LOCUS       sequence-154116-       11067 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nCas9(D10A) in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11067)
  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 11067)
  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(D10A)
FEATURES             Location/Qualifiers
     source          1..11067
                     /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"
     promoter        2852..3528
                     /gene="GAL1 and GAL10 genes of S. cerevisiae"
                     /label=GAL1,10 promoter
                     /note="divergent inducible promoter, regulated by Gal4"
     protein_bind    3080..3197
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3492..3515
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(3531..3557,3558..3560,3561..7634)
                     /codon_start=1
                     /product="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
                     /label=Cas9(D10A)
                     /note="generates RNA-guided single strand nicks in DNA due 
                     to the D10A mutation in the RuvC catalytic domain"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             3531..7634
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             7647..7667
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     terminator      7683..7930
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(7699..7717)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     primer_bind     complement(7948..7967)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7949..7967)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7977..7994)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7977..7993)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7986..8008)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      8138..8593
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(8225..8244)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8435..8456
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        8893..9297
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             9310..10404
                     /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(10898..10917)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11017..11039
                     /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 aaagctggta ccgggcccga attctctcct tctcttaggt
     2821 ggcagagcag gtggagggtc gaccatacta gtttcaaaaa ttcttacttt ttttttggat
     2881 ggacgcaaag aagtttaata atcatattac atggcattac caccatatac atatccatat
     2941 acatatccat atacatatcc atatctaatc ttacttatat gttgtggaaa tgtaaagagc
     3001 cccattatct tagcctaaaa aaaccttctc tttggaactt tcagtaatac gcttaactgc
     3061 tcattgctat attgaagtac ggattagaag ccgccgagcg ggtgacagcc ctccgaagga
     3121 agactctcct ccgtgcgtcc tcgtcttcac cggtcgcgtt cctgaaacgc agatgtgcct
     3181 cgcgccgcac tgctccgaac aataaagatt ctacaatact agcttttatg gttatgaaga
     3241 ggaaaaattg gcagtaacct ggccccacaa accttcaaat gaacgaatca aattaacaac
     3301 cataggatga taatgcgatt agttttttag ccttatttct ggggtaatta atcagcgaag
     3361 cgatgatttt tgatctatta acagatatat aaatgcaaaa actgcataac cactttaact
     3421 aatactttca acattttcgg tttgtattac ttcttattca aatgtaataa aagtatcaac
     3481 aaaaaattgt taatatacct ctatacttta acgtcaagga gaaaaaaccc atggacaaga
     3541 agtactccat tgggctcgct atcggcacaa acagcgtcgg ttgggccgtc attacggacg
     3601 agtacaaggt gccgagcaaa aaattcaaag ttctgggcaa taccgatcgc cacagcataa
     3661 agaagaacct cattggcgcc ctcctgttcg actccgggga gacggccgaa gccacgcggc
     3721 tcaaaagaac agcacggcgc agatataccc gcagaaagaa tcggatctgc tacctgcagg
     3781 agatctttag taatgagatg gctaaggtgg atgactcttt cttccatagg ctggaggagt
     3841 cctttttggt ggaggaggat aaaaagcacg agcgccaccc aatctttggc aatatcgtgg
     3901 acgaggtggc gtaccatgaa aagtacccaa ccatatatca tctgaggaag aagcttgtag
     3961 acagtactga taaggctgac ttgcggttga tctatctcgc gctggcgcat atgatcaaat
     4021 ttcggggaca cttcctcatc gagggggacc tgaacccaga caacagcgat gtcgacaaac
     4081 tctttatcca actggttcag acttacaatc agcttttcga agagaacccg atcaacgcat
     4141 ccggagttga cgccaaagca atcctgagcg ctaggctgtc caaatcccgg cggctcgaaa
     4201 acctcatcgc acagctccct ggggagaaga agaacggcct gtttggtaat cttatcgccc
     4261 tgtcactcgg gctgaccccc aactttaaat ctaacttcga cctggccgaa gatgccaagc
     4321 ttcaactgag caaagacacc tacgatgatg atctcgacaa tctgctggcc cagatcggcg
     4381 accagtacgc agaccttttt ttggcggcaa agaacctgtc agacgccatt ctgctgagtg
     4441 atattctgcg agtgaacacg gagatcacca aagctccgct gagcgctagt atgatcaagc
     4501 gctatgatga gcaccaccaa gacttgactt tgctgaaggc ccttgtcaga cagcaactgc
     4561 ctgagaagta caaggaaatt ttcttcgatc agtctaaaaa tggctacgcc ggatacattg
     4621 acggcggagc aagccaggag gaattttaca aatttattaa gcccatcttg gaaaaaatgg
     4681 acggcaccga ggagctgctg gtaaagctta acagagaaga tctgttgcgc aaacagcgca
     4741 ctttcgacaa tggaagcatc ccccaccaga ttcacctggg cgaactgcac gctatcctca
     4801 ggcggcaaga ggatttctac ccctttttga aagataacag ggaaaagatt gagaaaatcc
     4861 tcacatttcg gataccctac tatgtaggcc ccctcgcccg gggaaattcc agattcgcgt
     4921 ggatgactcg caaatcagaa gagaccatca ctccctggaa cttcgaggaa gtcgtggata
     4981 agggggcctc tgcccagtcc ttcatcgaaa ggatgactaa ctttgataaa aatctgccta
     5041 acgaaaaggt gcttcctaaa cactctctgc tgtacgagta cttcacagtt tataacgagc
     5101 tcaccaaggt caaatacgtc acagaaggga tgagaaagcc agcattcctg tctggagagc
     5161 agaagaaagc tatcgtggac ctcctcttca agacgaaccg gaaagttacc gtgaaacagc
     5221 tcaaagaaga ctatttcaaa aagattgaat gtttcgactc tgttgaaatc agcggagtgg
     5281 aggatcgctt caacgcatcc ctgggaacgt atcacgatct cctgaaaatc attaaagaca
     5341 aggacttcct ggacaatgag gagaacgagg acattcttga ggacattgtc ctcaccctta
     5401 cgttgtttga agatagggag atgattgaag aacgcttgaa aacttacgct catctcttcg
     5461 acgacaaagt catgaaacag ctcaagaggc gccgatatac aggatggggg cggctgtcaa
     5521 gaaaactgat caatgggatc cgagacaagc agagtggaaa gacaatcctg gattttctta
     5581 agtccgatgg atttgccaac cggaacttca tgcagttgat ccatgatgac tctctcacct
     5641 ttaaggagga catccagaaa gcacaagttt ctggccaggg ggacagtctt cacgagcaca
     5701 tcgctaatct tgcaggtagc ccagctatca aaaagggaat actgcagacc gttaaggtcg
     5761 tggatgaact cgtcaaagta atgggaaggc ataagcccga gaatatcgtt atcgagatgg
     5821 cccgagagaa ccaaactacc cagaagggac agaagaacag tagggaaagg atgaagagga
     5881 ttgaagaggg tataaaagaa ctggggtccc aaatccttaa ggaacaccca gttgaaaaca
     5941 cccagcttca gaatgagaag ctctacctgt actacctgca gaacggcagg gacatgtacg
     6001 tggatcagga actggacatc aatcggctct ccgactacga cgtggatcat atcgtgcccc
     6061 agtcttttct caaagatgat tctattgata ataaagtgtt gacaagatcc gataaaaata
     6121 gagggaagag tgataacgtc ccctcagaag aagttgtcaa gaaaatgaaa aattattggc
     6181 ggcagctgct gaacgccaaa ctgatcacac aacggaagtt cgataatctg actaaggctg
     6241 aacgaggtgg cctgtctgag ttggataaag ccggcttcat caaaaggcag cttgttgaga
     6301 cacgccagat caccaagcac gtggcccaaa ttctcgattc acgcatgaac accaagtacg
     6361 atgaaaatga caaactgatt cgagaggtga aagttattac tctgaagtct aagctggtct
     6421 cagatttcag aaaggacttt cagttttata aggtgagaga gatcaacaat taccaccatg
     6481 cgcatgatgc ctacctgaat gcagtggtag gcactgcact tatcaaaaaa tatcccaagc
     6541 ttgaatctga atttgtttac ggagactata aagtgtacga tgttaggaaa atgatcgcaa
     6601 agtctgagca ggaaataggc aaggccaccg ctaagtactt cttttacagc aatattatga
     6661 attttttcaa gaccgagatt acactggcca atggagagat tcggaagcga ccacttatcg
     6721 aaacaaacgg agaaacagga gaaatcgtgt gggacaaggg tagggatttc gcgacagtcc
     6781 ggaaggtcct gtccatgccg caggtgaaca tcgttaaaaa gaccgaagta cagaccggag
     6841 gcttctccaa ggaaagtatc ctcccgaaaa ggaacagcga caagctgatc gcacgcaaaa
     6901 aagattggga ccccaagaaa tacggcggat tcgattctcc tacagtcgct tacagtgtac
     6961 tggttgtggc caaagtggag aaagggaagt ctaaaaaact caaaagcgtc aaggaactgc
     7021 tgggcatcac aatcatggag cgatcaagct tcgaaaaaaa ccccatcgac tttctcgagg
     7081 cgaaaggata taaagaggtc aaaaaagacc tcatcattaa gcttcccaag tactctctct
     7141 ttgagcttga aaacggccgg aaacgaatgc tcgctagtgc gggcgagctg cagaaaggta
     7201 acgagctggc actgccctct aaatacgtta atttcttgta tctggccagc cactatgaaa
     7261 agctcaaagg gtctcccgaa gataatgagc agaagcagct gttcgtggaa caacacaaac
     7321 actaccttga tgagatcatc gagcaaataa gcgaattctc caaaagagtg atcctcgccg
     7381 acgctaacct cgataaggtg ctttctgctt acaataagca cagggataag cccatcaggg
     7441 agcaggcaga aaacattatc cacttgttta ctctgaccaa cttgggcgcg cctgcagcct
     7501 tcaagtactt cgacaccacc atagacagaa agcggtacac ctctacaaag gaggtcctgg
     7561 acgccacact gattcatcag tcaattacgg ggctctatga aacaagaatc gacctctctc
     7621 agctcggtgg agacagcagg gctgacccca agaagaagag gaaggtgtga tctcttctcg
     7681 agtcatgtaa ttagttatgt cacgcttaca ttcacgccct ccccccacat ccgctctaac
     7741 cgaaaaggaa ggagttagac aacctgaagt ctaggtccct atttattttt ttatagttat
     7801 gttagtatta agaacgttat ttatatttca aatttttctt ttttttctgt acagacgcgt
     7861 gtacgcatgt aacattatac tgaaaacctt gcttgagaag gttttgggac gctcgaaggc
     7921 tttaatttgc ggccggtacc caattcgccc tatagtgagt cgtattacgc gcgctcactg
     7981 gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt
     8041 gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct
     8101 tcccaacagt tgcgcagcct gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta
     8161 agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg
     8221 cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa
     8281 gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc
     8341 aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
     8401 cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
     8461 acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc
     8521 tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta
     8581 acgtttacaa tttcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     8641 gcatatcgac ggtcgaggag aacttctagt atatccacat acctaatatt attgccttat
     8701 taaaaatgga atcccaacaa ttacatcaaa atccacattc tcttcaaaat caattgtcct
     8761 gtacttcctt gttcatgtgt gttcaaaaac gttatattta taggataatt atactctatt
     8821 tctcaacaag taattggttg tttggccgag cggtctaagg cgcctgattc aagaaatatc
     8881 ttgaccgcag ttaactgtgg gaatactcag gtatcgtaag atgcaagagt tcgaatctct
     8941 tagcaaccat tatttttttc ctcaacataa cgagaacaca caggggcgct atcgcacaga
     9001 atcaaattcg atgactggaa attttttgtt aatttcagag gtcgcctgac gcatatacct
     9061 ttttcaactg aaaaattggg agaaaaagga aaggtgagag gccggaaccg gcttttcata
     9121 tagaatagag aagcgttcat gactaaatgc ttgcatcaca atacttgaag ttgacaatat
     9181 tatttaagga cctattgttt tttccaatag gtggttagca atcgtcttac tttctaactt
     9241 ttcttacctt ttacatttca gcaatatata tatatatttc aaggatatac cattctaatg
     9301 tctgccccta tgtctgcccc taagaagatc gtcgttttgc caggtgacca cgttggtcaa
     9361 gaaatcacag ccgaagccat taaggttctt aaagctattt ctgatgttcg ttccaatgtc
     9421 aagttcgatt tcgaaaatca tttaattggt ggtgctgcta tcgatgctac aggtgtccca
     9481 cttccagatg aggcgctgga agcctccaag aaggttgatg ccgttttgtt aggtgctgtg
     9541 ggtggtccta aatggggtac cggtagtgtt agacctgaac aaggtttact aaaaatccgt
     9601 aaagaacttc aattgtacgc caacttaaga ccatgtaact ttgcatccga ctctctttta
     9661 gacttatctc caatcaagcc acaatttgct aaaggtactg acttcgttgt tgtcagagaa
     9721 ttagtgggag gtatttactt tggtaagaga aaggaagacg atggtgatgg tgtcgcttgg
     9781 gatagtgaac aatacaccgt tccagaagtg caaagaatca caagaatggc cgctttcatg
     9841 gccctacaac atgagccacc attgcctatt tggtccttgg ataaagctaa tgttttggcc
     9901 tcttcaagat tatggagaaa aactgtggag gaaaccatca agaacgaatt ccctacattg
     9961 aaggttcaac atcaattgat tgattctgcc gccatgatcc tagttaagaa cccaacccac
    10021 ctaaatggta ttataatcac cagcaacatg tttggtgata tcatctccga tgaagcctcc
    10081 gttatcccag gttccttggg tttgttgcca tctgcgtcct tggcctcttt gccagacaag
    10141 aacaccgcat ttggtttgta cgaaccatgc cacggttctg ctccagattt gccaaagaat
    10201 aaggtcaacc ctatcgccac tatcttgtct gctgcaatga tgttgaaatt gtcattgaac
    10261 ttgcctgaag aaggtaaggc cattgaagat gcagttaaaa aggttttgga tgcaggtatc
    10321 agaactggtg atttaggtgg ttccaacagt accaccgaag tcggtgatgc tgtcgccgaa
    10381 gaagttaaga aaatccttgc ttaaaaagat tctctttttt tatgatattt gtacataaac
    10441 tttataaatg aaattcataa tagaaacgac acgaaattac aaaatggaat atgttcatag
    10501 ggtagacgaa actatatacg caatctacat acatttatca agaaggagaa aaaggaggat
    10561 gtaaaggaat acaggtaagc aaattgatac taatggctca acgtgataag gaaaaagaat
    10621 tgcactttaa cattaatatt gacaaggagg agggcaccac acaaaaagtt aggtgtaaca
    10681 gaaaatcatg aaactatgat tcctaattta tatattggag gattttctct aaaaaaaaaa
    10741 aaatacaaca aataaaaaac actcaatgac ctgaccattt gatggagttt aagtcaatac
    10801 cttcttgaac catttcccat aatggtgaaa gttccctcaa gaattttact ctgtcagaaa
    10861 cggccttaac gacgtagtcg atatggtgca ctctcagtac aatctgctct gatgccgcat
    10921 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
    10981 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
    11041 tttcaccgtc atcaccgaaa cgcgcga
//
