质粒名称: | pRDA_085 |
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出品公司: | 爱迪基因 |
目录编号: | 158583 |
存储实验室: | John Doench, David Root |
载体骨架基本信息 |
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载体名称: | pXPR |
载体出品公司: | -- |
载体抗性: | 氨苄青霉素 |
空载体大小: | -- |
完整质粒大小: | -- |
载体修饰: | -- |
载体类型: | 哺乳动物表达,慢病毒 |
筛选标记: | -- |
高拷贝/低拷贝: | 高拷贝 |
生长条件: | 37 ℃ |
插入基因信息 |
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插入基因名称: | SpCas9 |
物种来源: | Synthetic |
插入大小: | -- |
基因突变: | -- |
GenBank编号 | -- |
启动子 | -- |
标签/融合蛋白: | -- |
克隆方法: | 限制性内切酶 |
5' 克隆位点: | -- |
3' 克隆位点: | -- |
5' 测序引物 | -- |
3' 测序引物 | -- |
文献引用方法 |
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材料和方法部分: | pRDA_085 was a gift from John Doench & David Root (Addgene plasmid # 158583 ; http://n2t.net/addgene:158583 ; RRID:Addgene_158583) |
买家导航
pRDA085载体基本信息
pRDA085载体图谱质粒图谱和多克隆位点信息
pRDA085载体简介
pRDA085载体序列
LOCUS pRDA_085 12797 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Lentiviral expression of SpCas9; P2A site provides mKate2. ACCESSION . VERSION . KEYWORDS pRDA_085 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 12797) AUTHORS Hanna RE, Hegde M, Fagre CR, DeWeirdt PC, Sangree AK, Szegletes Z, Griffith A, Feeley MN, Sanson KR, Baidi Y, Koblan LW, Liu DR, Neal JT, Doench JG TITLE Massively parallel assessment of human variants with base editor screens JOURNAL BioRxiv REFERENCE 2 (bases 1 to 12797) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..12797 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 87..1265 /label=EF-1-alpha promoter /note="strong constitutive promoter for human elongation factor EF-1-alpha" intron 318..1256 /label=EF-1-alpha intron A /note="intron upstream of the start codon of human EF-1-alpha" primer_bind 1213..1233 /label=EF1a-F /note="Human elongation factor-1a promoter, forward primer" CDS 1282..1302 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 1309..5409 /codon_start=1 /product="Cas9 (Csn1) endonuclease from the Streptococcus pyogenes Type II CRISPR/Cas system" /label=Cas9 /note="generates RNA-guided double strand breaks in DNA" /translation="DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD" CDS 5416..5463 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 5476..5529 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 5536..5931 /codon_start=1 /gene="Aspergillus terreus BSD" /product="blasticidin S deaminase" /label=BSD /note="confers resistance to blasticidin" /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI KAIVKDSDGQPTAVGIRELLPSGYVWEG" CDS 5932..5988 /codon_start=1 /product="2A peptide from porcine teschovirus-1 polyprotein" /label=P2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="ATNFSLLKQAGDVEENPGP" CDS 5995..6693 /codon_start=1 /product="monomeric far-red fluorescent protein (Shcherbo et al., 2009)" /label=mKate2 /note="mammalian codon-optimized" /translation="MVSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIK AVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLT ATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMAL KLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARY CDLPSKLGHR" misc_feature 6712..7300 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(6765..6785) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(7183..7194) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 7372..7605 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" polyA_signal 7683..7804 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(7720..7739) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 7774..7793 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" rep_origin 7844..7979 /label=SV40 ori /note="SV40 origin of replication" primer_bind 7906..7925 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" primer_bind complement(7999..8018) /label=T7 /note="T7 promoter, forward primer" promoter complement(8000..8018) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(8028..8045) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(8028..8044) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8037..8059) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 8186..8641 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8273..8292) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 8483..8504 /label=F1ori-F /note="F1 origin, forward primer" promoter 8667..8771 /gene="bla" /label=AmpR promoter CDS 8772..9632 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(8990..9009) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 9803..10391 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 10292..10311 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 10545..10562 /label=L4440 /note="L4440 vector, forward primer" protein_bind 10679..10700 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 10715..10745 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 10753..10769 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." primer_bind 10758..10780 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 10777..10793 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 10777..10793 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 10812..10832 /label=T3 /note="T3 promoter, forward primer" promoter 10814..10832 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" promoter 10860..11086 /label=RSV promoter /note="Rous sarcoma virus enhancer/promoter" LTR 11087..11267 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 11314..11439 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 11932..12165 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 12350..12394 /codon_start=1 /product="antigenic peptide corresponding to amino acids 655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik et al., 2013)" /label=gp41 peptide /note="recognized by the 2H10 single-chain llama nanobody" /translation="KNEQELLELDKWASL" misc_feature 12628..12745 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" ORIGIN 1 cccgggtgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct 61 tctaggtctt gaaaggagtg ggaattggct ccggtgcccg tcagtgggca gagcgcacat 121 cgcccacagt ccccgagaag ttggggggag gggtcggcaa ttgaaccggt gcctagagaa 181 ggtggcgcgg ggtaaactgg gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg 241 gtgggggaga accgtatata agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt 301 ttgccgccag aacacaggta agtgccgtgt gtggttcccg cgggcctggc ctctttacgg 361 gttatggccc ttgcgtgcct tgaattactt ccacctggct gcagtacgtg attcttgatc 421 ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt 481 cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt 541 ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa aatttttgat 601 gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc caagatctgc 661 acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca 721 catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc 781 aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg 841 cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg cttcccggcc 901 ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac 961 ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac tccacggagt 1021 accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg tcgtctttag 1081 gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg gagactgaag 1141 ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt gagtttggat 1201 cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt 1261 cgtgagggat ccaccatggc accaaagaag aagcggaagg tcgcagccga caagaagtac 1321 agcatcggcc tggacatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac 1381 aaggtgccca gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag 1441 aacctgatcg gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag 1501 agaaccgcca gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc 1561 ttcagcaacg agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc 1621 ctggtggaag aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag 1681 gtggcctacc acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc 1741 accgacaagg ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg 1801 ggccacttcc tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc 1861 atccagctgg tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc 1921 gtagacgcca aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg 1981 atcgcccagc tgcccggcga gaagaagaat ggcctgttcg gaaacctgat tgccctgagc 2041 ctgggcctga cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag 2101 ctgagcaagg acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag 2161 tacgccgacc tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc 2221 ctgagagtga acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac 2281 gacgagcacc accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag 2341 aagtacaaag agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc 2401 ggagccagcc aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc 2461 accgaggaac tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcgtacgttc 2521 gacaacggca gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg 2581 caggaagatt tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc 2641 ttccgcatcc cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg 2701 accagaaaga gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc 2761 gctagcgccc agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag 2821 aaggtgctgc ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc 2881 aaagtgaaat acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa 2941 aaggccatcg tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa 3001 gaggactact tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat 3061 cggttcaacg cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac 3121 ttcctggaca atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg 3181 tttgaggaca gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac 3241 aaagtgatga agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag 3301 ctgatcaacg gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc 3361 gacggcttcg ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa 3421 gaggacatcc agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc 3481 aatctggccg gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac 3541 gagctcgtga aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga 3601 gagaaccaga ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa 3661 gagggcatca aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag 3721 ctgcagaacg agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac 3781 caggaactgg acatcaaccg gctgtccgac tacgatgtgg accatatcgt gcctcagagc 3841 tttctgaagg acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc 3901 aagagcgaca acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag 3961 ctgctgaacg ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga 4021 ggcggcctga gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg 4081 cagatcacaa agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag 4141 aatgacaagc tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat 4201 ttccggaagg atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac 4261 gacgcctacc tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa 4321 agcgagttcg tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc 4381 gagcaggaaa tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt 4441 ttcaagaccg agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca 4501 aacggcgaaa ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa 4561 gtgctgagca tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc 4621 agcaaagagt ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac 4681 tgggacccta agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg 4741 gtggccaaag tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg 4801 atcaccatca tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag 4861 ggctacaaag aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag 4921 ctggaaaacg gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa 4981 ctggccctgc cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg 5041 aagggctccc ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac 5101 ctggacgaga tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct 5161 aatctggaca aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag 5221 gccgagaata tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag 5281 tactttgaca ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc 5341 accctgatcc accagagcat caccggcctg tacgagacac gaatcgattt gtctcagctg 5401 ggaggcgacg ccgcaaaaag gccggcggcc acgaaaaagg ccggccaggc aaaaaagaaa 5461 aaggcaggct caggcgaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat 5521 cctggcccaa gcgctatggc caagcctttg tctcaagaag aatccaccct cattgaaaga 5581 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct 5641 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct 5701 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt 5761 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag 5821 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg 5881 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg cgcaacaaac 5941 ttctctctgc tgaaacaagc cggagatgtc gaagagaatc ctggaccgac tagtatggtg 6001 agcgagctga ttaaggagaa catgcacatg aagctataca tggagggcac cgtgaacaac 6061 caccacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac ccagaccatg 6121 agaatcaagg cggtcgaggg cggccctctc cccttcgcct tcgacatcct ggctaccagc 6181 ttcatgtacg gcagcaaaac cttcatcaac cacacccagg gcatccccga cttctttaag 6241 cagtccttcc ccgagggctt cacatgggag agagtcacca catacgaaga cgggggcgtg 6301 ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa cgtcaagatc 6361 agaggggtga acttcccatc caacggccct gtgatgcaga agaaaacact cggctgggag 6421 gcctccaccg agaccctgta ccccgctgac ggcggcctgg aaggcagagc cgacatggcc 6481 ctgaagctcg tgggcggggg ccacctgatc tgcaacttga agaccacata cagatccaag 6541 aaacccgcta agaacctcaa gatgcccggc gtctactatg tggacagaag actggaaaga 6601 atcaaggagg ccgacaaaga gacctacgtc gagcagcacg aggtggctgt ggccagatac 6661 tgcgacctcc ctagcaaact ggggcacaga tgatagacgc gttaagtcga caatcaacct 6721 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg 6781 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc 6841 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt 6901 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc 6961 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg 7021 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact 7081 gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct cgcctgtgtt 7141 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg 7201 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc 7261 cctcagacga gtcggatctc cctttgggcc gcctccccgc gtcgacttta agaccaatga 7321 cttacaaggc agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc 7381 taattcactc ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag 7441 accagatctg agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat 7501 aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact 7561 agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtacg tatagtagtt 7621 catgtcatct tattattcag tatttataac ttgcaaagaa atgaatatca gagagtgaga 7681 ggaacttgtt tattgcagct tataatggtt acaaataaag caatagcatc acaaatttca 7741 caaataaagc atttttttca ctgcattcta gttgtggttt gtccaaactc atcaatgtat 7801 cttatcatgt ctggctctag ctatcccgcc cctaactccg cccatcccgc ccctaactcc 7861 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc 7921 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct 7981 agggacgtac ccaattcgcc ctatagtgag tcgtattacg cgcgctcact ggccgtcgtt 8041 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 8101 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 8161 ttgcgcagcc tgaatggcga atgggacgcg ccctgtagcg gcgcattaag cgcggcgggt 8221 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 8281 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 8341 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 8401 tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 8461 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 8521 atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 8581 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gcttacaatt 8641 taggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 8701 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 8761 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 8821 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 8881 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 8941 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 9001 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 9061 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 9121 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 9181 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 9241 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 9301 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 9361 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 9421 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 9481 agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 9541 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 9601 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 9661 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 9721 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 9781 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 9841 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 9901 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt 9961 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 10021 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 10081 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 10141 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 10201 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 10261 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 10321 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 10381 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 10441 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 10501 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 10561 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 10621 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 10681 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 10741 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 10801 acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat 10861 gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 10921 cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 10981 tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgcc 11041 gcattgcaga gatattgtat ttaagtgcct agctcgatac ataaacgggt ctctctggtt 11101 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 11161 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 11221 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac 11281 agggacttga aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct 11341 gaagcgcgca cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact 11401 agcggaggct agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt 11461 agatcgcgat gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa 11521 catatagtat gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa 11581 acatcagaag gctgtagaca aatactggga cagctacaac catcccttca gacaggatca 11641 gaagaactta gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata 11701 gagataaaag acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag 11761 accaccgcac agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa 11821 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc 11881 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt 11941 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac 12001 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc 12061 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc 12121 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg 12181 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc 12241 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta 12301 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca 12361 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg 12421 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt 12481 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca 12541 gacccacctc ccaaccccga ggggaccctg gtacccatgc atgcatgaat tctagatctt 12601 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag 12661 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa 12721 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg 12781 gcgccggctc gaggggg //