pdCas9-DNMT3A-EGFP (ANV)

LOCUS       pdCas9-DNMT3A-EGFP (ANV)       10265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of mutant human codon-optimized dCas9-DNMT3A fusion 
            (inactive catalytic domain of DNMT3A) with T2A-EGFP; cloning 
            backbone for sgRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10265)
  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 10265)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.biofeng.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res. 2016 Mar 11. pii: gkw159."
COMMENT     Sequence Label: pdCas9-DNMT3A-EGFP (ANV)
FEATURES             Location/Qualifiers
     source          1..10265
                     /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..1232
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1244..1253
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1253..1318
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1325..1345
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1370..1393,1394..1396,1397..3883,3884..3886,
                     3887..5470)
                     /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             1370..5470
                     /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             5471..5518
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             5546..6478
                     /codon_start=1
                     /product="catalytic domain of DNA methyltransferase 3A"
                     /label=DNMT3A catalytic domain
                     /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG
                     LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI
                     GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFANVVAMGVS
                     DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR
                     IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR
                     LARQRLLGRSWSVPVIRHLFAPLKEYFACV"
     CDS             6506..6559
                     /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             join(6560..6562,6563..7273)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6560..6562,6563..7273)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(6863..6882)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7210..7231
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(7306..7323)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7312..7519
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   7528..7668
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   7528..7657
                     /label=AAV2 ITR
     rep_origin      7743..8198
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7830..7849)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     8040..8061
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(8215..8234)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8334..8356
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8394..8412)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8480..8584
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(8585..8653,8654..9445)
                     /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(8803..8822)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      9616..10204
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     10105..10124
                     /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 cgcgccgggg gggggggggg gggggggggg gsggggsggg gsgrggcgga
      901 gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa gtttcctttt atggcgaggc
      961 ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg ggcgggagtc gctgcgcgct
     1021 gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg cccgccccgg ctctgactga
     1081 ccgcgttact cccacaggtg agcgggcggg acggcccttc tcctccgggc tgtaattagc
     1141 tgagcaagag gtaagggttt aagggatggt tggttggtgg ggtattaatg tttaattacc
     1201 tggagcacct gcctgaaatc actttttttc aggttggacc ggtgccacca tggactataa
     1261 ggaccacgac ggagactaca aggatcatga tattgattac aaagacgatg acgataagat
     1321 ggccccaaag aagaagcgga aggtcggtat ccacggagtc ccagcagccg acaagaagta
     1381 cagcatcggc ctggccatcg gcaccaactc tgtgggctgg gccgtgatca ccgacgagta
     1441 caaggtgccc agcaagaaat tcaaggtgct gggcaacacc gaccggcaca gcatcaagaa
     1501 gaacctgatc ggagccctgc tgttcgacag cggcgaaaca gccgaggcca cccggctgaa
     1561 gagaaccgcc agaagaagat acaccagacg gaagaaccgg atctgctatc tgcaagagat
     1621 cttcagcaac gagatggcca aggtggacga cagcttcttc cacagactgg aagagtcctt
     1681 cctggtggaa gaggataaga agcacgagcg gcaccccatc ttcggcaaca tcgtggacga
     1741 ggtggcctac cacgagaagt accccaccat ctaccacctg agaaagaaac tggtggacag
     1801 caccgacaag gccgacctgc ggctgatcta tctggccctg gcccacatga tcaagttccg
     1861 gggccacttc ctgatcgagg gcgacctgaa ccccgacaac agcgacgtgg acaagctgtt
     1921 catccagctg gtgcagacct acaaccagct gttcgaggaa aaccccatca acgccagcgg
     1981 cgtggacgcc aaggccatcc tgtctgccag actgagcaag agcagacggc tggaaaatct
     2041 gatcgcccag ctgcccggcg agaagaagaa tggcctgttc ggcaacctga ttgccctgag
     2101 cctgggcctg acccccaact tcaagagcaa cttcgacctg gccgaggatg ccaaactgca
     2161 gctgagcaag gacacctacg acgacgacct ggacaacctg ctggcccaga tcggcgacca
     2221 gtacgccgac ctgtttctgg ccgccaagaa cctgtccgac gccatcctgc tgagcgacat
     2281 cctgagagtg aacaccgaga tcaccaaggc ccccctgagc gcctctatga tcaagagata
     2341 cgacgagcac caccaggacc tgaccctgct gaaagctctc gtgcggcagc agctgcctga
     2401 gaagtacaaa gagattttct tcgaccagag caagaacggc tacgccggct acattgacgg
     2461 cggagccagc caggaagagt tctacaagtt catcaagccc atcctggaaa agatggacgg
     2521 caccgaggaa ctgctcgtga agctgaacag agaggacctg ctgcggaagc agcggacctt
     2581 cgacaacggc agcatccccc accagatcca cctgggagag ctgcacgcca ttctgcggcg
     2641 gcaggaagat ttttacccat tcctgaagga caaccgggaa aagatcgaga agatcctgac
     2701 cttccgcatc ccctactacg tgggccctct ggccagggga aacagcagat tcgcctggat
     2761 gaccagaaag agcgaggaaa ccatcacccc ctggaacttc gaggaagtgg tggacaaggg
     2821 cgcttccgcc cagagcttca tcgagcggat gaccaacttc gataagaacc tgcccaacga
     2881 gaaggtgctg cccaagcaca gcctgctgta cgagtacttc accgtgtata acgagctgac
     2941 caaagtgaaa tacgtgaccg agggaatgag aaagcccgcc ttcctgagcg gcgagcagaa
     3001 aaaggccatc gtggacctgc tgttcaagac caaccggaaa gtgaccgtga agcagctgaa
     3061 agaggactac ttcaagaaaa tcgagtgctt cgactccgtg gaaatctccg gcgtggaaga
     3121 tcggttcaac gcctccctgg gcacatacca cgatctgctg aaaattatca aggacaagga
     3181 cttcctggac aatgaggaaa acgaggacat tctggaagat atcgtgctga ccctgacact
     3241 gtttgaggac agagagatga tcgaggaacg gctgaaaacc tatgcccacc tgttcgacga
     3301 caaagtgatg aagcagctga agcggcggag atacaccggc tggggcaggc tgagccggaa
     3361 gctgatcaac ggcatccggg acaagcagtc cggcaagaca atcctggatt tcctgaagtc
     3421 cgacggcttc gccaacagaa acttcatgca gctgatccac gacgacagcc tgacctttaa
     3481 agaggacatc cagaaagccc aggtgtccgg ccagggcgat agcctgcacg agcacattgc
     3541 caatctggcc ggcagccccg ccattaagaa gggcatcctg cagacagtga aggtggtgga
     3601 cgagctcgtg aaagtgatgg gccggcacaa gcccgagaac atcgtgatcg aaatggccag
     3661 agagaaccag accacccaga agggacagaa gaacagccgc gagagaatga agcggatcga
     3721 agagggcatc aaagagctgg gcagccagat cctgaaagaa caccccgtgg aaaacaccca
     3781 gctgcagaac gagaagctgt acctgtacta cctgcagaat gggcgggata tgtacgtgga
     3841 ccaggaactg gacatcaacc ggctgtccga ctacgatgtg gacgctatcg tgcctcagag
     3901 ctttctgaag gacgactcca tcgacaacaa ggtgctgacc agaagcgaca agaaccgggg
     3961 caagagcgac aacgtgccct ccgaagaggt cgtgaagaag atgaagaact actggcggca
     4021 gctgctgaac gccaagctga ttacccagag aaagttcgac aatctgacca aggccgagag
     4081 aggcggcctg agcgaactgg ataaggccgg cttcatcaag agacagctgg tggaaacccg
     4141 gcagatcaca aagcacgtgg cacagatcct ggactcccgg atgaacacta agtacgacga
     4201 gaatgacaag ctgatccggg aagtgaaagt gatcaccctg aagtccaagc tggtgtccga
     4261 tttccggaag gatttccagt tttacaaagt gcgcgagatc aacaactacc accacgccca
     4321 cgacgcctac ctgaacgccg tcgtgggaac cgccctgatc aaaaagtacc ctaagctgga
     4381 aagcgagttc gtgtacggcg actacaaggt gtacgacgtg cggaagatga tcgccaagag
     4441 cgagcaggaa atcggcaagg ctaccgccaa gtacttcttc tacagcaaca tcatgaactt
     4501 tttcaagacc gagattaccc tggccaacgg cgagatccgg aagcggcctc tgatcgagac
     4561 aaacggcgaa accggggaga tcgtgtggga taagggccgg gattttgcca ccgtgcggaa
     4621 agtgctgagc atgccccaag tgaatatcgt gaaaaagacc gaggtgcaga caggcggctt
     4681 cagcaaagag tctatcctgc ccaagaggaa cagcgataag ctgatcgcca gaaagaagga
     4741 ctgggaccct aagaagtacg gcggcttcga cagccccacc gtggcctatt ctgtgctggt
     4801 ggtggccaaa gtggaaaagg gcaagtccaa gaaactgaag agtgtgaaag agctgctggg
     4861 gatcaccatc atggaaagaa gcagcttcga gaagaatccc atcgactttc tggaagccaa
     4921 gggctacaaa gaagtgaaaa aggacctgat catcaagctg cctaagtact ccctgttcga
     4981 gctggaaaac ggccggaaga gaatgctggc ctctgccggc gaactgcaga agggaaacga
     5041 actggccctg ccctccaaat atgtgaactt cctgtacctg gccagccact atgagaagct
     5101 gaagggctcc cccgaggata atgagcagaa acagctgttt gtggaacagc acaagcacta
     5161 cctggacgag atcatcgagc agatcagcga gttctccaag agagtgatcc tggccgacgc
     5221 taatctggac aaagtgctgt ccgcctacaa caagcaccgg gataagccca tcagagagca
     5281 ggccgagaat atcatccacc tgtttaccct gaccaatctg ggagcccctg ccgccttcaa
     5341 gtactttgac accaccatcg accggaagag gtacaccagc accaaagagg tgctggacgc
     5401 caccctgatc caccagagca tcaccggcct gtacgagaca cggatcgacc tgtctcagct
     5461 gggaggcgac aaaaggccgg cggccacgaa aaaggccgga caggccaaaa agaaaaagct
     5521 cgagggcgga ggcgggagcg gatccccctc ccggctccag atgttcttcg ctaataacca
     5581 cgaccaggaa tttgaccctc caaaggttta cccacctgtc ccagctgaga agaggaagcc
     5641 catccgggtg ctgtctctct ttgatggaat cgctacaggg ctcctggtgc tgaaggactt
     5701 gggcattcag gtggaccgct acattgcctc ggaggtgtgt gaggactcca tcacggtggg
     5761 catggtgcgg caccagggga agatcatgta cgtcggggac gtccgcagcg tcacacagaa
     5821 gcatatccag gagtggggcc cattcgatct ggtgattggg ggcagtccct gcaatgacct
     5881 ctccatcgtc aaccctgctc gcaagggcct ctacgagggc actggccggc tcttctttga
     5941 gttctaccgc ctcctgcatg atgcgcggcc caaggaggga gatgatcgcc ccttcttctg
     6001 gctctttgcg aatgtggtgg ccatgggcgt tagtgacaag agggacatct cgcgatttct
     6061 cgagtccaac cctgtgatga ttgatgccaa agaagtgtca gctgcacaca gggcccgcta
     6121 cttctggggt aaccttcccg gtatgaacag gccgttggca tccactgtga atgataagct
     6181 ggagctgcag gagtgtctgg agcatggcag gatagccaag ttcagcaaag tgaggaccat
     6241 tactacgagg tcaaactcca taaagcaggg caaagaccag cattttcctg tgttcatgaa
     6301 tgagaaagag gacatcttat ggtgcactga aatggaaagg gtatttggtt tcccagtcca
     6361 ctatactgac gtctccaaca tgagccgctt ggcgaggcag agactgctgg gccggtcatg
     6421 gagcgtgcca gtcatccgcc acctcttcgc tccgctgaag gagtattttg cgtgtgtgtc
     6481 cggccggccc gaattcggca gtggagaggg cagaggaagt ctgctaacat gcggtgacgt
     6541 cgaggagaat cctggcccag tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
     6601 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
     6661 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     6721 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
     6781 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     6841 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     6901 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     6961 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     7021 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     7081 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     7141 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     7201 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     7261 cgagctgtac aaggaattca attctaacta gagctcgctg atcagcctcg actgtgcctt
     7321 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     7381 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     7441 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaga
     7501 atagcaggca tgctggggag cggccgcagg aacccctagt gatggagttg gccactccct
     7561 ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct
     7621 ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc
     7681 ggtattttct ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata
     7741 gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac
     7801 cgctacactt gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc
     7861 cacgttcgcc ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt
     7921 tagtgcttta cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg
     7981 gccatcgccc tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag
     8041 tggactcttg ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt
     8101 ataagggatt ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt
     8161 taacgcgaat tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat
     8221 ctgctctgat gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc
     8281 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     8341 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt
     8401 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     8461 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     8521 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     8581 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     8641 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     8701 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     8761 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     8821 atcccgtatt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     8881 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     8941 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     9001 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     9061 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     9121 gatgcctgta gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     9181 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     9241 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     9301 aagccgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     9361 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     9421 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     9481 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     9541 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     9601 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     9661 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     9721 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     9781 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     9841 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     9901 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     9961 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
    10021 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
    10081 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
    10141 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
    10201 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
    10261 catgt
//