LOCUS       sequence-181436-       11637 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Targeted CpG methylation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 11637)
  AUTHORS   Lei Y, Zhang X, Su J, Jeong M, Gundry MC, Huang YH, Zhou Y, Li W, 
            Goodell MA
  TITLE     Targeted DNA methylation in vivo using an engineered dCas9-MQ1 
            fusion protein.
  JOURNAL   Nat Commun. 2017 Jul 11;8:16026. doi: 10.1038/ncomms16026.
  PUBMED    28695892
REFERENCE   2  (bases 1 to 11637)
  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: "2017-07-11"; volume: "8:16026. doi"; pages: " 
            10.1038/ncomms16026"
COMMENT     Sequence Label: pcDNA3.1-dCas9-MQ1(WT)-EGFP
FEATURES             Location/Qualifiers
     source          1..11637
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      988..997
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             997..1062
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             1069..1089
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1099..1125,1126..1128,1129..3612,3613..3618,3619..3684,
                     3685..3687,3688..5202)
                     /codon_start=1
                     /product="catalytically dead mutant of the Cas9 
                     endonuclease from the Streptococcus pyogenes Type II 
                     CRISPR/Cas system"
                     /label=Cas9m4
                     /note="RNA-guided DNA-binding protein that lacks 
                     endonuclease activity due to the mutations D10A, D839A, 
                     H840A, and N863A (Mali et al., 2013)"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             join(1099..1125,1126..1128,1129..3615,3616..3618,
                     3619..5202)
                     /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="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             1099..5202
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     CDS             5215..5235
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6406..6459
                     /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(6466..6468,6469..7179)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(6466..6468,6469..7179)
                     /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(6508..6529)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6769..6788)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     7116..7137
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             7186..7203
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(7226..7243)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    7232..7456
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      7502..7930
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7589..7608)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7799..7820
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(7939..7959)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        7944..8273
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      8124..8259
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     8186..8205
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             8340..9134
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(8394..8413)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     9004..9023
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    9310..9431
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(9347..9366)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     9401..9420
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(9480..9496)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(9480..9496)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(9493..9515)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    9504..9520
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(9528..9534,9535..9552,9553..9558))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    9573..9594
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(9711..9728)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(9882..10470)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(9962..9981)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(10641..11432,11433..11501))
                     /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     11264..11283
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(11502..11606)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagt
      901 taagcttggt accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca
      961 gcacagtggc ggccgctcga gtctagagcc accatggact acaaagacca tgacggtgat
     1021 tataaagatc atgacatcga ttacaaggat gacgatgaca agatggcccc caagaagaag
     1081 aggaaggtgg gccgcggaat ggacaagaag tactccattg ggctcgccat cggcacaaac
     1141 agcgtcggct gggccgtcat tacggacgag tacaaggtgc cgagcaaaaa attcaaagtt
     1201 ctgggcaata ccgatcgcca cagcataaag aagaacctca ttggcgccct cctgttcgac
     1261 tccggggaaa ccgccgaagc cacgcggctc aaaagaacag cacggcgcag atatacccgc
     1321 agaaagaatc ggatctgcta cctgcaggag atctttagta atgagatggc taaggtggat
     1381 gactctttct tccataggct ggaggagtcc tttttggtgg aggaggataa aaagcacgag
     1441 cgccacccaa tctttggcaa tatcgtggac gaggtggcgt accatgaaaa gtacccaacc
     1501 atatatcatc tgaggaagaa gcttgtagac agtactgata aggctgactt gcggttgatc
     1561 tatctcgcgc tggcgcatat gatcaaattt cggggacact tcctcatcga gggggacctg
     1621 aacccagaca acagcgatgt cgacaaactc tttatccaac tggttcagac ttacaatcag
     1681 cttttcgaag agaacccgat caacgcatcc ggagttgacg ccaaagcaat cctgagcgct
     1741 aggctgtcca aatcccggcg gctcgaaaac ctcatcgcac agctccctgg ggagaagaag
     1801 aacggcctgt ttggtaatct tatcgccctg tcactcgggc tgacccccaa ctttaaatct
     1861 aacttcgacc tggccgaaga tgccaagctt caactgagca aagacaccta cgatgatgat
     1921 ctcgacaatc tgctggccca gatcggcgac cagtacgcag accttttttt ggcggcaaag
     1981 aacctgtcag acgccattct gctgagtgat attctgcgag tgaacacgga gatcaccaaa
     2041 gctccgctga gcgctagtat gatcaagcgc tatgatgagc accaccaaga cttgactttg
     2101 ctgaaggccc ttgtcagaca gcaactgcct gagaagtaca aggaaatttt cttcgatcag
     2161 tctaaaaatg gctacgccgg atacattgac ggcggagcaa gccaggagga attttacaaa
     2221 tttattaagc ccatcttgga aaaaatggac ggcaccgagg agctgctggt aaagcttaac
     2281 agagaagatc tgttgcgcaa acagcgcact ttcgacaatg gaagcatccc ccaccagatt
     2341 cacctgggcg aactgcacgc tatcctcagg cggcaagagg atttctaccc ctttttgaaa
     2401 gataacaggg aaaagattga gaaaatcctc acatttcgga taccctacta tgtaggcccc
     2461 ctcgcccggg gaaattccag attcgcgtgg atgactcgca aatcagaaga gaccatcact
     2521 ccctggaact tcgaggaagt cgtggataag ggggcctctg cccagtcctt catcgaaagg
     2581 atgactaact ttgataaaaa tctgcctaac gaaaaggtgc ttcctaaaca ctctctgctg
     2641 tacgagtact tcacagttta taacgagctc accaaggtca aatacgtcac agaagggatg
     2701 agaaagccag cattcctgtc tggagagcag aagaaagcta tcgtggacct cctcttcaag
     2761 acgaaccgga aagttaccgt gaaacagctc aaagaagact atttcaaaaa gattgaatgt
     2821 ttcgactctg ttgaaatcag cggagtggag gatcgcttca acgcatccct gggaacgtat
     2881 cacgatctcc tgaaaatcat taaagacaag gacttcctgg acaatgagga gaacgaggac
     2941 attcttgagg acattgtcct cacccttacg ttgtttgaag atagggagat gattgaagaa
     3001 cgcttgaaaa cttacgctca tctcttcgac gacaaagtca tgaaacagct caagaggcgc
     3061 cgatatacag gatgggggcg gctgtcaaga aaactgatca atgggatccg agacaagcag
     3121 agtggaaaga caatcctgga ttttcttaag tccgatggat ttgccaaccg gaacttcatg
     3181 cagttgatcc atgatgactc tctcaccttt aaggaggaca tccagaaagc acaagtttct
     3241 ggccaggggg acagtcttca cgagcacatc gctaatcttg caggtagccc agctatcaaa
     3301 aagggaatac tgcagaccgt taaggtcgtg gatgaactcg tcaaagtaat gggaaggcat
     3361 aagcccgaga atatcgttat cgagatggcc cgagagaacc aaactaccca gaagggacag
     3421 aagaacagta gggaaaggat gaagaggatt gaagagggta taaaagaact ggggtcccaa
     3481 atccttaagg aacacccagt tgaaaacacc cagcttcaga atgagaagct ctacctgtac
     3541 tacctgcaga acggcaggga catgtacgtg gatcaggaac tggacatcaa tcggctctcc
     3601 gactacgacg tggatgccat cgtgccccag tcttttctca aagatgattc tattgataat
     3661 aaagtgttga caagatccga taaaaataga gggaagagtg ataacgtccc ctcagaagaa
     3721 gttgtcaaga aaatgaaaaa ttattggcgg cagctgctga acgccaaact gatcacacaa
     3781 cggaagttcg ataatctgac taaggctgaa cgaggtggcc tgtctgagtt ggataaagcc
     3841 ggcttcatca aaaggcagct tgttgagaca cgccagatca ccaagcacgt ggcccaaatt
     3901 ctcgattcac gcatgaacac caagtacgat gaaaatgaca aactgattcg agaggtgaaa
     3961 gttattactc tgaagtctaa gctggtctca gatttcagaa aggactttca gttttataag
     4021 gtgagagaga tcaacaatta ccaccatgcg catgatgcct acctgaatgc agtggtaggc
     4081 actgcactta tcaaaaaata tcccaagctt gaatctgaat ttgtttacgg agactataaa
     4141 gtgtacgatg ttaggaaaat gatcgcaaag tctgagcagg aaataggcaa ggccaccgct
     4201 aagtacttct tttacagcaa tattatgaat tttttcaaga ccgagattac actggccaat
     4261 ggagagattc ggaagcgacc acttatcgaa acaaacggag aaacaggaga aatcgtgtgg
     4321 gacaagggta gggatttcgc gacagtccgg aaggtcctgt ccatgccgca ggtgaacatc
     4381 gttaaaaaga ccgaagtaca gaccggaggc ttctccaagg aaagtatcct cccgaaaagg
     4441 aacagcgaca agctgatcgc acgcaaaaaa gattgggacc ccaagaaata cggcggattc
     4501 gattctccta cagtcgctta cagtgtactg gttgtggcca aagtggagaa agggaagtct
     4561 aaaaaactca aaagcgtcaa ggaactgctg ggcatcacaa tcatggagcg atcaagcttc
     4621 gaaaaaaacc ccatcgactt tctcgaggcg aaaggatata aagaggtcaa aaaagacctc
     4681 atcattaagc ttcccaagta ctctctcttt gagcttgaaa acggccggaa acgaatgctc
     4741 gctagtgcgg gcgagctgca gaaaggtaac gagctggcac tgccctctaa atacgttaat
     4801 ttcttgtatc tggccagcca ctatgaaaag ctcaaagggt ctcccgaaga taatgagcag
     4861 aagcagctgt tcgtggaaca acacaaacac taccttgatg agatcatcga gcaaataagc
     4921 gaattctcca aaagagtgat cctcgccgac gctaacctcg ataaggtgct ttctgcttac
     4981 aataagcaca gggataagcc catcagggag caggcagaaa acattatcca cttgtttact
     5041 ctgaccaact tgggcgcgcc tgcagccttc aagtacttcg acaccaccat agacagaaag
     5101 cggtacacct ctacaaagga ggtcctggac gccacactga ttcatcagtc aattacgggg
     5161 ctctatgaaa caagaatcga cctctctcag ctcggtggag acagcagggc tgaccccaag
     5221 aagaagagga aggtggctag ggacagcaaa gtggagaaca aaacaaagaa gctgagagtg
     5281 ttcgaagcct tcgccggcat tggcgcccag agaaaggccc tggagaaagt gaggaaggac
     5341 gagtacgaga tcgtgggact ggccgagtgg tatgtgcccg ccatcgtcat gtaccaggcc
     5401 atccataaca acttccacac caagctcgag tacaagtccg tcagcagaga ggagatgatc
     5461 gactacctcg agaacaagac cctgtcctgg aacagcaaga accccgtcag caacggatac
     5521 tggaagagga agaaggatga cgagctgaag atcatctaca acgccatcaa gctgtccgaa
     5581 aaggagggca acattttcga catcagggac ctctacaaga ggacactgaa gaacatcgac
     5641 ctgctcacct acagcttccc ttgccaggac ctgagccagc aaggcatcca gaagggcatg
     5701 aagaggggaa gcggcaccag atccggcctg ctctgggaga tcgaaagagc cctggactcc
     5761 accgagaaga acgacctgcc taagtacctc ctcatggaga acgtgggagc cctgctgcac
     5821 aagaagaacg aggaggagct gaaccaatgg aagcagaagc tggagagcct gggctaccag
     5881 aacagcatcg aagtcctcaa tgctgccgat ttcggatcca gccaggccag gaggagagtg
     5941 ttcatgatct ccaccctcaa tgagttcgtg gaactgccta agggcgacaa gaagcccaag
     6001 agcatcaaaa aggtgctgaa caagatcgtg agcgagaagg acatcctcaa caacctgctg
     6061 aaatacaacc tcaccgaatt caagaagacc aagtccaaca tcaacaaggc cagcctgatc
     6121 ggctactcca agttcaactc cgagggctat gtgtacgacc ccgagttcac aggccccaca
     6181 ctgacagcta gcggcgccaa ctccaggatc aagatcaagg acggcagcaa catcaggaag
     6241 atgaacagcg acgagacctt cctgtacatc ggctttgaca gccaggacgg caagagagtg
     6301 aacgaaatcg agttcctgac cgagaaccag aagatcttcg tgtgtggcaa cagcatcagc
     6361 gtggaggtgc tggaggccat cattgacaag atcggcggcc ctagcgaggg cagaggaagt
     6421 cttctaacat gcggtgacgt ggaggagaat cccggccctg gtaccgtgag caagggcgag
     6481 gagctgttca ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac
     6541 aagttcagcg tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag
     6601 ttcatctgca ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc
     6661 tacggcgtgc agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag
     6721 tccgccatgc ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac
     6781 tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg
     6841 aagggcatcg acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac
     6901 aacagccaca acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc
     6961 aagatccgcc acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac
     7021 acccccatcg gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc
     7081 gccctgagca aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc
     7141 gccgccggga tcactctcgg catggacgag ctgtacaaga ccggtcatca tcaccatcac
     7201 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     7261 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     7321 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     7381 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     7441 gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag ggggtatccc
     7501 cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc
     7561 gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc
     7621 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt
     7681 agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc acgtagtggg
     7741 ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt
     7801 ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc ttttgattta
     7861 taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt
     7921 aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     7981 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     8041 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     8101 tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc
     8161 cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc tctgcctctg
     8221 agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctcc
     8281 cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga tcgtttcgca
     8341 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     8401 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     8461 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     8521 aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     8581 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     8641 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     8701 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     8761 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     8821 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc atgcccgacg
     8881 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     8941 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     9001 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     9061 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     9121 acgagttctt ctgagcggga ctctggggtt cgcgaaatga ccgaccaagc gacgcccaac
     9181 ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     9241 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     9301 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     9361 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     9421 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     9481 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     9541 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     9601 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     9661 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     9721 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     9781 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     9841 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     9901 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     9961 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
    10021 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
    10081 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
    10141 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
    10201 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
    10261 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
    10321 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
    10381 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
    10441 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
    10501 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
    10561 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
    10621 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
    10681 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
    10741 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
    10801 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
    10861 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
    10921 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
    10981 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
    11041 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
    11101 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
    11161 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
    11221 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
    11281 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
    11341 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
    11401 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
    11461 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
    11521 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
    11581 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
