LOCUS       sequence-381333-       12955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of human spdCas9-Tet1 fusion for targeted DNA methylation
            in mammalian cell. EGFP-puromycin double marker under an independent
            CMV promoter. Cloning backbone for spgRNA (BbsI).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12955)
  AUTHORS   Pahlevan Kakhki M, Giordano A, Starvaggi Cucuzza C, Venkata S Badam 
            T, Samudyata S, Lemee MV, Stridh P, Gkogka A, Shchetynsky K, Harroud
            A, Gyllenberg A, Liu Y, Boddul S, James T, Sorosina M, Filippi M, 
            Esposito F, Wermeling F, Gustafsson M, Casaccia P, Hillert J, Olsson
            T, Kockum I, Sellgren CM, Golzio C, Kular L, Jagodic M
  TITLE     A genetic-epigenetic interplay at 1q21.1 locus underlies 
            CHD1L-mediated vulnerability to primary progressive multiple 
            sclerosis.
  JOURNAL   Nat Commun. 2024 Jul 30;15(1):6419. doi: 10.1038/s41467-024-50794-z.
  PUBMED    39079955
REFERENCE   2  (bases 1 to 12955)
  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: "Nat 
            Commun."; date: "2024-07-30"; volume: "15(1):6419. doi"; pages: " 
            10.1038/s41467-024-50794-z"
COMMENT     Sequence Label: P3-dCas9-Tet1-GFP-Puro
FEATURES             Location/Qualifiers
     source          1..12955
                     /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"
     primer_bind     complement(7735..7752)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7741..7948
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     polyA_signal    7962..8083
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7973..7992)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     8027..8046
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(8251..8850)
                     /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     8831..8850
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             complement(8851..8904)
                     /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             complement(join(8935..9645,9646..9648,9649..9651))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(8935..9645,9646..9648,9649..9651))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(8977..8998)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     9326..9345
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     regulatory      9648..9657
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(9696..9899)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(9725..9745)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        9900..10203
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     repeat_region   10218..10358
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   10218..10347
                     /label=AAV2 ITR
     rep_origin      10433..10888
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(10520..10539)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     10730..10751
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(10905..10924)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11024..11046
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(11084..11102)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        11170..11274
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11275..11343,11344..12135)
                     /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(11493..11512)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      12306..12894
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     12795..12814
                     /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 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 gatccctgcc cacctgcagc tgtcttgatc gagttataca
     5581 aaaagacaaa ggcccatatt atacacacct tggggcagga ccaagtgttg ctgctgtcag
     5641 ggaaatcatg gagaataggt atggtcaaaa aggaaacgca ataaggatag aaatagtagt
     5701 gtacaccggt aaagaaggga aaagctctca tgggtgtcca attgctaagt gggttttaag
     5761 aagaagcagt gatgaagaaa aagttctttg tttggtccgg cagcgtacag gccaccactg
     5821 tccaactgct gtgatggtgg tgctcatcat ggtgtgggat ggcatccctc ttccaatggc
     5881 cgaccggcta tacacagagc tcacagagaa tctaaagtca tacaatgggc accctaccga
     5941 cagaagatgc accctcaatg aaaatcgtac ctgtacatgt caaggaattg atccagagac
     6001 ttgtggagct tcattctctt ttggctgttc atggagtatg tactttaatg gctgtaagtt
     6061 tggtagaagc ccaagcccca gaagatttag aattgatcca agctctccct tacatgaaaa
     6121 aaaccttgaa gataacttac agagtttggc tacacgatta gctccaattt ataagcagta
     6181 tgctccagta gcttaccaaa atcaggtgga atatgaaaat gttgcccgag aatgtcggct
     6241 tggcagcaag gaaggtcgac ccttctctgg ggtcactgct tgcctggact tctgtgctca
     6301 tccccacagg gacattcaca acatgaataa tggaagcact gtggtttgta ccttaactcg
     6361 agaagataac cgctctttgg gtgttattcc tcaagatgag cagctccatg tgctacctct
     6421 ttataagctt tcagacacag atgagtttgg ctccaaggaa ggaatggaag ccaagatcaa
     6481 atctggggcc atcgaggtcc tggcaccccg ccgcaaaaaa agaacgtgtt tcactcagcc
     6541 tgttccccgt tctggaaaga agagggctgc gatgatgaca gaggttcttg cacataagat
     6601 aagggcagtg gaaaagaaac ctattccccg aatcaagcgg aagaataact caacaacaac
     6661 aaacaacagt aagccttcgt cactgccaac cttagggagt aacactgaga ccgtgcaacc
     6721 tgaagtaaaa agtgaaaccg aaccccattt tatcttaaaa agttcagaca acactaaaac
     6781 ttattcgctg atgccatccg ctcctcaccc agtgaaagag gcatctccag gcttctcctg
     6841 gtcccccaag actgcttcag ccacaccagc tccactgaag aatgacgcaa cagcctcatg
     6901 cgggttttca gaaagaagca gcactcccca ctgtacgatg ccttcgggta gactcagtgg
     6961 tgccaatgct gcagctgctg atggccctgg catttcacag cttggcgaag tggctcctct
     7021 ccccaccctg tctgctcctg tgatggagcc cctcattaat tctgagcctt ccactggtgt
     7081 gactgagccg ctaacgcctc atcagccaaa ccaccagccc tccttcctca cctctcctca
     7141 agaccttgcc tcttctccaa tggaagaaga tgagcagcat tctgaagcag atgagcctcc
     7201 atcagacgaa cccctatctg atgaccccct gtcacctgct gaggagaaat tgccccacat
     7261 tgatgagtat tggtcagaca gtgagcacat ctttttggat gcaaatattg gtggggtggc
     7321 catcgcacct gctcacggct cggttttgat tgagtgtgcc cggcgagagc tgcacgctac
     7381 cactcctgtt gagcacccca accgtaatca tccaacccgc ctctcccttg tcttttacca
     7441 gcacaaaaac ctaaataagc cccaacatgg ttttgaacta aacaagatta agtttgaggc
     7501 taaagaagct aagaataaga aaatgaaggc ctcagagcaa aaagaccagg cagctaatga
     7561 aggtccagaa cagtcctctg aagtaaatga attgaaccaa attccttctc ataaagcatt
     7621 aacattaacc catgacaatg ttgtcaccgt gtccccttat gctctcacac acgttgcggg
     7681 gccctataac cattgggtct gagaattcaa ttctaactag agctcgctga tcagcctcga
     7741 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc
     7801 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc
     7861 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt
     7921 gggaagagaa tagcaggcat gctggggagc ggccgccgcg ttaagataca ttgatgagtt
     7981 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     8041 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     8101 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     8161 ctacaaatgt ggtatggctg attatgatca gttatctaga tccggtggat cccgggcccg
     8221 cggtaccgtc gactgcagaa ttcgaagctt tcaggcaccg ggcttgcggg tcatgcacca
     8281 ggtgcgcggt ccttcgggca cctcgacgtc ggcggtgacg gtgaagccga gccgctcgta
     8341 gaaggggagg ttgtggggcg cggaggtctc caggaaggcg ggcaccccgg cgcgctcggc
     8401 cgcctccact ccggggagca cgacggcgct gcccagaccc ttgccctggt ggtcgggcga
     8461 gacgccgacg gtggccagga accacgcggg ctccttgggc cggtgcggcg ccaggaggcc
     8521 ttccatctgt tgctgcgcgg ccagccggga accgctcaac tcggccatgc gcgggccgat
     8581 ctcggcgaac accgcccccg cttcgacgct ctccggcgtg gtccagaccg ccaccgcggc
     8641 gccgtcgtcc gcgacccaca ccttgccgat gtcgagcccg acgcgcgtga ggaagagttc
     8701 ttgcagctcg gtgacccgct cgatgtggcg gtccggatcg acggtgtggc gcgtggcggg
     8761 gtagtcggcg aacgcggcgg cgagggtgcg tacggccctg gggacgtcgt cgcgggtggc
     8821 gaggcgcacc gtgggcttgt actcggtcat tgggccagga ttctcctcga cgtcaccgca
     8881 tgttagcaga cttcctctgc cctcagaagc ttgagctcga gatctgagtc cggacttgta
     8941 cagctcgtcc atgccgagag tgatcccggc ggcggtcacg aactccagca ggaccatgtg
     9001 atcgcgcttc tcgttggggt ctttgctcag ggcggactgg gtgctcaggt agtggttgtc
     9061 gggcagcagc acggggccgt cgccgatggg ggtgttctgc tggtagtggt cggcgagctg
     9121 cacgctgccg tcctcgatgt tgtggcggat cttgaagttc accttgatgc cgttcttctg
     9181 cttgtcggcc atgatataga cgttgtggct gttgtagttg tactccagct tgtgccccag
     9241 gatgttgccg tcctccttga agtcgatgcc cttcagctcg atgcggttca ccagggtgtc
     9301 gccctcgaac ttcacctcgg cgcgggtctt gtagttgccg tcgtccttga agaagatggt
     9361 gcgctcctgg acgtagcctt cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg
     9421 gtcggggtag cggctgaagc actgcacgcc gtaggtcagg gtggtcacga gggtgggcca
     9481 gggcacgggc agcttgccgg tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc
     9541 atcgccctcg ccctcgccgg acacgctgaa cttgtggccg tttacgtcgc cgtccagctc
     9601 gaccaggatg ggcaccaccc cggtgaacag ctcctcgccc ttgctcacca tggtggcgac
     9661 cggtagcgct agcggatctg acggttcact aaaccagctc tgcttatata gacctcccac
     9721 cgtacacgcc taccgcccat ttgcgtcaat ggggcggagt tgttacgaca ttttggaaag
     9781 tcccgttgat tttggtgcca aaacaaactc ccattgacgt caatggggtg gagacttgga
     9841 aatccccgtg agtcaaaccg ctatccacgc ccattgatgt actgccaaaa ccgcatcacc
     9901 atggtaatag cgatgactaa tacgtagatg tactgccaag taggaaagtc ccataaggtc
     9961 atgtactggg cacaatgcca ggcgggccat ttaccgtcat tgacgtcaat agggggcgta
    10021 cttggcatat gatacacttg atgtactgcc aagtgggcag tttaccgtaa atactccacc
    10081 cattgacgtc aatggaaagt ccctattggc gttactatgg gaacatacgt cattattgac
    10141 gtcaatgggc gggggtcgtt gggcggtcag ccaggcgggc catttaccgt aagttatgta
    10201 acgcggaacg cggccgcagg aacccctagt gatggagttg gccactccct ctctgcgcgc
    10261 tcgctcgctc actgaggccg ggcgaccaaa ggtcgcccga cgcccgggct ttgcccgggc
    10321 ggcctcagtg agcgagcgag cgcgcagctg cctgcagggg cgcctgatgc ggtattttct
    10381 ccttacgcat ctgtgcggta tttcacaccg catacgtcaa agcaaccata gtacgcgccc
    10441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
    10501 gccagcgcct tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
    10561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
    10621 cggcacctcg accccaaaaa acttgatttg ggtgatggtt cacgtagtgg gccatcgccc
    10681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
    10741 ttccaaactg gaacaacact caactctatc tcgggctatt cttttgattt ataagggatt
    10801 ttgccgattt cggtctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
    10861 tttaacaaaa tattaacgtt tacaatttta tggtgcactc tcagtacaat ctgctctgat
    10921 gccgcatagt taagccagcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct
    10981 tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt
    11041 cagaggtttt caccgtcatc accgaaacgc gcgagacgaa agggcctcgt gatacgccta
    11101 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
    11161 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
    11221 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
    11281 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
    11341 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
    11401 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
    11461 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
    11521 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
    11581 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
    11641 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
    11701 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
    11761 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
    11821 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
    11881 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
    11941 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg aagccgcggt
    12001 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
    12061 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
    12121 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
    12181 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
    12241 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
    12301 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
    12361 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
    12421 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
    12481 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
    12541 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
    12601 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
    12661 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
    12721 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
    12781 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
    12841 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
    12901 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgt
//
