LOCUS       sequence-438166-       12048 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses dCAS9-DNMT3A-KRAB fusion protein and double marker 
            GFP-T2A-Puro under independent promoters.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 12048)
  TITLE     Systemic comparison of dCas9-based DNA methylation editing systems 
            for specificity and stability
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 12048)
  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: 
            "Unpublished"
COMMENT     Sequence Label: M3-dCAS9-DNMT3A-KRAB (dCas9-3A-KRAB)
FEATURES             Location/Qualifiers
     source          1..12048
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(28..47)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      841..1429
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1330..1349
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1491..1739
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1491..1511
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1662..1681
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1758..1833
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     enhancer        1930..2215
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        2217..2493
                     /label=chicken beta-actin promoter
     intron          2494..2721
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      2733..2742
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2742..2807
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             2814..2834
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2859..2882,2883..2885,2886..5372,5373..5375,
                     5376..6959)
                     /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             2859..6959
                     /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             6960..7007
                     /codon_start=1
                     /product="bipartite nuclear localization signal from 
                     nucleoplasmin"
                     /label=nucleoplasmin NLS
                     /translation="KRPAATKKAGQAKKKK"
     CDS             7035..7967
                     /codon_start=1
                     /product="catalytic domain of DNA methyltransferase 3A"
                     /label=DNMT3A catalytic domain
                     /translation="PSRLQMFFANNHDQEFDPPKVYPPVPAEKRKPIRVLSLFDGIATG
                     LLVLKDLGIQVDRYIASEVCEDSITVGMVRHQGKIMYVGDVRSVTQKHIQEWGPFDLVI
                     GGSPCNDLSIVNPARKGLYEGTGRLFFEFYRLLHDARPKEGDDRPFFWLFENVVAMGVS
                     DKRDISRFLESNPVMIDAKEVSAAHRARYFWGNLPGMNRPLASTVNDKLELQECLEHGR
                     IAKFSKVRTITTRSNSIKQGKDQHFPVFMNEKEDILWCTEMERVFGFPVHYTDVSNMSR
                     LARQRLLGRSWSVPVIRHLFAPLKEYFACV"
     CDS             8034..8054
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             8079..8273
                     /codon_start=1
                     /product="Kruppel-associated box (KRAB) transcriptional 
                     repression domain from the human zinc finger protein ZNF10 
                     (Margolin et al., 1994)"
                     /label=KRAB
                     /translation="RTLVTFKDVFVDFTREEWKLLDTAQQIVYRNVMLENYKNLVSLGY
                     QLTKPDVILRLEKGEEPWLV"
     primer_bind     complement(8318..8335)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    8324..8531
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        8546..8849
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        8850..9053
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     9004..9024
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      9092..9101
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(9098..9100,9101..9103,9104..9814)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(9098..9100,9101..9103,9104..9814)
                     /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(9404..9423)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     9751..9772
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             9845..9898
                     /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             9899..10498
                     /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(9899..9918)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     polyA_signal    10666..10787
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(10703..10722)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     10757..10776
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   10801..10941
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   10801..10930
                     /label=AAV2 ITR
     rep_origin      11016..11471
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(11103..11122)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     11313..11334
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(11488..11507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     11607..11629
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(11667..11685)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        11753..11857
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(11858..11926,11927..12048,1..670)
                     /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"
ORIGIN
        1 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtattgacgc
       61 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
      121 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
      181 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
      241 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
      301 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgtagcaat
      361 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
      421 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
      481 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggaagcc gcggtatcat
      541 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
      601 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
      661 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
      721 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
      781 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
      841 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
      901 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      961 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
     1021 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     1081 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     1141 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     1201 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     1261 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     1321 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     1381 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     1441 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt gagggcctat
     1501 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     1561 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     1621 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat atgcttaccg
     1681 taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga cgaaacaccg
     1741 ggtcttcgag aagacctgtt ttagagctag aaatagcaag ttaaaataag gctagtccgt
     1801 tatcaacttg aaaaagtggc accgagtcgg tgcttttttg ttttagagct agaaatagca
     1861 agttaaaata aggctagtcc gtttttagcg cgtgcgccaa ttctgcagac aaatggctct
     1921 agaggtaccc gttacataac ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc
     1981 ccgcccattg acgtcaatag taacgccaat agggactttc cattgacgtc aatgggtgga
     2041 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
     2101 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tgtgcccagt acatgacctt
     2161 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtcga
     2221 ggtgagcccc acgttctgct tcactctccc catctccccc ccctccccac ccccaatttt
     2281 gtatttattt attttttaat tattttgtgc agcgatgggg gcgggggggg gggggggcgc
     2341 gcgccaggcg gggcggggcg gggcgagggg cggggcgggg cgaggcggag aggtgcggcg
     2401 gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg gcggcggcgg
     2461 cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg ccttcgcccc
     2521 gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac cgcgttactc
     2581 ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagct gagcaagagg
     2641 taagggttta agggatggtt ggttggtggg gtattaatgt ttaattacct ggagcacctg
     2701 cctgaaatca ctttttttca ggttggaccg gtgccaccat ggactataag gaccacgacg
     2761 gagactacaa ggatcatgat attgattaca aagacgatga cgataagatg gccccaaaga
     2821 agaagcggaa ggtcggtatc cacggagtcc cagcagccga caagaagtac agcatcggcc
     2881 tggccatcgg caccaactct gtgggctggg ccgtgatcac cgacgagtac aaggtgccca
     2941 gcaagaaatt caaggtgctg ggcaacaccg accggcacag catcaagaag aacctgatcg
     3001 gagccctgct gttcgacagc ggcgaaacag ccgaggccac ccggctgaag agaaccgcca
     3061 gaagaagata caccagacgg aagaaccgga tctgctatct gcaagagatc ttcagcaacg
     3121 agatggccaa ggtggacgac agcttcttcc acagactgga agagtccttc ctggtggaag
     3181 aggataagaa gcacgagcgg caccccatct tcggcaacat cgtggacgag gtggcctacc
     3241 acgagaagta ccccaccatc taccacctga gaaagaaact ggtggacagc accgacaagg
     3301 ccgacctgcg gctgatctat ctggccctgg cccacatgat caagttccgg ggccacttcc
     3361 tgatcgaggg cgacctgaac cccgacaaca gcgacgtgga caagctgttc atccagctgg
     3421 tgcagaccta caaccagctg ttcgaggaaa accccatcaa cgccagcggc gtggacgcca
     3481 aggccatcct gtctgccaga ctgagcaaga gcagacggct ggaaaatctg atcgcccagc
     3541 tgcccggcga gaagaagaat ggcctgttcg gcaacctgat tgccctgagc ctgggcctga
     3601 cccccaactt caagagcaac ttcgacctgg ccgaggatgc caaactgcag ctgagcaagg
     3661 acacctacga cgacgacctg gacaacctgc tggcccagat cggcgaccag tacgccgacc
     3721 tgtttctggc cgccaagaac ctgtccgacg ccatcctgct gagcgacatc ctgagagtga
     3781 acaccgagat caccaaggcc cccctgagcg cctctatgat caagagatac gacgagcacc
     3841 accaggacct gaccctgctg aaagctctcg tgcggcagca gctgcctgag aagtacaaag
     3901 agattttctt cgaccagagc aagaacggct acgccggcta cattgacggc ggagccagcc
     3961 aggaagagtt ctacaagttc atcaagccca tcctggaaaa gatggacggc accgaggaac
     4021 tgctcgtgaa gctgaacaga gaggacctgc tgcggaagca gcggaccttc gacaacggca
     4081 gcatccccca ccagatccac ctgggagagc tgcacgccat tctgcggcgg caggaagatt
     4141 tttacccatt cctgaaggac aaccgggaaa agatcgagaa gatcctgacc ttccgcatcc
     4201 cctactacgt gggccctctg gccaggggaa acagcagatt cgcctggatg accagaaaga
     4261 gcgaggaaac catcaccccc tggaacttcg aggaagtggt ggacaagggc gcttccgccc
     4321 agagcttcat cgagcggatg accaacttcg ataagaacct gcccaacgag aaggtgctgc
     4381 ccaagcacag cctgctgtac gagtacttca ccgtgtataa cgagctgacc aaagtgaaat
     4441 acgtgaccga gggaatgaga aagcccgcct tcctgagcgg cgagcagaaa aaggccatcg
     4501 tggacctgct gttcaagacc aaccggaaag tgaccgtgaa gcagctgaaa gaggactact
     4561 tcaagaaaat cgagtgcttc gactccgtgg aaatctccgg cgtggaagat cggttcaacg
     4621 cctccctggg cacataccac gatctgctga aaattatcaa ggacaaggac ttcctggaca
     4681 atgaggaaaa cgaggacatt ctggaagata tcgtgctgac cctgacactg tttgaggaca
     4741 gagagatgat cgaggaacgg ctgaaaacct atgcccacct gttcgacgac aaagtgatga
     4801 agcagctgaa gcggcggaga tacaccggct ggggcaggct gagccggaag ctgatcaacg
     4861 gcatccggga caagcagtcc ggcaagacaa tcctggattt cctgaagtcc gacggcttcg
     4921 ccaacagaaa cttcatgcag ctgatccacg acgacagcct gacctttaaa gaggacatcc
     4981 agaaagccca ggtgtccggc cagggcgata gcctgcacga gcacattgcc aatctggccg
     5041 gcagccccgc cattaagaag ggcatcctgc agacagtgaa ggtggtggac gagctcgtga
     5101 aagtgatggg ccggcacaag cccgagaaca tcgtgatcga aatggccaga gagaaccaga
     5161 ccacccagaa gggacagaag aacagccgcg agagaatgaa gcggatcgaa gagggcatca
     5221 aagagctggg cagccagatc ctgaaagaac accccgtgga aaacacccag ctgcagaacg
     5281 agaagctgta cctgtactac ctgcagaatg ggcgggatat gtacgtggac caggaactgg
     5341 acatcaaccg gctgtccgac tacgatgtgg acgctatcgt gcctcagagc tttctgaagg
     5401 acgactccat cgacaacaag gtgctgacca gaagcgacaa gaaccggggc aagagcgaca
     5461 acgtgccctc cgaagaggtc gtgaagaaga tgaagaacta ctggcggcag ctgctgaacg
     5521 ccaagctgat tacccagaga aagttcgaca atctgaccaa ggccgagaga ggcggcctga
     5581 gcgaactgga taaggccggc ttcatcaaga gacagctggt ggaaacccgg cagatcacaa
     5641 agcacgtggc acagatcctg gactcccgga tgaacactaa gtacgacgag aatgacaagc
     5701 tgatccggga agtgaaagtg atcaccctga agtccaagct ggtgtccgat ttccggaagg
     5761 atttccagtt ttacaaagtg cgcgagatca acaactacca ccacgcccac gacgcctacc
     5821 tgaacgccgt cgtgggaacc gccctgatca aaaagtaccc taagctggaa agcgagttcg
     5881 tgtacggcga ctacaaggtg tacgacgtgc ggaagatgat cgccaagagc gagcaggaaa
     5941 tcggcaaggc taccgccaag tacttcttct acagcaacat catgaacttt ttcaagaccg
     6001 agattaccct ggccaacggc gagatccgga agcggcctct gatcgagaca aacggcgaaa
     6061 ccggggagat cgtgtgggat aagggccggg attttgccac cgtgcggaaa gtgctgagca
     6121 tgccccaagt gaatatcgtg aaaaagaccg aggtgcagac aggcggcttc agcaaagagt
     6181 ctatcctgcc caagaggaac agcgataagc tgatcgccag aaagaaggac tgggacccta
     6241 agaagtacgg cggcttcgac agccccaccg tggcctattc tgtgctggtg gtggccaaag
     6301 tggaaaaggg caagtccaag aaactgaaga gtgtgaaaga gctgctgggg atcaccatca
     6361 tggaaagaag cagcttcgag aagaatccca tcgactttct ggaagccaag ggctacaaag
     6421 aagtgaaaaa ggacctgatc atcaagctgc ctaagtactc cctgttcgag ctggaaaacg
     6481 gccggaagag aatgctggcc tctgccggcg aactgcagaa gggaaacgaa ctggccctgc
     6541 cctccaaata tgtgaacttc ctgtacctgg ccagccacta tgagaagctg aagggctccc
     6601 ccgaggataa tgagcagaaa cagctgtttg tggaacagca caagcactac ctggacgaga
     6661 tcatcgagca gatcagcgag ttctccaaga gagtgatcct ggccgacgct aatctggaca
     6721 aagtgctgtc cgcctacaac aagcaccggg ataagcccat cagagagcag gccgagaata
     6781 tcatccacct gtttaccctg accaatctgg gagcccctgc cgccttcaag tactttgaca
     6841 ccaccatcga ccggaagagg tacaccagca ccaaagaggt gctggacgcc accctgatcc
     6901 accagagcat caccggcctg tacgagacac ggatcgacct gtctcagctg ggaggcgaca
     6961 aaaggccggc ggccacgaaa aaggccggac aggccaaaaa gaaaaagctc gagggcggag
     7021 gcgggagcgg atccccctcc cggctccaga tgttcttcgc taataaccac gaccaggaat
     7081 ttgaccctcc aaaggtttac ccacctgtcc cagctgagaa gaggaagccc atccgggtgc
     7141 tgtctctctt tgatggaatc gctacagggc tcctggtgct gaaggacttg ggcattcagg
     7201 tggaccgcta cattgcctcg gaggtgtgtg aggactccat cacggtgggc atggtgcggc
     7261 accaggggaa gatcatgtac gtcggggacg tccgcagcgt cacacagaag catatccagg
     7321 agtggggccc attcgatctg gtgattgggg gcagtccctg caatgacctc tccatcgtca
     7381 accctgctcg caagggcctc tacgagggca ctggccggct cttctttgag ttctaccgcc
     7441 tcctgcatga tgcgcggccc aaggagggag atgatcgccc cttcttctgg ctctttgaga
     7501 atgtggtggc catgggcgtt agtgacaaga gggacatctc gcgatttctc gagtccaacc
     7561 ctgtgatgat tgatgccaaa gaagtgtcag ctgcacacag ggcccgctac ttctggggta
     7621 accttcccgg tatgaacagg ccgttggcat ccactgtgaa tgataagctg gagctgcagg
     7681 agtgtctgga gcatggcagg atagccaagt tcagcaaagt gaggaccatt actacgaggt
     7741 caaactccat aaagcagggc aaagaccagc attttcctgt gttcatgaat gagaaagagg
     7801 acatcttatg gtgcactgaa atggaaaggg tatttggttt cccagtccac tatactgacg
     7861 tctccaacat gagccgcttg gcgaggcaga gactgctggg ccggtcatgg agcgtgccag
     7921 tcatccgcca cctcttcgct ccgctgaagg agtattttgc gtgtgtgtcc ggccggccta
     7981 gcgctggagg aggtggaagc ggaggaggag gaagcggagg aggaggtagc ggacctaaga
     8041 aaaagaggaa ggtggcggcc gctggatccg gacgggctag aacactggtt acgttcaagg
     8101 acgtgtttgt ggactttaca cgtgaggagt ggaaattgct ggatactgcg caacaaattg
     8161 tgtatcgaaa tgtcatgctt gagaattaca agaacctcgt cagtctcgga taccagttga
     8221 cgaaaccgga tgtgatcctt aggctcgaaa agggggaaga accttggctg gtatagggcc
     8281 ggcccgaatt caattctaac tagagctcgc tgatcagcct cgactgtgcc ttctagttgc
     8341 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     8401 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     8461 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga gaatagcagg
     8521 catgctgggg agcggccgcg ttccgcgtta cataacttac ggtaaatggc ccgcctggct
     8581 gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc
     8641 caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact gcccacttgg
     8701 cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat gacggtaaat
     8761 ggcccgcctg gcattgtgcc cagtacatga ccttatggga ctttcctact tggcagtaca
     8821 tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac atcaatgggc
     8881 gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac gtcaatggga
     8941 gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac tccgccccat
     9001 tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga gctggtttag
     9061 tgaaccgtca gatccgctag cgctaccggt cgccaccatg gtgagcaagg gcgaggagct
     9121 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     9181 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     9241 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     9301 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     9361 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     9421 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     9481 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     9541 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     9601 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     9661 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     9721 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     9781 cgggatcact ctcggcatgg acgagctgta caagtccgga ctcagatctc gagctcaagc
     9841 ttctgagggc agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccaat
     9901 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     9961 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
    10021 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
    10081 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
    10141 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
    10201 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
    10261 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
    10321 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
    10381 gacctccgcg ccccacaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
    10441 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgaaa
    10501 gcttcgaatt ctgcagtcga cggtaccgcg ggcccgggat ccaccggatc tagataactg
    10561 atcataatca gccataccac atttgtagag gttttacttg ctttaaaaaa cctcccacac
    10621 ctccccctga acctgaaaca taaaatgaat gcaattgttg ttgttaactt gtttattgca
    10681 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
    10741 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttaacg cggcggccgc
    10801 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
    10861 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
    10921 gagcgcgcag ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg
    10981 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag
    11041 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc
    11101 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
    11161 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
    11221 aaaacttgat ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg
    11281 ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac
    11341 actcaactct atctcgggct attcttttga tttataaggg attttgccga tttcggtcta
    11401 ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac
    11461 gtttacaatt ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca
    11521 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
    11581 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
    11641 atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt
    11701 catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac
    11761 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
    11821 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
    11881 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
    11941 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
    12001 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttt
//
