LOCUS       sequence-327745-        9582 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription of SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9582)
  AUTHORS   Pelletier S, Gingras S, Green DR
  TITLE     Mouse genome engineering via CRISPR-Cas9 for study of immune 
            function.
  JOURNAL   Immunity. 2015 Jan 20;42(1):18-27. doi: 
            10.1016/j.immuni.2015.01.004.
  PUBMED    25607456
REFERENCE   2  (bases 1 to 9582)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.immuni.2015.01"; journalName: "Immunity"; date: 
            "2015-01-20- 20"; volume: "42"; issue: "1"; pages: "18-27"
COMMENT     Sequence Label: pCDNA3.3TOPO-T7-hCas9
FEATURES             Location/Qualifiers
     source          1..9582
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     primer_bind     680..699
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        680..698
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      722..731
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             728..4831
                     /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             728..4831
                     /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             728..4831
                     /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             4844..4864
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4919..4938)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4963..5011
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5213..5641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5300..5319)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5510..5531
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5650..5670)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5655..5984
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5835..5970
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5897..5916
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6051..6845
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6105..6124)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6715..6734
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    7021..7142
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7058..7077)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     7112..7131
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(7191..7207)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7191..7207)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7204..7226)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7215..7231
                     /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(join(7239..7245,7246..7263,7264..7269))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7284..7305
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7422..7439)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7593..8181)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7673..7692)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8352..9143,9144..9212))
                     /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     8975..8994
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(9213..9317)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9392..9411)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg gactctagat aatacgactc actataggga gatctagagg atcgaaccct
      721 tgccaccatg gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggctg
      781 ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac
      841 cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac
      901 ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg
      961 gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt
     1021 ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat
     1081 ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct
     1141 gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct
     1201 ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa
     1261 cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga
     1321 gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa
     1381 atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt
     1441 tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta acttcgacct
     1501 ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct
     1561 gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga
     1621 cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag
     1681 cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct
     1741 tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg
     1801 ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc
     1861 catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct
     1921 gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga
     1981 actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga
     2041 aaagattgag aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg
     2101 aaattccaga ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt
     2161 cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt
     2221 tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt
     2281 cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc
     2341 attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa
     2401 agttaccgtg aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt
     2461 tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct
     2521 gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga
     2581 cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac
     2641 ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg
     2701 atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga gtggaaagac
     2761 aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca
     2821 tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga
     2881 cagtcttcac gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact
     2941 gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa
     3001 tatcgttatc gagatggccc gagagaacca aactacccag aagggacaga agaacagtag
     3061 ggaaaggatg aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga
     3121 acacccagtt gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa
     3181 cggcagggac atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt
     3241 ggatcatatc gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac
     3301 aagatccgat aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa
     3361 aatgaaaaat tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga
     3421 taatctgact aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa
     3481 aaggcagctt gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg
     3541 catgaacacc aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct
     3601 gaagtctaag ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat
     3661 caacaattac caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat
     3721 caaaaaatat cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt
     3781 taggaaaatg atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt
     3841 ttacagcaat attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg
     3901 gaagcgacca cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag
     3961 ggatttcgcg acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac
     4021 cgaagtacag accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa
     4081 gctgatcgca cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac
     4141 agtcgcttac agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa
     4201 aagcgtcaag gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc
     4261 catcgacttt ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct
     4321 tcccaagtac tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg
     4381 cgagctgcag aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct
     4441 ggccagccac tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt
     4501 cgtggaacaa cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa
     4561 aagagtgatc ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag
     4621 ggataagccc atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt
     4681 gggcgcgcct gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc
     4741 tacaaaggag gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac
     4801 aagaatcgac ctctctcagc tcggtggaga cagcagggct gaccccaaga agaagaggaa
     4861 ggtgtgaaag ggttcgatcc ctaccggtta gtaatgagtt taaacggggg aggctaactg
     4921 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga
     4981 ataaaacgca cgggtgttgg gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg
     5041 cactctgtcg ataccccacc gagaccccat tggggccaat acgcccgcgt ttcttccttt
     5101 tccccacccc accccccaag ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg
     5161 gcaggccctg ccatagcaga tctgcgcagc tggggctcta gggggtatcc ccacgcgccc
     5221 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     5281 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     5341 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     5401 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     5461 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     5521 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     5581 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     5641 taattctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     5701 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     5761 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     5821 ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg
     5881 gctgactaat tttttttatt tatgcagagg ccgaggccgc ctctgcctct gagctattcc
     5941 agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagctc ccgggagctt
     6001 gtatatccat tttcggatct gatcaagaga caggatgagg atcgtttcgc atgattgaac
     6061 aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc ggctatgact
     6121 gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc
     6181 gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg caggacgagg
     6241 cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg
     6301 tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag gatctcctgt
     6361 catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg cggcggctgc
     6421 atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc atcgagcgag
     6481 cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa gagcatcagg
     6541 ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc
     6601 tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt
     6661 ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac atagcgttgg
     6721 ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt
     6781 acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt gacgagttct
     6841 tctgagcggg actctggggt tcgcgaaatg accgaccaag cgacgcccaa cctgccatca
     6901 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     6961 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     7021 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     7081 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     7141 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     7201 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     7261 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     7321 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     7381 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     7441 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     7501 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     7561 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     7621 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     7681 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     7741 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     7801 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     7861 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     7921 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     7981 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     8041 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     8101 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     8161 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     8221 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     8281 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     8341 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     8401 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     8461 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     8521 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     8581 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     8641 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     8701 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     8761 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     8821 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     8881 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     8941 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     9001 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     9061 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     9121 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     9181 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     9241 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     9301 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     9361 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     9421 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     9481 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     9541 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg tt
//
