LOCUS Exported 14873 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION Replaces original lentiCRISPRv1 (Addgene Plasmid 49535) and produces ~10-fold higher titer virus. 3rd generation lentiviral backbone.. ACCESSION . VERSION . KEYWORDS lentiCRISPR v2 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 14873) AUTHORS Sanjana NE, Shalem O, Zhang F TITLE Improved vectors and genome-wide libraries for CRISPR screening. JOURNAL Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. PUBMED 25075903 REFERENCE 2 (bases 1 to 14873) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..14873 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..199 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 154..174 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" LTR 217..397 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from HIV-1" misc_feature 444..569 /label=HIV-1 Psi /note="packaging signal of human immunodeficiency virus type 1" misc_feature 1062..1295 /label=RRE /note="The Rev response element (RRE) of HIV-1 allows for Rev-dependent mRNA export from the nucleus to the cytoplasm." CDS 1480..1524 /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 1822..1939 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 1990..2230 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1990..2010 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 2161..2180 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 4120..4195 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" promoter 4257..4468 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 4487..4496 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 4493..8596 /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="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL DATLIHQSITGLYETRIDLSQLGGD" CDS 8597..8644 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 8645..8668 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 8678..8734 /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 8735..9331 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind 9228..9248 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 9347..9935 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(9400..9420) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9818..9829) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" LTR 10007..10240 /label=3' LTR (Delta-U3) /note="self-inactivating 3' long terminal repeat (LTR) from HIV-1" primer_bind complement(10266..10283) /label=BGH-rev /note="Bovine growth hormone terminator, reverse primer. Also called BGH reverse" polyA_signal 10272..10496 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" rep_origin 10542..10970 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(10629..10648) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10839..10860 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(10979..10999) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 10984..11313 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 11164..11299 /label=SV40 ori /note="SV40 origin of replication" primer_bind 11226..11245 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" promoter 11361..11408 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 11427..11801 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" polyA_signal 11931..12052 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(11968..11987) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 12022..12041 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(12101..12117) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(12101..12117) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(12114..12136) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 12125..12141 /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)." promoter complement(12149..12179) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 12194..12215 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(12332..12349) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(12503..13091) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(12583..12602) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(13262..14122) /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 13885..13904 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(14123..14227) /gene="bla" /label=AmpR promoter primer_bind complement(14302..14321) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" enhancer 14493..14872 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" ORIGIN 1 tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc 61 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact 121 ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt 181 gggaggtcta tataagcagc gcgttttgcc tgtactgggt ctctctggtt agaccagatc 241 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg 301 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc 361 ctcagaccct tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga 421 aagcgaaagg gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca 481 cggcaagagg cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct 541 agaaggagag agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat 601 gggaaaaaat tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat 661 gggcaagcag ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag 721 gctgtagaca aatactggga cagctacaac catcccttca gacaggatca gaagaactta 781 gatcattata taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag 841 acaccaagga agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac 901 agcaagcggc cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt 961 gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca 1021 aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg 1081 ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc 1141 agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg 1201 caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg 1261 gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa 1321 ctcatttgca ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag 1381 atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta 1441 atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg 1501 gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat 1561 ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta 1621 ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc 1681 ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac 1741 agagacagat ccattcgatt agtgaacgga tcggcactgc gtgcgccaat tctgcagaca 1801 aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt acagtgcagg 1861 ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac aaaaacaaat 1921 tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccag tttggttaat 1981 taaggtaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct 2041 gttagagaga taattagaat taatttgact gtaaacacaa agatattagt acaaaatacg 2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg 2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg 2221 tggaaaggac gaaacaccgg agacggttgt aaatgagcac acaaaataca catgctaaaa 2281 tattatattc tatgaccttt ataaaatcaa ccaaaatctt ctttttaata actttagtat 2341 caataattag aatttttatg ttcctttttg caaactttta ataaaaatga gcaaaataaa 2401 aaaacgctag ttttagtaac tcgcgttgtt ttcttcacct ttaataatag ctactccacc 2461 acttgttcct aagcggtcag ctcctgcttc aatcattttt tgagcatctt caaatgttct 2521 aactccacca gctgctttaa ctaaagcatt gtctttaaca actgacttca ttagtttaac 2581 atcttcaaat gttgcacctg attttgaaaa tcctgttgat gttttaacaa attctaatcc 2641 agcttcaaca gctatttcac aagctttcat gatttcttct tttgttaata aacaattttc 2701 cataatacat ttaacaacat gtgatccagc tgcttttttt acagctttca tgtcttctaa 2761 aactaattca taatttttgt cttttaatgc accaatattt aataccatat caatttctgt 2821 tgcaccatct ttaattgctt cagaaacttc gaatgctttt gtagctgttg tgcatgcacc 2881 tagaggaaaa cctacaacat ttgttattcc tacatttgtg ccttttaata attctttaca 2941 atagcttgtt caatatgaat taacacaaac tgttgcaaaa tcaaattcaa ttgcttcatc 3001 acataattgt ttaatttcag ctttcgtagc atcttgtttt aataatgtgt gatctatata 3061 tttgtttagt ttcatttttt ctcctatata ttcattttta attttaattc tttaataatt 3121 tcgtctactt taactttagc gttttgaaca gattcaccaa cacctataaa ataaattttt 3181 agtttaggtt cagttccact tgggcgaaca gcaaatcatg acttatcttc taaataaaat 3241 tttagtaagt cttgtcctgg catattatac attccatcga tgtagtcttc aacattaaca 3301 actttaagtc cagcaatttg agttaagggt gttgctctca atgatttcat taatggttca 3361 atttttaatt tcttttcttc tggtttaaaa ttcaagttta aagtgaaagt gtaatatgca 3421 cccatttctt taaataaatc ttctaaatag tctactaatg ttttattttg ttttttataa 3481 aatcaagcag cctctgctat taatatagaa gcttgtattc catctttatc tctagctgag 3541 tcatcaatta catatccata actttcttca taagcaaaaa caaaatttaa tccgttatct 3601 tcttctttag caatttctct acccattcat ttaaatccag ttaaagtttt tacaatatta 3661 actccatatt tttcatgagc gattctatca cccaaatcac ttgttacaaa acttgaatat 3721 agagccggat tttttggaat gctatttaag cgttttagat ttgataattt tcaatcaatt 3781 aaaattggtc ctgtttgatt tccatctaat cttacaaaat gaccatcatg ttttattgcc 3841 attccaaatc tgtcagcatc tgggtcattc ataataataa tatctgcatc atgtttaata 3901 ccatattcaa gcggtatttt tcatgcagga tcaaattctg gatttggatt tacaacattt 3961 ttaaatgttt catcttcaaa tgcatgctct tcaacctcaa taacgttata tcctgattca 4021 cgtaatattt ttggggtaaa tttagttcct gttccattaa ctgcgctaaa aataattttt 4081 aaatcttttt tagcttcttg ctcttttttg tacgtctctg ttttagagct agaaatagca 4141 agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 4201 tgaattcgct agctaggtct tgaaaggagt gggaattggc tccggtgccc gtcagtgggc 4261 agagcgcaca tcgcccacag tccccgagaa gttgggggga ggggtcggca attgatccgg 4321 tgcctagaga aggtggcgcg gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct 4381 ttttcccgag ggtgggggag aaccgtatat aagtgcagta gtcgccgtga acgttctttt 4441 tcgcaacggg tttgccgcca gaacacagga ccggttctag agcgctgcca ccatggacaa 4501 gaagtacagc atcggcctgg acatcggcac caactctgtg ggctgggccg tgatcaccga 4561 cgagtacaag gtgcccagca agaaattcaa ggtgctgggc aacaccgacc ggcacagcat 4621 caagaagaac ctgatcggag ccctgctgtt cgacagcggc gaaacagccg aggccacccg 4681 gctgaagaga accgccagaa gaagatacac cagacggaag aaccggatct gctatctgca 4741 agagatcttc agcaacgaga tggccaaggt ggacgacagc ttcttccaca gactggaaga 4801 gtccttcctg gtggaagagg ataagaagca cgagcggcac cccatcttcg gcaacatcgt 4861 ggacgaggtg gcctaccacg agaagtaccc caccatctac cacctgagaa agaaactggt 4921 ggacagcacc gacaaggccg acctgcggct gatctatctg gccctggccc acatgatcaa 4981 gttccggggc cacttcctga tcgagggcga cctgaacccc gacaacagcg acgtggacaa 5041 gctgttcatc cagctggtgc agacctacaa ccagctgttc gaggaaaacc ccatcaacgc 5101 cagcggcgtg gacgccaagg ccatcctgtc tgccagactg agcaagagca gacggctgga 5161 aaatctgatc gcccagctgc ccggcgagaa gaagaatggc ctgttcggaa acctgattgc 5221 cctgagcctg ggcctgaccc ccaacttcaa gagcaacttc gacctggccg aggatgccaa 5281 actgcagctg agcaaggaca cctacgacga cgacctggac aacctgctgg cccagatcgg 5341 cgaccagtac gccgacctgt ttctggccgc caagaacctg tccgacgcca tcctgctgag 5401 cgacatcctg agagtgaaca ccgagatcac caaggccccc ctgagcgcct ctatgatcaa 5461 gagatacgac gagcaccacc aggacctgac cctgctgaaa gctctcgtgc ggcagcagct 5521 gcctgagaag tacaaagaga ttttcttcga ccagagcaag aacggctacg ccggctacat 5581 tgacggcgga gccagccagg aagagttcta caagttcatc aagcccatcc tggaaaagat 5641 ggacggcacc gaggaactgc tcgtgaagct gaacagagag gacctgctgc ggaagcagcg 5701 gaccttcgac aacggcagca tcccccacca gatccacctg ggagagctgc acgccattct 5761 gcggcggcag gaagattttt acccattcct gaaggacaac cgggaaaaga tcgagaagat 5821 cctgaccttc cgcatcccct actacgtggg ccctctggcc aggggaaaca gcagattcgc 5881 ctggatgacc agaaagagcg aggaaaccat caccccctgg aacttcgagg aagtggtgga 5941 caagggcgct tccgcccaga gcttcatcga gcggatgacc aacttcgata agaacctgcc 6001 caacgagaag gtgctgccca agcacagcct gctgtacgag tacttcaccg tgtataacga 6061 gctgaccaaa gtgaaatacg tgaccgaggg aatgagaaag cccgccttcc tgagcggcga 6121 gcagaaaaag gccatcgtgg acctgctgtt caagaccaac cggaaagtga ccgtgaagca 6181 gctgaaagag gactacttca agaaaatcga gtgcttcgac tccgtggaaa tctccggcgt 6241 ggaagatcgg ttcaacgcct ccctgggcac ataccacgat ctgctgaaaa ttatcaagga 6301 caaggacttc ctggacaatg aggaaaacga ggacattctg gaagatatcg tgctgaccct 6361 gacactgttt gaggacagag agatgatcga ggaacggctg aaaacctatg cccacctgtt 6421 cgacgacaaa gtgatgaagc agctgaagcg gcggagatac accggctggg gcaggctgag 6481 ccggaagctg atcaacggca tccgggacaa gcagtccggc aagacaatcc tggatttcct 6541 gaagtccgac ggcttcgcca acagaaactt catgcagctg atccacgacg acagcctgac 6601 ctttaaagag gacatccaga aagcccaggt gtccggccag ggcgatagcc tgcacgagca 6661 cattgccaat ctggccggca gccccgccat taagaagggc atcctgcaga cagtgaaggt 6721 ggtggacgag ctcgtgaaag tgatgggccg gcacaagccc gagaacatcg tgatcgaaat 6781 ggccagagag aaccagacca cccagaaggg acagaagaac agccgcgaga gaatgaagcg 6841 gatcgaagag ggcatcaaag agctgggcag ccagatcctg aaagaacacc ccgtggaaaa 6901 cacccagctg cagaacgaga agctgtacct gtactacctg cagaatgggc gggatatgta 6961 cgtggaccag gaactggaca tcaaccggct gtccgactac gatgtggacc atatcgtgcc 7021 tcagagcttt ctgaaggacg actccatcga caacaaggtg ctgaccagaa gcgacaagaa 7081 ccggggcaag agcgacaacg tgccctccga agaggtcgtg aagaagatga agaactactg 7141 gcggcagctg ctgaacgcca agctgattac ccagagaaag ttcgacaatc tgaccaaggc 7201 cgagagaggc ggcctgagcg aactggataa ggccggcttc atcaagagac agctggtgga 7261 aacccggcag atcacaaagc acgtggcaca gatcctggac tcccggatga acactaagta 7321 cgacgagaat gacaagctga tccgggaagt gaaagtgatc accctgaagt ccaagctggt 7381 gtccgatttc cggaaggatt tccagtttta caaagtgcgc gagatcaaca actaccacca 7441 cgcccacgac gcctacctga acgccgtcgt gggaaccgcc ctgatcaaaa agtaccctaa 7501 gctggaaagc gagttcgtgt acggcgacta caaggtgtac gacgtgcgga agatgatcgc 7561 caagagcgag caggaaatcg gcaaggctac cgccaagtac ttcttctaca gcaacatcat 7621 gaactttttc aagaccgaga ttaccctggc caacggcgag atccggaagc ggcctctgat 7681 cgagacaaac ggcgaaaccg gggagatcgt gtgggataag ggccgggatt ttgccaccgt 7741 gcggaaagtg ctgagcatgc cccaagtgaa tatcgtgaaa aagaccgagg tgcagacagg 7801 cggcttcagc aaagagtcta tcctgcccaa gaggaacagc gataagctga tcgccagaaa 7861 gaaggactgg gaccctaaga agtacggcgg cttcgacagc cccaccgtgg cctattctgt 7921 gctggtggtg gccaaagtgg aaaagggcaa gtccaagaaa ctgaagagtg tgaaagagct 7981 gctggggatc accatcatgg aaagaagcag cttcgagaag aatcccatcg actttctgga 8041 agccaagggc tacaaagaag tgaaaaagga cctgatcatc aagctgccta agtactccct 8101 gttcgagctg gaaaacggcc ggaagagaat gctggcctct gccggcgaac tgcagaaggg 8161 aaacgaactg gccctgccct ccaaatatgt gaacttcctg tacctggcca gccactatga 8221 gaagctgaag ggctcccccg aggataatga gcagaaacag ctgtttgtgg aacagcacaa 8281 gcactacctg gacgagatca tcgagcagat cagcgagttc tccaagagag tgatcctggc 8341 cgacgctaat ctggacaaag tgctgtccgc ctacaacaag caccgggata agcccatcag 8401 agagcaggcc gagaatatca tccacctgtt taccctgacc aatctgggag cccctgccgc 8461 cttcaagtac tttgacacca ccatcgaccg gaagaggtac accagcacca aagaggtgct 8521 ggacgccacc ctgatccacc agagcatcac cggcctgtac gagacacgga tcgacctgtc 8581 tcagctggga ggcgacaagc gacctgccgc cacaaagaag gctggacagg ctaagaagaa 8641 gaaagattac aaagacgatg acgataaggg atccggcgca acaaacttct ctctgctgaa 8701 acaagccgga gatgtcgaag agaatcctgg accgaccgag tacaagccca cggtgcgcct 8761 cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt tcgccgacta 8821 ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca ccgagctgca 8881 agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg cggacgacgg 8941 cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg tgttcgccga 9001 gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc aacagatgga 9061 aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca ccgtcggagt 9121 ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg gagtggaggc 9181 ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca acctcccctt 9241 ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag gaccgcgcac 9301 ctggtgcatg acccgcaagc ccggtgcctg aacgcgttaa gtcgacaatc aacctctgga 9361 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg 9421 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt 9481 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag 9541 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc 9601 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga 9661 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa 9721 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac 9781 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct 9841 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca 9901 gacgagtcgg atctcccttt gggccgcctc cccgcgtcga ctttaagacc aatgacttac 9961 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt 10021 cactcccaac gaagacaaga tctgcttttt gcttgtactg ggtctctctg gttagaccag 10081 atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc 10141 ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga 10201 tccctcagac ccttttagtc agtgtggaaa atctctagca gggcccgttt aaacccgctg 10261 atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc 10321 ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc 10381 atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa 10441 gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc 10501 tgaggcggaa agaaccagct ggggctctag ggggtatccc cacgcgccct gtagcggcgc 10561 attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg ccagcgccct 10621 agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg gctttccccg 10681 tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac ggcacctcga 10741 ccccaaaaaa cttgattagg gtgatggttc acgtagtggg ccatcgccct gatagacggt 10801 ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt tccaaactgg 10861 aacaacactc aaccctatct cggtctattc ttttgattta taagggattt tgccgatttc 10921 ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt aattctgtgg 10981 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa 11041 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc 11101 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg 11161 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt 11221 ttttttattt atgcagaggc cgaggccgcc tctgcctctg agctattcca gaagtagtga 11281 ggaggctttt ttggaggcct aggcttttgc aaaaagctcc cgggagcttg tatatccatt 11341 ttcggatctg atcagcacgt gttgacaatt aatcatcggc atagtatatc ggcatagtat 11401 aatacgacaa ggtgaggaac taaaccatgg ccaagttgac cagtgccgtt ccggtgctca 11461 ccgcgcgcga cgtcgccgga gcggtcgagt tctggaccga ccggctcggg ttctcccggg 11521 acttcgtgga ggacgacttc gccggtgtgg tccgggacga cgtgaccctg ttcatcagcg 11581 cggtccagga ccaggtggtg ccggacaaca ccctggcctg ggtgtgggtg cgcggcctgg 11641 acgagctgta cgccgagtgg tcggaggtcg tgtccacgaa cttccgggac gcctccgggc 11701 cggccatgac cgagatcggc gagcagccgt gggggcggga gttcgccctg cgcgacccgg 11761 ccggcaactg cgtgcacttc gtggccgagg agcaggactg acacgtgcta cgagatttcg 11821 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 11881 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 11941 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 12001 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 12061 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 12121 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 12181 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 12241 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 12301 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 12361 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 12421 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 12481 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 12541 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 12601 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 12661 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 12721 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 12781 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 12841 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 12901 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 12961 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 13021 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 13081 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 13141 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 13201 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 13261 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 13321 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 13381 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 13441 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 13501 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 13561 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 13621 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 13681 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 13741 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 13801 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 13861 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 13921 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 13981 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 14041 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 14101 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 14161 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 14221 ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga 14281 tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct 14341 gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt aagctacaac 14401 aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc gttttgcgct 14461 gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata 14521 gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact 14581 tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat 14641 gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta 14701 tttacggtaa actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc 14761 tattgacgtc aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg 14821 ggactttcct acttggcagt acatctacgt attagtcatc gctattacca tgg //