LOCUS Exported 11497 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION PiggyBac cargo vector expressing doxycycline inducible Cas9. ACCESSION . VERSION . KEYWORDS PB_tre_Cas9 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 11497) AUTHORS Schertzer MD, Thulson E, Braceros KCA, Lee DM, Hinkle ER, Murphy RM, Kim SO, Vitucci EM, Calabrese JM TITLE A piggyBac-based toolkit for inducible genome editing in mammalian cells. JOURNAL RNA. 2019 May 17. pii: rna.068932.118. doi: 10.1261/rna.068932.118. PUBMED 31101683 REFERENCE 2 (bases 1 to 11497) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..11497 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 1..315 /label=tight TRE promoter /note="Tet-responsive promoter PTight, consisting of seven tet operator sequences followed by the minimal CMV promoter" protein_bind 9..27 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 45..63 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 80..98 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 116..134 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 152..170 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 187..205 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" protein_bind 223..241 /gene="tetO" /label=tet operator /bound_moiety="tetracycline repressor TetR" /note="bacterial operator O2 for the tetR and tetA genes" primer_bind 288..312 /label=LNCX /note="Human CMV promoter, forward primer" regulatory 320..329 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 329..394 /codon_start=1 /product="three tandem FLAG(R) epitope tags, followed by an enterokinase cleavage site" /label=3xFLAG /translation="DYKDHDGDYKDHDIDYKDDDDK" CDS 401..421 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 446..4546 /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 4547..4594 /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /translation="KRPAATKKAGQAKKKK" polyA_signal 4628..4852 /label=bGH poly(A) signal /note="bovine growth hormone polyadenylation signal" promoter 4980..5070 /label=cat promoter /note="promoter of the E. coli cat gene encoding chloramphenicol acetyltransferase" promoter 5118..5329 /label=EF-1-alpha core promoter /note="core promoter for human elongation factor EF-1-alpha" LTR 5342..5610 /label=5' LTR (truncated) /note="truncated 5' long terminal repeat (LTR) from human T-cell leukemia virus (HTLV) type 1" CDS 5647..6672 /codon_start=1 /gene="aph(4)-Ia" /product="aminoglycoside phosphotransferase from E. coli" /label=HygR /note="confers resistance to hygromycin" /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK E" polyA_signal 6701..6822 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(6738..6757) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 6792..6811 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 7585..7619 /label=piggyBac left (5') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" primer_bind complement(8110..8126) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(8110..8126) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(8123..8145) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 8134..8150 /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(8158..8188) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 8203..8224 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(8341..8358) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(8512..9100) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8592..8611) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9271..10131) /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 9894..9913 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10132..10236) /gene="bla" /label=AmpR promoter rep_origin complement(10262..10717) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(10399..10420) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 10611..10630 /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 10844..10866 /label=M13/pUC Forward /note="In lacZ gene" primer_bind 10858..10875 /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind 10859..10875 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" repeat_region 11005..11067 /label=piggyBac right (3') inverted repeat /note="piggyBac transposon-specific inverted terminal repeat sequence (ITR)" ORIGIN 1 gagtttactc cctatcagtg atagagaacg tatgtcgagt ttactcccta tcagtgatag 61 agaacgatgt cgagtttact ccctatcagt gatagagaac gtatgtcgag tttactccct 121 atcagtgata gagaacgtat gtcgagttta ctccctatca gtgatagaga acgtatgtcg 181 agtttatccc tatcagtgat agagaacgta tgtcgagttt actccctatc agtgatagag 241 aacgtatgtc gaggtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg 301 aaccgtcaga tcgcctggag ccaccatgga ctataaggac cacgacggag actacaagga 361 tcatgatatt gattacaaag acgatgacga taagatggcc ccaaagaaga agcggaaggt 421 cggtatccac ggagtcccag cagccgacaa gaagtacagc atcggcctgg acatcggcac 481 caactctgtg ggctgggccg tgatcaccga cgagtacaag gtgcccagca agaaattcaa 541 ggtgctgggc aacaccgacc ggcacagcat caagaagaac ctgatcggag ccctgctgtt 601 cgacagcggc gaaacagccg aggccacccg gctgaagaga accgccagaa gaagatacac 661 cagacggaag aaccggatct gctatctgca agagatcttc agcaacgaga tggccaaggt 721 ggacgacagc ttcttccaca gactggaaga gtccttcctg gtggaagagg ataagaagca 781 cgagcggcac cccatcttcg gcaacatcgt ggacgaggtg gcctaccacg agaagtaccc 841 caccatctac cacctgagaa agaaactggt ggacagcacc gacaaggccg acctgcggct 901 gatctatctg gccctggccc acatgatcaa gttccggggc cacttcctga tcgagggcga 961 cctgaacccc gacaacagcg acgtggacaa gctgttcatc cagctggtgc agacctacaa 1021 ccagctgttc gaggaaaacc ccatcaacgc cagcggcgtg gacgccaagg ccatcctgtc 1081 tgccagactg agcaagagca gacggctgga aaatctgatc gcccagctgc ccggcgagaa 1141 gaagaatggc ctgttcggaa acctgattgc cctgagcctg ggcctgaccc ccaacttcaa 1201 gagcaacttc gacctggccg aggatgccaa actgcagctg agcaaggaca cctacgacga 1261 cgacctggac aacctgctgg cccagatcgg cgaccagtac gccgacctgt ttctggccgc 1321 caagaacctg tccgacgcca tcctgctgag cgacatcctg agagtgaaca ccgagatcac 1381 caaggccccc ctgagcgcct ctatgatcaa gagatacgac gagcaccacc aggacctgac 1441 cctgctgaaa gctctcgtgc ggcagcagct gcctgagaag tacaaagaga ttttcttcga 1501 ccagagcaag aacggctacg ccggctacat tgacggcgga gccagccagg aagagttcta 1561 caagttcatc aagcccatcc tggaaaagat ggacggcacc gaggaactgc tcgtgaagct 1621 gaacagagag gacctgctgc ggaagcagcg gaccttcgac aacggcagca tcccccacca 1681 gatccacctg ggagagctgc acgccattct gcggcggcag gaagattttt acccattcct 1741 gaaggacaac cgggaaaaga tcgagaagat cctgaccttc cgcatcccct actacgtggg 1801 ccctctggcc aggggaaaca gcagattcgc ctggatgacc agaaagagcg aggaaaccat 1861 caccccctgg aacttcgagg aagtggtgga caagggcgct tccgcccaga gcttcatcga 1921 gcggatgacc aacttcgata agaacctgcc caacgagaag gtgctgccca agcacagcct 1981 gctgtacgag tacttcaccg tgtataacga gctgaccaaa gtgaaatacg tgaccgaggg 2041 aatgagaaag cccgccttcc tgagcggcga gcagaaaaag gccatcgtgg acctgctgtt 2101 caagaccaac cggaaagtga ccgtgaagca gctgaaagag gactacttca agaaaatcga 2161 gtgcttcgac tccgtggaaa tctccggcgt ggaagatcgg ttcaacgcct ccctgggcac 2221 ataccacgat ctgctgaaaa ttatcaagga caaggacttc ctggacaatg aggaaaacga 2281 ggacattctg gaagatatcg tgctgaccct gacactgttt gaggacagag agatgatcga 2341 ggaacggctg aaaacctatg cccacctgtt cgacgacaaa gtgatgaagc agctgaagcg 2401 gcggagatac accggctggg gcaggctgag ccggaagctg atcaacggca tccgggacaa 2461 gcagtccggc aagacaatcc tggatttcct gaagtccgac ggcttcgcca acagaaactt 2521 catgcagctg atccacgacg acagcctgac ctttaaagag gacatccaga aagcccaggt 2581 gtccggccag ggcgatagcc tgcacgagca cattgccaat ctggccggca gccccgccat 2641 taagaagggc atcctgcaga cagtgaaggt ggtggacgag ctcgtgaaag tgatgggccg 2701 gcacaagccc gagaacatcg tgatcgaaat ggccagagag aaccagacca cccagaaggg 2761 acagaagaac agccgcgaga gaatgaagcg gatcgaagag ggcatcaaag agctgggcag 2821 ccagatcctg aaagaacacc ccgtggaaaa cacccagctg cagaacgaga agctgtacct 2881 gtactacctg cagaatgggc gggatatgta cgtggaccag gaactggaca tcaaccggct 2941 gtccgactac gatgtggacc atatcgtgcc tcagagcttt ctgaaggacg actccatcga 3001 caacaaggtg ctgaccagaa gcgacaagaa ccggggcaag agcgacaacg tgccctccga 3061 agaggtcgtg aagaagatga agaactactg gcggcagctg ctgaacgcca agctgattac 3121 ccagagaaag ttcgacaatc tgaccaaggc cgagagaggc ggcctgagcg aactggataa 3181 ggccggcttc atcaagagac agctggtgga aacccggcag atcacaaagc acgtggcaca 3241 gatcctggac tcccggatga acactaagta cgacgagaat gacaagctga tccgggaagt 3301 gaaagtgatc accctgaagt ccaagctggt gtccgatttc cggaaggatt tccagtttta 3361 caaagtgcgc gagatcaaca actaccacca cgcccacgac gcctacctga acgccgtcgt 3421 gggaaccgcc ctgatcaaaa agtaccctaa gctggaaagc gagttcgtgt acggcgacta 3481 caaggtgtac gacgtgcgga agatgatcgc caagagcgag caggaaatcg gcaaggctac 3541 cgccaagtac ttcttctaca gcaacatcat gaactttttc aagaccgaga ttaccctggc 3601 caacggcgag atccggaagc ggcctctgat cgagacaaac ggcgaaaccg gggagatcgt 3661 gtgggataag ggccgggatt ttgccaccgt gcggaaagtg ctgagcatgc cccaagtgaa 3721 tatcgtgaaa aagaccgagg tgcagacagg cggcttcagc aaagagtcta tcctgcccaa 3781 gaggaacagc gataagctga tcgccagaaa gaaggactgg gaccctaaga agtacggcgg 3841 cttcgacagc cccaccgtgg cctattctgt gctggtggtg gccaaagtgg aaaagggcaa 3901 gtccaagaaa ctgaagagtg tgaaagagct gctggggatc accatcatgg aaagaagcag 3961 cttcgagaag aatcccatcg actttctgga agccaagggc tacaaagaag tgaaaaagga 4021 cctgatcatc aagctgccta agtactccct gttcgagctg gaaaacggcc ggaagagaat 4081 gctggcctct gccggcgaac tgcagaaggg aaacgaactg gccctgccct ccaaatatgt 4141 gaacttcctg tacctggcca gccactatga gaagctgaag ggctcccccg aggataatga 4201 gcagaaacag ctgtttgtgg aacagcacaa gcactacctg gacgagatca tcgagcagat 4261 cagcgagttc tccaagagag tgatcctggc cgacgctaat ctggacaaag tgctgtccgc 4321 ctacaacaag caccgggata agcccatcag agagcaggcc gagaatatca tccacctgtt 4381 taccctgacc aatctgggag cccctgccgc cttcaagtac tttgacacca ccatcgaccg 4441 gaagaggtac accagcacca aagaggtgct ggacgccacc ctgatccacc agagcatcac 4501 cggcctgtac gagacacgga tcgacctgtc tcagctggga ggcgacaaaa ggccggcggc 4561 cacgaaaaag gccggccagg caaaaaagaa aaagtaagaa ttcctagagc tcgccaatgc 4621 gtttcgactg tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc 4681 ttgaccctgg aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg 4741 cattgtctga gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg 4801 gaggattggg aagacaatag caggcatgct ggggatgcgg tgggctctat ggaaacacgc 4861 ggcgacgcgt tcgaccgaat aaaacctgtg acggaagatc acttcgcaga ataaataaat 4921 cctggtgtcc ctgttgatac cgggaagccc tgggccaact tttggcgaaa atgagacgtt 4981 gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact accgggcgta 5041 ttttttgagt tgtcgagatt ttcaggagct aaagatctgg aagctaagga tctgcgatcg 5101 ctccggtgcc cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg 5161 aggggtcggc aattgaacgg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga 5221 tgtcgtgtac tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt 5281 agtcgccgtg aacgttcttt ttcgcaacgg gtttgccgcc agaacacagc tgaagcttcg 5341 aggggctcgc atctctcctt cacgcgcccg ccgccctacc tgaggccgcc atccacgccg 5401 gttgagtcgc gttctgccgc ctcccgcctg tggtgcctcc tgaactgcgt ccgccgtcta 5461 ggtaagttta aagctcaggt cgagaccggg cctttgtccg gcgctccctt ggagcctacc 5521 tagactcagc cggctctcca cgctttgcct gaccctgctt gctcaactct acgtctttgt 5581 ttcgttttct gttctgcgcc gttacagatc caagctgtga ccggcgccta caagctgcta 5641 gccaccatga aaaagcctga actcaccgcg acgtctgtcg agaagtttct gatcgaaaag 5701 ttcgacagcg tctccgacct gatgcagctc tcggagggcg aagaatctcg tgctttcagc 5761 ttcgatgtag gagggcgtgg atatgtcctg cgggtaaata gctgcgccga tggtttctac 5821 aaagatcgtt atgtttatcg gcactttgca tcggccgcgc tcccgattcc ggaagtgctt 5881 gacattgggg agttcagcga gagcctgacc tattgcatct cccgccgtgc acagggtgtc 5941 acgttgcaag acctgcctga aaccgaactg cccgctgttc tgcagccggt cgcggaggcc 6001 atggatgcga tcgctgcggc cgatcttagc cagacgagcg ggttcggccc attcggaccg 6061 caaggaatcg gtcaatacac tacatggcgt gatttcatat gcgcgattgc tgatccccat 6121 gtgtatcact ggcaaactgt gatggacgac accgtcagtg cgtccgtcgc gcaggctctc 6181 gatgagctga tgctttgggc cgaggactgc cccgaagtcc ggcacctcgt gcacgcggat 6241 ttcggctcca acaatgtcct gacggacaat ggccgcataa cagcggtcat tgactggagc 6301 gaggcgatgt tcggggattc ccaatacgag gtcgccaaca tcttcttctg gaggccgtgg 6361 ttggcttgta tggagcagca gacgcgctac ttcgagcgga ggcatccgga gcttgcagga 6421 tcgccgcggc tccgggcgta tatgctccgc attggtcttg accaactcta tcagagcttg 6481 gttgacggca atttcgatga tgcagcttgg gcgcagggtc gatgcgacgc aatcgtccga 6541 tccggagccg ggactgtcgg gcgtacacaa atcgcccgca gaagcgcggc cgtctggacc 6601 gatggctgtg tagaagtact cgccgatagt ggaaaccgac gccccagcac tcgtccgagg 6661 gcaaaggaat agttttacgt attgagtatt taactaacta aacttgttta ttgcagctta 6721 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact 6781 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct ggaattgact 6841 caaatgatgt caattagtct atcagaagct catctggtct cccttccggg ggacaagaca 6901 tccctgttta atatttaaac agcagtgttc ccaaactggg ttcttatatc ccttgctctg 6961 gtcaaccagg ttgcagggtt tcctgtcctc acaggaacga agtccctaaa gaaacagtgg 7021 cagccaggtt tagccccgga attgactgga ttcctttttt agggcccatt ggtatggctt 7081 tttccccgta tccccccagg tgtctgcagg ctcaaagagc agcgagaagc gttcagagga 7141 aagcgatccc gtgccacctt ccccgtgccc gggctgtccc cgcacgctgc cggctcgggg 7201 atgcgggggg agcgccggac cggagcggag ccccgggcgg ctcgctgctg ccccctagcg 7261 ggggagggac gtaattacat ccctgggggc tttggggggg ggctgtccct gatatctata 7321 acaagaaaat atatatataa taagttatca cgtaagtaga acatgaaata acaatataat 7381 tatcgtatga gttaaatctt aaaagtcacg taaaagataa tcatgcgtca ttttgactca 7441 cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat tgacaagcac gcctcacggg 7501 agctccaagc ggcgactgag atgtcctaaa tgcacagcga cggattcgcg ctatttagaa 7561 agagagagca atatttcaag aatgcatgcg tcaattttac gcagactatc tttctagggt 7621 taatctagct gcatcaggat catatcgtcg ggtctttttt ccggctcagt catcgcccaa 7681 gctggcgcta tctgggcatc ggggaggaag aagcccgtgc cttttcccgc gaggttgaag 7741 cggcatggaa agagtttgcc gaggatgact gctgctgcat tgacgttgag cgaaaacgca 7801 cgtttaccat gatgattcgg gaaggtgtgg ccatgcacgc ctttaacggt gaactgttcg 7861 ttcaggccac ctgggatacc agttcgtcgc ggcttttccg gacacagttc cggatggtca 7921 gcccgaagcg catcagcaac ccgaacaata ccggcgacag ccggaactgc cgtgccggtg 7981 tgcagattaa tgacagcggt gcggcgctgg gatattacgt cagcgaggac gggtatcctg 8041 gctggatgcc gcagaaatgg acatggatac cccgtgagtt acccggcggg cgcgcttggc 8101 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa 8161 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac 8221 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca 8281 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc 8341 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 8401 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 8461 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 8521 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 8581 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 8641 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 8701 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 8761 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 8821 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 8881 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 8941 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 9001 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 9061 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 9121 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 9181 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 9241 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 9301 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 9361 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 9421 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 9481 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 9541 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 9601 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 9661 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 9721 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 9781 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 9841 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 9901 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 9961 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 10021 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 10081 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 10141 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 10201 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 10261 taaattgtaa gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca 10321 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag 10381 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc 10441 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc 10501 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc 10561 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa 10621 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc 10681 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccatt caggctgcgc 10741 aactgttggg aagggcgatc ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg 10801 ggatgtgctg caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt 10861 aaaacgacgg ccagtgagcg cgcctcgttc attcacgttt ttgaacccgt ggaggacggg 10921 cagactcgcg gtgcaaatgt gttttacagc gtgatggagc agatgaagat gctcgacacg 10981 ctgcagaaca cgcagctaga ttaaccctag aaagataatc atattgtgac gtacgttaaa 11041 gataatcatg tgtaaaattg acgcatgtgt tttatcggtc tgtatatcga ggtttattta 11101 ttaatttgaa tagatattaa gttttattat atttacactt acatactaat aataaattca 11161 acaaacaatt tatttatgtt tatttattta ttaaaaaaaa caaaaactca aaatttcttc 11221 tataaagtaa caaaactttt atgagggaca gccccccccc aaagccccca gggatgtaat 11281 tacgtccctc ccccgctagg gggcagcagc gagccgcccg gggctccgct ccggtccggc 11341 gctccccccg catccccgag ccggcagcgt gcggggacag cccgggcacg gggaaggtgg 11401 cacgggatcg ctttcctctg aacgcttctc gctgctcttt gagcctgcag acacctgggg 11461 ggatacgggg aaaaggcctc cacggccaga ctagttc //