LOCUS       sequence-154115-       12029 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses nCas9(D10A)-PmCDA1 in yeast cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12029)
  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 12029)
  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: pRS315e_pGal-nCas9(D10A)-PmCDA1
FEATURES             Location/Qualifiers
     source          1..12029
                     /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
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFFHNMGDHVTRLDRW
                     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     2488..2505
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2622..2643
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     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"
     promoter        2757..2775
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        3092..3533
                     /gene="S. cerevisiae GAL1"
                     /label=GAL1 promoter
                     /note="inducible promoter, regulated by Gal4"
     protein_bind    3092..3209
                     /label=UAS
                     /bound_moiety="Gal4"
                     /note="upstream activating sequence mediating 
                     Gal4-dependent induction"
     primer_bind     3504..3527
                     /label=GAL1
                     /note="S. cerevisiae GAL1 promoter, forward primer"
     CDS             join(3543..3569,3570..3572,3573..7646)
                     /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             3543..7646
                     /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             7659..7679
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             7887..7952
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             7959..8582
                     /codon_start=1
                     /product="cytidine deaminase from the sea lamprey 
                     Petromyzon marinus (Rogozin et al., 2007)"
                     /label=PmCDA1
                     /translation="MTDAEYVRIHEKLDIYTFKKQFFNNKKSVSHRCYVLFELKRRGER
                     RACFWGYAVNKPQSGTERGIHAEIFSIRKVEEYLRDNPGQFTINWYSSWSPCADCAEKI
                     LEWYNQELRGNGHTLKIWACKLYYEKNARNQIGLWNLRDNGVGLNVMVSEHYQCCRKIF
                     IQSSHNQLNENRWLEKTLKRAEKRRSELSIMIQVKILHTTKSPAV"
     terminator      8613..8800
                     /gene="S. cerevisiae ADH1"
                     /label=ADH1 terminator
                     /note="transcription terminator for the S. cerevisiae 
                     alcohol dehydrogenase 1 (ADH1) gene"
     primer_bind     complement(8668..8692)
                     /label=pHybLex-R
                     /note="ADH terminator, reverse primer"
     terminator      complement(8837..8882)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     complement(8918..8937)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(8919..8937)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(8944..8961)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8944..8960)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8953..8975)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      9101..9556
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(9188..9207)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     9398..9419
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        9856..10260
                     /gene="S. cerevisiae LEU2"
                     /label=LEU2 promoter
     CDS             10273..11367
                     /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
                     GLYEPCHGSAPDLPKNKVDPIATILSAAMMLKLSLNLPEEGKAIEDAVKKVLDAGIRTG
                     DLGGSNSTTEVGDAVAEEVKKILA"
     primer_bind     complement(11860..11879)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11979..12001
                     /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 ttttcacaac 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 ggcagtcagg 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 cgtgagcatt gagaaagcgc cacgcttccc
     2161 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2221 agggagcttc caggggggaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2281 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc cgagcctatg 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 ttagctcact
     2641 cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg
     2701 agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa gctcgaaatt
     2761 aaccctcact aaagggaaca aaagctggta ccgggcccga attctctcct tctcttaggt
     2821 ggcagagcag gtggagggtc gaccatacta gtttcaaaaa ttcttacttt ttttttggat
     2881 ggacgcaaag aagtttaata atcatattac atggcattac caccatatac atatccatat
     2941 acatatccat atacatatcc atatacatat ccatatctaa tcttacttat atgttgtgga
     3001 aatgtaaaga gccccattat cttagcctaa aaaaaccttc tctttggaac tttcagtaat
     3061 acgcttaact gctcattgct atattgaagt acggattaga agccgccgag cgggtgacag
     3121 ccctccgaag gaagactctc ctccgtgcgt cctcgtcttc accggtcgcg ttcctgaaac
     3181 gcagatgtgc ctcgcgccgc actgctccga acaataaaga ttctacaata ctagctttta
     3241 tggttatgaa gaggaaaaat tggcagtaac ctggccccac aaaccttcaa atgaacgaat
     3301 caaattaaca accataggat gataatgcga ttagtttttt agccttattt ctggggtaat
     3361 taatcagcga agcgatgatt tttgatctat taacagatat ataaatgcaa aaactgcata
     3421 accactttaa ctaatacttt caacattttc ggtttgtatt acttcttatt caaatgtaat
     3481 aaaagtatca acaaaaaatt gttaatatac ctctatactt taacgtcaag gagaaaaaac
     3541 ccatggacaa gaagtactcc attgggctcg ctatcggcac aaacagcgtc ggttgggccg
     3601 tcattacgga cgagtacaag gtgccgagca aaaaattcaa agttctgggc aataccgatc
     3661 gccacagcat aaagaagaac ctcattggcg ccctcctgtt cgactccggg gagacggccg
     3721 aagccacgcg gctcaaaaga acagcacggc gcagatatac ccgcagaaag aatcggatct
     3781 gctacctgca ggagatcttt agtaatgaga tggctaaggt ggatgactct ttcttccata
     3841 ggctggagga gtcctttttg gtggaggagg ataaaaagca cgagcgccac ccaatctttg
     3901 gcaatatcgt ggacgaggtg gcgtaccatg aaaagtaccc aaccatatat catctgagga
     3961 agaagcttgt agacagtact gataaggctg acttgcggtt gatctatctc gcgctggcgc
     4021 atatgatcaa atttcgggga cacttcctca tcgaggggga cctgaaccca gacaacagcg
     4081 atgtcgacaa actctttatc caactggttc agacttacaa tcagcttttc gaagagaacc
     4141 cgatcaacgc atccggagtt gacgccaaag caatcctgag cgctaggctg tccaaatccc
     4201 ggcggctcga aaacctcatc gcacagctcc ctggggagaa gaagaacggc ctgtttggta
     4261 atcttatcgc cctgtcactc gggctgaccc ccaactttaa atctaacttc gacctggccg
     4321 aagatgccaa gcttcaactg agcaaagaca cctacgatga tgatctcgac aatctgctgg
     4381 cccagatcgg cgaccagtac gcagaccttt ttttggcggc aaagaacctg tcagacgcca
     4441 ttctgctgag tgatattctg cgagtgaaca cggagatcac caaagctccg ctgagcgcta
     4501 gtatgatcaa gcgctatgat gagcaccacc aagacttgac tttgctgaag gcccttgtca
     4561 gacagcaact gcctgagaag tacaaggaaa ttttcttcga tcagtctaaa aatggctacg
     4621 ccggatacat tgacggcgga gcaagccagg aggaatttta caaatttatt aagcccatct
     4681 tggaaaaaat ggacggcacc gaggagctgc tggtaaagct taacagagaa gatctgttgc
     4741 gcaaacagcg cactttcgac aatggaagca tcccccacca gattcacctg ggcgaactgc
     4801 acgctatcct caggcggcaa gaggatttct accccttttt gaaagataac agggaaaaga
     4861 ttgagaaaat cctcacattt cggataccct actatgtagg ccccctcgcc cggggaaatt
     4921 ccagattcgc gtggatgact cgcaaatcag aagagaccat cactccctgg aacttcgagg
     4981 aagtcgtgga taagggggcc tctgcccagt ccttcatcga aaggatgact aactttgata
     5041 aaaatctgcc taacgaaaag gtgcttccta aacactctct gctgtacgag tacttcacag
     5101 tttataacga gctcaccaag gtcaaatacg tcacagaagg gatgagaaag ccagcattcc
     5161 tgtctggaga gcagaagaaa gctatcgtgg acctcctctt caagacgaac cggaaagtta
     5221 ccgtgaaaca gctcaaagaa gactatttca aaaagattga atgtttcgac tctgttgaaa
     5281 tcagcggagt ggaggatcgc ttcaacgcat ccctgggaac gtatcacgat ctcctgaaaa
     5341 tcattaaaga caaggacttc ctggacaatg aggagaacga ggacattctt gaggacattg
     5401 tcctcaccct tacgttgttt gaagataggg agatgattga agaacgcttg aaaacttacg
     5461 ctcatctctt cgacgacaaa gtcatgaaac agctcaagag gcgccgatat acaggatggg
     5521 ggcggctgtc aagaaaactg atcaatggga tccgagacaa gcagagtgga aagacaatcc
     5581 tggattttct taagtccgat ggatttgcca accggaactt catgcagttg atccatgatg
     5641 actctctcac ctttaaggag gacatccaga aagcacaagt ttctggccag ggggacagtc
     5701 ttcacgagca catcgctaat cttgcaggta gcccagctat caaaaaggga atactgcaga
     5761 ccgttaaggt cgtggatgaa ctcgtcaaag taatgggaag gcataagccc gagaatatcg
     5821 ttatcgagat ggcccgagag aaccaaacta cccagaaggg acagaagaac agtagggaaa
     5881 ggatgaagag gattgaagag ggtataaaag aactggggtc ccaaatcctt aaggaacacc
     5941 cagttgaaaa cacccagctt cagaatgaga agctctacct gtactacctg cagaacggca
     6001 gggacatgta cgtggatcag gaactggaca tcaatcggct ctccgactac gacgtggatc
     6061 atatcgtgcc ccagtctttt ctcaaagatg attctattga taataaagtg ttgacaagat
     6121 ccgataaaaa tagagggaag agtgataacg tcccctcaga agaagttgtc aagaaaatga
     6181 aaaattattg gcggcagctg ctgaacgcca aactgatcac acaacggaag ttcgataatc
     6241 tgactaaggc tgaacgaggt ggcctgtctg agttggataa agccggcttc atcaaaaggc
     6301 agcttgttga gacacgccag atcaccaagc acgtggccca aattctcgat tcacgcatga
     6361 acaccaagta cgatgaaaat gacaaactga ttcgagaggt gaaagttatt actctgaagt
     6421 ctaagctggt ctcagatttc agaaaggact ttcagtttta taaggtgaga gagatcaaca
     6481 attaccacca tgcgcatgat gcctacctga atgcagtggt aggcactgca cttatcaaaa
     6541 aatatcccaa gcttgaatct gaatttgttt acggagacta taaagtgtac gatgttagga
     6601 aaatgatcgc aaagtctgag caggaaatag gcaaggccac cgctaagtac ttcttttaca
     6661 gcaatattat gaattttttc aagaccgaga ttacactggc caatggagag attcggaagc
     6721 gaccacttat cgaaacaaac ggagaaacag gagaaatcgt gtgggacaag ggtagggatt
     6781 tcgcgacagt ccggaaggtc ctgtccatgc cgcaggtgaa catcgttaaa aagaccgaag
     6841 tacagaccgg aggcttctcc aaggaaagta tcctcccgaa aaggaacagc gacaagctga
     6901 tcgcacgcaa aaaagattgg gaccccaaga aatacggcgg attcgattct cctacagtcg
     6961 cttacagtgt actggttgtg gccaaagtgg agaaagggaa gtctaaaaaa ctcaaaagcg
     7021 tcaaggaact gctgggcatc acaatcatgg agcgatcaag cttcgaaaaa aaccccatcg
     7081 actttctcga ggcgaaagga tataaagagg tcaaaaaaga cctcatcatt aagcttccca
     7141 agtactctct ctttgagctt gaaaacggcc ggaaacgaat gctcgctagt gcgggcgagc
     7201 tgcagaaagg taacgagctg gcactgccct ctaaatacgt taatttcttg tatctggcca
     7261 gccactatga aaagctcaaa gggtctcccg aagataatga gcagaagcag ctgttcgtgg
     7321 aacaacacaa acactacctt gatgagatca tcgagcaaat aagcgaattc tccaaaagag
     7381 tgatcctcgc cgacgctaac ctcgataagg tgctttctgc ttacaataag cacagggata
     7441 agcccatcag ggagcaggca gaaaacatta tccacttgtt tactctgacc aacttgggcg
     7501 cgcctgcagc cttcaagtac ttcgacacca ccatagacag aaagcggtac acctctacaa
     7561 aggaggtcct ggacgccaca ctgattcatc agtcaattac ggggctctat gaaacaagaa
     7621 tcgacctctc tcagctcggt ggagacagca gggctgaccc caagaagaag aggaaggtgg
     7681 gtggaggagg ttctggaggt ggaggttctg cagagtatgt gcgggccctc tttgacttta
     7741 atgggaatga tgaagaagat cttcccttta agaaaggaga catcctgaga atccgggata
     7801 agcctgaaga gcagtggtgg aatgcagagg acagcgaagg aaagaggggg atgattcctg
     7861 tcccttacgt ggagaagtat tccggagact ataaggacca cgacggagac tacaaggatc
     7921 atgatattga ttacaaagac gatgacgata agtctaggat gaccgacgct gagtacgtga
     7981 gaatccatga gaagttggac atctacacgt ttaagaaaca gtttttcaac aacaaaaaat
     8041 ccgtgtcgca tagatgctac gttctctttg aattaaaacg acggggtgaa cgtagagcgt
     8101 gtttttgggg ctatgctgtg aataaaccac agagcgggac agaacgtggc attcacgccg
     8161 aaatctttag cattagaaaa gtcgaagaat acctgcgcga caaccccgga caattcacga
     8221 taaattggta ctcatcctgg agtccttgtg cagattgcgc tgaaaagatc ttagaatggt
     8281 ataaccagga gctgcggggg aacggccaca ctttgaaaat ctgggcttgc aaactctatt
     8341 acgagaaaaa tgcgaggaat caaattgggc tgtggaacct cagagataac ggggttgggt
     8401 tgaatgtaat ggtaagtgaa cactaccaat gttgcaggaa aatattcatc caatcgtcgc
     8461 acaatcaatt gaatgagaat agatggcttg agaagacttt gaagcgagct gaaaaacgac
     8521 ggagcgagtt gtccattatg attcaggtaa aaatactcca caccactaag agtcctgctg
     8581 tttctagagg ctccggataa ggatcctaat aagcgaattt cttatgattt atgattttta
     8641 ttattaaata agttataaaa aaaataagtg tatacaaatt ttaaagtgac tcttaggttt
     8701 taaaacgaaa attcttattc ttgagtaact ctttcctgta ggtcaggttg ctttctcagg
     8761 tatagcatga ggtcgctctt attgaccaca cctctaccgg catgccgagc aaatgcctgc
     8821 aaatcgctcc cgggcaaaaa accccctcaa gacccgttta gaggccccaa ggggttatgc
     8881 tatgcatacc aagcttccgc ggtggagctc caattcgccc tatagtgagt cgtattacaa
     8941 ttcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa
     9001 tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga
     9061 tcgcccttcc caacagttgc gcagcctgaa tggcgaatgg acgcgccctg tagcggcgca
     9121 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     9181 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     9241 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     9301 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     9361 tttcgccctt tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga
     9421 acaacactca accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg
     9481 gcctattggt taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata
     9541 ttaacgttta caatttcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     9601 accgcatatc gaccctcgag gagaacttct agtatatcca catacctaat attattgcct
     9661 tattaaaaat ggaatcggaa caattacatc aaaatccaca ttctcttcaa aatcaattgt
     9721 cctgtacttc cttgttcatg tgtgttcaaa aacgttatat ttataggata attatactct
     9781 atttctcaac aagtaattgg ttgtttggcc gagcggtcta aggcgcctga ttcaagaaat
     9841 atcttgaccg cagttaactg tgggaatact caggtatcgt aagatgcaag agttcgaatc
     9901 tcttagcaac cattattttt ttcctcaaca taacgagaac acacaggggc gctatcgcac
     9961 agaatcaaat tcgatgactg gaaatttttt gttaatttca gaggtcgcct gacgcatata
    10021 cctttttcaa ctgaaaaatt gggagaaaaa ggaaaggtga gaggccggaa ccggcttttc
    10081 atatagaata gagaagcgtt catgactaaa tgcttgcatc acaatacttg aagttgacaa
    10141 tattatttaa ggacctattg ttttttccaa taggtggtta gcaatcgtct tactttctaa
    10201 cttttcttac cttttacatt tcagcaatat atatatatat ttcaaggata taccattcta
    10261 atgtctgccc ctatgtctgc ccctaagaag atcgtcgttt tgccaggtga ccacgttggt
    10321 caagaaatca cagccgaagc cattaaggtt cttaaagcta tttctgatgt tcgttccaat
    10381 gtcaagttcg atttcgaaaa tcatttaatt ggtggtgctg ctatcgatgc tacaggtgtc
    10441 ccacttccag atgaggcgct ggaagcctcc aagaaggttg atgccgtttt gttaggtgct
    10501 gtgggtggtc ctaaatgggg taccggtagt gttagacctg aacaaggttt actaaaaatc
    10561 cgtaaagaac ttcaattgta cgccaactta agaccatgta actttgcatc cgactctctt
    10621 ttagacttat ctccaatcaa gccacaattt gctaaaggta ctgacttcgt tgttgtcaga
    10681 gaattagtgg gaggtattta ctttggtaag agaaaggaag acgatggtga tggtgtcgct
    10741 tgggatagtg aacaatacac cgttccagaa gtgcaaagaa tcacaagaat ggccgctttc
    10801 atggccctac aacatgagcc accattgcct atttggtcct tggataaagc taatgttttg
    10861 gcctcttcaa gattatggag aaaaactgtg gaggaaacca tcaagaacga gttccctaca
    10921 ttgaaggttc aacatcaatt gattgattct gccgccatga tcctagttaa gaacccaacc
    10981 cacctaaatg gtattataat caccagcaac atgtttggtg atatcatctc cgatgaagcc
    11041 tccgttatcc caggttcctt gggtttgttg ccatctgcgt ccttggcctc tttgccagac
    11101 aagaacaccg catttggttt gtacgaacca tgccacggtt ctgctccaga tttgccaaag
    11161 aataaggttg accctatcgc cactatcttg tctgctgcaa tgatgttgaa attgtcattg
    11221 aacttgcctg aagaaggtaa ggccattgaa gatgcagtta aaaaggtttt ggatgcaggt
    11281 atcagaactg gtgatttagg tggttccaac agtaccaccg aagtcggtga tgctgtcgcc
    11341 gaagaagtta agaaaatcct tgcttaaaaa gattctcttt ttttatgata tttgtacata
    11401 aactttataa atgaaattca taatagaaac gacacgaaat tacaaaatgg aatatgttca
    11461 tagggtagac gaaactatat acgcaatcta catacattta tcaagaagga gaaaaaggag
    11521 gatagtaaag gaatacaggt aagcaaattg atactaatgg ctcaacgtga taaggaaaaa
    11581 gaattgcact ttaacattaa tattgacaag gaggagggca ccacacaaaa agttaggtgt
    11641 aacagaaaat catgaaacta cgattcctaa tttgatattg gaggattttc tctaaaaaaa
    11701 aaaaaataca acaaataaaa aacactcaat gacctgacca tttgatggag tttaagtcaa
    11761 taccttcttg aaccatttcc cataatggtg aaagttccct caagaatttt actctgtcag
    11821 aaacggcctt acgacgtagt cgatatggtg cactctcagt acaatctgct ctgatgccgc
    11881 atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
    11941 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
    12001 gttttcaccg tcatcaccga aacgcgcga
//
