LOCUS Exported 11659 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION For cloning sgRNA to knockout a specific gene in piggybac plasmid. ACCESSION . VERSION . KEYWORDS PB-CRISPR SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11659) TITLE Lian Lab PB plasmids JOURNAL Unpublished REFERENCE 2 (bases 1 to 11659) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11659 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin complement(83..538) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(220..241) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 432..451 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 665..687 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 679..696 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 680..696 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind 706..725 /label=T7 /note="T7 promoter, forward primer" promoter 706..724 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" repeat_region 754..788 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" promoter 1078..1318 /label=U6 promoter /note="RNA polymerase III promoter for human U6 snRNA" primer_bind 1078..1098 /label=hU6-F /note="Human U6 promoter, forward primer" primer_bind 1249..1268 /label=LKO.1 5' /note="Human U6 promoter, forward primer" misc_RNA 3208..3283 /label=gRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" misc_feature 3331..3448 /label=cPPT/CTS /note="central polypurine tract and central termination sequence of HIV-1" promoter 3517..3728 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" regulatory 3748..3757 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3757..3780 /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" CDS 3787..3807 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 3832..7932 /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 7933..7980 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" CDS 7996..8052 /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 8053..8652 /codon_start=1 /gene="pac from Streptomyces alboniger" /product="puromycin N-acetyltransferase" /label=PuroR /note="confers resistance to puromycin" /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA" primer_bind complement(8053..8072) /label=Puro-R /note="Puromycin resistance gene, reverse primer. Also called puro-variant-R" primer_bind 8549..8569 /label=Puro-F /note="Puromycin resistance gene, forward primer" misc_feature 8668..9256 /label=WPRE /note="woodchuck hepatitis virus posttranscriptional regulatory element" primer_bind complement(8721..8741) /label=WPRE-R /note="WPRE, reverse primer" CDS complement(9139..9150) /codon_start=1 /product="Factor Xa recognition and cleavage site" /label=Factor Xa site /translation="IEGR" repeat_region 9438..9500 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(9551..9571) /label=T3 /note="T3 promoter, forward primer" promoter complement(9551..9569) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind complement(9590..9606) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(9590..9606) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(9603..9625) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 9614..9630 /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(9638..9668) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 9683..9704 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(9821..9838) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(9992..10580) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(10072..10091) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(10751..11611) /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 11374..11393 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(11612..57) /gene="bla" /label=AmpR promoter ORIGIN 1 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 61 atttccccga aaagtgccac ctaaattgta agcgttaata ttttgttaaa attcgcgtta 121 aatttttgtt aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat 181 aaatcaaaag aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca 241 ctattaaaga acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc 301 ccactacgtg aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta 361 aatcggaacc ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg 421 gcgagaaagg aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg 481 gtcacgctgc gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc 541 cattcgccat tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta 601 ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg 661 ttttcccagt cacgacgttg taaaacgacg gccagtgagc gcgcgtaata cgactcacta 721 tagggcgaat tggggcgcgc cattctagat taaccctaga aagatagtct gcgtaaaatt 781 gacgcatgca ttcttgaaat attgctctct ctttctaaat agcgcgaatc cgtcgctgtg 841 catttaggac atctcagtcg ccgcttggag ctcccgtgag gcgtgcttgt caatgcggta 901 agtgtcactg attttgaact ataacgaccg cgtgagtcaa aatgacgcat gattatcttt 961 tacgtgactt ttaagattta actcatacga taattatatt gttatttcat gttctactta 1021 cgtgataact tattatatat atattttctt gttatagata tcaactagaa tgctagcgag 1081 ggcctatttc ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata 1141 attagaatta atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag 1201 taataatttc ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg 1261 cttaccgtaa cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga 1321 aacaccggag acggttgtaa atgagcacac aaaatacaca tgctaaaata ttatattcta 1381 tgacctttat aaaatcaacc aaaatcttct ttttaataac tttagtatca ataattagaa 1441 tttttatgtt cctttttgca aacttttaat aaaaatgagc aaaataaaaa aacgctagtt 1501 ttagtaactc gcgttgtttt cttcaccttt aataatagct actccaccac ttgttcctaa 1561 gcggtcagct cctgcttcaa tcattttttg agcatcttca aatgttctaa ctccaccagc 1621 tgctttaact aaagcattgt ctttaacaac tgacttcatt agtttaacat cttcaaatgt 1681 tgcacctgat tttgaaaatc ctgttgatgt tttaacaaat tctaatccag cttcaacagc 1741 tatttcacaa gctttcatga tttcttcttt tgttaataaa caattttcca taatacattt 1801 aacaacatgt gatccagctg ctttttttac agctttcatg tcttctaaaa ctaattcata 1861 atttttgtct tttaatgcac caatatttaa taccatatca atttctgttg caccatcttt 1921 aattgcttca gaaacttcga atgcttttgt agctgttgtg catgcaccta gaggaaaacc 1981 tacaacattt gttattccta catttgtgcc ttttaataat tctttacaat agcttgttca 2041 atatgaatta acacaaactg ttgcaaaatc aaattcaatt gcttcatcac ataattgttt 2101 aatttcagct ttcgtagcat cttgttttaa taatgtgtga tctatatatt tgtttagttt 2161 cattttttct cctatatatt catttttaat tttaattctt taataatttc gtctacttta 2221 actttagcgt tttgaacaga ttcaccaaca cctataaaat aaatttttag tttaggttca 2281 gttccacttg ggcgaacagc aaatcatgac ttatcttcta aataaaattt tagtaagtct 2341 tgtcctggca tattatacat tccatcgatg tagtcttcaa cattaacaac tttaagtcca 2401 gcaatttgag ttaagggtgt tgctctcaat gatttcatta atggttcaat ttttaatttc 2461 ttttcttctg gtttaaaatt caagtttaaa gtgaaagtgt aatatgcacc catttcttta 2521 aataaatctt ctaaatagtc tactaatgtt ttattttgtt ttttataaaa tcaagcagcc 2581 tctgctatta atatagaagc ttgtattcca tctttatctc tagctgagtc atcaattaca 2641 tatccataac tttcttcata agcaaaaaca aaatttaatc cgttatcttc ttctttagca 2701 atttctctac ccattcattt aaatccagtt aaagttttta caatattaac tccatatttt 2761 tcatgagcga ttctatcacc caaatcactt gttacaaaac ttgaatatag agccggattt 2821 tttggaatgc tatttaagcg ttttagattt gataattttc aatcaattaa aattggtcct 2881 gtttgatttc catctaatct tacaaaatga ccatcatgtt ttattgccat tccaaatctg 2941 tcagcatctg ggtcattcat aataataata tctgcatcat gtttaatacc atattcaagc 3001 ggtatttttc atgcaggatc aaattctgga tttggattta caacattttt aaatgtttca 3061 tcttcaaatg catgctcttc aacctcaata acgttatatc ctgattcacg taatattttt 3121 ggggtaaatt tagttcctgt tccattaact gcgctaaaaa taatttttaa atctttttta 3181 gcttcttgct cttttttgta cgtctctgtt ttagagctag aaatagcaag ttaaaataag 3241 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg aattctagat 3301 cttgagacaa atggcagtat tcatccacaa ttttaaaaga aaagggggga ttggggggta 3361 cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 3421 aaaacaaatt acaaaaattc aaaattttcg ggtttattac agggacagca gagatccact 3481 ttggcgccgg ctcgagtggc tccggtgccc gtcagtgggc agagcgcaca tcgcccacag 3541 tccccgagaa gttgggggga ggggtcggca attgaaccgg tgcctagaga aggtggcgcg 3601 gggtaaactg ggaaagtgat gtcgtgtact ggctccgcct ttttcccgag ggtgggggag 3661 aaccgtatat aagtgcagta gtcgccgtga acgttctttt tcgcaacggg tttgccgcca 3721 gaacacaggt gtcgtgacgc gggatccgcc accatggatt acaaagacga tgacgataag 3781 atggccccaa agaagaagcg gaaggtcggt atccacggag tcccagcagc cgacaagaag 3841 tacagcatcg gcctggacat cggcaccaac tctgtgggct gggccgtgat caccgacgag 3901 tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca ccgaccggca cagcatcaag 3961 aagaacctga tcggagccct gctgttcgac agcggcgaaa cagccgaggc cacccggctg 4021 aagagaaccg ccagaagaag atacaccaga cggaagaacc ggatctgcta tctgcaagag 4081 atcttcagca acgagatggc caaggtggac gacagcttct tccacagact ggaagagtcc 4141 ttcctggtgg aagaggataa gaagcacgag cggcacccca tcttcggcaa catcgtggac 4201 gaggtggcct accacgagaa gtaccccacc atctaccacc tgagaaagaa actggtggac 4261 agcaccgaca aggccgacct gcggctgatc tatctggccc tggcccacat gatcaagttc 4321 cggggccact tcctgatcga gggcgacctg aaccccgaca acagcgacgt ggacaagctg 4381 ttcatccagc tggtgcagac ctacaaccag ctgttcgagg aaaaccccat caacgccagc 4441 ggcgtggacg ccaaggccat cctgtctgcc agactgagca agagcagacg gctggaaaat 4501 ctgatcgccc agctgcccgg cgagaagaag aatggcctgt tcggaaacct gattgccctg 4561 agcctgggcc tgacccccaa cttcaagagc aacttcgacc tggccgagga tgccaaactg 4621 cagctgagca aggacaccta cgacgacgac ctggacaacc tgctggccca gatcggcgac 4681 cagtacgccg acctgtttct ggccgccaag aacctgtccg acgccatcct gctgagcgac 4741 atcctgagag tgaacaccga gatcaccaag gcccccctga gcgcctctat gatcaagaga 4801 tacgacgagc accaccagga cctgaccctg ctgaaagctc tcgtgcggca gcagctgcct 4861 gagaagtaca aagagatttt cttcgaccag agcaagaacg gctacgccgg ctacattgac 4921 ggcggagcca gccaggaaga gttctacaag ttcatcaagc ccatcctgga aaagatggac 4981 ggcaccgagg aactgctcgt gaagctgaac agagaggacc tgctgcggaa gcagcggacc 5041 ttcgacaacg gcagcatccc ccaccagatc cacctgggag agctgcacgc cattctgcgg 5101 cggcaggaag atttttaccc attcctgaag gacaaccggg aaaagatcga gaagatcctg 5161 accttccgca tcccctacta cgtgggccct ctggccaggg gaaacagcag attcgcctgg 5221 atgaccagaa agagcgagga aaccatcacc ccctggaact tcgaggaagt ggtggacaag 5281 ggcgcttccg cccagagctt catcgagcgg atgaccaact tcgataagaa cctgcccaac 5341 gagaaggtgc tgcccaagca cagcctgctg tacgagtact tcaccgtgta taacgagctg 5401 accaaagtga aatacgtgac cgagggaatg agaaagcccg ccttcctgag cggcgagcag 5461 aaaaaggcca tcgtggacct gctgttcaag accaaccgga aagtgaccgt gaagcagctg 5521 aaagaggact acttcaagaa aatcgagtgc ttcgactccg tggaaatctc cggcgtggaa 5581 gatcggttca acgcctccct gggcacatac cacgatctgc tgaaaattat caaggacaag 5641 gacttcctgg acaatgagga aaacgaggac attctggaag atatcgtgct gaccctgaca 5701 ctgtttgagg acagagagat gatcgaggaa cggctgaaaa cctatgccca cctgttcgac 5761 gacaaagtga tgaagcagct gaagcggcgg agatacaccg gctggggcag gctgagccgg 5821 aagctgatca acggcatccg ggacaagcag tccggcaaga caatcctgga tttcctgaag 5881 tccgacggct tcgccaacag aaacttcatg cagctgatcc acgacgacag cctgaccttt 5941 aaagaggaca tccagaaagc ccaggtgtcc ggccagggcg atagcctgca cgagcacatt 6001 gccaatctgg ccggcagccc cgccattaag aagggcatcc tgcagacagt gaaggtggtg 6061 gacgagctcg tgaaagtgat gggccggcac aagcccgaga acatcgtgat cgaaatggcc 6121 agagagaacc agaccaccca gaagggacag aagaacagcc gcgagagaat gaagcggatc 6181 gaagagggca tcaaagagct gggcagccag atcctgaaag aacaccccgt ggaaaacacc 6241 cagctgcaga acgagaagct gtacctgtac tacctgcaga atgggcggga tatgtacgtg 6301 gaccaggaac tggacatcaa ccggctgtcc gactacgatg tggaccatat cgtgcctcag 6361 agctttctga aggacgactc catcgacaac aaggtgctga ccagaagcga caagaaccgg 6421 ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga agatgaagaa ctactggcgg 6481 cagctgctga acgccaagct gattacccag agaaagttcg acaatctgac caaggccgag 6541 agaggcggcc tgagcgaact ggataaggcc ggcttcatca agagacagct ggtggaaacc 6601 cggcagatca caaagcacgt ggcacagatc ctggactccc ggatgaacac taagtacgac 6661 gagaatgaca agctgatccg ggaagtgaaa gtgatcaccc tgaagtccaa gctggtgtcc 6721 gatttccgga aggatttcca gttttacaaa gtgcgcgaga tcaacaacta ccaccacgcc 6781 cacgacgcct acctgaacgc cgtcgtggga accgccctga tcaaaaagta ccctaagctg 6841 gaaagcgagt tcgtgtacgg cgactacaag gtgtacgacg tgcggaagat gatcgccaag 6901 agcgagcagg aaatcggcaa ggctaccgcc aagtacttct tctacagcaa catcatgaac 6961 tttttcaaga ccgagattac cctggccaac ggcgagatcc ggaagcggcc tctgatcgag 7021 acaaacggcg aaaccgggga gatcgtgtgg gataagggcc gggattttgc caccgtgcgg 7081 aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga ccgaggtgca gacaggcggc 7141 ttcagcaaag agtctatcct gcccaagagg aacagcgata agctgatcgc cagaaagaag 7201 gactgggacc ctaagaagta cggcggcttc gacagcccca ccgtggccta ttctgtgctg 7261 gtggtggcca aagtggaaaa gggcaagtcc aagaaactga agagtgtgaa agagctgctg 7321 gggatcacca tcatggaaag aagcagcttc gagaagaatc ccatcgactt tctggaagcc 7381 aagggctaca aagaagtgaa aaaggacctg atcatcaagc tgcctaagta ctccctgttc 7441 gagctggaaa acggccggaa gagaatgctg gcctctgccg gcgaactgca gaagggaaac 7501 gaactggccc tgccctccaa atatgtgaac ttcctgtacc tggccagcca ctatgagaag 7561 ctgaagggct cccccgagga taatgagcag aaacagctgt ttgtggaaca gcacaagcac 7621 tacctggacg agatcatcga gcagatcagc gagttctcca agagagtgat cctggccgac 7681 gctaatctgg acaaagtgct gtccgcctac aacaagcacc gggataagcc catcagagag 7741 caggccgaga atatcatcca cctgtttacc ctgaccaatc tgggagcccc tgccgccttc 7801 aagtactttg acaccaccat cgaccggaag aggtacacca gcaccaaaga ggtgctggac 7861 gccaccctga tccaccagag catcaccggc ctgtacgaga cacggatcga cctgtctcag 7921 ctgggaggcg acaagcgtcc tgctgctact aagaaagctg gtcaagctaa gaaaaagaaa 7981 gctagcggca gcggcgccac caacttcagc ctgctgaagc aggccggcga cgtggaggag 8041 aaccccggcc ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 8101 cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 8161 gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 8221 gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 8281 accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 8341 gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 8401 cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga ccaccagggc 8461 aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 8521 cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 8581 accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 8641 cccggtgcct gaacgcgtta agtcgacaat caacctctgg attacaaaat ttgtgaaaga 8701 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg 8761 cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt gtataaatcc 8821 tggttgctgt ctctttatga ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc 8881 actgtgtttg ctgacgcaac ccccactggt tggggcattg ccaccacctg tcagctcctt 8941 tccgggactt tcgctttccc cctccctatt gccacggcgg aactcatcgc cgcctgcctt 9001 gcccgctgct ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg 9061 aaatcatcgt cctttccttg gctgctcgcc tgtgttgcca cctggattct gcgcgggacg 9121 tccttctgct acgtcccttc ggccctcaat ccagcggacc ttccttcccg cggcctgctg 9181 ccggctctgc ggcctcttcc gcgtcttcgc cttcgccctc agacgagtcg gatctccctt 9241 tgggccgcct ccccgcacta gttaaaagtt ttgttacttt atagaagaaa ttttgagttt 9301 ttgttttttt ttaataaata aataaacata aataaattgt ttgttgaatt tattattagt 9361 atgtaagtgt aaatataata aaacttaata tctattcaaa ttaataaata aacctcgata 9421 tacagaccga taaaacacat gcgtcaattt tacgcatgat tatctttaac gtacgtcaca 9481 atatgattat ctttctaggg ttaatctagt atacgcgtat gcggccgctt aattaatcca 9541 gcttttgttc cctttagtga gggttaattg cgcgcttggc gtaatcatgg tcatagctgt 9601 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa 9661 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac 9721 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg 9781 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc 9841 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat 9901 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca 9961 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc 10021 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 10081 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg 10141 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta 10201 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg 10261 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac 10321 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag 10381 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat 10441 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat 10501 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc 10561 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt 10621 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct 10681 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt 10741 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 10801 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac 10861 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat 10921 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg 10981 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata 11041 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta 11101 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 11161 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag 11221 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa 11281 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc 11341 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt 11401 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc 11461 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 11521 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa 11581 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca 11641 tttatcaggg ttattgtct //