LOCUS       sequence-471631-        8056 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid for SpCas9.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8056)
  AUTHORS   Kleinstiver BP, Prew MS, Tsai SQ, Topkar VV, Nguyen NT, Zheng Z, 
            Gonzales AP, Li Z, Peterson RT, Yeh JJ, Aryee MJ, Joung JK
  TITLE     Engineered CRISPR-Cas9 nucleases with altered PAM specificities.
  JOURNAL   Nature. 2015 Jun 22. doi: 10.1038/nature14592.
  PUBMED    26098369
REFERENCE   2  (bases 1 to 8056)
  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"; journalName: "Nature. 
            2015 Jun 22. doi: 10.1038/nature14592."
COMMENT     Sequence Label: BPK764
FEATURES             Location/Qualifiers
     source          1..8056
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    3..27
                     /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)."
     CDS             join(71..3472,3473..3475,3476..4072,4073,4074..4076,
                     4077..4078,4079..4081,4082..4174)
                     /codon_start=1
                     /product="Streptococcus pyogenes Cas9 endonuclease with the
                     D1135V/R1335Q/T1337R mutations"
                     /label=Cas9 VQR
                     /note="has an altered recognition specificity for the 
                     protospacer adjacent motif (PAM) (Kleinstiver et al., 
                     2015)"
                     /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             71..4174
                     /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             71..4174
                     /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             4181..4201
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4208..4273
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     promoter        4343..4361
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_RNA        4417..4492
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(4494..4512)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4508..4555
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(4776..5435)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     5350..5369
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(5436..5538)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(5930..5947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6064..6609)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6144..6163)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6834..7916)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     7878..7897
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7917..7994)
                     /gene="lacI"
                     /label=lacI promoter
     promoter        join(8040..8056,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggggaattgt gagcggataa caattcccct gtagaaataa ttttgtttaa ctttaataag
       61 gagatatacc atggataaaa agtattctat tggtttagac atcggcacta attccgttgg
      121 atgggctgtc ataaccgatg aatacaaagt accttcaaag aaatttaagg tgttggggaa
      181 cacagaccgt cattcgatta aaaagaatct tatcggtgcc ctcctattcg atagtggcga
      241 aacggcagag gcgactcgcc tgaaacgaac cgctcggaga aggtatacac gtcgcaagaa
      301 ccgaatatgt tacttacaag aaatttttag caatgagatg gccaaagttg acgattcttt
      361 ctttcaccgt ttggaagagt ccttccttgt cgaagaggac aagaaacatg aacggcaccc
      421 catctttgga aacatagtag atgaggtggc atatcatgaa aagtacccaa cgatttatca
      481 cctcagaaaa aagctagttg actcaactga taaagcggac ctgaggttaa tctacttggc
      541 tcttgcccat atgataaagt tccgtgggca ctttctcatt gagggtgatc taaatccgga
      601 caactcggat gtcgacaaac tgttcatcca gttagtacaa acctataatc agttgtttga
      661 agagaaccct ataaatgcaa gtggcgtgga tgcgaaggct attcttagcg cccgcctctc
      721 taaatcccga cggctagaaa acctgatcgc acaattaccc ggagagaaga aaaatgggtt
      781 gttcggtaac cttatagcgc tctcactagg cctgacacca aattttaagt cgaacttcga
      841 cttagctgaa gatgccaaat tgcagcttag taaggacacg tacgatgacg atctcgacaa
      901 tctactggca caaattggag atcagtatgc ggacttattt ttggctgcca aaaaccttag
      961 cgatgcaatc ctcctatctg acatactgag agttaatact gagattacca aggcgccgtt
     1021 atccgcttca atgatcaaaa ggtacgatga acatcaccaa gacttgacac ttctcaaggc
     1081 cctagtccgt cagcaactgc ctgagaaata taaggaaata ttctttgatc agtcgaaaaa
     1141 cgggtacgca ggttatattg acggcggagc gagtcaagag gaattctaca agtttatcaa
     1201 acccatatta gagaagatgg atgggacgga agagttgctt gtaaaactca atcgcgaaga
     1261 tctactgcga aagcagcgga ctttcgacaa cggtagcatt ccacatcaaa tccacttagg
     1321 cgaattgcat gctatactta gaaggcagga ggatttttat ccgttcctca aagacaatcg
     1381 tgaaaagatt gagaaaatcc taacctttcg cataccttac tatgtgggac ccctggcccg
     1441 agggaactct cggttcgcat ggatgacaag aaagtccgaa gaaacgatta ctccctggaa
     1501 ttttgaggaa gttgtcgata aaggtgcgtc agctcaatcg ttcatcgaga ggatgaccaa
     1561 ctttgacaag aatttaccga acgaaaaagt attgcctaag cacagtttac tttacgagta
     1621 tttcacagtg tacaatgaac tcacgaaagt taagtatgtc actgagggca tgcgtaaacc
     1681 cgcctttcta agcggagaac agaagaaagc aatagtagat ctgttattca agaccaaccg
     1741 caaagtgaca gttaagcaat tgaaagagga ctactttaag aaaattgaat gcttcgattc
     1801 tgtcgagatc tccggggtag aagatcgatt taatgcgtca cttggtacgt atcatgacct
     1861 cctaaagata attaaagata aggacttcct ggataacgaa gagaatgaag atatcttaga
     1921 agatatagtg ttgactctta ccctctttga agatcgggaa atgattgagg aaagactaaa
     1981 aacatacgct cacctgttcg acgataaggt tatgaaacag ttaaagaggc gtcgctatac
     2041 gggctgggga cgattgtcgc ggaaacttat caacgggata agagacaagc aaagtggtaa
     2101 aactattctc gattttctaa agagcgacgg cttcgccaat aggaacttta tgcagctgat
     2161 ccatgatgac tctttaacct tcaaagagga tatacaaaag gcacaggttt ccggacaagg
     2221 ggactcattg cacgaacata ttgcgaatct tgctggttcg ccagccatca aaaagggcat
     2281 actccagaca gtcaaagtag tggatgagct agttaaggtc atgggacgtc acaaaccgga
     2341 aaacattgta atcgagatgg cacgcgaaaa tcaaacgact cagaaggggc aaaaaaacag
     2401 tcgagagcgg atgaagagaa tagaagaggg tattaaagaa ctgggcagcc agatcttaaa
     2461 ggagcatcct gtggaaaata cccaattgca gaacgagaaa ctttacctct attacctaca
     2521 aaatggaagg gacatgtatg ttgatcagga actggacata aaccgtttat ctgattacga
     2581 cgtcgatcac attgtacccc aatccttttt gaaggacgat tcaatcgaca ataaagtgct
     2641 tacacgctcg gataagaacc gagggaaaag tgacaatgtt ccaagcgagg aagtcgtaaa
     2701 gaaaatgaag aactattggc ggcagctcct aaatgcgaaa ctgataacgc aaagaaagtt
     2761 cgataactta actaaagctg agaggggtgg cttgtctgaa cttgacaagg ccggatttat
     2821 taaacgtcag ctcgtggaaa cccgccaaat cacaaagcat gttgcacaga tactagattc
     2881 ccgaatgaat acgaaatacg acgagaacga taagctgatt cgggaagtca aagtaatcac
     2941 tttaaagtca aaattggtgt cggacttcag aaaggatttt caattctata aagttaggga
     3001 gataaataac taccaccatg cgcacgacgc ttatcttaat gccgtcgtag ggaccgcact
     3061 cattaagaaa tacccgaagc tagaaagtga gtttgtgtat ggtgattaca aagtttatga
     3121 cgtccgtaag atgatcgcga aaagcgaaca ggagataggc aaggctacag ccaaatactt
     3181 cttttattct aacattatga atttctttaa gacggaaatc actctggcaa acggagagat
     3241 acgcaaacga cctttaattg aaaccaatgg ggagacaggt gaaatcgtat gggataaggg
     3301 ccgggacttc gcgacggtga gaaaagtttt gtccatgccc caagtcaaca tagtaaagaa
     3361 aactgaggtg cagaccggag ggttttcaaa ggaatcgatt cttccaaaaa ggaatagtga
     3421 taagctcatc gctcgtaaaa aggactggga cccgaaaaag tacggtggct tcgatagccc
     3481 tacagttgcc tattctgtcc tagtagtggc aaaagttgag aagggaaaat ccaagaaact
     3541 gaagtcagtc aaagaattat tggggataac gattatggag cgctcgtctt ttgaaaagaa
     3601 ccccatcgac ttccttgagg cgaaaggtta caaggaagta aaaaaggatc tcataattaa
     3661 actaccaaag tatagtctgt ttgagttaga aaatggccga aaacggatgt tggctagcgc
     3721 cggagagctt caaaagggga acgaactcgc actaccgtct aaatacgtga atttcctgta
     3781 tttagcgtcc cattacgaga agttgaaagg ttcacctgaa gataacgaac agaagcaact
     3841 ttttgttgag cagcacaaac attatctcga cgaaatcata gagcaaattt cggaattcag
     3901 taagagagtc atcctagctg atgccaatct ggacaaagta ttaagcgcat acaacaagca
     3961 cagggataaa cccatacgtg agcaggcgga aaatattatc catttgttta ctcttaccaa
     4021 cctcggcgct ccagccgcat tcaagtattt tgacacaacg atagatcgca aacgatacac
     4081 ttctaccaag gaggtgctag acgcgacact gattcaccaa tccatcacgg gattatatga
     4141 aactcggata gatttgtcac agcttggggg tgacggatcc cccaagaaga agaggaaagt
     4201 ctcgagcgac tacaaagacc atgacggtga ttataaagat catgacatcg attacaagga
     4261 tgacgatgac aagtgaagcg gccgcataat gcttaagtcg aacagaaagt aatcgtattg
     4321 tacacggccg cataatcgaa attaatacga ctcactatag ggagacccat gccatagcgt
     4381 tgttcggaac agattcacca acacctagtg gtctccgttt tagagctaga aatagcaagt
     4441 taaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctccgctga
     4501 gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa
     4561 acctcaggca tttgagaagc acacggtcac actgcttccg gtagtcaata aaccggtaaa
     4621 ccagcaatag acataagcgg ctatttaacg accctgccct gaaccgacga ccgggtcgaa
     4681 tttgctttcg aatttctgcc attcatccgc ttattatcac ttattcaggc gtagcaacca
     4741 ggcgtttaag ggcaccaata actgccttaa aaaaattacg ccccgccctg ccactcatcg
     4801 cagtactgtt gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga
     4861 tgaacctgaa tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccata
     4921 gtgaaaacgg gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa
     4981 ctcacccagg gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag
     5041 gccaggtttt caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa
     5101 tcgtcgtggt attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg
     5161 taacaagggt gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaac
     5221 tccggatgag cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc
     5281 ttatttttct ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag
     5341 gtacattgag caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata
     5401 tcaacggtgg tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat
     5461 ctcgataact caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa
     5521 cctcttacgt gccgatcaac gtctcatttt cgccaaaagt tggcccaggg cttcccggta
     5581 tcaacaggga caccaggatt tatttattct gcgaagtgat cttccgtcac aggtatttat
     5641 tcggcgcaaa gtgcgtcggg tgatgctgcc aacttactga tttagtgtat gatggtgttt
     5701 ttgaggtgct ccagtggctt ctgtttctat cagctgtccc tcctgttcag ctactgacgg
     5761 ggtggtgcgt aacggcaaaa gcaccgccgg acatcagcgc tagcggagtg tatactggct
     5821 tactatgttg gcactgatga gggtgtcagt gaagtgcttc atgtggcagg agaaaaaagg
     5881 ctgcaccggt gcgtcagcag aatatgtgat acaggatata ttccgcttcc tcgctcactg
     5941 actcgctacg ctcggtcgtt cgactgcggc gagcggaaat ggcttacgaa cggggcggag
     6001 atttcctgga agatgccagg aagatactta acagggaagt gagagggccg cggcaaagcc
     6061 gtttttccat aggctccgcc cccctgacaa gcatcacgaa atctgacgct caaatcagtg
     6121 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggcg gctccctcgt
     6181 gcgctctcct gttcctgcct ttcggtttac cggtgtcatt ccgctgttat ggccgcgttt
     6241 gtctcattcc acgcctgaca ctcagttccg ggtaggcagt tcgctccaag ctggactgta
     6301 tgcacgaacc ccccgttcag tccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6361 ccaacccgga aagacatgca aaagcaccac tggcagcagc cactggtaat tgatttagag
     6421 gagttagtct tgaagtcatg cgccggttaa ggctaaactg aaaggacaag ttttggtgac
     6481 tgcgctcctc caagccagtt acctcggttc aaagagttgg tagctcagag aaccttcgaa
     6541 aaaccgccct gcaaggcggt tttttcgttt tcagagcaag agattacgcg cagaccaaaa
     6601 cgatctcaag aagatcatct tattaatcag ataaaatatt tctagatttc agtgcaattt
     6661 atctcttcaa atgtagcacc tgaagtcagc cccatacgat ataagttgta attctcatgt
     6721 tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg
     6781 gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc
     6841 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6901 ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc
     6961 aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg
     7021 gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag
     7081 ctgtcttcgg tatcgtcgta tcccactacc gagatgtccg caccaacgcg cagcccggac
     7141 tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg
     7201 ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag
     7261 tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca
     7321 gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg
     7381 tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata
     7441 atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag
     7501 gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg
     7561 acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct
     7621 accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca
     7681 atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt
     7741 ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct
     7801 tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg
     7861 gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc
     7921 accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc
     7981 cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaaatt
     8041 aatacgactc actata
//
