LOCUS       sequence-148774-       10793 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nCas9(H840A) in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10793)
  AUTHORS   Nishida K, Arazoe T, Yachie N, Banno S, Kakimoto M, Tabata M, 
            Mochizuki M, Miyabe A, Araki M, Hara KY, Shimatani Z, Kondo A
  TITLE     Targeted nucleotide editing using hybrid prokaryotic and vertebrate 
            adaptive immune systems.
  JOURNAL   Science. 2016 Aug 4. pii: aaf8729.
  PUBMED    27492474
REFERENCE   2  (bases 1 to 10793)
  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: "Science. 
            2016 Aug 4. pii: aaf8729."
COMMENT     Sequence Label: pRS415_pGal-nCas9(H840A)
FEATURES             Location/Qualifiers
     source          1..10793
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(10..28)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     misc_feature    70..573
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an 
                     autonomously replicating sequence"
     promoter        610..714
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(715..783,784..1575)
                     /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     complement(933..952)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1746..2334
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2235..2254
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(2658..2663,2664..2681,2682..2688)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2696..2712
                     /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)."
     primer_bind     2701..2723
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2720..2736
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2720..2736
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2755..2775
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     3184..3207
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     primer_bind     3228..3244
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3228..3244
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     CDS             join(3258..5774,5775..5777,5778..7361)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(H840A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the H840A mutation in the HNH catalytic domain"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3258..7361
                     /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
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7374..7394
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7410..7657
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7426..7444)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7675..7694)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7676..7694)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7704..7721)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7704..7720)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7713..7735)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      7865..8320
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7952..7971)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8162..8183
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8620..9024
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             9037..10131
                     /codon_start=1
                     /gene="S. cerevisiae LEU2"
                     /product="3-isopropylmalate dehydrogenase, required for 
                     leucine biosynthesis"
                     /label=LEU2
                     /note="yeast auxotrophic marker"
                     /translation="MSAPKKIVVLPGDHVGQEITAEAIKVLKAISDVRSNVKFDFENHL
                     IGGAAIDATGVPLPDEALEASKKVDAVLLGAVGGPKWGTGSVRPEQGLLKIRKELQLYA
                     NLRPCNFASDSLLDLSPIKPQFAKGTDFVVVRELVGGIYFGKRKEDDGDGVAWDSEQYT
                     VPEVQRITRMAAFMALQHEPPLPIWSLDKANVLASSRLWRKTVEETIKNEFPTLKVQHQ
                     LIDSAAMILVKNPTHLNGIIITSNMFGDIISDEASVIPGSLGLLPSASLASLPDKNTAF
                     GLYEPCHGSAPDLPKNKVNPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(10624..10643)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     10743..10765
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 gacgaaaggg cctcgtgata cgcctatttt tataggttaa tgtcatgata ataatggttt
       61 cttaggacgg atcgcttgcc tgtaacttac acgcgcctcg tatcttttaa tgatggaata
      121 atttgggaat ttactctgtg tttatttatt tttatgtttt gtatttggat tttagaaagt
      181 aaataaagaa ggtagaagag ttacggaatg aagaaaaaaa aataaacaaa ggtttaaaaa
      241 atttcaacaa aaagcgtact ttacatatat atttattaga caagaaaagc agattaaata
      301 gatatacatt cgattaacga taagtaaaat gtaaaatcac aggattttcg tgtgtggtct
      361 tctacacaga caagatgaaa caattcggca ttaatacctg agagcaggaa gagcaagata
      421 aaaggtagta tttgttggcg atccccctag agtcttttac atcttcggaa aacaaaaact
      481 attttttctt taatttcttt ttttactttc tatttttaat ttatatattt atattaaaaa
      541 atttaaatta taattatttt tatagcacgt gatgaaaagg acccaggtgg cacttttcgg
      601 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      661 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      721 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      781 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      841 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      901 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      961 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     1021 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1081 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1141 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1201 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1261 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1321 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1381 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1441 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1501 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1561 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1621 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1681 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1741 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1801 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1861 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     1921 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1981 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     2041 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2101 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2341 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2401 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2461 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2521 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac
     2581 aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttacctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc ctatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt
     2761 aaccctcact aaagggaaca aaagctggag ctcgtgacag ccctccgaag gaagactctc
     2821 ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac gcagatgtgc ctcgcgccgc
     2881 actgctccga acaataaaga ttctacaata ctagctttta tggttatgaa gaggaaaaat
     2941 tggcagtaac ctggccccac aaaccttcaa atgaacgaat caaattaaca accataggat
     3001 gataatgcga ttagtttttt agccttattt ctggggtaat taatcagcga agcgatgatt
     3061 tttgatctat taacggatat ataaatgcaa aaactgcata accactttaa ctaatacttt
     3121 caacattttc ggtttgtatt acttcttatt caaatgtaat aaaagtatca acaaaaaatt
     3181 gttaatatac ctctatactt taacgtcaag gagaaaaaac cccggattct agaactagtg
     3241 gatcccccgg gaaaaaaatg gacaagaagt actccattgg gctcgatatc ggcacaaaca
     3301 gcgtcggttg ggccgtcatt acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc
     3361 tgggcaatac cgatcgccac agcataaaga agaacctcat tggcgccctc ctgttcgact
     3421 ccggggagac ggccgaagcc acgcggctca aaagaacagc acggcgcaga tatacccgca
     3481 gaaagaatcg gatctgctac ctgcaggaga tctttagtaa tgagatggct aaggtggatg
     3541 actctttctt ccataggctg gaggagtcct ttttggtgga ggaggataaa aagcacgagc
     3601 gccacccaat ctttggcaat atcgtggacg aggtggcgta ccatgaaaag tacccaacca
     3661 tatatcatct gaggaagaag cttgtagaca gtactgataa ggctgacttg cggttgatct
     3721 atctcgcgct ggcgcatatg atcaaatttc ggggacactt cctcatcgag ggggacctga
     3781 acccagacaa cagcgatgtc gacaaactct ttatccaact ggttcagact tacaatcagc
     3841 ttttcgaaga gaacccgatc aacgcatccg gagttgacgc caaagcaatc ctgagcgcta
     3901 ggctgtccaa atcccggcgg ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga
     3961 acggcctgtt tggtaatctt atcgccctgt cactcgggct gacccccaac tttaaatcta
     4021 acttcgacct ggccgaagat gccaagcttc aactgagcaa agacacctac gatgatgatc
     4081 tcgacaatct gctggcccag atcggcgacc agtacgcaga cctttttttg gcggcaaaga
     4141 acctgtcaga cgccattctg ctgagtgata ttctgcgagt gaacacggag atcaccaaag
     4201 ctccgctgag cgctagtatg atcaagcgct atgatgagca ccaccaagac ttgactttgc
     4261 tgaaggccct tgtcagacag caactgcctg agaagtacaa ggaaattttc ttcgatcagt
     4321 ctaaaaatgg ctacgccgga tacattgacg gcggagcaag ccaggaggaa ttttacaaat
     4381 ttattaagcc catcttggaa aaaatggacg gcaccgagga gctgctggta aagcttaaca
     4441 gagaagatct gttgcgcaaa cagcgcactt tcgacaatgg aagcatcccc caccagattc
     4501 acctgggcga actgcacgct atcctcaggc ggcaagagga tttctacccc tttttgaaag
     4561 ataacaggga aaagattgag aaaatcctca catttcggat accctactat gtaggccccc
     4621 tcgcccgggg aaattccaga ttcgcgtgga tgactcgcaa atcagaagag accatcactc
     4681 cctggaactt cgaggaagtc gtggataagg gggcctctgc ccagtccttc atcgaaagga
     4741 tgactaactt tgataaaaat ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt
     4801 acgagtactt cacagtttat aacgagctca ccaaggtcaa atacgtcaca gaagggatga
     4861 gaaagccagc attcctgtct ggagagcaga agaaagctat cgtggacctc ctcttcaaga
     4921 cgaaccggaa agttaccgtg aaacagctca aagaagacta tttcaaaaag attgaatgtt
     4981 tcgactctgt tgaaatcagc ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc
     5041 acgatctcct gaaaatcatt aaagacaagg acttcctgga caatgaggag aacgaggaca
     5101 ttcttgagga cattgtcctc acccttacgt tgtttgaaga tagggagatg attgaagaac
     5161 gcttgaaaac ttacgctcat ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc
     5221 gatatacagg atgggggcgg ctgtcaagaa aactgatcaa tgggatccga gacaagcaga
     5281 gtggaaagac aatcctggat tttcttaagt ccgatggatt tgccaaccgg aacttcatgc
     5341 agttgatcca tgatgactct ctcaccttta aggaggacat ccagaaagca caagtttctg
     5401 gccaggggga cagtcttcac gagcacatcg ctaatcttgc aggtagccca gctatcaaaa
     5461 agggaatact gcagaccgtt aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata
     5521 agcccgagaa tatcgttatc gagatggccc gagagaacca aactacccag aagggacaga
     5581 agaacagtag ggaaaggatg aagaggattg aagagggtat aaaagaactg gggtcccaaa
     5641 tccttaagga acacccagtt gaaaacaccc agcttcagaa tgagaagctc tacctgtact
     5701 acctgcagaa cggcagggac atgtacgtgg atcaggaact ggacatcaat cggctctccg
     5761 actacgacgt ggatgctatc gtgccccagt cttttctcaa agatgattct attgataata
     5821 aagtgttgac aagatccgat aaaaatagag ggaagagtga taacgtcccc tcagaagaag
     5881 ttgtcaagaa aatgaaaaat tattggcggc agctgctgaa cgccaaactg atcacacaac
     5941 ggaagttcga taatctgact aaggctgaac gaggtggcct gtctgagttg gataaagccg
     6001 gcttcatcaa aaggcagctt gttgagacac gccagatcac caagcacgtg gcccaaattc
     6061 tcgattcacg catgaacacc aagtacgatg aaaatgacaa actgattcga gaggtgaaag
     6121 ttattactct gaagtctaag ctggtctcag atttcagaaa ggactttcag ttttataagg
     6181 tgagagagat caacaattac caccatgcgc atgatgccta cctgaatgca gtggtaggca
     6241 ctgcacttat caaaaaatat cccaagcttg aatctgaatt tgtttacgga gactataaag
     6301 tgtacgatgt taggaaaatg atcgcaaagt ctgagcagga aataggcaag gccaccgcta
     6361 agtacttctt ttacagcaat attatgaatt ttttcaagac cgagattaca ctggccaatg
     6421 gagagattcg gaagcgacca cttatcgaaa caaacggaga aacaggagaa atcgtgtggg
     6481 acaagggtag ggatttcgcg acagtccgga aggtcctgtc catgccgcag gtgaacatcg
     6541 ttaaaaagac cgaagtacag accggaggct tctccaagga aagtatcctc ccgaaaagga
     6601 acagcgacaa gctgatcgca cgcaaaaaag attgggaccc caagaaatac ggcggattcg
     6661 attctcctac agtcgcttac agtgtactgg ttgtggccaa agtggagaaa gggaagtcta
     6721 aaaaactcaa aagcgtcaag gaactgctgg gcatcacaat catggagcga tcaagcttcg
     6781 aaaaaaaccc catcgacttt ctcgaggcga aaggatataa agaggtcaaa aaagacctca
     6841 tcattaagct tcccaagtac tctctctttg agcttgaaaa cggccggaaa cgaatgctcg
     6901 ctagtgcggg cgagctgcag aaaggtaacg agctggcact gccctctaaa tacgttaatt
     6961 tcttgtatct ggccagccac tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga
     7021 agcagctgtt cgtggaacaa cacaaacact accttgatga gatcatcgag caaataagcg
     7081 aattctccaa aagagtgatc ctcgccgacg ctaacctcga taaggtgctt tctgcttaca
     7141 ataagcacag ggataagccc atcagggagc aggcagaaaa cattatccac ttgtttactc
     7201 tgaccaactt gggcgcgcct gcagccttca agtacttcga caccaccata gacagaaagc
     7261 ggtacacctc tacaaaggag gtcctggacg ccacactgat tcatcagtca attacggggc
     7321 tctatgaaac aagaatcgac ctctctcagc tcggtggaga cagcagggct gaccccaaga
     7381 agaagaggaa ggtgtgatct cttctcgagt catgtaatta gttatgtcac gcttacattc
     7441 acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac ctgaagtcta
     7501 ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta tatttcaaat
     7561 ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga aaaccttgct
     7621 tgagaaggtt ttgggacgct cgaaggcttt aatttgcggc cggtacccaa ttcgccctat
     7681 agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga ctgggaaaac
     7741 cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat
     7801 agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg
     7861 cgcgacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     7921 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     7981 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     8041 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt
     8101 gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat
     8161 agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat
     8221 ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa
     8281 tttaacgcga attttaacaa aatattaacg tttacaattt cctgatgcgg tattttctcc
     8341 ttacgcatct gtgcggtatt tcacaccgca tatcgacggt cgaggagaac ttctagtata
     8401 tccacatacc taatattatt gccttattaa aaatggaatc ccaacaatta catcaaaatc
     8461 cacattctct tcaaaatcaa ttgtcctgta cttccttgtt catgtgtgtt caaaaacgtt
     8521 atatttatag gataattata ctctatttct caacaagtaa ttggttgttt ggccgagcgg
     8581 tctaaggcgc ctgattcaag aaatatcttg accgcagtta actgtgggaa tactcaggta
     8641 tcgtaagatg caagagttcg aatctcttag caaccattat ttttttcctc aacataacga
     8701 gaacacacag gggcgctatc gcacagaatc aaattcgatg actggaaatt ttttgttaat
     8761 ttcagaggtc gcctgacgca tatacctttt tcaactgaaa aattgggaga aaaaggaaag
     8821 gtgagaggcc ggaaccggct tttcatatag aatagagaag cgttcatgac taaatgcttg
     8881 catcacaata cttgaagttg acaatattat ttaaggacct attgtttttt ccaataggtg
     8941 gttagcaatc gtcttacttt ctaacttttc ttacctttta catttcagca atatatatat
     9001 atatttcaag gatataccat tctaatgtct gcccctatgt ctgcccctaa gaagatcgtc
     9061 gttttgccag gtgaccacgt tggtcaagaa atcacagccg aagccattaa ggttcttaaa
     9121 gctatttctg atgttcgttc caatgtcaag ttcgatttcg aaaatcattt aattggtggt
     9181 gctgctatcg atgctacagg tgtcccactt ccagatgagg cgctggaagc ctccaagaag
     9241 gttgatgccg ttttgttagg tgctgtgggt ggtcctaaat ggggtaccgg tagtgttaga
     9301 cctgaacaag gtttactaaa aatccgtaaa gaacttcaat tgtacgccaa cttaagacca
     9361 tgtaactttg catccgactc tcttttagac ttatctccaa tcaagccaca atttgctaaa
     9421 ggtactgact tcgttgttgt cagagaatta gtgggaggta tttactttgg taagagaaag
     9481 gaagacgatg gtgatggtgt cgcttgggat agtgaacaat acaccgttcc agaagtgcaa
     9541 agaatcacaa gaatggccgc tttcatggcc ctacaacatg agccaccatt gcctatttgg
     9601 tccttggata aagctaatgt tttggcctct tcaagattat ggagaaaaac tgtggaggaa
     9661 accatcaaga acgaattccc tacattgaag gttcaacatc aattgattga ttctgccgcc
     9721 atgatcctag ttaagaaccc aacccaccta aatggtatta taatcaccag caacatgttt
     9781 ggtgatatca tctccgatga agcctccgtt atcccaggtt ccttgggttt gttgccatct
     9841 gcgtccttgg cctctttgcc agacaagaac accgcatttg gtttgtacga accatgccac
     9901 ggttctgctc cagatttgcc aaagaataag gtcaacccta tcgccactat cttgtctgct
     9961 gcaatgatgt tgaaattgtc attgaacttg cctgaagaag gtaaggccat tgaagatgca
    10021 gttaaaaagg ttttggatgc aggtatcaga actggtgatt taggtggttc caacagtacc
    10081 accgaagtcg gtgatgctgt cgccgaagaa gttaagaaaa tccttgctta aaaagattct
    10141 ctttttttat gatatttgta cataaacttt ataaatgaaa ttcataatag aaacgacacg
    10201 aaattacaaa atggaatatg ttcatagggt agacgaaact atatacgcaa tctacataca
    10261 tttatcaaga aggagaaaaa ggaggatgta aaggaataca ggtaagcaaa ttgatactaa
    10321 tggctcaacg tgataaggaa aaagaattgc actttaacat taatattgac aaggaggagg
    10381 gcaccacaca aaaagttagg tgtaacagaa aatcatgaaa ctatgattcc taatttatat
    10441 attggaggat tttctctaaa aaaaaaaaaa tacaacaaat aaaaaacact caatgacctg
    10501 accatttgat ggagtttaag tcaatacctt cttgaaccat ttcccataat ggtgaaagtt
    10561 ccctcaagaa ttttactctg tcagaaacgg ccttacgacg tagtcgatat ggtgcactct
    10621 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
    10681 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
    10741 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cga
//
