag tcaaaatagt ggatttaatg cagaagcaaa cataaaaatc 56700
cagtggtctt ctaatgacac agacaagaaa gaggcttgca atcatggaca atgtaaaaag 56760
ctccactttt cacactaccg tgattactat gggaagtaat tattttcaca tgaaatgcat 56820
gggttattca tattcctcta aaaaataaat ttaaaatgtt tattatttca agtatggaaa 56880
atatctattg aagtaacaaa acacatatcc ctgaggacct cactaatttg taaaactgta 56940
taaatactct gagtaaaatg tttggaaatc gctgagctca cagcccctgg cccagaactc 57000
agagagacag ggtgacaaag agctttctga gcagaagaag caaaaccagg gcagagtccc 57060
agggccccgg cagggtgtgg ctctggggtc tcaggctcca gggcggcgtc tgggcgggga 57120
ggcgccgtgg tggggagtcc ccgtgtcccc agtttcactt ctccgtctcc caacctgtat 57180
ggggccgtcc tgcccggaca ctcgtgatgc ggccccactt ctctctccta ttgcgtgtcc 57240
ggtttctgga gaagccaatc agcgccaccg cggttcccgg ttataaactc tccacgcacc 57300
cgcctcgaca cagaatctcc gcagattcca aagatgcggg tcaggggccc tcaagccatc 57360
ctcattctgc tgtcgggggc cctggccctg accgggacct gggcgggtga gtgcggggtc 57420
gggggacaag gccgctgccg ggaggagcaa gggaaccgcc cgggggtcgg atgggggcag 57480
gacccacagg gaagatgaga ctctccctgc ccggtccggg ccccaccacc tcgcactcgg 57540
ggccccgcgc cgagaggagg gtcgtgtctc accctcccgc ccccaggtcc ccactccctg 57600
agctatttct ccaccgccgt gtcccggccc gaccgcgggg agccccgctt catcgccgtc 57660
ggctacgtgg acgacacgca gttcgtgcgg ttcgacagcg acgcccccaa tccgcggatg 57720
gagccgcggg cgccgtggat acagcaggag gggcaggact attgggatcg ggagacgcgg 57780
aacgtcatgg gcagcgcaca gactgaccga gtgaacctga agaccctgcg cggctactac 57840
aaccagagcg aggccg 57856
<210> 2
<211> 20
<212> DNA
<213> Sus scrofa
<400> 2
gggcaggact attgggatcg 20
<210> 3
<211> 20
<212> DNA
<213> Sus scrofa
<400> 3
cgtctggtcc caagtagcag 20
<210> 4
<211> 21
<212> DNA
<213> Sus scrofa
<400> 4
gcccctgggc ctatcctaga a 21
<210> 5
<211> 23
<212> DNA
<213> Sus scrofa
<400> 5
ctgggatctc tggggtcttg gtt 23
<210> 6
<211> 23
<212> DNA
<213> Sus scrofa
<400> 6
tagcagcggg aacagtactg cag 23
<210> 7
<211> 22
<212> DNA
<213> Sus scrofa
<400> 7
gcctgctggg gtggaacctg gt 22

Claims (5)

1.一种检测猪皮下脂肪或肌内脂肪含量的试剂，其特征在于，试剂包含一对用于荧光定量PCR检测的引物，所述引物序列为SEQ ID NO.2和SEQ ID NO.3，所述猪的品种为大白猪或莱芜猪。
2.权利要求1所述的试剂在预测猪皮下脂肪或肌内脂肪含量中的应用，所述猪的品种为大白猪或莱芜猪。
3.根据权利要求2所述的应用，其特征在于，所述预测为辅助预测。
4.权利要求1所述的试剂在制备预测猪皮下脂肪或肌内脂肪含量试剂中的应用，所述猪的品种为大白猪或莱芜猪。
5.根据权利要求4所述的应用，其特征在于，所述预测为辅助预测。
CN201810590056.5A 2018-06-08 2018-06-08 猪肉质性状相关的circRNA及其应用 Active CN108893539B (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810590056.5A CN108893539B (zh) 2018-06-08 2018-06-08 猪肉质性状相关的circRNA及其应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810590056.5A CN108893539B (zh) 2018-06-08 2018-06-08 猪肉质性状相关的circRNA及其应用

Publications (2)

Publication Number Publication Date
CN108893539A CN108893539A (zh) 2018-11-27
CN108893539B true CN108893539B (zh) 2021-11-19

Family

ID=64344486

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810590056.5A Active CN108893539B (zh) 2018-06-08 2018-06-08 猪肉质性状相关的circRNA及其应用

Country Status (1)

Country Link
CN (1) CN108893539B (zh)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109628446B (zh) * 2018-12-05 2022-05-13 中国农业科学院北京畜牧兽医研究所 circRNA_27455及其检测试剂与应用
CN109517904A (zh) * 2018-12-05 2019-03-26 中国农业科学院北京畜牧兽医研究所 circRNA_14759及其互作基因的应用
CN109637588B (zh) * 2018-12-29 2022-07-15 北京百迈客生物科技有限公司 一种基于全转录组高通量测序构建基因调控网络的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105637093A (zh) * 2013-08-28 2016-06-01 荷兰皇家科学院 转导缓冲液
CN108103207A (zh) * 2017-12-28 2018-06-01 中国农业科学院北京畜牧兽医研究所 Brca1、jaml及其调控基因在品种选育中的应用
CN108103064A (zh) * 2017-12-28 2018-06-01 中国农业科学院北京畜牧兽医研究所 长链非编码rna及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105637093A (zh) * 2013-08-28 2016-06-01 荷兰皇家科学院 转导缓冲液
CN108103207A (zh) * 2017-12-28 2018-06-01 中国农业科学院北京畜牧兽医研究所 Brca1、jaml及其调控基因在品种选育中的应用
CN108103064A (zh) * 2017-12-28 2018-06-01 中国农业科学院北京畜牧兽医研究所 长链非编码rna及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
猪脂肪组织发育相关miRNA的鉴定及miR-103生物学功能的初步研究;李国喜;《中国博士学位论文全文数据库 农业科技辑》;20120315(第3期);第五章第1段、表5-1及表5-4 *

Also Published As

Publication number Publication date
CN108893539A (zh) 2018-11-27

Similar Documents

Publication Publication Date Title
KR102114412B1 (ko) 위장관췌장 신경내분비 신생물 (GEP-NENs)의 예측 방법
AU2023204606A1 (en) Genetically modified cells, tissues, and organs for treating disease
KR20190017985A (ko) 질환을 치료하기 위한 유전적으로 변형된 세포, 조직, 및 장기
KR101421326B1 (ko) 유방암 예후 예측을 위한 조성물 및 이를 포함하는 키트
CN108893539B (zh) 猪肉质性状相关的circRNA及其应用
KR20180093902A (ko) 태아와 임신 여성간에 상이하게 메틸화된 디엔에이 영역을 이용한 태아 염색체 이수성의 검출
KR20120082906A (ko) 자가포식현상-향상 유전자 생성물의 조절을 통한 자가포식현상의 조절 방법
CN101258249A (zh) 检测黑素瘤的方法和试剂
AU2023251413A1 (en) Assay for distinguishing between sepsis and systemic inflammatory response syndrome
KR20140140069A (ko) 전반적 발달장애의 진단 및 치료용 조성물 및 그 진단 및 치료 방법
AU2016202635B2 (en) Method for assessing embryotoxicity
CN110283850A (zh) 一种免疫缺陷裸鼠模型的制备方法及应用
KR20040065524A (ko) 백혈병을 검사 및 치료하는 방법
CN108841823B (zh) circRNA_26852及其应用
KR20160059446A (ko) 암 유전체 돌연변이 검출용 유전자 패널
JP2003159059A (ja) 痛みに関連する分子の同定及び使用
CN109680072A (zh) 检测猪肌内脂肪含量的分子标志物及应用
CN109628605A (zh) 一种环状rna及其在检测肌内脂肪中的应用
KR20230101839A (ko) 세이프 하버 유전자좌위
CN106967723A (zh) 与水牛泌乳相关基因scap及其作为分子标记的应用
JP2003156489A (ja) 痛みに関連する分子の同定及び使用
CN108893540A (zh) circRNA_14707及其在分子辅助育种中应用
CN115151558A (zh) 哺乳动物序列中的靶向整合增强基因表达
CN109628604A (zh) 与肌内脂肪相关的新circRNA及其应用
US20230035235A1 (en) Swi/snf family chromatin remodeling complexes and uses thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant