LOCUS       sequence-129541-       10152 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human codon-optimized dCas9-DNMT3A fusion with 
            T2A-PuroR for targeted DNA methylation in mammalian cells; cloning 
            backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10152)
  AUTHORS   Vojta A, Dobrinic P, Tadic V, Bockor L, Korac P, Julg B, Klasic M, 
            Zoldos V
  TITLE     Repurposing the CRISPR-Cas9 system for targeted DNA methylation.
  JOURNAL   Nucleic Acids Res. 2016 Mar 11. pii: gkw159.
  PUBMED    26969735
REFERENCE   2  (bases 1 to 10152)
  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: "Nucleic 
            Acids Res. 2016 Mar 11. pii: gkw159."
COMMENT     Sequence Label: pdCas9-DNMT3A-PuroR
FEATURES             Location/Qualifiers
     source          1..10152
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        268..343
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        440..725
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        727..1004
                     /label=chicken beta-actin promoter
     intron          1005..1233
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1245..1254
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1254..1319
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1326..1346
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1371..1394,1395..1397,1398..3884,3885..3887,
                     3888..5471)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=dCas9
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the D10A mutation in the RuvC 
                     catalytic domain and the H840A mutation in the HNH 
                     catalytic domain"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             1371..5471
                     /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="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     CDS             5472..5519
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5547..6479
                     /codon_start=1
                     /product="catalytic domain of DNA methyltransferase 3A"
                     /label=DNMT3A catalytic domain
                     /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG
                     LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI
                     GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS
                     DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR
                     IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR
                     LARQRLLGRSWSVPVIRHLFAPLKEYFACV"
     CDS             6507..6560
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             6561..7160
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(6561..6580)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     complement(7193..7210)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7199..7406
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7415..7555
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7415..7544
                     /label=AAV2 ITR
     rep_origin      7630..8085
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7717..7736)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7927..7948
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8102..8121)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8221..8243
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8281..8299)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8367..8471
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8472..8540,8541..9332)
                     /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(8690..8709)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9503..10091
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     9992..10011
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag
      301 gctagtccgt tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct
      361 agaaatagca agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac
      421 aaatggctct agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      481 ccaacgaccc ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc
      541 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
      601 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt
      661 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
      721 ccatggtcga ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac
      781 ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg
      841 ggggggggcg cgcgccaggc ggggcggggc ggggcgaggg gcggggcggg gcgaggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgacgc
     1021 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
     1081 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
     1141 ctgagcaaga ggtaagggtt taagggatgg ttggttggtg gggtattaat gtttaattac
     1201 ctggagcacc tgcctgaaat cacttttttt caggttggac cggtgccacc atggactata
     1261 aggaccacga cggagactac aaggatcatg atattgatta caaagacgat gacgataaga
     1321 tggccccaaa gaagaagcgg aaggtcggta tccacggagt cccagcagcc gacaagaagt
     1381 acagcatcgg cctggccatc ggcaccaact ctgtgggctg ggccgtgatc accgacgagt
     1441 acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac cgaccggcac agcatcaaga
     1501 agaacctgat cggagccctg ctgttcgaca gcggcgaaac agccgaggcc acccggctga
     1561 agagaaccgc cagaagaaga tacaccagac ggaagaaccg gatctgctat ctgcaagaga
     1621 tcttcagcaa cgagatggcc aaggtggacg acagcttctt ccacagactg gaagagtcct
     1681 tcctggtgga agaggataag aagcacgagc ggcaccccat cttcggcaac atcgtggacg
     1741 aggtggccta ccacgagaag taccccacca tctaccacct gagaaagaaa ctggtggaca
     1801 gcaccgacaa ggccgacctg cggctgatct atctggccct ggcccacatg atcaagttcc
     1861 ggggccactt cctgatcgag ggcgacctga accccgacaa cagcgacgtg gacaagctgt
     1921 tcatccagct ggtgcagacc tacaaccagc tgttcgagga aaaccccatc aacgccagcg
     1981 gcgtggacgc caaggccatc ctgtctgcca gactgagcaa gagcagacgg ctggaaaatc
     2041 tgatcgccca gctgcccggc gagaagaaga atggcctgtt cggcaacctg attgccctga
     2101 gcctgggcct gacccccaac ttcaagagca acttcgacct ggccgaggat gccaaactgc
     2161 agctgagcaa ggacacctac gacgacgacc tggacaacct gctggcccag atcggcgacc
     2221 agtacgccga cctgtttctg gccgccaaga acctgtccga cgccatcctg ctgagcgaca
     2281 tcctgagagt gaacaccgag atcaccaagg cccccctgag cgcctctatg atcaagagat
     2341 acgacgagca ccaccaggac ctgaccctgc tgaaagctct cgtgcggcag cagctgcctg
     2401 agaagtacaa agagattttc ttcgaccaga gcaagaacgg ctacgccggc tacattgacg
     2461 gcggagccag ccaggaagag ttctacaagt tcatcaagcc catcctggaa aagatggacg
     2521 gcaccgagga actgctcgtg aagctgaaca gagaggacct gctgcggaag cagcggacct
     2581 tcgacaacgg cagcatcccc caccagatcc acctgggaga gctgcacgcc attctgcggc
     2641 ggcaggaaga tttttaccca ttcctgaagg acaaccggga aaagatcgag aagatcctga
     2701 ccttccgcat cccctactac gtgggccctc tggccagggg aaacagcaga ttcgcctgga
     2761 tgaccagaaa gagcgaggaa accatcaccc cctggaactt cgaggaagtg gtggacaagg
     2821 gcgcttccgc ccagagcttc atcgagcgga tgaccaactt cgataagaac ctgcccaacg
     2881 agaaggtgct gcccaagcac agcctgctgt acgagtactt caccgtgtat aacgagctga
     2941 ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc cttcctgagc ggcgagcaga
     3001 aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa agtgaccgtg aagcagctga
     3061 aagaggacta cttcaagaaa atcgagtgct tcgactccgt ggaaatctcc ggcgtggaag
     3121 atcggttcaa cgcctccctg ggcacatacc acgatctgct gaaaattatc aaggacaagg
     3181 acttcctgga caatgaggaa aacgaggaca ttctggaaga tatcgtgctg accctgacac
     3241 tgtttgagga cagagagatg atcgaggaac ggctgaaaac ctatgcccac ctgttcgacg
     3301 acaaagtgat gaagcagctg aagcggcgga gatacaccgg ctggggcagg ctgagccgga
     3361 agctgatcaa cggcatccgg gacaagcagt ccggcaagac aatcctggat ttcctgaagt
     3421 ccgacggctt cgccaacaga aacttcatgc agctgatcca cgacgacagc ctgaccttta
     3481 aagaggacat ccagaaagcc caggtgtccg gccagggcga tagcctgcac gagcacattg
     3541 ccaatctggc cggcagcccc gccattaaga agggcatcct gcagacagtg aaggtggtgg
     3601 acgagctcgt gaaagtgatg ggccggcaca agcccgagaa catcgtgatc gaaatggcca
     3661 gagagaacca gaccacccag aagggacaga agaacagccg cgagagaatg aagcggatcg
     3721 aagagggcat caaagagctg ggcagccaga tcctgaaaga acaccccgtg gaaaacaccc
     3781 agctgcagaa cgagaagctg tacctgtact acctgcagaa tgggcgggat atgtacgtgg
     3841 accaggaact ggacatcaac cggctgtccg actacgatgt ggacgctatc gtgcctcaga
     3901 gctttctgaa ggacgactcc atcgacaaca aggtgctgac cagaagcgac aagaaccggg
     3961 gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa gatgaagaac tactggcggc
     4021 agctgctgaa cgccaagctg attacccaga gaaagttcga caatctgacc aaggccgaga
     4081 gaggcggcct gagcgaactg gataaggccg gcttcatcaa gagacagctg gtggaaaccc
     4141 ggcagatcac aaagcacgtg gcacagatcc tggactcccg gatgaacact aagtacgacg
     4201 agaatgacaa gctgatccgg gaagtgaaag tgatcaccct gaagtccaag ctggtgtccg
     4261 atttccggaa ggatttccag ttttacaaag tgcgcgagat caacaactac caccacgccc
     4321 acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat caaaaagtac cctaagctgg
     4381 aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt gcggaagatg atcgccaaga
     4441 gcgagcagga aatcggcaag gctaccgcca agtacttctt ctacagcaac atcatgaact
     4501 ttttcaagac cgagattacc ctggccaacg gcgagatccg gaagcggcct ctgatcgaga
     4561 caaacggcga aaccggggag atcgtgtggg ataagggccg ggattttgcc accgtgcgga
     4621 aagtgctgag catgccccaa gtgaatatcg tgaaaaagac cgaggtgcag acaggcggct
     4681 tcagcaaaga gtctatcctg cccaagagga acagcgataa gctgatcgcc agaaagaagg
     4741 actgggaccc taagaagtac ggcggcttcg acagccccac cgtggcctat tctgtgctgg
     4801 tggtggccaa agtggaaaag ggcaagtcca agaaactgaa gagtgtgaaa gagctgctgg
     4861 ggatcaccat catggaaaga agcagcttcg agaagaatcc catcgacttt ctggaagcca
     4921 agggctacaa agaagtgaaa aaggacctga tcatcaagct gcctaagtac tccctgttcg
     4981 agctggaaaa cggccggaag agaatgctgg cctctgccgg cgaactgcag aagggaaacg
     5041 aactggccct gccctccaaa tatgtgaact tcctgtacct ggccagccac tatgagaagc
     5101 tgaagggctc ccccgaggat aatgagcaga aacagctgtt tgtggaacag cacaagcact
     5161 acctggacga gatcatcgag cagatcagcg agttctccaa gagagtgatc ctggccgacg
     5221 ctaatctgga caaagtgctg tccgcctaca acaagcaccg ggataagccc atcagagagc
     5281 aggccgagaa tatcatccac ctgtttaccc tgaccaatct gggagcccct gccgccttca
     5341 agtactttga caccaccatc gaccggaaga ggtacaccag caccaaagag gtgctggacg
     5401 ccaccctgat ccaccagagc atcaccggcc tgtacgagac acggatcgac ctgtctcagc
     5461 tgggaggcga caaaaggccg gcggccacga aaaaggccgg acaggccaaa aagaaaaagc
     5521 tcgagggcgg aggcgggagc ggatccccct cccggctcca gatgttcttc gctaataacc
     5581 acgaccagga atttgaccct ccaaaggttt acccacctgt cccagctgag aagaggaagc
     5641 ccatccgggt gctgtctctc tttgatggaa tcgctacagg gctcctggtg ctgaaggact
     5701 tgggcattca ggtggaccgc tacattgcct cggaggtgtg tgaggactcc atcacggtgg
     5761 gcatggtgcg gcaccagggg aagatcatgt acgtcgggga cgtccgcagc gtcacacaga
     5821 agcatatcca ggagtggggc ccattcgatc tggtgattgg gggcagtccc tgcaatgacc
     5881 tctccatcgt caaccctgct cgcaagggcc tctacgaggg cactggccgg ctcttctttg
     5941 agttctaccg cctcctgcat gatgcgcggc ccaaggaggg agatgatcgc cccttcttct
     6001 ggctctttga gaatgtggtg gccatgggcg ttagtgacaa gagggacatc tcgcgatttc
     6061 tcgagtccaa ccctgtgatg attgatgcca aagaagtgtc agctgcacac agggcccgct
     6121 acttctgggg taaccttccc ggtatgaaca ggccgttggc atccactgtg aatgataagc
     6181 tggagctgca ggagtgtctg gagcatggca ggatagccaa gttcagcaaa gtgaggacca
     6241 ttactacgag gtcaaactcc ataaagcagg gcaaagacca gcattttcct gtgttcatga
     6301 atgagaaaga ggacatctta tggtgcactg aaatggaaag ggtatttggt ttcccagtcc
     6361 actatactga cgtctccaac atgagccgct tggcgaggca gagactgctg ggccggtcat
     6421 ggagcgtgcc agtcatccgc cacctcttcg ctccgctgaa ggagtatttt gcgtgtgtgt
     6481 ccggccggcc cgaattcggc agtggagagg gcagaggaag tctgctaaca tgcggtgacg
     6541 tcgaggagaa tcctggccca atgaccgagt acaagcccac ggtgcgcctc gccacccgcg
     6601 acgacgtccc cagggccgta cgcaccctcg ccgccgcgtt cgccgactac cccgccacgc
     6661 gccacaccgt cgatccggac cgccacatcg agcgggtcac cgagctgcaa gaactcttcc
     6721 tcacgcgcgt cgggctcgac atcggcaagg tgtgggtcgc ggacgacggc gccgcggtgg
     6781 cggtctggac cacgccggag agcgtcgaag cgggggcggt gttcgccgag atcggcccgc
     6841 gcatggccga gttgagcggt tcccggctgg ccgcgcagca acagatggaa ggcctcctgg
     6901 cgccgcaccg gcccaaggag cccgcgtggt tcctggccac cgtcggcgtc tcgcccgacc
     6961 accagggcaa gggtctgggc agcgccgtcg tgctccccgg agtggaggcg gccgagcgcg
     7021 ccggggtgcc cgccttcctg gagacctccg cgccccacaa cctccccttc tacgagcggc
     7081 tcggcttcac cgtcaccgcc gacgtcgagg tgcccgaagg accgcgcacc tggtgcatga
     7141 cccgcaagcc cggtgcctga gaattcaatt ctaactagag ctcgctgatc agcctcgact
     7201 gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg
     7261 gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg
     7321 agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg
     7381 gaagagaata gcaggcatgc tggggagcgg ccgcaggaac ccctagtgat ggagttggcc
     7441 actccctctc tgcgcgctcg ctcgctcact gaggccgggc gaccaaaggt cgcccgacgc
     7501 ccgggctttg cccgggcggc ctcagtgagc gagcgagcgc gcagctgcct gcaggggcgc
     7561 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat acgtcaaagc
     7621 aaccatagta cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     7681 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     7741 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     7801 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgatttgggt gatggttcac
     7861 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     7921 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg ggctattctt
     7981 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     8041 aaaaatttaa cgcgaatttt aacaaaatat taacgtttac aattttatgg tgcactctca
     8101 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
     8161 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
     8221 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg agacgaaagg
     8281 gcctcgtgat acgcctattt ttataggtta atgtcatgat aataatggtt tcttagacgt
     8341 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     8401 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     8461 aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat
     8521 tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc
     8581 agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga
     8641 gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg
     8701 cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc
     8761 agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag
     8821 taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc
     8881 tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg
     8941 taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg
     9001 acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac
     9061 ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac
     9121 cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg
     9181 agcgtggaag ccgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg
     9241 tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg
     9301 agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac
     9361 tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg
     9421 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     9481 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     9541 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     9601 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     9661 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     9721 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     9781 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     9841 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     9901 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     9961 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
    10021 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
    10081 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
    10141 ttgctcacat gt
//
